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Park: New Genus Scolizona Lecithoceridae (Lepidoptera, Gelechioidea)303 LECITHOCERIDAE (LEPIDOPTERA, GELECHIOIDEA) OF NEW GUINEA, PART III: A NEW GENUS SCOLIZONA WITH DESCRIPTION OF TWO NEW SPECIES K YU -T EK P ARK The Korean Academy of Science and Technology, #7-1 Gumi-dong, Seongnam, Gyounggi Prov., 463-808 Korea McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida, Gainesville, FL 32611 USA E-mail:ktpark02@gmail.com A BSTRACT As the third part of a serial study on the family Lecithoceridae (Lepidoptera, Gelechioidea) of New Guinea, a new genus, Scolizona gen. nov. is described, with the type species, S. rhinoceros (Diakonoff 1954) comb. nov. and two additional new species: S. ulnaformis sp. nov. and S. palinoides sp. nov. Adults, wing venations, the male genitalia of these three species, and the female genitailia of S. ulnaformis sp. nov. are illustrated, and a key to the species of the new genus is given. K ey Words: Taxonomy, new genus, new species, Scolizona New Guinea R ESUMEN En la tercera parte de una serie de estudios sobre la familia Lecithoceridae (Lepidoptera, Gelechioidea) de Nueva Guinea, se describe un nuevo gnero, Scolizona gen. nov. con la especie tipo S. rhinoceros (Diakonoff 1954) comb. nov. y dos addicionales nuevas especies: S. ulnaformis sp. nov. y S. palinoides sp. nov. Se ilustran los adultos, la nervadura de las alas y los genitales de los mac hos de estas tres especies, los genitales de la hembra de S. ulnaformis sp. nov. y se provee una clave de las especies en este nuevo gnero. The family Lecithoceridae (Gelechioidea) on the island of New Guinea (including West Papua (Irian Jaya) of Indonesia in the western part, and Papua New Guinea in the eastern part) is poorly known as well as other micro-moths. The family is mostly distributed in the Oriental and Australian Regions, comprising more than 1,100 species. The family is characterized by the very long antenna, usually longer than the forewing, and the male genitalia with gnathos bent downwards or absent. These characters are useful to differentiate from other gelechioid-moths. With respect to Lecithoceridae biology, the larva of Crocanthes prasinopis Meyrick (type species of the genus Crocanthes Meyrick) is reported to feed only on eucalypt lea ves in Australia (Common 1990). New Guinea is geographically close to Australia, but New Guineas Lecithoceridae fauna differs greatly from that of Australia, with a very high endemism in the composition of the species, i.e., more than 90% of Crocanthes are unique to New Guinea. Gielis (2003), in his recent study for the family Pterophoridae of the island, also mentioned that the majorities of species of the 2 areas do not overlap in distribution. These results may, of course, inadequately reect the actual diversity of species in the region, because it has been based on limited collecting results and some fragmentary reports by the few early workers, i.e., Walker (1864); Durrant (1915); and Meyrick (1910, 1918, 1925, 1929, 1931, 1938). Diakonoff (1954) published a comprehensive study for the microlepidoptera of New Guinea, listing 78 species of Lecithoceridae, as a part of Gelechiidae: 31 species of Lecithocera 40 species of Crocanthes and 7 species of little known genera, i.e., Gonaepa Meyrick, Periphorectis Meyrick, Spenocrates Meyrick, and Asmenistis Meyrick. His study was based on material collected in the western part of the island, Irian J aya, Indonesia by the American-Netherlands-Indian Expedition (the 3rd Archbold Expedition) in 1938 and 1939 (Fig. 1). Park (2010) recently reported a new Thubana species for the rst time from New Guinea and at the same time he reviewed the genus Telephata Meyrick, describing two new species In addition, Park and Byun (2010) described, Neopectinimura Park, along with descriptions of 6 new species from P apua New Guinea. The aim of this study was to identify undetermined materials of the family collected from Papua New Guinea since 1975 and that are preserved in the National Museum of Natural History (USNM), USA, and those from Irian Jaya of Indonesia in 2005 that are in the Zoological Museum Amsterdam (ZMAN), the Netherlands. In an earlier report as part of this serial study on the Lecithoceridae (Lepidoptera, Gelechioidea)

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304 Florida Entomologist 94(2) June 2011 of New Guinea (Irian Jaya of Indonesia and Papua New Guinea), a new genus, Onnuria Park, gen. nov. including three new species, and Hamatina Park, gen. nov. including four new species were describes (P ark 2011b, c). M ATERIALS AND M ETHODS This study is based (i) on specimens deposited in the National Museum of Natural History (USNM), Washington, D.C., USA, collected from Papua New Guinea by G. F. Hevel and R. E. Dietz IV in 1976, Scott E. and Pamela Miller in 1983, and V. O. Becker in 1992, and (ii) specimens in the Zoological Museum Amsterdam (ZMAN), The Netherlands, collected from Irian Jaya of Indonesia by Drs. Rob de Vos and colleagues in several expeditions during last two decades. All species described herein were compared with the types of Lecithocera described by Diakonff (1954), which are deposited in the Rijksmuseum van Natuurlijke Historie (RMNH), Leiden, The Netherlands. The new species also were compared with the original descriptions of some early known species of Lecithocera described by Durrant (1915) and Meyric k (1910, 1918, 1929, 1931, 1938) from New Guinea, the types of which could not be found. Indeed they probably are lost or have been destroyed (Clarke 1955: 31). The types of L. deloma Durrant, 1915 and L. strigosa Durrant, 1915 are known to be deposited in The Natural History Museum, London (BMNH), but they are not found. The locations of the types of 6 species described by Meyrick (i.e., autodyas coleasta praecentrix prudens squamifera and tamiodes ) are unknown, and the types of strepsicrena Meyric k, stelophanes Meyrick, and staurophora Meyric k are in the BMNH. Lecithocera invariellla Walker (1846) was listed in the key by Diakonoff (1954), but it was not described from New Guinea, but was erroneously cited from there. The new species of Scolizona gen. nov. described in this paper are easily distinguished from any of the above species by ha ving a characteristically specialized labial palpus as a diagnostic character in their description. The wingspan is measured from the left apex to the right apex of the forewing. Images of genitalia and wings were captured with the Automontage Microscopic System at the Florida State of Collection of Arthropods, Division of Plant Industry, Gainesville, Florida, USA. The color standard for the description of adults follows Kornerup and Wanscher (1978), and the morphological termiFig. 1. Map of New Guinea (Indonesian part and Papua New Guinea) with the area of the 3rd Archbold Expedition in 1938-1939.

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Park: New Genus Scolizona Lecithoceridae (Lepidoptera, Gelechioidea)305 nology follows Park (1999). Types are deposited in the USNM or ZMAN on indenite loan from Papua New Guinea or Indonesia. S YSTEMATICS Genus Scolizona Park, gen. nov. Type species: Lecithocera rhinoceros Diakonoff, 1954: 47. The new genus is one of the genera related to Lecithocera Herrich-Schffer by having a similar venation and the male genital c haracter. However, the genus is characterized by the uniquely specialized labial palpus: 1st segment relatively long; 2nd segment remarkably stout, strongly recurved backwards and exceeding vertex, with long hair-pencils apically; 3rd segment considerably variable in the size and shape, as long as the 2nd or much longer than 2nd segment, with long hair-pencils. External Morphology. Head roughly scaled, with yellowish-brown to dark-brown scales dorsally. Antenna longer than forewing, with slender basal joint, without pectin; agellum sometimes with blackish basal and preapical parts, with usually whitish apex. Labial palpus very stout, with appressed or rough scales; rst segment relatively long, often about half the length of 2nd segment; 2nd segment attened laterally, strongly recurved, longitudinally furrowed on inner surface with hair-like, long scale-tufts apically; 3rd segment as long as the 2nd, or extremely long, with hair-like, long scale-tufts, these hairs usually appressed, but sometimes erect (Figs. 3a and 4c). Forewing irregularly covered with dark brown scales, more densely scattered in base of costal area, with a pair of large blackish discal spots before middle and near end of cell, usually anterior one larger, elongate; apex more or less obtuse; termen sinuate; fringe usually with paleorange basal line; venation with R 1 arising before middle of cell; distance R 1 and R 2 more than 1.5 times than that of R 2 and R 3 ; R 3 and R 4+5 stalked before middle; R 4 and R 5 stalked for more than 2/ 3 length; R 5 reaching termen; M 1 close to R 3+4+5 ; M 2 approximate to M 3 at base; CuA 1 and CuA 2 shortstalked; anal vein well developed; cell closed with weak cross vein. Hindwing pale gray, slightly broader than forewing, nearly trapezoidal; apex more or less acute; termen slightly sinuate; venation with Rs and M 1 connate or short-stalked; M 2 well developed, closely approximated to M 3 at base or stalked with M 3 +CuA 1 ; M 3 and CuA 1 short stalked; CuA 2 arising from near lower corner of cell; cell partly closed. Hind tibia roughly scaled all around. Abdomen has no spinous zones on tergites. Male Genitalia. Basal lobes of uncus usually ovate, directed outwardly. Gnathos strongly bent preapically. Costal bar sharply angulated at middle. Valva broad basally; cucullus elongate, with one or double stout spikes under a row of comb in 2/3 length on ventral margin and dense bristles along ventral margin. Juxta deeply or slightly concave on caudal margin. Aedeagus very stout, bent medially, as long as valve or slightly longer, with complex of heavily sclerotized plates and broad plate with numerous spicules dorsally. Seventh sternite with long hair-pencils. Distribution. Irian Jaya of Indonesia and Papua New Guinea. Etymology. The generic name is derived from the Greek, scoli (= curved) and zona (= belt) referring to the strongly recurved labial palpus Remarks. This genus is remarkable for its large, recurved palpus which resembles those of many deltoids of Noctuidae. KEY TO SPECIES OF THE GENUS SCOLIZONA PARK1.Second segment of labial palpus curved, as long as 3rd; 3rd segment stout, nearly straight (Figs. 2a, 3a, b, c, and 5). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 Second segment of labial palpus slightly curved, less than 1/3 length of 3rd segment; 3rd segment extremely long, slender, bent before middle (Figs. 4a, b, and 6) . . . . . . . . . . .S palinoides Park, sp. nov 2.Flagellum of antenna blackish in basal 1/8 and apical 1/8, grayish orange speckled with dark-brown scales between them; second segment of labial palpus strongly bent anteriorly; forewing brownish yellow, with R4and R5 stalked beyond 3 and CuA1 stalked for 1/3 length; male genitalia with more slender cucullus, with double spikes at 2/3 on ventral edge . . . . . . . . . . . . . . . . . . S. rhinoceros (Diakonoff) agellum of antenna blackish wholly, except orange white between apical 8th and 9th, with white apex; Second segment of labial palpus weakly bent; forewing yellowish brown, with R4 and R5 stalked beyond 2/3 (Fig. 7); hindwing venation with M3 and CuA1 stalked for 1/4 length; male genitalia with less slender cucullus, with a single spike at 2/3 on ventral edge . . . . . . . . . . . . . . . . S ulnaformis Park, sp. nov Scolizona rhinoceros (Diakonoff 1954), comb. nov. (Figs. 2, 2a, 9, and 9a-b) Lecithocera rhinoceros Diakonoff, 1954. Microl. New Guinea, 4: 47.Diagnosis. Wingspan, 19-21 mm. This species is hardly distinguishable from S. ulnaformis sp.

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306 Florida Entomologist 94(2)June 2011nov by external characters, but it has a slightly larger and darker forewing. The venation of both wings also differs slightly: R4 and R5 stalked for more than 2/3 length in the forewing and CuA1and CuA2 with longer stalk than that of the latter. The male genitalia are also similar, but can be distinguished by the following description for the male genitalia: the cucullus more elongated, with nearly straight costal margin, with longer spike on ventral margin; and the aedeagus with cornutus-complex bearing elongate apical process and more heavily sclerotized dorsal projection. Female. Unknown. Male Genitalia (Figs. 9, 9a, and 9b): Very similar to those of S. ulnaformis sp. nov but differs as follows: cucullus more elongated; costal edge nearly straight; spikes at 2/3 on ventral edge double, longer than comb; juxta with shorter caudal lobes; cornutus-complex with more elongate apical lobe and strong spike-like projection before middle dorsally; 7th sternite triangularly convex medially on posterior margin, whereas truncate medially in S. ulnaformis sp. nov (Fig. 9b). Aedeagus with cornutus-complex consists of heavily sclerotized plates, with elongated apical process and a short, spike-like dorsal projection before Figs. 2-4. Adults (a: head in dorsal respect; b: labial palpus in lateral respect): 2, S. rhinoceros (Diakonoff), holotype in RMNH; 2a, ditto, dorsal aspect of head part; 2b, ditto, labial palpus; 3, S. ulnaformis sp. nov ., paratype 1; 3a, ditto, dorsal aspect of head part; 3b, ditto, labial palpus of male; 3c, lateral aspect of labial palpus of female; 4, S. palinoides sp. nov ., paratype; 4a, ditto, dorsal aspect of head part; 4b, ditto, labial palpus.

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Park: New Genus Scolizona Lecithoceridae (Lepidoptera, Gelechioidea)307middle; a broad plate with numerous spicules dorsally. Material Examined. Male (holotype in Rijksmuseum of Natuurlijke Historie (RMNH), Leiden), Araucaria Camp, 800 m, 21 iii 1939, slide no. 962 D.; 1 Indonesia, Irian Jaya, Kokamatan Oksibil, Mobilabol 1,340 m, 4S 140E, 21-25 ii 2005, disturbed montane forest, UNCENZMA Expedition Papua Indonesia 2005, gen. slide no. CIS-5951/Park Distribution. Irian Jaya (Indonesia). Remarks. After the species was described by Diakonoff (1954), an additional male was newly found in the area not far from the type locality.Scolizona ulnaformis Park, sp. nov (Figs. 3, 3a-c, 5, 7, 10, 10a-b, and 12)Diagnosis. This new species is externally very similar to the type species, S. rhinoceros (Diakonoff), but can be distinguished by the wholly blackish antenna with whitish color between apical 8th and 9th; the forewing brownish yellow, with venation R4 and R5 stalked beyond and CuA1 with longer stalk in the hindwing; the backish basal part of agellum shorter, the 2nd segment of labial palpus less bent anteriorly; the male genitalia with a single spike at 2/3 on ventral edge, whereas double spikes in S. rhinoceros. The new species is distinguished from the following new species by the labial palpus with shorter thickened 3rd segment, whereas S. palinoides sp. nov has an extremely long 3rd segment, as illustrated in Figs. 4a and 4b. Description. Male & Female (Figs. 3, 3a-c, 5, and 7). Wingspan, 18.0-19.0 mm. External morphology: Head brownish yellow to yellowish brown. Antenna with basal joint slender, blackish dorsally, grayish orange ventroapically, without pectin; agellum wholly blackish except orange white part between apical 8th and 9th, with white apex. Labial palpus (Fig. 5) very stout; 1st segment relatively long, about half length of 2nd segment; 2nd segment thickened, brownish orange, speckled with dark-brown scales on outer surface, orange white and longitudinally furrowed on inner surface with hair-like, long scale-tuft apically; 3rd segment as long as 2nd, darker than 2nd on outer surface, with hair-like, long scale-tuft on inner surface, these hairs usually appressed, but sometimes erect (Figs. 3a and 5); apex obtuse. Tegula and thorax brownish yellow to yellowish brown. Forewing uniformly covered with brownFigs. 5-8. Labial palpus (5-6): 5, S. ulnaformis sp. nov ., paratype; 6, S. palinoides sp. nov ., paratype. Wing venation (7-8): 7, S. ulnaformis sp. nov ., paratype; 8, S. palinoides sp. nov ., paratype.

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308 Florida Entomologist 94(2)June 2011 Figs. 9-12. Male genitalia (a: aedeagus; b: 7th-8th abdominal segments): 9. S. rhinoceros Diakonoff: leftholotype wit h aedeagus; rightgen. slide no. CIS-5951; 10, S. ulnaformis sp. nov ., gen. slide no. CIS-5707; 11, S. palinoides sp. nov ., gen. slide no. CIS-5703; 12, Female genitalia of S. ulnaformis sp. nov ., gen. slide no. CIS-5960; 12a, Close-up signum.

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Park: New Genus Scolizona Lecithoceridae (Lepidoptera, Gelechioidea)309ish scales throughout; a pair of large blackish discal spots before middle and at end of cell, usually middle one larger; apex more or less obtuse; termen sinuate; fringe yellowish brown, with paleorange basal line; venation (Fig. 7) with R1 arising before middle of cell; distance R1-R2 about 1.5 times as long as R2-R3; R3 and R4+5 stalked for about 1/3 length; R4 and R5 stalked for 2/3 length; R5 reaching termen; M1 closed to R3+4+5; M2 nearly parallel to M1, closer to M3 at base; CuA1 and CuA2stalked for 1/5 length of CuA1; anal vein well developed; cell closed with weak cross vein. Hindwing pale gray, slightly broader than forewing, nearly trapezoidal; apex more or less acute; termen slightly sinuate; fringe yellowish brown, with pale orange basal line; venation with Rs and M1 connate; M2 well developed, closed to M3 at base; M3 and CuA1 stalked for 2/5 length; CuA2arising from near lower corner of cell; cell open. Abdomen brownish yellow dorsally; spinous zones on tergites absent. Male Genitalia (Figs. 10 and 10a-b). Basal lobes of uncus ovate, directed outwardly, forming Y-shape. Gnathos strongly bent preapically. Costal bar sharply angulated at middle. Valva broad basally, concave medially; cucullus nearly ovate, with gently arched costal margin, with dense bristles along ventral margin; with a single, small spike under a row of comb in 2/3 length on ventral margin, length of spike shorter than comb. Juxta deeply concave on caudal margin; caudal lobes long, about half the length of juxta. Aedeagus stout, bent medially; cornuti consist of complex of heavily sclerotized plates, with strongly curved apical process; median spike-like projection absent; with a broad plate with numerous spicules dorsally. Seventh sternite with long hair-pencil, truncate medially on posterior margin. Female Genitalia (Fig. 12). Apophyses anteriores about 1/2 length of apophyses posteriores. Ostial plate wide, membranous. Ductus bursae narrow in posterior 1/3, broad in anterior 2/3; ductus seminalis arising beyond middle, as broad as anterior part of ductus bursae, with large accessory sac. Corpus bursae ovate, relatively small; signum long, with transverse median groove. Holotype: Male, Papua New Guinea, Morobe Pr., Wau, Wau Ecol. Inst., 12-24 vii, 1983, S.E. & P.M. Miller, 1,200 m, Second Montane For., gen. slide No. CIS-5707/Park. Paratypes: 1 same locality as the holotype, 25-31 vii 1983, S.E. & P.M. Miller, 1,200 m, secondary Montane For., gen. slide No. CIS-5703/Park; 3 Morobe Prov., Wau, 8-14 xii 1976, mercury vapor light, C. F. Havel && R. F. Dietze; 9 2 Morobe, 17-30 IX 1992, V. O. Becker, Col. Becker, PNG. 840, gen. slide No. CIS5660/Park (male); 2 Papua New Guinea, Madang, Brahman Mission, 200 m, 11-15, X 1992, V. O. Becker Col.; Col. Becker, PNG 2991, gen. slide No. CIS-5704/Park. Distribution. Papua New Guinea (Morobe). Etymology. The species name is derived from Latin, ulna (= elbow) and formis (= forma), referring to the shape of labial palpus.Scolizona palinoides Park, sp. nov (Figs. 4, 4a-b, 6, 8, 11, and 11a-b)Diagnosis. The new species is distinguished from the preceding new species, S. ulnaformis sp. nov ., by the extremely long 3rd segment of labial palpus as in the gures 4b and 6. The structure of the male genitalia also can be a good separation character, with a nail-like projection apically in the aedeagus. Description. Male (Figs. 4, 4a-b, 6, and 8). Wingspan, 18.0-20.0 mm. External characters: Head yellowish brown. Antenna with slender, blackish basal segment, without pectin; agellum blackish in basal 1/8 length and in apical 1/8 length, orange white wholly between them. Labial palpus characteristic, with exceptionally unusual shape: 1st segment relatively long, about 1/ 3 as long as 2nd segment; 2nd segment thickened, triangularly dilated apically, dark brown on ventro-outer surface; 3rd segment more than 3 times as long as 2nd, basal half thickened, yellowish white, with long hair-like scale tuft ventrally, then slightly bent, narrowed toward apex, with acute apex (Fig. 6). Tegula and thorax yellowish brown. Forewing with dark-brown scales irregularly scattered, especially dense in basal area and below discal spots; a pair of large blackish discal spots before middle and near end of cell, usually middle one elongate; apex more or less acute; termen oblique, sinuate; fringe yellowish brown, with pale-orange basal line; venation (Fig. 8) similar to that of ulnaformis sp. nov Hindwing pale gray, with dark-brown scales sparsely scattered in the lower part of discal cell, broader than forewing, nearly trapezoidal; apex more or less acute; termen slightly sinuate; fringe yellowish brown, with pale orange basal line; venation with M2 and M3+CuA1 stalked for 1/4 length, whereas approximated in the latter as shown in the Fig. 7. Fore leg dark brown on femur and tibia ventrally; mid leg dark brown on femur and basal half of tibia, then yellowish white speckling with brownish scales; hind tibia slender, yellowish white. Abdomen pale orange; anal tuft orange; spinous zones on tergites absent. Male Genitalia (Figs. 11 and 11a-b). Uncus similar to that of S. ulnaformis and gnathos more slender. Costal bar less sharply angulated at middle. Valva broad basally, concave medially; cucullus with slightly concave or nearly straight costal margin; bristles on ventral margin more dense; spike at base of comb on ventral margin very small, about half length of that of S. ulnaformis Juxta slightly concave on caudal margin, with

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310 Florida Entomologist 94(2)June 2011small emargination at middle; caudal lobes not developed. Aedeagus stout, bent at basal 1/3; cornuti consist of two heavily sclerotized long plates, a plate with dense spicules, and with a nail-like strong spike apically. Holotype: Male, Papua New Guinea, Morobe Pr., Wau, Wau Ecol Inst., 25-31 vii, 1983, S. E. & P. M. Miller, 1,200m. UV Light, Montane For., gen. slide No. CIS-5703/Park. Paratypes: 6 same locality, 12-24 vii 1983, gen. slide No. CIS5747/Park, -5749/Park; 2 same locality, 1-10 viii 1983; 1 same locality, 23-31 viii 1983. Distribution. Papua New Guinea (Morobe). Etymology. The species name is derived from the Greek, palin (=backward) with sufx oides. Remarks. This new species has some differences from S. ulnaformis sp. nov by the extremely long 3rd segment of the labial palpus, and the hindwing venation with M2 stalked with M3 and CuA1, whereas they are free in S. ulnaformis sp. nov However, the author placed tentatively these two species in the same new genus Scolizona gen. nov. because they are very similar in other external and male genital characters. It is needed a further study when additional species are found. ACKNOWLEDGMENTSI am indebted to John Brown, Systematic Entomology Laboratory, U.S. Department of Agriculture, National Museum of Natural History, Washington D.C., and Rov de Vos, Zoological Museum, Amsterdam (ZMAN). The Netherlands for the loan of specimens; E. J. van Nieukerken, Rijksmuseum of Natuurlijke Historie (RMNH), Leiden, The Netherlands, for allowing me to examine Diakonoffs type specimens related to this study during my visit in August, 2010; John B. Heppner, McGuire Center, for his corrections and suggestions that improved the clarity of the manuscript.REFERENCES CITEDCOMMON, I. B. F. 1990. Superfamily Gelechioidea, 217266 pp. In I. B. F. Common [ed.], Moths of Australia. Melboune University Press, Melbourne. DIAKONOFF, A. [ed.] 1954. Gelechiidae In Microlepidoptera of New Guinea. Results of the third Archbold expedition (American-Netherlands Indian Expedition 1938-1939), Part 4, Tweede Reeks, Deel L, No. 1. North-Holland Pub. Co., Amsterdam. 4161pp. DURRANT, J. H. 1915. Microlepidoptera collected by the British Ornithologists Union and Wollaston Expeditions in the Snow Mountains. Southern Dutch New Guinea by John Hartley Durrant, F. E. S. Lepidoptera B.O.U. & Woll. Expedition Snow Mts. 2: 151-166. GIELIS, C. 1990. Review of the Pterophoridae from New Guinea, with descriptions of eight new species (Lepidoptera). Zoologische Mededelingen 77(21): 349391. KORNERUP, A., AND J. H. WANSCHER. 1978. Methuen Handbook of Colour. 2nd ed., Methuen, London. 252 pp. MEYRICK, E. 1910. Description of Malayan Micro-Lepidoptera. Trans. Royal Entomol. Soc. London 1910: 445. MEYRICK, E. 1918. Exotic Microlepidoptera 2:102-111. Marlborough, Wilts. MEYRICK, E. 1929. Exotic Microlepidoptera 3: 522-525. Marlborough, Wilts. MEYRICK, E. 1931. Exotic Microlepidoptera 4: 78-82. Marlborough, Wilts. MEYRICK, E. 1938. Papuan Microlepidoptera. Trans. Royal Entomol. Soc. London 87: 513. PARK, K. T. 1999. Lecithoceridae (Lepidoptera) of Taiwan I: Subfamily Lecithocerinae: genera Homaloxestis Meyrick and Lecithocera Herrich-Schffer. Zoological Studies 38(2): 238-256. PARK, K. T. 2010. First Record of Torodora Species from New Guinea, describing a new species (Lepidoptera, Lecithoceridae). Proc. Entomol. Soc. Washington. 112(3): 404-409. PARK, K. T. 2011a. Two new species of the genus Telephata Meyrick (Lepidoptera, Lecithoceridae ) from Papua New Guinea with notes on T. nitens (Diakonoff), comb. nov. Entomol. Science 14: 82-86. PARK, K. T. 2011b. Lecithoceridae (Lepidoptera, Gelechioidea) of New Guinea, Part I: Onnuria gen. nov. with descriptions of three new species. Proc. Entomol. Soc. Wash. 113: (in press). PARK, K. T. 2011c. Lecithoceridae (Lepidoptera, Gelechioidea) of New Guinea, Part II: Hamatina gen. nov. with descriptions of four new species. J. AsiaPacic Entomol. 14: 205-211. PARK, K. T., AND B. K. BYUN. 2008. A new genus Pectinimura (Lepidoptera, Gelechioidea, Lecithoceridae), with four species from Thailand and the Philippines. Florida Entomol. 91 (1): 110115. PARK, K. T., AND B. K. BYUN. 2010. A new genus Neopectinimura (Lepidoptera, Lecithoceridae) with descriptions of ve new species. Florida Entomol. 93(2): 298-307. WALKER, F. 1864. List of the specimens of Lepidoptera insects in the collection of the British Museum. Part 29. Tineites 29: 641.



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Kendra et al.: Ambrosia Beetle Diversity 123 DIVERSITY OF SCOLYTINAE (COLEOPTERA: CURCULIONIDAE) ATTRACTED TO AVOCADO, LYCHEE, AND ESSENTIAL OIL LURES P AUL E. K ENDRA 1 J ORGE S. S ANCHEZ 1 W AYNE S. M ONTGOMERY 1 K ATHERINE E. O KINS 2 J EROME N IOGRET 1 J ORGE E. P EA 3 N ANCY D. E PSKY 1 AND R OBERT R. H EATH 1 1 USDA-ARS, Subtropical Horticulture Research Station, Miami, FL 33158 2 Florida Department of Agriculture and Consumer Services, DPI, CAPS, Gainesville, FL 32608 3 University of Florida, Tropical Research and Education Center, Homestead, FL 33031 *Corresponding author; E-mail: paul.kendra@ars.usda.gov A BSTRACT The redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae), is an exotic wood-boring insect that vectors laurel wilt, a lethal vascular disease of trees in the Lauraceae, including avocado ( Persea americana ) and native Persea species (redba y, swampbay). As part of research to identify host-based attractants for X. glabratus, we discovered that a diverse arra y of non-target ambrosia beetles was attracted to the same substrates as X. glabratus During Sep-Dec 2009, several eld tests were conducted in north Florida (in woodlands with advanced stages of laurel wilt) with traps baited with commer cial lures of the essential oils, manuka and phoebe, and with freshly-cut wood bolts of avocado (a known host) and lychee ( Litchi chinensis a non-host high in the sesquiterpene copaene a putative host attractant). In addition, manuka-baited traps were deployed in avocado groves in south Florida to monitor for potential spread of X. glabratus. The combined trapping results indicated that none of these substrates w as specic in attraction of X. glabratus Numerous non-target ambrosia beetles were captured, including 17 species representative of 4 tribes within the subfamily Scolytinae This report provides photodocumentation and data on the species diversity and relative abundance for a group of poorly-studied beetles, the scolytine community in Florida Persea habitats. K ey Words: ambrosia beetles, Persea americana Litchi chinensis manuka oil, phoebe oil R ESUMEN El escarabajo de la ambrosa del laurel rojo (redbay), Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae), es un insecto extico barrenador de madera que transmite la marchitez del laurel, una enfermedad vascular mortal de rboles de la familia Lauraceae, los cuales inc luyen el aguacate ( Persea americana ) y las especies nativas del gnero Persea (redba y, swampbay). Como parte de la investigacin para identicar las substancias qumicas que atraen a X. glabratus fue descubierto que un arsenal diverso de otros escarabajos de la ambrosa que no eran de inters econmico fue atrado a las mismas substancias Entre septiembre y diciembre del 2009, se realizaron varias pruebas en el norte de la Florida (en arboledas con etapas a vanzadas de la marchitez del laurel) usando trampas con cebos comerciales de los aceites esenciales manuka y de phoebe y con madera de aguacate recien cortada (un husped conocido) y del lychee ( Litchi chinensis que no es husped, pero es alto en el sesquiterpeno -copaene, una substancia qumica atractiva). Adems, trampas con manuka fueron desplegadas en arboledas de aguacate en el sur de la Florida para supervisar la extensin potencil del X. glabratus Los resultados de las capturas combinados indicaron que ninguna de estas substancias eran especcas en la atraccin del X. glabratus Numerosos escarabajos de la ambrosa fueron capturados incluyendo 17 especies que representan cuatro tribus dentro de la subfamilia Scolytinae. Este informe proporciona la foto-documentacin y datos en la diversidad de la especie y la abundancia relativa para un grupo de escarabajos poco estudiado, la comunidad del Scolytinae en los hbitats de Persea de la Florida. Translation provided by the authors. The redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae), is an exotic wood-boring insect that vectors laurel wilt, a lethal vascular disease of trees in the Lauraceae (Fraedrich et al. 2008). Haploid males are ightless and remain within the host tree, while diploid females (typically siblingmated) disperse to colonize new hosts. Unlike most ambrosia beetles, female X. glabratus are primary colonizers capable of attacking healthy

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124 Florida Entomologist 94(2) June 2011 unstressed trees. During gallery excavation, females introduce spores of a symbiotic fungus, Raffaelea lauricola T.C. Harr., Fraedrich & Agha yeva (Harrington et al. 2008), carried in mycangial pouches located at the base of the mandibles (Fraedrich et al. 2008). The fungus provides food for both larvae and adults, but it also invades the host vascular system and results in systemic wilt and ultimately tree death. Native to southeastern Asia, X. glabratus was rst detected in the U .S. in 2002 near Port Wentworth, Georgia (Rabaglia et al. 2006). Since then, the vector-disease complex has spread along the coastal plain into South Carolina and Florida, and has been reported from a single county in Mississippi (USDA-FS 2010). In northern Florida, high mortality has occurred in native Persea species, inc luding redbay ( P. borbonia (L.) Spreng.) and sw ampbay ( P. palustris (Raf.) Sarg.), and the rapid southw ard spread of the pest complex currently threatens commercial groves of avocado ( P. americana Mill.), a conrmed susceptible host (Ma yeld et al. 2008). Floridas avocado production, centered in Miami-Dade County, is worth $13 million annually (USDA-NASS 2010), and replacement costs of all avocado trees (commercial and backyard) in Miami-Dade, Broward, Palm Beach, and Lee Counties have been estimated at $429 million (Evans & Crane 2008). Due to the serious economic threat posed by X. glabratus there is a critical need for effective attractants to detect, monitor, and control the spread of this invasive pest. Preliminary research provided no evidence of an aggregation pheromone and no strong attraction to its fungal symbiont, to its frass, or to ethanol (a standard attractant for ambrosia beetles); suggesting that host tree volatiles are the primary attractants for dispersing females (Hanula et al. 2008). Additional studies identied manuka and phoebe oils (essential oil extracts from the tea tree, Leptospermum scoparium Forst. & Forst., and the Brazilian walnut, Phoebe porosa Mez., respectively) as effective baits for eld monitoring of X. glabratus in South Carolina (Hanula & Sullivan 2008). Based on comparisons of volatile chemicals emitted from chipped redbay wood, manuka oil, and phoebe oil, Hanula & Sullivan (2008) hypothesized that 2 sesquiterpenes, -copaene and calamenene, were likely the primary host attractants While conducting research to evaluate attraction of female X. glabratus to wood volatiles and essential oil lures we discovered that a diverse number of non-target ambrosia beetles (both endemics and exotics established in Florida) were attracted to the same substrates as X. glabratus Several eld tests were conducted in north-central Florida (Alac hua and Marion Counties) in natural stands of redbay and swampbay with known infestations of X. glabratus and visible signs of laurel wilt disease We used 4-funnel Lindgren traps and/or sticky panels baited with commercially available essential oil lures (manuka and phoebe) or with freshly-cut wood bolts of avocado (a conrmed host) and lychee ( Litchi chinensis Sonn., a presumed non-host). L ychee is in the family Sapindacaeae; it lacks the typical aromatic laurel volatiles, but it has a high content of -copaene (Niogret et al. unpublished). During that same period, monitoring traps (Lindgren traps baited with manuka lures) were deployed in avocado groves in south Florida (Miami-Dade County). This report summarizes and illustrates the ambrosia beetles captured over a 4-month period (Sep-Dec 2009) in Florida to (1) provide a tool for action agencies and eld scientists to facilitate identication of non-target species captured in X. glabratus monitoring traps, (2) document the species diversity and relative abundance for the scolytine community in Persea habitats and (3) identify potential secondary colonizers of Persea hosts subsequent to initial attack by X. glabratus. M ATERIALS AND M ETHODS Field test 1 was conducted in Citra, Marion County, FL at the University of Florida Agricultural Experiment Station (PSREU). The back edge of the station bordered an upland wooded area dominated by mature live oak ( Quercus virginiana Mill.) with an understory that included redba y trees symptomatic for laurel wilt. Test 1 was run from 27 Aug-22 Oct 2009 and consisted of 6 treatments: a commercial manuka lure (Synergy Semiochemicals, Burnaby, BC), wood bolts from 3 avocado cultivars representative of the 3 horticultural races (Simmonds, West Indian race; Brooks Late, Guatemalan race; and Seedless Mexican, Mexican race), bolts from lychee (cv. Hanging Green), and an unbaited control. Wood bolts were collected from the USDA-ARS germplasm collection at the Subtropical Horticulture Research Station (SHRS), Miami, FL 1 d prior to test deployment. The ends of the bolts were coated with wax to prevent desiccation, and then both ends re-cut when used as baits at the start of the test. All baits were deployed in fourfunnel Lindgren traps (BioQuip, Rancho Dominguez, CA) with 300 mL of an aqueous solution of 10% propylene glycol (Low-Tox antifreeze; Prestone, Danbury, CT) added to the collection cup. For the manuka treatment, a single lure was hung from the trap lid by a wire twist tie. For the wood substrates, 2 freshly-cut bolts (5 cm diam 15 cm length) were suspended with wire from the lid on opposite sides of the trap Experimental design was a randomized complete block, with 5 replicate blocks arranged in a linear array along the fence at the back of the research station. Within a block, traps were spaced 10 m apart, 1.5 m above the ground, and spacing was 50 m between repli-

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Kendra et al.: Ambrosia Beetle Diversity 125 cate blocks. Traps were checked every 2 weeks for a total of 8 weeks. At each sampling date, the retention solutions (with insect captures) were collected, a thin layer was sawed from the lower end of each bolt (to renew release of wood volatiles), the collection cups were relled, and trap positions were rotated sequentially within each block to minimize potential positional effects on beetle capture. Field tests 2 and 3 were conducted in Cross Creek, Alachua County, FL at the Lochloosa Wildlife Conservation Area (St. Johns River Water Management District). The study site consisted of mesic atwoods composed of an overstory of slash pine ( Pinus elliottii Englem) with a mixed understory that inc luded numerous swampbay trees exhibiting advanced stages of laurel wilt. Test 2 was conducted from 7 Oct-2 Dec 2009 and evaluated the same 6 treatments described above. Test 3 was conducted from 5 Nov-29 Dec (at a site adjacent to test 2) and contained a commercial phoebe lure (Synergy Semiochemicals) in addition to the 6 treatments above. However, with tests 2 and 3 there were differences in trap type and trap layout. The essential oil lures were still deployed in four-funnel Lindgren traps, but the wood bolts were paired and hung vertically with 2 white sticky panels (23 cm 28 cm, Sentry wing trap bottoms; Great Lakes IPM, Vestaburg, MI) stapled back-to-back at the bottom of the bolts. Sticky panels were further secured with several binder clips around the edges. Tests 2 and 3 followed randomized complete block design, with 5 replicate blocks arranged in a rectangular grid. Each block consisted of a row of traps hung ~2 m high in non-host trees, with a minimum of 10 m spacing between adjacent traps in a row, and with 50 m spacing between rows. Both tests were 8 weeks in duration and checked every 2 weeks. At each check, the retention solutions and sticky panels were collected, a thin layer was sawed from the bottom of each bolt, the solutions/panels were replaced, and the trap positions were rotated sequentially within each row. In addition to the eld tests conducted in north Florida, monitoring/survey traps were deployed in several avocado groves in Miami-Dade County, FL during the fall of 2009. All monitoring traps consisted of four-funnel Lindgren traps baited with manuka lures, which were hung ~2 m above ground within the canopy of avocado trees. Traps were checked at 2-week intervals and sites included the SHRS avocado germplasm collection in Miami and 3 commercial groves in Homestead. All sample collections (from monitoring traps and eld tests) were sorted in the laboratory at SHRS, and scolytine species were counted, photographed, and stored in 70% ethanol. Specimens removed from sticky panels were soaked overnight in histological clearing agent (Histo-clear II; National Diagnostics, Atlanta, GA) prior to storage in alcohol. Beetle identications were conrmed at FDACS-DPI (Gainesville, FL) by K. E. Okins, and voucher specimens were deposited at both DPI and SHRS. R ESULTS The combined trapping results from eld tests and monitoring traps totaled 659 ambrosia beetles, consisting of 17 species from 4 tribes within the subfamily Scolytinae (Table 1). More than 90% of the captures were from the tribe Xyleborini, and only 1 specimen ( Coccotrypes distinctus (Motshulsky)) was representative of the tribe Dryocoetini. With the exception of X. glabratus (Fig. 1), most beetles were captured in fairly low numbers with many species represented by only a single capture, despite signicant trapping efforts with a variety of host-based attractants. Xyleborus glabratus comprised the majority of captures in north Florida, as expected due to its invasive pest status, but the percentages varied by site (15% in Marion County with test 1; 75.4% and 86.2% in Alachua County with tests 2 and 3, respectively). Four other species of Xyleborus were captured (Fig. 2), with X. ferrugineus (Fabricius) (F ig. 2A) and X. afnis (Eichhoff) (Fig. 2B) the 2 most abundant species after X. glabratus There were several other representatives within the tribe Xyleborini (F ig. 3), with Ambrosiodmus obliquus (LeConte) (Fig. 3A) a dominant species at both the Alachua site and in the Miami-Dade avocado groves. The tribe Cryphalini was represented by Hypothenemus dissimilis (Zimmerman) and several other Hypothenemus species difcult to discern to species level (Fig. 4). Hypothenemus beetles were major components at the Marion site and in Miami-Dade County Within the tribe Corthylini, 5 species were captured, of which 4 are presented in Fig. 5. Two of those ambrosia beetles had distinctive morphological features. Females of Corthylus papulans Eichhoff (Fig. 5C) have greatly enlarged terminal antennal segments whic h bear several long, recurved setae (Fig 5E); males of C. papulans lack the characteristic setae. In Pityoborus comatus (Zimmerman) (Fig. 5D), females are unique in that the mycangia are located on the pronotum, and consist of a pair of large shallow depressions covered with dense pubescence (Fig. 5F; Furniss et al. 1987). D ISCUSSION The subfamily Scolytinae contains 2 functionally distinct groups of beetles bark beetles which feed on phloem from the inner bark of host trees, and ambrosia beetles which cultivate and feed on symbiotic fungi within the xylem layers (Rabaglia 2002). Among the bark beetles, there are major forest pests which have been well studied, includ-

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126 Florida Entomologist 94(2) June 2011 ing the southern pine beetle, Dendroctonus frontalis Zimmerman (Chellman & Wilkinson 1980), the western pine engra ver, Ips pini (Say) (Kegley et al. 1997), and several other Ips spp. found in the southeastern U .S. (Conner & Wilkinson 1998). In contrast, the ambrosia beetles are generally not of economic importance and consequently have received less attention. They are minute beetles, spend the majority of their life concealed within host trees, and typically attack stressed or dying trees. Despite the large number of described species (e.g., >500 currently recognized Xyleborus spp. worldwide, Rabaglia et al. 2006), much is unknown regarding the basic biology, ecology, host range, fungal symbionts, and population dynamics of many endemic ambrosia beetles. Far less is known about exotic invasive species, which are not pests in their native lands but may acquire pest status when introduced into new environments, as is the case with X. glabratus in the U.S. Although our researc h was focused on identication of attractants specically for detection and control of X. glabratus information was obtained concurrently on the species diversity and relative abundance for the ambrosia beetles found in native Persea habitats in north-central Florida. In south Florida the trapping effort w as less intensive, but preliminary data was also obtained for the species composition in avocado groves. This summary report identies the species of Scolytinae most likely to be encountered while monitoring for X. glabratus and the photo-documentation provides fellow researc hers (non-taxonomists) and action agency personnel with a convenient tool for preliminary identication of nontarget captures. Some of the non-target beetles identied herein are species that may potentially function as secondary vectors of the laurel wilt pathogen. Once healthy trees are attacked by X. glabratus the stressed trees are susceptible to further attac k by secondary colonizers that can contribute to the rapid mortality seen in laurel hosts. Observations made on dead swampbay trees at the Lochloosa Conservation Area (Kendra et al. unpublished) indicated that multiple wood-boring species attacked those Persea trees, as evidencedTABLE 1.AMBROSIA BEETLES (CURCULIONIDAE: SCOLYTINAE) CAPTURED IN MARION, ALACHUA, AND MIAMI-DADE COUNTIES, FL FROM SEP-DEC 2009, ARRANGED ACCORDING TO LAWRENCE & NEWTON (1995). Marion Co.Alachua Co.Miami-Dade Co Test 1aTest 2bTest 3bMonitoringcTribe Dryocoetini Coccotrypes distinctus (Motschulsky)1 Tribe Xyleborini Ambrosiodmus lecontei Hopkins1 Ambrosiodmus obliquus (LeConte)61422 Premnobius cavipennis Eichhoff1 Theoborus ricini (Eggers) 1 Xyleborus afnis (Eichhoff)2114 Xyleborus californicus Wood 2 1 Xyleborus ferrugineus (Fabricius) 3 3422 1 Xyleborus glabratus Eichhoff 3193287 Xyleborus volvulus (Fabricius) 73 Tribe Cryphalini Hypothenemus dissimilis (Zimmerman) 1 1 Hypothenemus spp. 9 11 22 Tribe Corthylini Subtribe Corthylina Corthylus papulans Eichhoff 1 Monarthrum mali (Fitch) 1 Subtribe Pityophthorina Pityoborus comatus (Zimmerman) 1 Pseudopityophthorus minutissimus (Zimmerman) 1 Pseudopityophthorus pruinosus (Eichhoff) 1a8-wk field test in redbay; Lindgren traps baited with wood bolts (avocado, lychee) or manuka oil lures.b8-wk field test in swampbay; Sticky traps baited with wood bolts (avocado, lychee); Lindgren traps baited with manuka/phoebe oil lures.cMonitoring in avocado groves; Lindgren traps baited with manuka oil lures.

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Kendra et al.: Ambrosia Beetle Diversity 127 Fig. 1. The redbay ambrosia beetle Xyleborus glabratus Eichhoff vector of a lethal wilt fungus ( Raffaellea lauricola) causing high mortality of trees in the Lauraceae in the southeastern U.S. A. Female. B. Male. (Note: Males of X. glabratus are ightless; this specimen was obtained from host wood, not from a ight trap.) Fig. 2. Four species of Xyleborus not of economic importance. A. X. ferrugineus (Fabricius). B. X. afnis (Eichhoff). C. X. volvulus (Fabricius). D. X. californicus Wood. (All specimens female.)

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128 Florida Entomologist 94(2)June 2011by bore holes of various diameters. These secondary colonizers may potentially pick up Raffaelea from the host xylem and transport it to new trees, accelerating the spread of laurel wilt. In north Florida, X. ferrugineus X. afnis and A. obliquus were the most abundant species in native Persea habitats; in avocado A. obliquus and Hypothenemus spp. were dominant. In South Carolina, Hanula et al. (2008) found that Xylosandrus crassiusculus (Motschulsky) was attracted to wounded redbay trees. Further research should evaluate these additional beetle species to (a) determine if stressed (diseased) trees in the Lauraceae can serve as hosts, and if so, then (b) determine if Raffaelea spores can be recovered from the mycangia of ambrosia beetles that developed within Raffaelea -infected hosts. In other systems, there is evidence that exchange or cross contamination of symbiotic fungi may occur among ambrosia beetle species that occupy a common breeding site (Gebhardt et al. 2004). Alternatively, Raffaelea spores may potentially be transported passively by the setae and/or cuticular asperities (protuberances) commonly found on the anterior slope of the female pronotum, as has been demonstrated for Hypothenemus hampei (Ferrari) and spores of Fusarium solani (Martius) (Morales-Ramos et al. 2000). The commercial lures currently available for X. glabratus are non-specic in attraction, so high numbers of non-target captures are likely to be encountered with the monitoring system (manuka-baited Lindgren traps) employed by the State of Florida. Manuka and phoebe oil lures were originally developed for eld monitoring of another (phylogenetically distant) wood-boring beetle, the emerald ash borer Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) (Crook et al. 2008). Preliminary research (Kendra et al. unpublished) indicated that these essential oil lures were not only non-specic, but may have limited eld life for attraction of X. glabratus With the data set presented here, approximately 30% of the captures were non-target species of Scolytinae. Development of effective strategies for early detection and control (i.e., attract-and-kill systems) of X. glabratus is contingent on identication of specic attractants. In the absence of species-specic pheromones or food-based attractants for X. glabratus (Hanula et al. 2008), this will be a difcult challenge. CONCLUSIONSMultiple trapping studies targeting the redbay ambrosia beetle, Xyleborus glabratus, effectively generated a survey of the overall scolytine community resident in Florida Persea habitats. These ambrosia beetle species that co-occur with X. glabratus are attracted to the same host-based volatile chemicals; and they are the non-target species likely to be encountered in traps set out to monitor for X. glabratus Those species that can function as secondary colonizers of Persea hosts should be evaluated as potential secondary vectors for transmission of the laurel wilt pathogen, Raffaelea lauricola. ACKNOWLEDGMENTSWe gratefully acknowledge David Long, Mike Winterstein (USDA-ARS; Miami, FL), Rita Duncan (Univ. Florida; Homestead, FL), Gurpreet Brar, and Stephen McLean (Univ. Florida; Gainesville, FL) for technical assistance; Patti Anderson (FDACS-DPI, Gainesville, FL) for Persea identications; Bud Mayeld (USDAForest Service; Asheville, NC) for advice on eld trapping; Ray Schnell (USDA-ARS; Miami, FL) for advice on avocado germplasm samples; David Jenkins (USDA-ARS; Mayagez, PR) and 2 anonymous reviewers for suggestions with the manuscript; Pansy Vzquez-Kendra and Elena Schnell for translation of the abstract; and Connie Rightmire (St. Johns River Fig. 3. Ambrosia beetles within the tribe Xyleborini. A Ambrosiodmus obliquus (LeConte). B. Theoborus ricini (Eggers). C. Premnobius cavipennis Eichhoff. (All specimens female.)

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Kendra et al.: Ambrosia Beetle Diversity 129Water Management District) for assistance in obtaining a special use permit for the Lochloosa Wildlife Conservation Area. This work was supported in part by the USDA-ARS National Plant Disease Recovery System and the Florida Avocado Administrative Committee. This report presents the results of research only; mention of a proprietary product does not constitute an endorsement by the USDA. Fig. 4. Ambrosia beetles within the tribe Cryphalini. A. Hypothenemus dissimilis (Zimmerman). B. Hypothenemus sp. (Both specimens female.) Fig. 5. Ambrosia beetles within the tribe Corthylini. A. Monarthrum mali (Fitch). B. Pseudopityophthorus pruinosus (Eichhoff). C. Corthylus papulans Eichhoff. D. Pityoborus comatus (Zimmerman). E. Detail of C. papulans (anterior end; ventral view) showing enlarged terminal antennal segments bearing long recurved setae. F. Detail of P. comatus (anterior end; dorsal view on left, lateral view on right) showing pronotal mycangia (oval pits covered with dense setae), the storage site for symbiotic fungal spores. (All specimens female.)

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130 Florida Entomologist 94(2)June 2011REFERENCES CITEDCHELLMAN, C. W., AND WILKINSON, R. C. 1980. Southern pine beetle outbreaks in Florida since 1974. Florida Entomol. 63: 515. CONNER, M. D., AND WILKINSON, R. C. 1998. Ips Bark Beetles in the South. Forest insect and disease leaflet 129. United States Department of Agriculture, Forest Service. CROOK, D. J., KHRIMIAN, A., FRANCESE, J. A., FRASER, I., POLAND, T. M., SAWYER, A. J., AND MASTRO, V. C. 2008. Development of a host-based semiochemical lure for trapping emerald ash borer Agrilus planipennis (Coleoptera: Buprestidae). Environ. Entomol. 37: 356-365. EVANS, E. A., AND CRANE, J. H. 2008. Estimation of the Replacement Costs of Commercial and Backyard Avocado Trees in South Florida. Circular FE 825. University of Florida, Department of Food and Resource Economics. FRAEDRICH, S. W., HARRINGTON, T. C., RABAGLIA, R. J., ULYSHEN, M. D., MAYFIELD III, A. E., HANULA, J. L., EICKWORT, J. M., AND MILLER, D. R. 2008. A fungal symbiont of the redbay ambrosia beetle causes a lethal wilt in redbay and other Lauraceae in the southeastern United States. Plant Dis. 92: 215-224. FURNISS, M. M., WOO, J. Y., DEYRUP, M. A., AND ATKINSON, T. H. 1987. Prothoracic mycangium on pine-infesting Pityoborus spp. (Coleoptera: Scolytidae). Ann. Entomol. Soc. America 80: 692-696. GEBHARDT, H., BEGEROW, D., AND OBERWINKLER, F. 2004. Identication of the ambrosia fungus of Xyleborus monographus and X. dryographus (Coleoptera: Curculionidae, Scolytinae). Mycol. Progress 3: 95-102. HANULA, J. L., AND SULLIVAN, B. 2008. Manuka oil and phoebe oil are attractive baits for Xyleborus glabratus (Coleoptera: Scolytinae), the vector of laurel wilt. Environ. Entomol. 37: 1403-1409. HANULA, J. L., MAYFIELD III, A. E., FRAEDRICH, S. W.,AND RABAGLIA, R. J. 2008. Biology and host associations of the redbay ambrosia beetle (Coleoptera: Curculionidae: Scolytinae), exotic vector of laurel wilt killing redbay trees in the southeastern United States. J. Econ. Entomol. 101: 1276-1286. HARRINGTON, T. C., FRAEDRICH, S. W., AND AGHAYEVA, D. N. 2008. Raffaelea lauricola, a new ambrosia beetle symbiont and pathogen on the Lauraceae. Mycotaxon 104: 399-404. KEGLEY, S. J., LIVINGSTON, R. L., AND GIBSON, K. E. 1997. Pine Engraver, Ips pini (Say), in the Western United States. Forest insect and disease leaet 122. United States Department of Agriculture, Forest Service. LAWRENCE, J. F., AND NEWTON JR., A. F. 1995. Families and subfamilies of Coleoptera (with selected genera, notes, references and data on family-group names), pp. 779-1006 In J. Pakalud and S. A. Slipinski [eds.], Biology, Phylogeny, and Classication of Coleoptera: Papers Celebrating the 80th Birthday of Roy A. Crowson. Muzeum I Instytyt Zoologii PAN, Waszawa. MAYFIELD III, A. E., PEA, J. E., CRANE, J. H., SMITH, J. A., BRANCH, C. L., OTTOSON, E. D., AND HUGHES, M. 2008. Ability of the redbay ambrosia beetle (Coleoptera: Curculionidae: Scolytinae) to bore into young avocado (Lauraceae) plants and transmit the laurel wilt pathogen (Raffaelea sp.). Florida Entomol. 91: 485-487. MORALES-RAMOS, J. A., ROJAS, M. G., SITTERTZ-BHATKAR, H., AND SALDAA, G. 2000. Symbiotic relationship between Hypothenemus hampei (Coleoptera: Scolytidae) and Fusarium solani (Moniliales: Tuberculariaceae). Ann. Entomol. Soc. America 93: 541547. RABAGLIA, R. J. 2002. XVII. Scolytinae Latreille 1807, pp. 792-805 In R. H. Arnett, Jr., M. C. Thomas, P. E. Skelley, and J. H. Frank [eds.], American Beetles, vol. 2, Polyphaga: Scarabaeoidea through Curculionoidea. CRC Press, Boca Raton, FL. RABAGLIA, R. J., DOLE, S. A., AND COGNATA, A. I. 2006. Review of American Xyleborina (Coleoptera: Curculionidae: Scolytinae) occurring north of Mexico, with an illustrated key. Ann. Entomol. Soc. America 99: 1034-1056. USDA-FS. 2010. Laurel Wilt Distribution. United States Department of Agriculture, Forest Service, Forest Health Protection, Southern Region. USDA-NASS. 2010. Noncitrus Fruits and Nuts: 2009 preliminary summary, Fr Nt 1-3 (10) a. United States Department of Agriculture, National Agricultural Statistic Service.

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Ferreira & Scheffrahn: Light Attraction Behavior of a Drywood Termite131 LIGHT ATTRACTION AND SUBSEQUENT COLONIZATION BEHAVIORS OF ALATES AND DEALATES OF THE WEST INDIAN DRYWOOD TERMITE (ISOPTERA: KALOTERMITIDAE) M ARIA T ERESA F ERREIRA 1,2 AND R UDOLF H. S CHEFFRAHN 1,2 1 Fort Lauderdale Research and Education Center, University of Florida,3205 College Avenue, Davie, FL 33314 2 Azorean Biodiversity Group (CITA-A), Departamento de Cincias Agrrias, Universidade dos Aores, Pico da Urze, Angra do Herosmo, Portugal A BSTRACT Laboratory studies were conducted during the 2007 and 2008 dispersal seasons of the West Indian drywood termite Cryptotermes brevis (Walker), a serious urban pest of wooden structures Attraction to light and subsequent colonization of this species were studied by observing the response of alates to lit and dark chambers. Several intensities of light were tested to determine if light intensity had a role in the alates attraction to light and subsequent colonization. A bioassay was conducted with semi-shaded wood blocks to quantify negative phototaxis for the dealates. We found that the alates of C. brevis preferred ying into lit areas for colonization, and that the number of colonizations was highest in the high light intensity treatments. Negative phototaxis of the dealates was observed because these preferred to colonize in the dark habitat treatments. This information is important when deciding what control methods may be used to prevent C. brevis from colonizing wood structures. Traps with a high intensity light to attract C. brevis alates and to prevent infestation may be a way to monitor and control this urban pest. K ey Words: Cryptotermes brevis, phototaxis, colonization, alates, dealates R ESUMO Estudos num laboratrio foram realizados durante a poca de voo de disperso de 2007 e 2008 para a trmita da madeira seca Cryptotermes brevis (Walker), uma praga urbana de estruturas de madeira sria. A atraco luz e subsequente colonizao desta espcie foi estudada, observando a resposta dos alados a cmaras de preferncia de luz. Vrias intensidades de luz foram testadas para determinar se a intensidade da luz tinha um papel na atraco dos alados pela luz e subsequente colonizao. Um ensaio usando blocos de madeira semi-cobertos para quanticar o comportamento fototctico negativo dos dealados foi conduzido. Ns observmos que os alados de C. brevis preferem reas com maior iluminao para colonizarem, e que o nmero de colonizaes era maior no tratamento com maior intensidade de luz. O comportamento fototctico negativo dos dealados foi observado porque os dealados preferem colonizar nos tratamentos de habitats escuros. Esta informao importante quando se tem de decidir que mtodos de controlo podem ser usados para prevenir a trmita C. brevis de colonizar estruturas de madeira. Usar armadilhas com uma intensidade luminosa elevada para atrair alados de C. brevis e prevenir uma infestao poder ser uma forma de monitorizar e controlar esta praga. Translation provided by the authors. The West Indian drywood termite Cryptotermes brevis (Walker) (WIDT) is a serious urban pest that causes signicant levels of damage to wooden structures This termite was rst described in Jamaica in 1853 and has a tropicopolitan urban distribution except in Asia (Scheffrahn et al. 2008). Like most Kalotermitidae the WIDT nests in its food source, wood, where it spends most of its life cycle. A colony of drywood termites can vary in size from hundreds to a few thousand termites (Nutting 1970), and several colonies can be found inside a single piece of wood. Myles et al. (2007), for example, found as many as 30 colonies of WIDT in a single oor board. Drywood termites are major pests, accounting for about 20% of the budget spent on termite control in the United States (Su & Scheffrahn 1990). One of the main methods for controlling these pests has been the use of fumigation to eliminate existing colonies. Fumigation, however, does not prevent new infestations, and therefore it is benecial to combine fumigation with additional preventative control methods. Preventing this species from founding a new colony during the dispersal ight season is an important technique for the control of this pest. This study aims to develop a better understanding of the behavior of the WIDT during the dispersal ight season, the time

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132 Florida Entomologist 94(2) June 2011 when WIDT is most accessible to physical, chemical, or behavioral management efforts. The life cycle of WIDT includes a dispersal ight where the mature winged forms (alates) leave their previous colony to form new colonies. The dispersal ights are the only occasion when this species is found outside of wood (Kofoid 1934) because it never leaves the nest to forage for new food sources (Korb & Katrantzis 2004). After ying, the alates shed their wings and associate as pairs of female and male dealates. These pairs will crawl on the substrata in search for a suitable place to start a new colony (Snyder 1926; Wilkinson 1962; Nutting 1969; Minnick 1973). According to Light (1934a) most termites castes are negatively phototactic, but the alates, attracted to light, seek to emerge into openings and y toward light. However, there are no data correlating colonization sites with lighting conditions. Positive phototaxis of C. brevis and congenus alates is followed by negative phototaxis of the dealates (W ilkinson 1962; Minnick1973), although no data have been produced to conrm this observation. The present study quantied the phototactic colony site selection of C. brevis alates and dealates The rst hypothesis tested was that favorable colonization sites in lighted areas are more likely to be selected by alates of C.brevis and that colonization will be higher at higher light intensities The second hypothesis tested was that dealates search for darker areas on the substrate to colonize. M ATERIALS AND M ETHODS Termites Experiments were conducted at the University of Florida, Fort Lauderdale Research and Education Center (FLREC), Davie, Florida, in a room partially lled with wood infested by C. brevis that originated from several sites around South Florida. The wood was stored in a dark room at ambient temperature (average 25.6C) and ambient relative humidity (average 73.5%). The termite alates used in the experiments dispersed naturally from the infested wood. The room remained dark except for the time the experiments were conducted, and the alates were free to y anywhere in the room. The rst experiments took place between Apr and Jul 2007, and the second experiments between Apr and Jun 2008. Light Intensity Experiments Twenty four transparent plastic boxes (36 23 28 cm) served as light preference chambers. The boxes were wrapped in aluminum foil to isolate the light box from adjacent boxes The boxes were placed with the open side facing the infested wood and a hole was cut in the side of each box in order to t (Fig. 1) an electrical cord for a set of white Light Emitting Diodes (LED) (HolidayLEDS model No TS-70) strung in a series of 5 single light bulbs attached with tape and hung through the hole. The LEDs were all connected to each other in a continuous string of lights mounted in a series and connected to a single power source. Electrical tape was used to position the light bulbs in place as well as prevent light from dispersing through the cut hole, so that the only light source was inside each box. The LED sets were randomly distributed among 12 lit boxes and 12 dark boxes. The lit boxes had a light intensity of approximately 40 lux as measured by a light meter (Extech Instruments model No 403125) and the dark boxes had approximately 0.11 lux (due to some contaminating light from nearby experiments occurring at the same time). A cube of wood (5 5 5 cm) with 6 drilled holes w as placed in the center of each box (Fig. 1). Each 2.3 mm diam hole was 1.5 cm deep, and there was a single hole per face of the block. Four thumb push pins were placed on the underside of the block to allow for enough space (3 mm) for the termite to access the hole on the underside. The difference in colonization between different light intensities was analyzed with the same 24 boxes previously described. The LED lights were also used but this time there were 4 different set ups for the different light intensities with 6 replicates per light intensity (measured by the light meter): 6 of the boxes were dark with ( 0.11 lux); 6 boxes had 1 LED light bulb with an intensity of approximately 11 lux; 6 boxes had 5 LEDs together ( 40 lux); 6 boxes had 10 LEDs attached with an intensity of approximately 480 lux. The boxes with the different light intensities were randomly distributed. A block of wood (15x2x9 cm) with 24 holes (12 on top and 12 on the bottom, and each 1.5 cm deep and 2.3 mm diam) was placed in the center of each box. After 3 months, the blocks were collected and the number of colonized holes per block was counted. A hole was considered to be colonized when a complete fecal seal (covering of the hole with hardened fecal material from the termites) was present. Negative Phototaxis Experiment The negative phototaxis of the dealates was studied with a white PVC pipe (51 cm 7 cm inside diam) wrapped with electrical tape and c losed on one end. A 102 2 5 cm board with 40 holes each 2.3-mm diam and drilled 2.5 cm apart, was placed inside the pipe. The outermost holes were 1 cm from the edge of the board. Of the 40 holes, 20 were always exposed to light (regular uorescent indoor lighting) and 20 were exposed to decreasing levels of light toward the closed end of the PVC pipe (Fig. 2). The board was visually

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Ferreira & Scheffrahn: Light Attraction Behavior of a Drywood Termite133 divided into 10-cm sections (excluding 1 cm at each end). Each section had 4 holes available for colonization. A board with the same dimensions and number of holes was used as a control and was completely exposed to the same light intensity. The 10-cm sections were lettered from A to J with sections A, B, C, D, and E inside the PVC pipe and sections F, G, H, I, and J outside (Table 1). This experimental protocol was replicated 4 times. Light measurements were made inside and outside the PVC pipe (Table 1). After dispersal ight season was over the number of colonized holes was counted for each 10-cm section based on the previously described colonization criteria. Statistical Analysis The data for the lit versus dark boxes were analyzed by a non-parametric Wilcoxon Matched Pairs test (SAS Institute 2003) to test whether the numbers of colonizations in the dark and lit areas were different. Colonization differences between the light intensities were tested with Students t -test (SAS Institute 2003). A Chi-squared test for independence (SAS Institute 2003) w as used to test whether the distribution of colonizations was dependent on the light intensity. A Students ttest for dependent samples w as used to determine whether the differences between the numbers of colonizations in each 10-cm section were signicant, (SAS Institute 2003). R ESULTS Light Intensity Experiments Lit vs dark boxes. A total of 43 holes were colonized. There were signicantly ( t-t est = 4.01E05, P < 0.0001) more holes colonized in the lighted boxes (2.8 0.3, mean SEM) than in the dark areas (0.8 0.2 (mean SEM)). Fig. 1. Arena for light intensity experiments. Aluminum foil isolated the box and the LED lights were placed above the block of wood.

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134 Florida Entomologist 94(2) June 2011 Light intensity A total of 76 holes were colonized in the light intensity experiment. A higher number of colonizations was recorded in high light intensity boxes. There were signicant ( ttest, P < 0.05) differences in colonizations among the various light intensities (F ig. 3). Negative Phototaxis Experiment A total of 175 holes were colonized during the negative phototaxis experiment. There were no signicant differences in number of colonizations between the 10-cm sections ( P 0.29) in the control boards The distribution of number of colonizations was independent of the section where the colonizations occurred (Chi-squared = 5.9541, P = 0.75; n.s.) with no section of the control board preferred over any other section. In the boards placed in the PVC pipes colonization distribution was not independent of the section where it occurred (Chi-squared = 33.3939, P = 0.001) with a signicantly higher number of colonizations occurring in the dark sections (F ig. 3). Sections A, B, C, and D inside the PVC pipe had a signicantly higher number of colonizations than sections G, H, I, and J outside the PVC pipe ( P < 0.05). Section E (inside the PVC pipe) was not signicantly different ( P 0.16) from sections G-J (outside the PVC pipe); and section F (outside the PVC pipe) was not signicantly different ( P 0.08) from sections A-D (inside the PVC pipe) (Fig. 4). D ISCUSSION The results of the light versus dark experiment conrmed the hypothesis that colonization of the alates occur more frequently in lit areas. The termite C. brevis in South Florida ies mainly between 1:00 and 2:00 AM (unpublished data) when it is very dark. These results showed that colonization sites located in areas that are lit during the night may be more susceptible to infestation by C. brevis during dispersal ights. The presence of articial lights ma y cause a change of behavior for some species of animals (Longcore & Rich 2004), but articial lights may be benecial to structure infesting termites like C. brevis. The attraction of these termites to articial lights puts structures that ha ve a continuous nighttime light source at higher risk of being infested than structures near by that are not lit. The light intensity experiment showed that increasing light intensities increased the number of termite colonizations (Fig. 3). Minnick (1973) reported differences in the wavelength of light preferred by C. brevis, and Guerreiro et al. (2007) reported differences in color preference for the alates Neither of them reported results based on light intensity. The fact that we found that 11 lux of light intensity was signicantly different from 480 lux shows that the increase in light intensity caused an increase in colonization of the wood blocks by the termites. Cryptotermes brevis has Fig. 2. Arena for negative phototaxis experiment. PVC pipe wrapped in black tape covered half of the wood block. Controls had no PVC pipe cover. T ABLE 1.D ISTANCES OF 10CM WOOD SECTIONS FROM THE CLOSED END OF 51CM PVC PIPE AND THE LIGHT INTENSITY AT THE CENTER OF EACH SECTION Section Inside of PVC pipe Outside of PVC pipe ABCDEABCDE Distance from closed end of PVC pipe (cm)102030405060708090100 Light Intensity (lux) 0.010.040.080.200.70600600600600600

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Ferreira & Scheffrahn: Light Attraction Behavior of a Drywood Termite135 been observed to have as many as 2 founded colonies in a total of 22 nuptial chambers (Scheffrahn et al. 2001) which means that there is a 9% success rate of colonization. This shows that investing in preventing C. brevis alates from founding colonies is important because their success rate is high enough to make prevention methods necessary The attraction to light by alates can be used to create light traps as a form of preventing infestations and re-infestations by C. brevis alates and as a means of partial control of this species Such light traps inside structures that are already infested may minimize the spread of the infestation, and the results of this study showed that more intense the light used the more alates will be attracted; thereby making the use of light a possible alternative to use of chemicals for prevention. Further studies with different light wavelengths can help improve light traps as an alternative method to prevent the founding of colonies by C. brevis Previous experiments with C. havilandi (Sjstedt) (W ilkinson 1962) and C. brevis (Minnick 1973) showed negative phototaxis in dealates of these species After landing, the dealates search and colonize dark areas. Due to the nature of wood structures, it could be argued that this behavior is not really negative phototaxis but that because the cracks and holes are usually hidden and in dark areas the dealates end up colonizing there. If so, the behavior would be dependent not on light intensity but on the locations of a good places to colonize. However, the present study showed that negative phototaxis of dealates did occur. Independent distributions of colonizations occurred in the controls, but independent colonizations did not occur in the semi-shaded blocks; this conrmed the negative phototaxis hypothesis. The lighter segment of the wood block inserted in the PVC pipe had signicantly less colonizations than the darker segment. However, the numbers of colonizations in section F (immediately outside the PVC pipe) were not signicantly different from the numbers in the darker areas inside the PVC pipe. Colonization of section F might have occurred because the termites colonizing that area had searched for colonizing sites in the dark area inside the PVC pipe where earlier colonizers had already taken all suitable sites, i.e., a site saturation. Also they may have landed near the PVC pipe cueing in on the darker area nearby and colonizing the sites near that dark area. However, further studies on this are needed to understand why the section closest to the PVC pipe on the light side was signicantly more colonized than the section immediately inside the dark PVC pipe; where it would be expected considering the negative phototaxis behavior. One way to approach this might be to use a higher density of colonizing holes or fewer termites, so that the number of holes is not a limiting factor. In termites, both the alates and dealates have well developed compound eyes (Light 1934b) and their behavior during ight season has shown that they do respond to light while in ight (positive phototaxis) and to have negative phototaxis after landing. Further studies on the behavior of C. brevis during the ight season can help improve different methods to prevent colonization and subsequent infestation by this species This study has shown that alates y to and colonize more in higher light intensity areas, while the dealates have an opposite behavior colonizing more in darker areas. This knowledge is useful to improve light traps as a control method against colony foundation from C. brevis A CKNOWLEDGMENTS We thank the late Boudanath Maharajh for technical support, and Roxanne Connelly, Jonathan F. Day, and Paulo A. V. Borges for reviewing an early version of this manuscript. We also thank Dr. James L. Nation and anonymous reviews whose invaluable critical comments helped improve this manuscript. Financial support for this research was provided in part by the University of Florida and the Portugal Foundation for Science and Technology (FCT-SFRH/BD/29840/2006). R EFERENCES C ITED G UERREIRO O., M YLES T. G., F ERREIRA M., B ORGES, A., AND BORGES, P. A. V. 2007. Voo e fundao de Fig. 3. Average number of holes colonized by C. brevis per light intensity ( n = 43). Different letters represent signicant differences at P < 0.05. Fig. 4. Average number of C. brevis colonized holes per section (n = 175). Sections A, B, C, D, and E were inside the PVC pipe and sections F, G, H, I, and J were outside the PVC pipe. Bars with the same letter were not signicantly different at P < 0.05.

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136 Florida Entomologist 94(2)June 2011colnias pelas trmitas dos Aores, com nfase na Cryptotermes brevis, pp. 29-46 In P. Borges and T. Myles [eds.], Trmitas dos Aores. Principia, Estoril, 127 pp. KOFOID, C. A. 1934. Biological backgrounds of the termite problem, pp. 1-12 In C. A. Kofoid, S. F. Light, A. C. Horner, M. Randall, W. B. Herms, and E. E. Bowe [eds.], Termites and Termite Control. Univ. California Press, Berkeley, California, 795 pp. KORB, J., AND KATRANTZIS, S. 2004. Inuence of environmental conditions on the expression of the sexual dispersal phenotype in a lower termite: implications for the evolution of workers in termites. Evol. Develop. 6: 342-352. LIGHT, S. F. 1934a. The constitution and development of the termite colony, pp. 22-41 In C. A. Kofoid, S. F. Light, A. C. Horner, M. Randall, W. B. Herms, and E. E. Bowe [eds.], Termites and Termite Control. Univ. California Press, Berkeley, California, 795 pp. LIGHT, S. F. 1934b. The external anatomy of termites, pp. 50-57 In C. A. Kofoid, S. F. Light, A. C. Horner, M. Randall, W. B. Herms, and E. E. Bowe [eds.], Termites and Termite Control. Univ. California Press, Berkeley, California, 795 pp. LONGCORE, T., AND RICH, C. 2004. Ecological light pollution. Front. Ecol. Environ. 2: 191-198. MINNICK, D. R. 1973. The ight and courtship behavior of the drywood termite, Cryptotermes brevis J. Environ. Entomol. 2: 587-591. MYLES, T. G., BORGES, P. A. V., FERREIRA, M., GUERREIRO O. BORGES, A., AND RODRIGUES, C. 2007. Filogenia, biogeograa e ecologia das trmitas dos Aores, pp. 15-28 In P. Borges and T. Myles [eds.], Trmitas dos Aores. Principia, Estoril, 127 pp. NUTTING, W. L. 1969. Flight and colony foundation, pp. 233-282 In K. Krishna and F. M. Weesner [eds.], Biology of Termites.Volume I. Academic Press, London and New York, 598 pp. NUTTING, W. L. 1970. Composition and size of some termite colonies in Arizona and Mexico. Ann. Entomol. Soc. America 63: 1105-1110. SAS. 2003. Version 9.1. SAS Institute, Cary, North Carolina. SCHEFFRAHN, R. H., BUSEY, P., EDWARDS, J. K., KRECEK, J., MAHARAJH, B., AND SU, N-Y. 2001. Chemical prevention of colony foundation by Cryptotermes brevis (Isoptera: Kalotermitidae) in attic modules. J. Econ. Entom. 94: 915-919. SCHEFFRAHN, R. H., KRECEK, J., RIPA, R., AND LUPPICHINI, P. 2008. Endemic origin and vast anthropogenic dispersal of the West Indian drywood termite. Biol. Invasions. http://www.springerlink.com/content/ 914p8530t781632n/fulltext.html. Accessed July 2008. SNYDER, T. E. 1926. The biology of the termite castes. Quart. Rev. Biol. 1: 522-552. SU, N.-Y., AND SCHEFFRAHN, R. H. 1990. Economically important termites in the United States and their control. Sociobiology 17: 77-94. WILKINSON, W. 1962. Dispersal of alates and establishment of new colonies in Cryptotermes havilandi (Sjstedt) (Isoptera, Kalotermitidae). Bull. Entomol. Res. 53: 265-288.

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Pereira et al.: Male A. suspensa Lipid and Protein Content 137 INFLUENCE OF METHOPRENE AND DIETARY PROTEIN ON MALE ANASTREPHA SUSPENSA (DIPTERA: TEPHRITIDAE) LIPID AND PRO TEIN CONTENT R UI P EREIRA 1,2 J OHN S IVINSKI 2 J EFFREY P. S HAPIRO 2 AND P ETER E. A. T EAL 2 1 Entomology and Nematology Department, University of Florida, Gainesville, Florida 2 Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, Florida A BSTRACT Because both the application of a juvenile hormone analog, methoprene, and the addition of protein to the adult diet increased the sexual success of male Caribbean fruit y, Anastrepha suspensa (Loew) (Diptera: Tephritidae), it was hypothesized that both might also impact male nutritional status Total content of lipid and of protein in A. suspensa males were measured to discover if there w as an effect of these treatments alone or in combination on the content of each of these subtstances. In the rst 24 hours following adult emergence, 6 different treatments were applied (all possible combinations of methoprene in acetone solution or acetone alone, and protein-diet enrichment). Adult weight was determined for all treatments at 5, 10, 15, 20, 25, 30 and 35 d post-emergence. Dietary protein had a positive effect on the weight and total lipid and protein contents during the rst 35 d of adult male life. There were minimal negative impacts from methoprene applications. Even though males were more active sexually, there was no signicant change in weight or protein content during the study period. However, total lipid content decreased with age. The usefulness of methoprene to enhance the sexual performance of mass-reared tephritids destined for sterile release appears to outweigh any physiological costs/limitations that such treatment might confer. Key Words: adult age, adult weight, Caribbean fruit y, hydrolyzed yeast, juvenile hormone, sexual maturation R ESUMEN Debido a que tanto la aplicacin de un anlogo de la hormona juvenil, metopreno, y la adicin de protenas a la dieta del adulto aumento el xito sexual del macho de la mosca de la fruta de Caribe, Anastrepha suspensa (Loew) (Diptera: Tephritidae), se plante la hiptesis de que ambos tambin podran afectar el estatus nutricional masculino Se midi el contenido total de lpidos y de protenas en los machos de A. suspensa para descubrir si haba un efecto de estos tratamientos solos o en combinacin sobre el contenido de cada una de estas sustancias En las primeras 24 horas despus de la emergencia de adultos, se aplicaron 6 diferentes tratamientos (todas las combinaciones posibles de metopreno en solucin de acetona en solucin o solo acetona y con el enriquecimiento de protenas en la dieta). Se determin el peso adulto para todos los tratamientos a los 5, 10, 15, 20, 25, 30 y 35 das despus de la emergencia. Las protenas dietticas tuvo un efecto positivo sobre el peso y el total de lpidos y protenas durante los primeros 35 das de vida de los machos adultos. Hubo un mnimo de impactos negativos de las aplicaciones de metopreno. A pesar de que los machos eran ms activos sexualmente, no hubo ningn cambio signicativo en el peso o el contenido de protena durante el perodo de estudio. Sin embargo, el contenido de lpidos totales disminuyeron con la edad. La utilidad de metopreno para mejorar el desempeo sexual de moscas tefrtidas criadas en masa destinadas para programas que liberan los machos estriles parece superar los costos siolgicos y las limitaciones que dicho tratamiento puede conferir. Topical application of the juvenile hormone analog, methoprene, on the dorsal surface of adult male Caribbean fruit ies, Anastrepha suspensa (Loew) (Diptera: Tephritidae), increases male sexual success (P ereira et al. 2009), apparently because it increases the production of male sex pheromone. In addition it accelerates sexual maturation by several days (Teal & Gomez-Simuta 2002). The addition of protein to the adult diet has a similar effect on sexual performance, but the underlying cause(s) has yet to be investigated experimentally. When methoprene and protein are combined there is an additive increase in male sexual performance, and males are ~ 4 times more likely to mate than males not exposed to methoprene nor given access to protein (Pereira et al. 2010). Presumably, increased pheromone production occurring at an earlier age, as well as accelerated sexual activity, is energetically demanding and

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138 Florida Entomologist 94(2) June 2011 may affect the balance of the metabolic compounds (Teal et al. 2000). One hypothesis frequently mentioned for the relatively long, sometimes more than 2 weeks, pre-reproductive period found in many adult frugivorous Tephritidae is that the time is used to acquire resources needed for reproduction (Sivinski et al. 2000). Decreases in resource foraging time due to accelerated sexual maturation and increases in body nutrients resource expenditure might be expected to result in substantive changes in a fruit ys nutritional status that could affect longevity and long-term sexual performance. We further supposed that these expenses could be particularly difcult to incur in the absence of a protein enriched adult diet. As a result, we hypothesized that the nutritional effects of methoprene and diet, both alone and in combination, might have an important effect on sexual performance of male ies reared for sterile insect technique (SIT) programs. It is from this perspective that we address the following questions: (1) What inuence does methoprene treatment have on male A. suspensa weight and lipid/protein nutrient stores over a period of 35 d; ~ 33% of males survive to this age under laboratory conditions (Sivinski 1993); and (2) do protein enriched and protein deprived diets affect male weight and lipid/protein content, and is there an interaction between diet and methoprene treatment? Considering the potential importance of adult diet to SIT, relatively little nutritional work has been done with A. suspensa The rate and the temporal patterns of consumption of carbohydrates proteins, and amino acids by adults (Sharp & Chambers 1984; Landolt & Davis-Hernandez 1993), as well as the role of food availability and quality on male pheromone production have been studied to some extent (Epsky & Heath 1993; Teal et al. 2000; Teal & Gomez-Simuta 2002). A positive inuence of sucrose on male pheromone calling (Landolt & Sivinski 1992) and survival (Teal et al. 2004) has been reported. However, no work has been done specically on the nutritional impact of protein incorporation into adult diets. This is the rst study of male tephritid nutritional balance challenged by articially elevated juvenile hormone titers to improve sexual performance. M ATERIAL AND M ETHODS Insects The Caribbean fruit ies used in this study were obtained from laboratory colony at the Center for Medical, Agricultural and Veterinary Entomology (CMAVE) USDA-ARS, at Gainesville, FL. At the time of the study, the colony was 3 years old and had been produced according to the conventional mass rearing protocols (FDACS 1995). Pupae were collected from the colony and sorted by size in a pupal sorting machine (FAO/IAEA/ USDA 2003). Pupal size was homogenized to reduce male size and weight variability; large males have been shown to have a sexual advantage over smaller males (Burk & Webb 1983; Burk 1984; Webb et al. 1984; Sivinski & Dodson 1992; Sivinski 1993). Males used for this experiment were from pupal size class of 10.9 0.71 mg ( n = 30) in weight. This is considered a mid-size pupal weight for eld collected A. suspensa males in infested gua va fruits (Hendrichs 1986). Throughout the experiment ies were maintained in a laboratory room with a photoperiod of 13L:11D (light from 0700 to 2000 h), a light intensity of 550 50 lux, a temperature of 25 1C and a relative humidity of 55 5%. Diet and Hormonal Treatments Following emergence, males were subjected to 1 of the following 6 diet and hormonal treatments: M + P + : topical methoprene in acetone; access to sugar and hydrolyzed yeast M + P : topical methoprene in acetone; access to sugar M P + : topical acetone; access to sugar and hydrolyzed yeast M P : topical acetone; access to sugar P + : no topical application; access to sugar and hydrolyzed yeast P : no topical application; access to sugar. Methoprene (5 g in 1 L acetone) was applied topically within the rst 24h after emergence. Controls consisted of application of 1 L acetone only (M ) or no topical application (P + and P ). In order to conduct the topical application, males were immobilized in a net bag, and the solution was applied through the mesh on the dorsal surface of the thorax from a micro-pipette. No anaesthesia was used to immobilize the ies. Precautions were taken to avoid cross contaminations among experimental subjects. Male ies exposed to the different treatments were maintained in screen cages (30 cm by 30 cm by 30 cm), with a maximum male density of 200 ies/cage. Flies were allowed free access to food (according to above treatments) and water. In protein-deprived treatments (P ) ies were only provided with sugar. Protein was provided to the ies in the form of hydrolyzed yeast mixed with sugar (1:3 parts, respectively). This mixture is considered a high quality diet for Anastrepha species (Jcome et al. 1995; Aluja et al. 2001). Experimental cages were maintained for up to 35 d. For weight and chemical analysis, male ies

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Pereira et al.: Male A. suspensa Lipid and Protein Content 139 were sampled at the following ages: 5, 10, 15, 20, 25, 30, and 35 d of adult age. For each age and treatment, 5 ies were randomly sampled and stored at -84C until used for analysis. In order to obtain the base line information after emergence, 5 newly emerged (without access to any food or water) and untreated ies were collected as well. Because lipid content in Ceratitis capitata (Wied.) has been found to vary according to the time of the da y, due to the different activities in which males were engaged (Warburg & Yuval 1997), we sampled males at the same time each day (16:30 h, immediately before the beginning of the calling period). Flies were weighed individually prior to homogenization for lipid and protein determination. Quantication of Lipids and Proteins Individual male ies were homogenized in a solution of PBS buffer at pH 7.25 (8.77 g of 0.15 M NaCl and 7.1 g of 50 mM Na 2 HPO 4 in 1 L of water). The homogenate was then brought up to 4.0 mL with PBS. Lipids were extracted from the homogenate by adding 40 mg of Na 2 SO 4 to half of the initial volume, and 3.75 mL of chloroform: methanol (1:2) (Bligh & Dyer 1959) was used to separate polar and non-polar constituents of the homogenate. An additional 1.25 mL of chloroform was added to the homogenate and vortexed for 4 min at 4,000 rpm. The non-polar chloroform phase was collected. The remaining solution was re-extracted with chloroform (1.875 mL), vortexed and collected. Chloroform was evaporated in a Speed Vac device (Thermo Savant, San Jose, CA). Lipid contents were determined by the vanillin reagent method (Van Handel 1985; Warburg & Yuval 1996), with triolein being used as a standard. Quantication of lipids was done by reacting 10 L of sample with 190 L of vanillin reagent. Lipid content was determined colorimetrically at 530 nm in a spectrophotometer (Bio-tek Instruments, Winooski, VT). Protein determination was done according the Pierce BCA protein assay (Pierce, Rockford, IL). One mL of the polar fraction of the homogenate was centrifuged for 1 min at 14,000 rpm. Half the volume was mixed with 100 L of sodium deoxychoate reagent (0.15 w/v) and 100 L of 72% (w/v) tricloroacetic acid (TCA) to precipitate the proteins. After incubation at room temperature for 10 min and centrifugation for 10 min at 14,000 rpm the supernatant was discarded. The precipitate was dissolved and reacted with 50 L of 5% (w/v) sodium dodecyl sulfate (SDS) and 1 mL of Pierce micro BCA TM protein assay reagent (Pierce, 1999). After incubation in a water bath at 37C for 30 min, proteins in samples and standards were determined colorimetrically at 562 nm in a spectrophotometer (Bio-Tek Instruments, Winooski, VT). Statistical Analyses Data were analyzed by two-way analysis of variance (ANOVA) to detect the interactions between age and treatment for the parameters studied, independently (weight, lipid content, and protein content). These analyses were followed by an ANOVA to detect differences between means in the treatments. Tukeys test was used to separate means (Ott & Longnecker 2001). Statistical analyses were performed with R software (version 2.1.0, www.r-project.org). R ESULTS Male weight Average adult weight varied between 5.8 mg and 11.6 mg (Fig. 1). There was no interaction between treatment and adult age ( F 35,192 = 1.16, P = 0.256), and no effect of age ( F 7,192 = 1.59, P = 0.140; T able 1) on adult weight. There was, however, a signicant effect of treatment ( F 5,192 = 24.46, P < 0.05). Protein-fed males generally had signicantly higher fresh weights than sugar fed males (Fig. 1, Table 2). Lipid content Signicant effects of treatment ( F 5,192 = 131.37, P < 0.001), adult age ( F 7,192 = 83.14, P < 0.001), and the interaction of adult age and treatment ( F 35,192 = 6.34, P < 0.001) were found. Male lipid content per treatment per age (F ig. 2) differed both among ages and for different treatments (Table 1) and among treatments for different ages (Table 2). In protein-deprived males, lipid contents dropped at 5 d after emergence, while protein-fed males maintained stable lipid levels during the rst 10 d of adult life (Fig. 2). Afterwards, lipids dropped to lower levels. Methoprene treatment did not affect lipid levels in either proteinfed or protein-deprived male ies. Protein Content Signicant effects of treatment ( F 5,192 = 44.63, P < 0.001), adult age ( F 7,192 = 15.00, P < 0.05), and the interaction of adult age and treatment ( F 35,192 = 4.77, P < 0.001) were found. Signicant differences in protein content among the different ages were found within eac h treatment except for treatment M P + (Fig. 3, Table 1), and among treatments at all ages (Table 2). Protein-fed males maintained higher protein levels than protein-deprived males (Fig. 3). In protein-fed males, protein content steadily increased through time, while in protein-deprived males, protein levels declined. Methoprene did not affect the level of protein in either protein-fed or protein-deprived males.

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140 Florida Entomologist 94(2) June 2011 D ISCUSSION In male A. suspensa there was a clear effect of a protein-enric hed diet on weight, total lipid, and total protein content over the rst 35 d of adult life. In contrast, there was no effect of methoprene or acetone application on the studied parameters. Regardless of diet type, weight and total protein content were relatively stable during adult life. In contrast, total lipid content steadily decreased with age. This decline began later, however, in ies fed a protein-enriched diet (10 d after emergence). In all the treatments, consumption of protein resulted in insects able to regulate their weight, protein levels, and lipid content at higher level than insects without a protein food source. This is broadly consistent with what has been observed in Tephritidae in general and other Anastrepha spp. in particular (Aluja et al. 2001). In nature, adult tephritids feed on a variety of carbohydrates and proteins derived from fruit juices honeydew, and bird feces (Hendrichs et al. 1991; Warburg & Yuval 1997; Yuval & Hendrichs 2000). Protein enhances reproductive performance in C. Fig 1. Mean (SD) adult weight ( n = 5) of male Caribbean fruit ies at different adult ages among the 6 treatments featuring methoprene (M) and protein (P) and their various combinations. T ABLE 1.A NALYSIS OF VARIANCE (ANOVA) FOR MALE CARIBBEAN FRUIT FLY WEIGHT TOTAL LIPIDS AND TOTAL PROTEINS AMONG DIFFERENT AGES IN 6 DIFFERENT TREATMENTS FEATURING METHOPRENE (M) AND PROTEIN (P)AND THEIR VARIOUS COMBINATIONS (NS, NON SIGNIFICANT DIFFERENCES, P > 0.05; 0.01< P < 0.05; *** P< 0.001). TreatmentsMale weightTotal lipidsTotal proteins M+P+F7,32 = 1.1559 (ns) F7,32 = 7.5932 *** F7,32 = 3.2711 M+PF7,32 = 1.9367 (ns) F7,32 = 32.76 *** F7,32 = 8.6007 *** M-P+ F7,32 = 1.8041 (ns) F7,32 = 11.124 *** F7,32 = 2.0186 (ns) M-PF7,32 = 2.0277 (ns) F7,32 = 35.906 *** F7,32 = 7.7853 *** P+ F7,32 = 1.0071 (ns) F7,32 = 12.504 *** F7,32 = 2.9685 PF7,32 = 0.1291 (ns) F7,32 = 20.805 *** F7,32 = 4.8732 ***

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Pereira et al.: Male A. suspensa Lipid and Protein Content 141capitata (Warburg & Yuval 1997; Kaspi et al. 2000; Shelly & Kennelly 2002; Shelly et al. 2002; Yuval et al. 2002), and protein-fed males start to call earlier in life (Papadopoulos et al. 1998). Protein-fed males are more competitive in terms of post copulatory sexual selection as well (Taylor & Yuval 1999). In Bactrocera dorsalis (Hendel), incorporation of protein into adult diet signicantly increases survival and mating success (Shelly el al. 2005). Among Anastrepha species, Aluja et al. (2001) evaluated the effects of different adult nutrients, including protein and sugar, on male sexual performance in adults of 4 species, ( A. ludens (Loew), A. obliqua (Macquart), A. serpentina (Wied.), A. striata Schiner). Overall, protein-fed males were more sexually successful than protein-deprived, except for A. ludens where no differences were found. Neither did male diet inuence A. ludens female reproductive potential following trophalaxis (Mangan 2003). However, in a more recent study protein did improve A. ludens sexual performance (Aluja et al. 2008). Thus, perhaps not surprisingly, protein-enhanced diets typically, but not always, enhance Fig. 2. Mean (SD) total lipid content ( n = 5) of male Caribbean fruit ies at different adult ages among the 6 treatments featuring methoprene (M) and protein (P) and their various combinations. TABLE 2.ANALYSIS OF VARIANCE (ANOVA) FOR MALE CARIBBEAN FRUIT FLY WEIGHT, TOTAL LIPIDS, AND TOTAL PROTEINS AMONG TREATMENTS FOR DIFFERENT AGES FEATURING JUVENILE HORMONE (JH) AND PROTEIN (P)AND THEIR VARIOUS COMBINATIONS (* 0.01< P < 0.05; ** 0.001< P < 0.01; *** P < 0.001). Adult age (days)Male weightTotal lipidsTotal proteins 5F5,24 = 4.546 ** F5,24 = 26.285 *** F5,24 = 3.5202 10 F5,24 = 4.566 ** F5,24 = 28.881 *** F5,24 = 26.629 *** 15 F5,24 = 5.521 ** F5,24 = 12.548 *** F5,24 = 10.277 *** 20 F5,24 = 2.624 F5,24 = 13.873 *** F5,24 = 8.9948 *** 25 F5,24 = 4.370 ** F5,24 = 50.275 *** F5,24 = 4.2223 ** 30 F5,24 = 4.429 ** F5,24 = 21.339 *** F5,24 = 8.9493 *** 35 F5,24 = 5.120 ** F5,24 = 36.197 *** F5,24 = 9.5048 ***

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142 Florida Entomologist 94(2)June 2011sexual success. However, there are perhaps revealing differences in physiology and foraging tactics for food and mates among males of different species with different diets and activities. For instance, unlike A. suspensa protein-fed C. capitata males have lower lipid content than those that are protein-deprived (Kaspi et al. 2000). In addition, while lekking males are heavier and contain signicantly more protein and sugar than resting males, they do not contain more lipids (Yuval et al. 1998). Lipids (fatty acids, phospholipids, and sterols) have a nutritional role distinct from carbohydrates and proteins. Yuval et al. (1994) described lipids metaphorically as an energetic trust fund, whereas carbohydrates are comparable to a readily accessible cash account. Perhaps the difference between A. suspensa and C. capitata in their use of protein for lipogenesis reects a difference in energy use patterns, with A. suspensa putting more reserves in long-term accounts for future use. This in turn might reect more predictably encountered food sources for C. capitata or a lower daily chance of mortality for A. suspensa that leads in turn to planning for the future. Many kinds of fatty acids and phospholipids are synthesized by insects, but all insects require sterols in their diet (Chapman 1998). Reduction of total lipid content with age in A. suspensa can be the result of somatic activities, since lipids represent stored energy, even if some restoration of lipid reserves occurs by lipogenesis (Warburg & Yuval 1996). In male A. suspensa at least in the rst 10 days of adult life of protein-fed males, there is a slight increase in lipid content. The same phenomenon occurs in C. capitata (Warburg & Yuval 1996) and A. serpentina (Jcome et al. 1995). Total lipid content declined following male A. suspensa sexual maturation. Sharp decreases indicate that males started to utilize their metabolic reserves, and this seems likely to correspond to the energetic requirements of any number of sexual and agonistic behaviors and processes (e.g., Sivinski et al. 2000). One of these potential expenditures that can be indirectly examined with the present data is pheromone production. Nestel et al. (1986) suggested that lipid reserves in male C. capitata may play an important role in the regulation and production of sex pheromone. Nestel et al. (2005) found a decrease in lipid body content after sexual maturation (as the present data reveal for A. suspensa ), but later on the content displayed a harmonic pattern where total lipid content increased and decreased at a periodicity of 10 days. In A. suspensa male pheromone production increases when methoprene is Fig. 3. Mean (SD) total protein content ( n = 5) of male Caribbean fruit ies at different adult ages among the 6 treatments featuring methoprene (M) and protein (P) and their various combinations.

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Pereira et al.: Male A. suspensa Lipid and Protein Content 143applied (Teal et al. 2000). However, we found that application of methoprene did not affect lipid content of ies maintained on different diet treatments. The differences in total protein content between protein-fed and protein-deprived males may be inuenced by ingested protein in the gut. However, the gradual increase in total protein content over time in both protein-deprived (after 10 d as adult) and protein-fed males is both difcult to explain and inconsistent with articialdiet protein alone accounting for the difference. Tephritids are known to feed on animal excrement (Prokopy et al. 1993; Epsky et al. 1997), and perhaps the consumption of bacteria from their own feces or bacteria growing on dead ies or on food sources, inadvertently provided them with a protein source. Regardless of the origin of this additional protein, the difference in protein contents on the different diets suggests it was not sufcient to completely satisfy nutritional requirements. The ndings of this study have implications for SIT programs. Among the most important is that while the addition of methoprene has male sexual advantages it appears to have no immediate nutritional detriments. Thus it is a relatively costfree means of improving the performance of mass-reared and released ies. The incorporation of dietary protein has a positive effect on adult weight and lipid and protein content all of which are plausibly related to performance as well (Yuval et al. 1998). Due to these effects, the incorporation of protein in adult diet for SIT programs is also recommended. ACKNOWLEDGMENTSWe thank David Nestel (IPP-The Volcani Center, Beit-Dagan, Israel), Nikos Papadopoulos (University of Thessaly, Magnisia, Greece), and Steve Ferkovich (CMAVE, USDA-ARS, Gainesville-FL, USA) for critical reviews of an earlier version of this manuscript. This project was funded in part by the International Atomic Energy Agency (Research Contract 12863). Financial support was provided to RP by the Centro de Cincia e Tecnologia da Madeira through the Ph.D. grant BD I/ 2002-004.REFERENCES CITEDALUJA, M., JCOME, I., AND MACAS-ORDEZ, R. 2001. Effect of adult nutrition on male sexual performance in four neotropical fruit y species of the genus Anastrepha. (Diptera: Tephritidae). J. Insect Behav. 14: 759-775. ALUJA, M., PEREZ-STAPLES, D., SIVINSKI, J., SANCHEZ, A., AND PIERO, J. 2008. Effects of male condition on tness in two tropical tephritid ies with contrasting life histories. Animal Behav. 76: 1997-2009. BLIGH, E. G., AND DYER, W. J. 1959. A rapid method of total lipid extraction and purication. Canadian J. Biochem. Physiol. 37: 911-917. BURK, T. 1984. Male-male interactions in Caribbean fruit ies, Anastrepha suspensa (Loew) (Diptera: Tephritidae): territorial ghts and signaling stimulation. Florida Entomol. 67: 542-547. BURK, T., AND WEBB, J. C. 1983. Effect of male size on calling propensity, song parameters, and mating success in Caribbean fruit ies, Anastrepha suspensa (Loew) (Diptera: Tephritidae). Ann. Entomol. Soc. America 76: 678-682. CHAPMAN, R. F. 1998. The Insects: Structure and Function. 4th ed. Cambridge University Press, Cambridge, UK. EPSKY, N. D., AND HEATH, R. R. 1993. Food availability and pheromone production by males of Anastrepha suspensa (Diptera: Tephritidae). Environ. Entomol. 22: 942-947. EPSKY, N. D., DUEBEN, B. D., HEATH, R. R., LAUZON, C. R., AND PROKOPY, R. J. 1997. Attraction of Anastrepha suspensa (Diptera: Tephritidae) to volatiles from avian fecal material. Florida Entomol. 80: 270277. FAO/IAEA/USDA. 2003. Manual for Product Quality Control and Shipping Procedures for Sterile MassReared Tephritid Fruit Flies, Version 5.0, Vienna, Austria. FDACS (FLORIDA DEPARTMENT OF AGRICULTURE ANDCONSUMER SERVICES). 1995. Procedures Manual for Mass Rearing the Caribbean Fruit Fly Anastrepha suspensa (Loew) (Diptera: Tephritidae). Bureau of Methods Development and Biological Control Caribbean Fruit Fly Mass Rearing Facility, Gainesville, Florida. HENDRICHS, J. 1986. Sexual Selection in Wild and Sterile Caribbean Fruit Fly, Anastrepha suspensa (Loew) (Diptera: Tephritidae). M.Sc. thesis, University of Florida, Gainesville, Florida. HENDRICHS, J., KATSOYANNOS, B. I., PAPAJ, D. R., ANDPROKOPY, R. J. 1991. Sex differences in movement between natural feeding and mating sites and tradeoffs between food consumption, mating success and predator evasion in Mediterranean fruit ies (Diptera: Tephritidae). Oecologia 86: 223-231. JCOME, I., ALUJA, M., LIEDO, P., AND NESTEL, D. 1995. The inuence of adult diet and age on lipid reserves in the tropical fruit y Anastrepha serpentina (Diptera: Tephritidae). J. Insect Physiol. 41: 10791086. KASPI, R., TAYLOR, P. W., AND YUVAL, B. 2000. Diet and size inuence sexual advertisement and copulatory success of males in Mediterranean fruit y leks. Ecol. Entomol. 25: 279-284. LANDOLT, P. J., AND DAVIS-HERNANDEZ, K. M. 1993. Temporal patterns of feeding by Caribbean fruit ies (Diptera: Tephritidae) on sucrose and hydrolyzed yeast. Ann. Entomol. Soc. America 86: 749755. LANDOLT, P. J., AND SIVINSKI, J.1992. Effects of time of day, adult food, and host fruit on incidence of calling by male Caribbean fruit ies (Diptera: Tephritidae). Environ. Entomol. 21: 382-387. MANGAN, R. L. 2003. Adult diet and male-female contact effects on female reproductive potential in Mexican fruit y ( Anastrepha ludens Loew) (Diptera: Tephritidae). J. Econ. Entomol. 96: 341-347. NESTEL, D., GALUN, R., AND FRIEDMAN, S. 1986. Balance energtico en el adulto irradiado de Ceratitis capitata (Wied.) (Diptera: Tephritidae). Folia Entomol. Mexicana 70: 75-85.

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144 Florida Entomologist 94(2)June 2011NESTEL, D., PAPADOPOULOS, N. T., LIEDO, P., GONZALEZ-CERON, A. L., AND CAREY, J. R. 2005. Trends in lipid and protein content during Medy aging: A harmonic path to death. Arch. Insect Biochem. Physiol. 60: 130-139. OTT, R. L., AND LONGNEAKER, M. 2001. An introduction of statistics methods and data analyses, 5th ed. Duxbury Publishers, Pacic Grove, CA. PAPADOPOULOS, N. T., KATSOYANNOS, B. I., KOULOUSSIS, N. A., ECONOMOPOULOS, A. P., AND CAREY, J. R. 1998. Effect of adult age, food, and time of the day on sexual calling incidence of wild and mass reared Ceratitis capitata males. Entomol. Exp. Appl. 89: 175182. PEREIRA, R., SIVINSKI, J., AND TEAL, P. E. A. 2009. Inuence of methoprene and dietary protein on male Anastrepha suspensa (Diptera: Tephritidae) mating aggregations. J. Insect Physiol. 55: 328-335. PEREIRA, R., SIVINSKI, J., AND TEAL, P. E. A. 2010. Inuence of a juvenile hormone analog and dietary protein on male Anastrepha suspensa (Diptera: Tephritidae) sexual success. J. Econ. Entomol. 103: 40-46. PIERCE. 1999. Instructions for Micro BCA Protein Assay Reagent Kit. Pierce Chemical Company, Rockford, IL. PROKOPY, R. J., HSU, C. L., AND VARGAS, R. I. 1993. Effect of source and conditions of animal excrement on attractiveness to adults of Ceratitis capitata (Diptera: Tephritidae). Environ. Entomol. 22: 453458. SIVINSKI, J. 1993. Longevity and fecundity in the Caribbean fruit y (Diptera: Tephritidae): effects of mating, strain and body size. Florida Entomol. 76: 635644. SIVINSKI, J., AND DODSON, G. 1992. Sexual dimorphism in Anastrepha suspensa (Loew) and other tephritid ies: possible roles of developmental rate, fecundity and dispersal. J. Insect Behav. 5: 491-506. SIVINSKI, J., M. ALUJA, M., DODSON, G., FREIDBERG, A., HEADRICK, D., KANESHIRO, K., AND LANDOLT, P. 2000. Topics in the evolution of sexual behaviour in the Tephritidae, pp. 751-792 In M. Aluja, and A. L. Norrbom [eds.], Fruit Flies (Tephritidae): Phylogeny and Evolution of Behaviour. CRC Press, Boca Raton, Florida. SHARP, J. L., AND CHAMBERS, D. L. 1984. Consumption of carbohydrates, proteins and amino acids by Anastrepha suspensa (Loew) (Diptera: Tephritidae) in the laboratory. Environ. Entomol. 13: 768-773. SHELLY, T. E., AND KENNELLY, S. 2002 Inuence of male diet on male mating success and longevity and female remating in the Mediterranean fruit y (Diptera: Tephritidae) under laboratory conditions. Florida Entomol. 85: 572-579. SHELLY, T. E., KENNELLY, S., AND MCINNIS, D. O. 2002. Effect of adult diet on signalling activity, mate attraction, and mating success in male Mediterranean fruit ies (Diptera: Tephritidae). Florida Entomol. 85: 150-155. SHELLY, T. E., EDU, J., AND PAHIO, E. 2005. Inuence of diet and methyl eugenol on the mating success of males of the oriental fruit y, Bactrocera dorsalis (Diptera: Tephritidae). Florida Entomol. 88: 307313. TAYLOR, P. W., AND YUVAL, B. 1999. Postcopulatory sexual selection in Mediterranean fruit ies: advantages for large and protein-fed males. Animal Behav. 58: 247-254. TEAL, P. E. A., AND GOMEZ-SIMUTA, Y. 2002. Juvenile hormone: action in regulation of sexual maturity in Caribbean fruit ies and potential use in improving efcacy of sterile insect control technique for Tephritid fruit ies. IOBC-WPRS Bull. 25: 167-180. TEAL, P. E. A., GOMEZ-SIMUTA, Y., AND PROVEAUX, A. T. 2000. Mating experience and juvenile hormone enhance sexual signaling and mating in male Caribbean fruit ies. Proc. Natl. Acad. Sci., USA 97: 37083712. TEAL, P. E. A., GAVILANEZ-SLONE, J. M., AND DUEBEN, B. D. 2004. Effects of sucrose in adult diet mortality of males of Anastrepha suspensa (Diptera: Tephritidae). Florida Entomol. 87: 487-491. VAN HANDEL, E. 1985. Rapid determination of total lipids in mosquitoes. J. American Mosquito Control Assoc. 1: 302-304. WARBURG, M. S., AND YUVAL, B. 1996. Effect of diet and activity on lipid levels of adult Mediterranean fruit ies. Physiol. Entomol. 21: 151-158. WARBURG, M. S., AND YUVAL, B. 1997. Effects on energetic reserves on behavioral patterns of Mediterranean fruit ies. Oecologia 112: 314-319. WEBB, J. C., SIVINSKI, J., AND LITZKOW, C. 1984. Acoustical behavior and sexual success in the Caribbean fruit y, Anastrepha suspensa (Loew) (Diptera: Tephritidae). Environ. Entomol. 13: 650-656. YUVAL, B., AND HENDRICHS, J. 2000. Behavior of ies in the genus Ceratitis pp. 429-456 In M. Aluja, and A. L. Norrbom [eds.], Fruit Flies (Tephritidae): Phylogeny and Evolution of Behaviour. CRC Press, Boca Raton, Florida. YUVAL, B., HOLLIDAY-HANSON, M., AND WASHINO, R. K. 1994. Energy budget of swarming male mosquitoes. Ecol. Entomol. 19: 74-78. YUVAL, B., KASPI, R., SHOLMIT, S., AND WARBURG, M. S. 1998. Nutritional reserves regulates male participation in Mediterranean fruit y leks. Ecol. Entomol. 23: 211-215. YUVAL, B., KASPI, R., FIELD, S. A., BLAY, S., AND TAYLOR, P. 2002. Effects of post-teneral nutrition on reproductive success of male Mediterranean fruit ies (Diptera: Tephritidae). Florida Entomol. 85: 165170.

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Lee & Thomas: Taxonomic Status of Cucujus clavipes 145 CLARIFICATION OF THE TAXONOMIC STATUS OF CUCUJUS CLAVIPES WITH DESCRIPTIONS OF THE LARVAE OF C. C. CLAVIPES AND C. C. PUNICEUS (COLEOPTERA: CUCUJIDAE) J ONGEUN L EE 1 AND M ICHAEL C. T HOMAS 2 1 Department of Biological Science, College of Natural Sciences, Andong National University, Andong, 760749 E-mail: Korea jelee@andong.ac.kr 2 Florida State Collection of Arthropods, Division of Plant Industry, Gainesville, Florida 326147100, USA E-mail: michael.thomas@freshfromorida.com A BSTRACT The larvae of Cucujus c. clavipes Fabricius and C. c. puniceus Mannerheim are fully described and illustrated in detail for the rst time Based on larval and adult morphology the present recognition of two subspecies is maintained. Key Words: taxonomy, Cucujus larva, North America R ESUMEN Por primera vez se describen e ilustran las larvas de Cucujus c. clavipes Fabricius y C. c. puniceus Mannerheim. Basndose en la morfologa larval, se acepta el reconocimiento de las dos subespecies Translation provided by the authors. Cucujus clavipes Fabricius (1781) was described from America boreali. Cucujus puniceus Mannerheim (1843) was described from insula Sitkha, now Baranof Island in southeastern Alaska and the site of the modern city of Sitka. Both descriptions are of adults only, are based on the adult stage and are brief and relatively uninformative. Of C. clavipes Fabricius wrote: ruber, thorace fuscato, femoribus clavatis rus (red, thorax dark, femora clavate, red); of C. puniceus Mannerheim wrote: elongatus depressus, laete sanguineus, antennis nigrofuscis, pectore abdomineque rufoferrugineis, thorax subrotundato, lateribus leviter denticulato, supra obsolete bisulcato (Elongate, depressed, rich red, antennae nigro-fuscus, abdomen rufo-ferrugineous; thorax rounded, laterally weakly denticulate, above obsoletely bisulcate). LeConte (1854, 1861, 1863) consistently treated C. puniceus as a valid species. Casey (1884) reduced it to a variety of C. clavipes and said of it: The body is more elongated, and usually of a brighter color. The rst joint of the antennae is usually of a dark testaceous, while in clavipes it is blac k. The antennae are slightly longer, and the neck slightly narrower in puniceus . Leng (1920) treated C. puniceus as either a variety or subspecies of C. clavipes [In the Leng Catalogue, a lettered taxon following a numbered species name could be . . variety, subspecies, race, etc. (Leng 1920: v)] and Hetschko (1930) followed Casey in treating it as a variety of C. clavipes Schaeffer (1931) described Cucujus clavipes subnitens as a variety from Arizona and Utah. Thomas (1993) in a list of Nearctic Cucujidae treated C. puniceus as a subspecies of C. clavipes and Schaeffers taxon as a variety as previously described. In an effort to resolve the status of Cucujus clavipes we examined adults and larvae from both eastern and western North America. Larvae Larvae of Japanese Cucujus coccinatus Lewis were described and illustrated by Hayashi (1980, 1986) and the larva of C. mniszechi Grouvelle was described by Lee and Sato (2007). Larvae of C. clavipes Fabricius were briey and partially illustrated (head and mandible) by Bving and Craighead (1931) and Klausnitzer (2001). P eterson (1951) provided extensive illustrations of C. clavipes but provided only a brief description. In neither case w as the origin of the specimen illustrated provided. Lawrence (1991) re-used Petersons illustrations and added scanning electron micrographs of mouthparts of a specimen from California. The larva of both North American subspecies of C. clavipes Fabricius are fully illustrated and described for the rst time in the present paper The larva of C. clavipes is similar to C. mniszech i (Lee and Sato 2007), but can be distinguished by absence of a distinct epicranial stem and presence of a sharp prostheca. In C. mniszechi the epicranial stem is present and the prostheca is blunt.

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146 Florida Entomologist 94(2) June 2011 Larvae of C. clavipes are reported to be predaceous (Smith and Sears 1982) or facultatively predaceous (La wrence 1991). Their extreme cold tolerance, which increases with increasing latitude, has been extensively studied (Sformo et al. 2010, and references therein). M ATERIALS AND M ETHODS The larvae were preserved in 70% ethyl alcohol, cleared in 10% KOH solution for 1 hour, rinsed in water, and dissected under a stereoscopic microscope (Leica MS5). Slide mounting procedures were carried out according to LeSage (1984), and the larval terminology follows Lawrence (1991). Specimens were measured with an ocular micrometer and the measurements were transferred to graph paper. The illustrations were then sketched in pencil, the sketches inked, and assembled into plates, which were optically scanned and cleaned up in a graphics editor. Specimens examined are deposited in the Florida State Collection of Arthropods (FSCA) and the University of Alberta E. H. Strickland Entomological Museum (UASM). Descriptions Cucujus clavipes clavipes Fabricius, 1781 (Fig. 1 AJ) Diagnosis : See this section under C. c. puniceus Material examined : 37 total from: INDIANA : Morgan Co.: Martinsville (10); Tippecanoe Co. (1); OHIO : Champaign Co. (1); Columbiana Co. (1); WISCONSIN : Calumet Co.: Forest Junction (1); Ingham Co .: Dansville State Game Area (1); Shawano Co.: Shawano (16); Shawnee Co.: Tilleda (6) (all deposited in the FSCA). Description: Late instar (Fig. 1A). Body 22.0 26.0 mm long, elongate, subparallel, strongly dorsoventrally attened with strongly forked median process at abdominal apex (Fig. 1A). Head and abdominal segment 8 moderately sclerotized, yellowishbrown to brown, tergite of abdominal segment 9 strongly sclerotized and brown. Head (Fig. 1B): prognathous, strongly transverse and dorsoventrally attened. Lateral margin rounded. Median endocarina absent; epicranial stem present but very short; frontal sutures lyriform, strongly curved; bases contiguous. Stemmata well-developed, 6 on each side of head (Peterson (1951) reported 5 on each side; we count 6 but 1 is small and difcult to see). Frontoclypeal suture absent. Fronotoclypeal region with 3 long setae anterior to angles of frontal arms, 1 pair anterior to the apex of the frontal arms on each side of the head, 1 pair medially between the frontal arms, and 1 pair at the apex of the frontoclypeal region near the clypeolabral suture. Clypeolabral suture complete. Labrum (Fig. 1G) free, with 3 pairs of setae and anterior border mbriate. Epipharynx glabrous medially, with 5 anterior setae on each side. Antennae 3segmented, ratio of lengths of antennomeres 1, 2, and 3 about 1.0: 1.2: 1.0. Mandibles (Fig. 1H) heavily sclerotized, symmetrical, apices bidentate with a smaller subapical tooth; with 2 dorsolateral mandibular setae; prostheca acuminate, spinelike, with a broad base; mola with numerous setae medially and penicillus posteriorly (The scanning electron micrographs in Lawrence (1991: 464, gs. 34.528, cf) show a conspicuous patch of microtrichia on both the dorsal and ventral surfaces of the mandible near the base; these are virtually invisible in liquid and are not illustrated here). Maxilla (Fig. 1E) with cardo triangular, divided by an internal ridge, basal portion trapezoidal, 1 moderately elongate seta near latero-basal margin; stipes elongate; mala falciform with 5 apical spines and a medial brush composed of several thick setae; maxillary palpus 3segmented, segment 1 asetose, segment 2 with 2 setae, segment 3 with 4 minute apical setae. Labium (Fig. 1F) with conspicuous mentum and prementum; mentum about as long as wide, with 2 pairs of setae and prementum with 1 pair of setae and 1 pair of sensilla; ligula rounded anteriorly, 1 pair of setae and microtrichia anteriorly; labial palpi 2-segmented and widely separated at base. Thorax: Meso and metathorax tergites, and abdominal tergites and ventrites 18 each with 1 transverse ridge near anterior margin, ridge on ventral surface of abdominal segment 1 lightly sclerotized. Prothorax subquadrate, transverse, 0.5 times as long as wide, sides slightly curved, dorsal surface smooth; prosternal surface smooth, 3 setae (1 elongate) at anterolateral angles and 2 short setae at posterolateral angles; prosternum trapezoidal, sides oblique, posterior margin straight, pair of medial setae present posterior to posterior margin of presternum. Mesoand metathorax transverse, both 0.5 times as long as wide, sides curved, dorsal surface of both tergites smooth with 3 short setae at anterolateral angles and 2 short setae at posterolateral angles; both sterna without well-dened subdivisions, each smooth with a pair of discal setae near anterior margin; spiracular sclerite projecting strongly from lateral margin, spiracles (Fig. 1C) annular and angled posterolaterally. Legs (Fig. 1D) moderately long, 5segmented; claw falciform, large. Abdomen: Segments 17 transverse, tergite surface smooth with 2 setae anterior to spiracles and 2 setae posterior to spiracles; ventrite surface with 3 setae, 2 anteriorly and 1 posteriorly. Segment 8 slightly enlarged, tergite (Fig. 1I) with a stout spicule at each posterolateral margin, posterolateral angles with 4 long and 4 short setae, 3 pairs of short setae anteromedially; sternite (Fig. 1J) with 7 pairs of setae and with large stout pro-

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Lee & Thomas: Taxonomic Status of Cucujus clavipes 147 Fig. 1. Larva of Cucujus c. clavipes A, habitus, dorsal view; B, head, dorsal view; C, A7 spiracle, D, prothoracic leg; E, left maxilla, dorsal view; F, labium, ventral view; G, labrum, dorsal view; H, left mandible, dorsal view; I, abdominal segments 89, dorsal view; J, same, ventral view.

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148 Florida Entomologist 94(2) June 2011 cess posteriorly with many minute setae apically. Tergum 9 with a basally forked process, directed dorsad; base of process with a pair of short, apically forked processes, 1 short seta at apex of forked process; anterior margin with laterally curved processes projecting from tergum 8; ventrite 9 reduced and concealed from above. Cucujus clavipes puniceus Mannerheim (F ig. 2 AJ) Diagnosis. The larva of this species is very similar to that of Cucujus c. clavipes but can be distinguished by the ratio of the 8th abdominal segment length vs length of the forked process (4:3 in C. c. puniceus ; 1:1 in C. c. Clavipes ), and the ratio of the 8th abdominal segment width vs the width of forked process (measured at tips) (5:3 in C. c. puniceus ; 3:2 in C. c. clavipes ). Material examined: 7 total, from: CANADA: ALBERTA: George Lake (2, UASM); USA: CALIFORNIA: El Dorado Co.: Blodgett Forest (1, FSCA); Tulare Co.: Sequoia National Park, Stoney Cr. Picnic Area (2, FSCA);UTAH: Cache Co.: Logan Valley (2, FSCA) Description: Late instar larva (Fig. 2A). Body 21.0-24.0 mm long, elongate, subparallel, strongly dorsoventrally attened with forked median process at abdominal apex (Fig. 2A). Head and abdominal segment 8 moderately sclerotized, brown, tergum 9 strongly sclerotized and dark brown. Head (Fig. 2B): prognathous, strongly transverse and dorsoventrally attened. Lateral margin rounded. Hind corners of epicranium slightly produced posteriorly. Median endocarina and epicranial stem very short; frontal sutures lyriform, strongly curved; bases contiguous. Stemmata well-developed, 6 present on each side of head. Frontoclypeal suture absent. Fronotoclypeal region with 3 long setae anterior to angles of frontal arms, 1 pair anterior to the apex of the frontal arms on each side of the head, 1 pair medially between the frontal arms, and 1 pair at the apex of the frontoclypeal region near the clypeolabral suture. Clypeolabral suture complete. Labrum free (Fig. 2G), with 5 pairs of setae. Epipharynx medially glabrous, 6 anterior setae on each side. Antennae 3segmented, ratio of lengths of antennomeres 1, 2, and 3 about 1.0: 1.4: 1.0. Mandibles (Fig. 2H) heavily sclerotized, symmetrical, apices bidentate with a smaller subapical tooth; with 2 dorsolateral mandibular setae present; prostheca acuminate, spinelike, with a broad base; mola with numerous setae medially and posteriorly. Maxilla (Fig. 2E) with cardo, divided by an internal ridge, basal portion trapezoidal, with 1 moderately elongate seta near basal margin; stipes elongate; mala falciform, mala falciform with 5 apical spines and a medial brush composed of several thick setae; maxillary palpus 3segmented, segment 1 asetose, segment 2 with 3 setae, segment 3 with 1 seta and 4 minute apical setae. Labium (Fig. 2F) with conspicuous mentum and prementum; mentum about as long as wide, with 3 pairs of setae, prementum with 3 pairs of setae; ligula transverse, with anterior microtrichia; labial palpi 2 segmented. Thorax: Meso and metathorax tergites, and abdominal tergites and ventrites 18 each with 1 transverse ridge near anterior margin, ridge on ventral surface of abdominal segment 1 smaller lightly sclerotized. Prothorax subquadrate, transverse, 0.5 times as long as wide, sides curved, dorsal surface smooth; prosternal surface smooth, 3 setae (1 elongate) at anterolateral angles and 2 short setae at posterolateral angles; prosternum trapezoidal, sides oblique, posterior margin straight, a pair of medial setae present posterior to posterior margin of presternum. Mesoand metathorax transverse, both 0.5 times as long as wide, sides curved, surface of both tergites smooth with 3 short seta at anterolateral angles and 2 short setae at posterolateral angles; both sterna without welldened subdivisions, each smooth with a pair of discal setae near anterior margin; spiracular sclerite projecting strongly from lateral margin, spiracles (Fig. 2C) annular and angled posterolaterally. Legs (Fig. 2D) moderately long, 5segmented; claw falciform, with 2 setae. Abdomen: Segments 17 transverse, tergite surface smooth with 2 setae anterior to spiracles and 2 setae posterior to spiracles; ventrite surface with 3 setae, 2 anteriorly and 1 posteriorly. Segment 8 enlarged, tergite (Fig. 2I) with a stout spicule at each posterolateral margin, posterolateral angles with 8 short setae, 3 pairs of short setae anteromedially, 2 pairs of short setae posteromedially. Ventrite (Fig. 2J) with 9 pairs of setae and large stout process posteriorly with numerous minute setae apically. Tergite 9 with a basally forked process, directed dorsad, as wide as long; base of process with a pair of short, apically forked processes, 1 short seta at apex of forked process; anterior margin with lateral curved processes projecting from tergite 8; sternite 9 reduced and concealed from above. Adults Given the differences discovered in the larvae of the 2 subspecies, we examined adults to determine if there were corresponding adult differences. We examined 120 adult specimens of C. c. clavipes in the FSCA from the following states and provinces: CANADA: Ontario; USA: Colorado, Illinois, Indiana, Iowa, Kansas, Maine, Maryland, Massachusetts, Michigan, Mississippi, Missouri, New York, New Jersey, North Carolina, Ohio, Pennsylvania, Virginia, Wisconsin. We examined 46 adult specimens of C. c. puniceus in the

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Lee & Thomas: Taxonomic Status of Cucujus clavipes 149 Fig. 2. Larva of Cucujus c. puniceus A, habitus, dorsal view; B, head, dorsal view; C, A7 spiracle, D, prothoracic leg; E, left maxilla, dorsal view; F, labium, ventral view; G, labrum, dorsal view; H, left mandible, dorsal view; I, abdominal segments 89, dorsal view; J, same, ventral view.

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150 Florida Entomologist 94(2) June 2011 FSCA from the following states and provinces: CANADA: Alberta, British Columbia; USA: Alaska, California, Idaho, Oregon. As noted in previous literature, C. c. clavipes has a black scape, while C. c. puniceus has a red scape However, specimens of C. c. puniceus from Alaska ha ve black scapes. We had formed the impression that individuals from the western U.S. were on average more elongate than those from the eastern part of the country. Measurements of series from both populations revealed considerable overlap in body proportions, with specimens of the C. c. puniceus slightly more elongate, ranging in size from 12.5mm to 16.6mm, while specimens of C. c. clavipes ranged in size from 9.5mm to 14.6mm. Lee and Sato (2007) found taxonomically useful genitalic differences among Asian species of Cucujus Male genitalia from specimens of C. clavipes from all parts of its distribution were examined and found to be indistinguishable C ONCLUSIONS Despite the larval differences, the lack of consistent and signicant morphological differences in the adults suggests that at this point given the state of our knowledge, the present treatment of these 2 populations as subspecies of the same species is valid. Research into molecular differences may prove useful in understanding the limits of both taxa. A CKNOWLEDGMENTS We thank Chi Feng Lee and John Marris and 2 anonymous reviewers for reviewing a previous draft of this manuscript. George Ball generously lent larvae from the University of Alberta collection. This is Entomology Contribution No. 1180 of the Bureau of Entomology, Nematology, and Plant Pathology, Florida Department of Agriculture and Consumer Services. This research was supported by a grant to the senior author from the 2008 Academic Exchange Program of Andong National University. R EFERENCES C ITED B VING A. G., AND C RAIGHEAD F. C. 1931. An illustrated synopsis of the principal larval forms of the order Coleoptera. Entomol. Am. (New Series) 11: 1351. C ASEY T. L. 1884. Revision of the Cucujidae of America North of Mexico. Trans. Am. Entomol. Soc. 11:69112. F ABRICIUS J. C 1781. Species insectorum, exhibentes eorum differentias specicas synonyma auctorum, loca natalia, metamorphosin, adjectis observationibus, descriptionibus. Tom. 1. Hamburgi et Kilonii, impensis C. E. Bohnii, 566 p. H AYASHI N. 1980. Illustrations for identication of larvae of Cucujoidea (Coleoptera) found living in dead trees in Japan. Mem. Education Inst. Private Schools Japan. No. 72, 53 pp. H AYASHI N. 1986. 113 plates of Coleopteran larvae, In K. Morimoto and N. Hayashi [eds.], The Coleoptera of J apan in Color, Vol. 1, Osaka: Hoikusha, vi+323 pp., 113 pls. H ETSCHKO A. 1930. Cucujidae. Coleopterorum Catalogus 15(109): 193. K LAUSNITZER B. 2001. Die Larven Der Kafer Mitteleuropas (6. Band Polyphaga). Spektrum Akad. Verlag Heidelberg, Berlin, 1301. L AWRENCE J. F. 1991. Cucujidae. pp. 463488 In F. W. Stehr, [ed.], Immature insects. Vol. 2. Dubuque, IA: Kendall/Hunt. LECONTE, J. L. 1854. Synopsis of the Cucujides of the United States. Proc. Acad. Nat. Sci. Philadelphia 7: 7379. LECONTE, J. L. 1861. Classication of the Coleoptera of North America. Prepared for the Smithsonian Institution. Part 1. Smithsonian Misc. Collection 136: 1208. LECONTE, J. L. 1863. List of the Coleoptera of North America. Prepared for the Smithsonian Institution. Part 1. Smithsonian Misc. Collection 140: 149. LEE, C. F., AND SATO, M 2007. A review of the genus Cucujus Fabricius (Insecta: Cucujoidea: Cucujidae) from Taiwan, Japan, and China, with descriptions of two new species and the larvae of Cucujus mniszechi Grouvelle. Zool. Studies 46: 311321. LENG, C. W. 1920. Catalogue of the Coleoptera of America, North of Mexico. Mount Vernon, NY. John D. Sherman, Jr. x + 470 pp. LESAGE, L. 1984. Immature stages of Canadian Neochlamisus Karren (Col.: Chrysomelidae). Can. Entomol. 116: 383409. PETERSON, A. 1951. Larvae of Insects. An Introduction to Nearctic Species. Part II. Coleoptera, Diptera, Neuroptera, Siphonaptera, Mecoptera, Trichoptera. Columbus, OH: A. Peterson. v + 435 pp. SCHAEFFER, C. 1931. On a few new and known Coleoptera. Bull. Broooklyn Entomol. Soc. 26: 174176. SFORMO, T., WALTERS, K., JEANNET, K., WOWK, B., FAHY, G. M., BARNES, B. M., AND DUMAN, J. G. 2010. Deep supercooling, vitrication and limited survival to -100C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae. J. Exper. Biol. 213: 502-509 SMITH, D. B., AND SEARS, M. K. 1982. Mandibular structure and feeding habits of three morphologically similar coleopterous larvae Cucujus clavipes (Cucujidae), Dendroides canadensis (Pyrochroidae), and Pytho depressus (Salpingidae). Can. Entomol. 114: 173175. THOMAS, M. C. 1993. The at bark beetles of Florida (Laemophloeidae, Passandridae, Silvanidae). Arthropods of Florida and Neighboring Land Areas 15: i-viii and 1-93.

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Garcia & Norrbom.: Tephritoid Flies and Their Host Plants in Southern Brazil151 TEPHRITOID FLIES (DIPTERA, TEPHRITOIDEA) AND THEIR PLANT HOSTS FROM THE STATE OF SANTA CATARINA IN SOUTHERN BRAZIL F LVIO R. M. G ARCIA 1 AND A LLEN L. N ORRBOM 2 1 Federal University of Pelotas, Institute of Biology, Department of Zoology and Genetics Laboratory of Insect Ecology, 96010-900 P.O. Box: 354, Pelotas, RS, Brazil E-mail: avio.garcia@pq.cnpq.br 2 Systematic Entomology Laboratory, c/o National Museum of Natural History, MRC-168, P.O. Box 37012, Washington, DC 20013-7012, USA E-mail: allen.norrbom@ars.usda.gov A BSTRACT A total of 12,540 ripe fruits belonging to 46 species in 25 plant families were sampled from either the trees or the ground in 6 municipalities in the state of Santa Catarina, Brazil between 2002 and 2006 to determine which fruit y species developed on various host plants. Each fruit was weighed and placed into a plastic ask lled with sterilized sand 7 cm deep, and the opening of the ask was covered with sheer fabric. The asks were kept under controlled conditions (25 3C, 70 10% RH and 12h photophase). After 7 d, the pupae were sifted from the sand and transferred to Petri dishes lined with lter paper. Twenty-one species of Tephritoidea were recovered consisting of 13 species of Tephritidae, 6 of Lonchaeidae, and 2 of Ulidiidae. We present new host records for some species of fruit ies. Key Words: Tephritidae, Lonchaeidae, Ulidiidae, fruit pests, new host records R ESUMEN Este trabajo dirigido a la evaluacin de las especies de moscas de la fruta y sus plantas hospederas en el estado de Santa Catarina Brasil. Un total de 12.540 frutos maduros que pertenecen a 46 especies y 25 familias de arboles o del suelo en seis municipios del estado de Santa Catarina, Brasil entre 2002 y 2006 fueran muestradas. Cada fruto fue pesado y se coloca en un frasco de plstico cubierto con Voil, con 7 cm de arena esterilizada. Los frascos fueron mantenidos en condiciones controladas (25 3C, UR 70 10% y 12h de photophase). Despus de siete das, la arena se tamiza y la pupas fueron transferidas a placas de petri con papel ltro como sustrato. Veintin especies de Tephritoidea fueron recuperados 13 especies de Tephritidae, seis especies de Lonchaeidae, y dos de Ulidiidae. Se presentan los registros de para algunas especies de fruta o moscas. Translation provided by the authors. Approximately 70 species of Tephritidae are considered important pests of fruit production worldwide. The majority of the species of economic importance belong to 5 genera: Anastrepha Bactrocera Ceratitis Dacus and Rhagoletis (Garcia 2009). The genus Neosilba of the family Lonc haeidae (McAlpine & Steyskal 1982) includes 16 described species (Strikis & Prado 2005), some of which cause severe damage to certain species of fruit crops in the American tropics. Field surveys of fruit ies (Tephritoidea) and their host plants and parasitoids are essential for understanding the bioecology of the economically important genera and species in this superfamily (Bateman 1972). The creation of the common market, Mercosul, involving Brazil, Argentina, Paraguay and Uruguay, has elevated the importance of such studies because knowledge of these pest species, their hosts and natural enemies is key to containing their destructive effects as trade in fruits between these countries expands. In Brazil, most of the pest tephritids belong to the genus Anastrepha, but host plants are known for only 44% of the species (Zucchi 2007). Santa Catarina has the most host plant records 81, for species of Tephritidae among the Brazilian states (Garcia 2011). However, only 46 plant species belonging to 18 families are recorded in the state as hosts for fruit ies in the genus Anastrepha (Nora et al. 2000). This work reports new information from a sur vey of fruit y species and their host plants in the state of Santa Catarina, Brazil. M ATERIALS AND M ETHODS Fruit Sampling Between 2002 and 2006, a total of 12,540 ripe fruits from 46 plant species belonging to 25 fami-

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152 Florida Entomologist 94(2) June 2011 lies were sampled. Fruits were picked from the plants, or freshly fallen fruits were gathered from the ground below them. Sampling occurred in 6 municipalities of Santa Catarina, Brazil: Anchieta (26 53S and 53 33W), Chapec (27 06S and 53 16W), Cunha Por (26 07S and 53W 16), Palmitos (27 06S and 53 16W), So Carlos (27 07 S and 53 00 W), and Xanxer (26 87 S and 52W 40). Each fruit was weighed and placed into a plastic ask containing 7 cm of sterilized sand, and the opening of the ask was covered with sheer fabric. The asks were kept under controlled conditions (25 3C, 70 10% RH and 12h photophase). After 7 d, the sand was sifted and the pupae transferred to Petri dishes with lter paper as substrate. Identication of fruit ies and host plants Characters of the females, primarily of the aculeus, and body and wing markings, were considered in identifying species of Anastrepha (Zucchi 2000) identied by Garcia and Zucc hi. Ceratitis capitata (Wiedemann) is the only species of Ceratitis in Brazil and was easily recognized by the description by Zucc hi (2000). Lonchaeidae were identied by Dr. Pedro Strikis, and other Tephritidae and Notogramma cimiciforme Loew (Ulidiidae) were identied by Norrbom. The host plant species were identied by the botanists Dr. Srgio Augusto de Loreto Bordignon, Dr. Rosiane Berenice Denardin, and Lcia Salengue. Some voucher specimens of fruit ies and host plants were deposited at the Zoobotanic Museum of the University of Chapec. Data Analysis The infestation indexes were calculated in 2 ways: (1) by dividing the total number of puparia obtained by the number of fruits in the sample (puparia/fruit); or (2) by dividing the total number of puparia by the total mass (kg) of fruits in the sample (puparia/kg). The host plants of Anastrepha obtained in this work were compared to the lists of hosts assembled by Norrbom (2004) and Zucc hi (2007, 2008) with the aim of providing new host records for Brazil. R ESULTS AND D ISCUSSION Twenty-one species of Tephritoidea were recovered: 13 species of Tephritidae, 6 of Lonchaeidae, and 2 of Ulidiidae (= Otitidae) (Table 1). The species, Parastenopa guttata Aczl and P. montana Aczl, are new records of fruit ies for the state of Santa Catarina, and the total number of known species of Tephritidae from the state is now 81 (Garcia 2011). The development of ies from the fruit of yerba mat, Ilex paraguariensis A. St. Hil., is reported for the rst time. Two species of the genus Parastenopa P. guttata and P. montana were reared. The only Parastenopa species previously known to attac k this plant were reared from stems or from leaf galls of the Paraguay tea psyllid, Gyropsylla spegazziniana Lizer & Trelles (Hemiptera, Psyllidae) (Blanchard 1929; psyllid as Metaphalara spegazziniana ), although the North American P. limata (Coquillett) breeds in the fruit of several Ilex species (Benjamin 1934; Phillips 1946). Araticum, Annona rugulosa (Schltdl.) H. Rainer (Annonaceae), Inga sellowiana Benth. (Fabaceae), and the iguana hac kberry, C. iguanaea (Jacq.) Sarg. (Ulmaceae) are recorded for the rst time as host plants of Anastrepha fraterculus (Wiedemann). Rio Grande c herry, Eugenia involucrata DC., is recorded for the rst time as a host plant of Anastrepha obliqua (Macquart); and sete-capas, Campomanesia guazumif olia (Cambess.) O. Berg. (Myrtaceae), is recorded as a host plant of Anastrepha sororcula Zucchi. Strawberry guava, P. cattleianum Sabine (Myrtaceae), is recorded for the rst time as host plant of both A. obliqua and A. sororcula in Brazil. Previously stra wberry guava had been reported as a host of A. obliqua in Guatemala (Eska & Cunningham, 1987). The greatest infestations based on the number of puparia per fruit were found in pumpkin, Cucurbita pepo L. (6.59), followed by pineapple gua va, Acca sellowiana (O. Berg) Burret (6.23), and common gua va, Psidium guajava L. (6.16). Regarding the parameter puparia/kg the greatest infestations occurred in strawberry guava, P. cattleianum (422), followed by pineapple guava, P. cattleianum (278), yerba mat, I. paraguariensis A. St. Hil. (260), and wild cherry, P. avium (L.) L. (232). Considering both parameters, pineapple gua va, P. cattleianum was the species most infested by fruit ies The highest number of plant hosts was recorded for A. fraterculus (20 plant species from 8 families) (T able 1); predominantly g, Ficus carica L. (Moraceae) (75.0% of the total of samples collected were infested); guavirova, Campomanesia xanthocarpa O. Berg, (60.7%); guaviju, Myrcianthes pungens (O. Berg) D. Legrand (57.1%); Surinam c herry, Eugenia uniora L. (55.3%); wild c herry, P. avium (L.) L. (Rosacae) (52.0%); pineapple gua va, P. cattleianum (51.7%); common guava, P. cattleianum (51.4%); guava (48.0%), Campomanesia guazumif olia (45.4%) (Myrtaceae); and carambola, Averrhoa carambola L. (Oxalidaceae), (35.3%). Nine new host plants of A. fraterculus were recorded in Brazil: araticum, A. rugulosa (Annonaceae); Inga sellowiana (Fabaceae); common g, F. carica (Moraceae); pineapple guava, P. cattleianum (Myrtaceae); jaboticaba, Myrciaria cauliora (Mart.) O. Berg (Myrtaceae); Campomanesia guazumif olia (Myrtaceae); wild cherry, P. avium (Rosaceae); bergamot orange, Citrus reticulata

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Garcia & Norrbom.: Tephritoid Flies and Their Host Plants in Southern Brazil153 T ABLE 1. P LANTS SAMPLED WITH THEIR RESPECTIVE ORIGIN (O), FRUIT WEIGHT (FW), NUMBER OF FRUITS SAMPLED ( N ), NUMBER OF PUPAE (P), AVERAGE NUMBER OF PUPAE PER FRUIT (P/N), AND AVERAGE NUMBER OF PUPAE PER KG (P/KG). N = NATIVE AND E = EXOTIC. NUMBER IN PARENTHESES FOLLOWING FLY SPECIES NAMES = NUMBER OF SPECIMENS REARED. Plant Species O FW (kg) # fruits n #pupae PP/n SEP/kg SETephritidaeLonchaeidae & Ulidiidae Annonaceae Araticum, Annona rugulosa N4.64102330.32 0.17.10 2,4 A. fraterculus (3) Neosilba zadolicha (18) Aquifoliaceae Erva-mate, Ilex paraguariensis N1.0024652590.11 0.1259.70 20.5 Parastenopa spp.(254) Cactaceae Pereskia aculeata N0.3750450.90 0.2121.62 10.4 A. barbiellinii (19) Cucurbitaceae Abbora, Cucurbita pepo E139.77684486.59 2.13.21 1,8 A. grandis (310) Dasiops sp. (8) Euxesta sp.(12) Neosilba padroi (40) Chuchu, Sechium edule E8.94120460.38 0.15.15 3.2 Euxesta sp. (22) Lonchaea sp. (12) Neosilba padroi (10) Melancia, Citrullus lanatus E58.301420.14 0.10.03 0.1 A. grandis (2) Melo, Cucumis melo E10.8013120.92 0.31.11 0.7 Neosilba padroi (9) Pepino, Cucumis sativus E8.2243110.26 0.11.34 0.8 Euxesta sp. (7) Ebenaceae Caqui, Diospyros kaki E9.471263672.91 1.138.74 12.0 A. fraterculus (11) C. capitata (293) Euphorbiaceae Mandioca, Manihot esculenta N0.5221020.01 0.03.83 1.3 A. montei (2) Fabaceae Ing, Inga sellowiana N1.75246490.20 0.227.97 6.2 A. fraterculus (5) Lonchaea sp. (12) C. capitata (4) Neosilba sp. (19) Moraceae Figo, Ficus carica E1.2252220.42 0.218.10 8.3 A. fraterculus (16) Myrtaceae Ara, Psidium cattleianum N5.6767023933.57 1.3421.99 25.1 A. fraterculus (1220) Neosilba zadolicha (5) C. capitata (10) Neosilba padroi (7) Neosilba sp. (6)

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154 Florida Entomologist 94(2)June 2011Cereja, Eugenia involucrata N2.855161550.30 0.154.47 13.0 A. fraterculus (79) Neosilba padroi (6) C. capitata (15) Goiaba, Psidium guajava N12.4723614546.16 2.4116.64 10.9 A. fraterculus (697) Neosilba padroi (29) A. obliqua (14) A. sororcula (7) C. capitata (13) Goiaba-do-campo, Acca sellowiana N1.79804986.23 3.2277.80 23.2 A. fraterculus (254) Guaviju, Myrcianthes pungens N0.2552210.40 0.184.31 13.3 A. fraterculus (12) Guavirova, Campomanesia xanthocarpa N2.61717530.07 0.020.27 6.8 A. fraterculus (32) Jabuticaba, Myrciaria cauliora N0.162530.12 0.118.75 7.7 A. fraterculus (3) Pitanga, Eugenia uniora N4.4916994060.24 0.190.37 15.6 A. fraterculus (223) Neosilba padroi (12) Sete-capotes, Campomanesia guazumifolia N4.513987992.01 1.0177.08 23.1 A. fraterculus (360) Neosilba padroi (5) A. obliqua (4) A. sororcula (5) Uvaia, Eugenia pyriformis N1.603341480.44 0.292.48 17.9 A. fraterculus (51) Neosilba padroi (3) C. capitata (43) Oxalidaceae Carambola, Averrhoa carambola E3.3165250.38 0.17.56 3.12 A. fraterculus (9) Neosilba padroi (12) A. obliqua (2) Passioraceae Maracuj, Passiora edulis N26.582986282.11 0.523.63 15.7 A. dissimilis (9) Lonchaea sp. (185) A. pseudoparallela (363) Neosilba padroi (29) C capitata (12) Rosaceae Ameixa, Prunus domestica E5.241482671.80 1.250.94 10.8 A. fraterculus (148) Neosilba sp. (14) Cereja-do-mato, Prunus avium E0.4036942.61 1.1232.45 27.9 A. fraterculus (47) Nspera, Eriobotrya japonica E12.79126312851.02 0.7100.44 30.1 A. fraterculus (218) C. capitata (816) Pera, Pyrus communis E9.8596520.54 0.25.28 2.6 A. fraterculus (33) Pssego, Prunus persica E27.3265211511.77 0.942.13 18.1 A. fraterculus (372) Lonchaea sp. (14) C. capitata (322) Neosilba zadolicha (43) Neosilba sp. (41) Rutaceae Bergamota, Citrus reticulata E8.67138440.32 0.15.07 3.3 A. fraterculus (12) Neosilba padroi (12) TABLE 1. (CONTINUED) PLANTS SAMPLED WITH THEIR RESPECTIVE ORIGIN (O), FRUIT WEIGHT (FW), NUMBER OF FRUITS SAMPLED (N), NUMBER OF PUPAE (P), AVERAGE NUMBER OF PUPAE PER FRUIT (P/N), AND AVERAGE NUMBER OF PUPAE PER KG (P/KG). N = NATIVE AND E = EXOTIC. NUMBER IN PARENTHESES FOLLOWING FLY SPECIES NAMES = NUMBER OF SPECIMENS REARED. Plant SpeciesO FW (kg) # fruits n #pupae PP/n SEP/kg SETephritidaeLonchaeidae & Ulidiidae

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Garcia & Norrbom.: Tephritoid Flies and Their Host Plants in Southern Brazil155Notogramma cimiciforme (9) Laranja, Citrus sinensis E17.201761050.60 0.26.10 4.0 Neosilba padroi (69) Notogramma cimiciforme (29) Sapindaceae Camboat-vermelho, Cupania vernalis N5.806320.03 0.00.34 0.2 Neosilba padroi (2) Sapotaceae Agua, Chrysophyllum gonocarpum N0.248790.10 0.137.50 12.3 A. elegans (7) Solanaceae Jo, Solanum sisimbrifolium N0.2232130.41 0.259.09 29.5 Neosilba padroi (12) Tomate, Lycopersicum esculentum E6.571931070.55 0.316.29 8.2 Neosilba padroi (52) Ulmaceae Esporo-de-galo, Celtis iguanaea N911.668076080.75 0.30.67 0.2 A. fraterculus (3) Neosilba padroi(3) R. pastranai (577) TABLE 1. (CONTINUED) PLANTS SAMPLED WITH THEIR RESPECTIVE ORIGIN (O), FRUIT WEIGHT (FW), NUMBER OF FRUITS SAMPLED (N), NUMBER OF PUPAE (P), AVERAGE NUMBER OF PUPAE PER FRUIT (P/N), AND AVERAGE NUMBER OF PUPAE PER KG (P/KG). N = NATIVE AND E = EXOTIC. NUMBER IN PARENTHESES FOLLOWING FLY SPECIES NAMES = NUMBER OF SPECIMENS REARED. Plant SpeciesO FW (kg) # fruits n #pupae PP/n SEP/kg SETephritidaeLonchaeidae & Ulidiidae

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156 Florida Entomologist 94(2)June 2011Blanco (Rutaceae); and iguana hackberry, C. iguanaea (Jacq.) Sarg. (Ulmaceae). Pereskia aculeata Mill., also known as Orapro-nobis or Barbados gooseberry, was found to be a host plant for Anastrepha barbiellinii Lima; and Campomanesia guazumifolia (Myrtaceae) was recorded for the first time as a host plant for both A. obliqua and A. sororcula. Native plant species served as hosts of 12 fruit y species from 4 genera of Tephritidae, whereas exotic plant species served as hosts of only 4 species from 2 genera. Ceratitis capitata developed in 9 plant species from 5 families, with the following order of predominance: khaki, Diospyros kaki Thunb. (Ebenaceae) (93.1% of the fruits sampled were infested); medlar, Eriobotrya japonica (Thunb.) Lindl. (Rosaceae) (63.5%); uvaia, Eugenia pyriformis Cambess. (Myrtaceae) (29.2%); and peach, P. persica (L.) Batsch (28.1%). Some fruit y species occurred exclusively in 1 plant species: Anastrepha barbiellinii in ora-pro-nobis, Pereskia aculeata; Anastrepha grandis (Macquart) only in pumpkin, C. pepo; Rhagoletotrypeta pastranai Aczl only in esporo-de-galo, Celtis iguaneae (Jacq.) Sarg.; Anastrepha dissimilis Stone and A. pseudoparallela (Loew) only in Passiora edulis Sims; Anastrepha montei Lima only in cassava, Manihot esculenta Crantz; and Parastenopa guttata and P. montana only in yerba mat, I. paraguariensis St. Hil. Lonchaeid ies were recorded from 22 host plant species from 9 families of which 12 were native and 10 exotic. Arajo & Zucchi (2002) have also described the indiscriminate infestation of native and exotic fruits by Lonchaeidae. Neosilba padroi, a species described recently by Strikis & Lerena (2009), had the highest number of host species (7 native, 15 exotic) belonging to 8 families; the lance y, Lonchaea sp., had 4 host species (2 native and 2 exotic) in 4 families; Neosilba zadolicha McAlpine & Steyskal had 3 host species (1 native and 2 exotic) in 3 families; and Dasiops sp. occurred only in C. pepo (exotic). Neosilba zadolicha occurred in araticum, A. rugulosa (Annonaceae), ara, P. cattleianum (Myrtaceae), and peach, P. persica (Rosaceae). Spondias spp. (Anacardiaceae) (Santos et al. 2004) and medlar, Eriobotrya japonica (Strikis & Prado 2009) also ma y serve as hosts of N. zadolicha The Ulidiidae occurred only in 5 exotic species of Rutaceae and Cucurbitaceae; Euxesta sp. occurred only in Cucurbitaceae and N. cimiciforme Loew only in Rutacaeae. Euxesta sp. occurred on 3 plant species, with predominance in chayote, Sechium edule (Jacq.) Sw., (48.9%). N. cimiciforme occurred only in bergamot orange, C. reticulata Blanco, and orange, Citrus sinensis (L.) Osbeck. This species has a wide geographic range in the New World and is a scavenger recorded from a wide variety of plants (Steyskal 1963). Unlike our results, Ucha-Fernandes et al. (2003) and Aguiar-Menezes et al. (2004) obtained specimens of N. cimiciforme in passion fruit ( Passiora sp.), with occurrences also in tangerine, C. reticulata, and orange, C. sinensis Such differences may be due to the interpopulation differences or seasonal availability of host plants in different regions (Selivon 2000). Pumpkin was infested by 4 species of ies belonging to 3 families. Guava, passion fruit, and peach were infested by 5 species each, and these fruits were found to support infestations only of species of Tephritidae and Lonchaeidae. Under the conditions in which this research was conducted, we conclude that a wide diversity of fruit-bearing plant species in the state of Santa Catarina was attacked by 22 species of tephritoid ies. The most predominant y was A. fraterculus and P. cattleianum was the host species most frequently infested by these ies. ACKNOWLEDGMENTSWe thank the National Council of Technological and Scientic Development of Brazil (CNPq) for the Scholarship of Research Productivity; Biologist Pedro Strikis from Unicamp for Lonchaeidae identications; Prof. Dr. Roberto Antonio Zucchi for some species of Tephritidae conrmations, and Professors Dr. Srgio Bordignon from Unilasalle, and Dra. Rosiane Denardin and Lcia Verona from Unochapec, for plant identications.REFERENCES CITEDAGUIAR-MENEZES, E., NASCIMENTO, R. J., AND MENEZES, E. B. 2004. Diversity of fly species (Diptera: Tephritoidea) from Passiflora spp. and their hymenopterous parasitoids in two municipalities of southeastern Brazil. Neotrop. Entomol. 33: 113-116. ARAJO, E. L., AND ZUCCHI, R. A. 2002. Hospedeiros e nveis de infestao de Neosilba pendula (Bezzi) (Diptera: Lonchaeidade) na regio de Mossor-Au, R.N. Arq. Inst. Biol. 69: 91-94. BATEMAN, M. A. 1972. The ecology of fruit flies. Annu. Rev. Entomol. 17: 493-518. BENJAMIN, F. H. 1934. Descriptions of some native trypetid flies with notes on their habits. U.S. Dep. Agric. Tech. Bull. 401: 95 p. BLANCHARD, E. E. 1929. Descriptions of Argentine Diptera. Physis 9: 458-465. ESKAFI, F. M., AND CUNNINGHAM, R. T. 1987. Host plants of fruit ies (Diptera: Tephritidae) of economic importance in Guatemala. Florida Entomol. 70: 116-123. GARCIA, F. R. M. 2009. Fruit flies: biological and ecological aspects, pp. 1-35 In R. R. Bandeira [ed.], Current Trends in Fruit Flies Control on Perennial Crops and Research Prospects. Transworld Research Network, Kerala. GARCIA, F. R. M. 2011. Santa Catarina, in press In A. Malavasi et al. [eds.], Biologia, monitoramento, controle e distribuio de moscas-das-frutas no Brasil. Holos, Ribeiro Preto. MCALPINE, J. F., AND STEYSKAL, G. C. 1982. A revision of Neosilba McAlpine with a key to world genera of Lonchaeidae (Diptera). Canadian Entomol. 114: 105-137.

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Garcia & Norrbom.: Tephritoid Flies and Their Host Plants in Southern Brazil157NORA, I., HICKEL, E. R., AND PRANDO, H. F. 2000. Santa Catarina, pp. 320-327 In A. Malavasi and R. A. Zucchi [eds.], Mosca-das-frutas de Importncia Econmica no Brasil; Conhecimento Bsico e Aplicado. Holos, Ribeiro Preto. NORRBOM, A. L. 2004. Host plant database for Anastrepha and Toxotrypana (Diptera: Tephritidae: Toxotrypanini). Diptera Data Dissemination Disk (CDROM) 2. PHILLIPS, V. T. 1946. The Biology and Identification of Trypetid Larvae (Diptera: Trypetidae). Mem. Amer. Entomol. Soc. 12: [ii] + 161 + xvi p. SANTOS, W. S, CARVALHO, C. A. L., AND MARQUES, O. M. 2004. Registro de Neosilba zadolicha McAlpine & Steyskal (Diptera: Lonchaeidae) em umb-caj (Anacardiaceae). Neotrop. Entomol. 33: 653-654. SELIVON, D. 2000. Relaes com as plantas hospedeiras, pp.87-91 In A. Malavasi and R. A. Zucchi [eds.], Mosca-das-frutas de Importncia Econmica no Brasil; Conhecimento Bsico e Aplicado. Holos, Ribeiro Preto. STEYSKAL, G. C. 1963. The genus Notogramma Loew (Diptera: Acalyptratae, Otitidae). Proc. Entomol. Soc. Washington 65: 195-200. STRIKIS, P. C., AND PRADO, A. P. 2005. A new species of genus Neosilba (Diptera: Lonchaeidae). Zootaxa 828: 1-5. STRIKIS, P. C., AND PRADO, A. P. 2009. Lonchaeidae associados a frutos de nspera, Eryobotria japonica (Thunb.) Lindley (Rosaceae), com descrio de uma nova espcie de Neosilba (Diptera: Tephritoidea). Arq. Inst. Biol. 76: 49-54. STRIKIS, P. C., AND LERENA, M. L. M. A. 2009 A new species of Neosilba (Diptera: Lonchaeidae) from Brazil. Iheringia, Srie Zool. 99: 273-275. UCHA-FERNANDES, M., OLIVEIRA, I., MOLINA, R. M. S.,AND ZUCCHI, R. A. 2003. Biodiversity of frugivorous flies (Diptera: Tephritoidea) captured in Citrus groves in Mato Grosso do Sul, Brazil. Neotrop. Entomol. 32: 239-246. ZUCCHI, R. A. 2007. Diversidad, distribucin y hospederos del gnero Anastrepha en Brasil, pp. 77-100 In V. Hernndez-Ortiz [ed.], Moscas de la Fruta en Latinoamrica (Diptera: Tephritidae): Diversidad, Biologa y Manejo. S y G editores, Pedregal de Santo Domingo. ZUCCHI, R. A. 2008. Fruit flies in Brazil Anastrepha species and their hosts plants. Piracicaba, 2008. Acessado em: 20. Nov. 2009. Online. Disponvel em: www.lef.esalq.usp.br/anastrepha/. ZUCCHI, R. A. 2000. Taxonomia, pp. 13-24 In A. Malavasi and R. A. Zucchi [eds.], Moscas-das-frutas de Importncia Econmica no Brasil, Conhecimento Bsico e Aplicado. Holos, Ribeiro Preto.

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158 Florida Entomologist 94(2) June 2011 SUSCEPTIBILITY OF GENERA AND CULTIVARS OF TURFGRASS TO SOUTHERN CHINCH BUG BLISSUS INSULARIS (HEMIPTERA: BLISSIDAE) J AMES A. R EINERT A MBIKA C HANDRA AND M. C. E NGELKE Texas AgriLife Research & Extension Center, Texas A&M System, 17360 Coit Road, Dallas, TX 75252-6599 USA E-mail: j-reinert@tamu.edu A BSTRACT The southern chinch bug ( Blissus insularis Barber) is the most damaging insect pest of St. Augustinegrass ( Stenotaphrum secundatum Walt. Kuntze), across the southern U.S.A. Susceptibility to the southern c hinch bug and reproductive potential of the bugs on 24 cultivars from 7 genera in 8 turfgrasses were evaluated under greenhouse conditions. Stenotaphrum secundatum (Raleigh, Texas Common, and Captiva) cultivars were the most susceptible among all the turfgrass genera and eac h produced populations 97.5 bugs per 15-cm diameter plant within the 11-week test period from J ul to Sep 2008. Substantial populations also developed on zoysiagrass ( Zoysia spp.) (Emerald, Empire, Palisades, and Zorro) cultivars and on buffalograss ( Buchlo dactyloides (Nutt.) Engelm.). Low population development w as recorded on cultivars of bermudagrass ( Cynodon spp.), centipedegrass ( Eremochloa ophiuroides (Munro) Hack.), seashore paspalum ( Paspalum vaginatum Swartz), bahiagrass ( Paspalum notatum Flugge), and tall fescue ( Festuca arundinacea Schreb.). K ey Words: turfgrass pests, host plant resistance, host range, pest management, host plants R ESUMEN La chinche surea del pasto ( Blissus insularis Barber) es el insecto mas daino para St. Augustingrass ( Stenotaphrum secundatum Walt. Kuntze), en el sur de U.S.A. La susceptibilidad de materiales y el potencial reproductivo del insecto fueron evaluados en 24 cultivares pertenecientes a siete gneros de pasto para csped en condiciones de invernadero Los cultivares de S. secundatum (Raleigh, Texas Common, y Captiva) fueron los mas susceptibles con poblaciones de 97.5 chinches por planta en maceta de 15 cm de dimetro, en 11 semanas de evaluacion de julio a septiembre 2008. Considerables niveles de poblaciones tambin fueron registrados en zoysiagrass ( Zoysia spp.) (cultivares Emerald, Empire, Palisades, y Zorro), as como en el cultivar de buffalograss ( Buchloe dactyloides (Nutt.) Engelm.). Bajos niveles de desarrollo se registraron en cultivares de bermudagrass ( Cynodon spp.), centipedegrass ( Eremochloa ophiuroides (Munro) Hack.), seashore paspalum ( Paspalum vaginatum Swartz), bahiagrass ( Paspalum notatum Flugge), y tall fescue ( Festuca arundinacea Schreb.). The authors provided the translation by Carlos Campos The southern chinch bug (SCB) ( Blissus insularis Barber) (Hemiptera: Blissidae) is the most damaging insect pest of St. Augustinegrass ( Stenotaphrum secundatum Walt. Kuntze), across the southern U .S.A., Bermuda, Mexico, and throughout the Caribbean Archipelago (Henry & Froeschner 1988; Sweet 2000). In the U.S.A. it is found from South Carolina to Florida, westward to Oklahoma and along the Gulf Coast to Texas and in California, Hawaii, Puerto Rico, and Guam (Reinert et al. 1995; Mortorell 1976; Vittum et al. 1999). This pest begins to damage St. Augustine lawns as early as Mar in parts of Southern Florida and Texas and rst instars have been found during all 12 months in Southern Florida (Reinert, unpublished data). Damage begins as small patches of dead grass early in the season, with entire lawns killed as the summer progresses. During heavy infestations, large populations will progress from one lawn to another as they move from one city block to the next (Reinert & Kerr 1973). According to Painter (1928) B. leucopterous L. damages grasses by removal of the synergic food-bearing solutions whic h ow to the roots by way of the phloem; the stopping up of the sieve tubes, and perhaps also the removal of water from the xylem, together with the stoppage of the tracheids. It is believed that this same process takes place when SCB feeds at the node and the crown area of Stenotaphrum which mimics the effects of a toxin being injected into the plant. SCB infestations soon turn the grass yellow, brown, and it eventually dies within a few days. Both nymphs and adults feed in aggregates in localized areas early in the season, with these areas coalescing

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Reinert et al.: Susceptibility of Turfgrasses to Southern Chinch Bug159 into large dead areas or entire lawns as the season progresses (Reinert et al. 1995). Stenotaphrum is cultivated extensively in subtropical and tropical climates around the world (Busey 2003; Sauer 1972). It is used widely across the southern U.S.A. as a turfgrass in urban landscapes, including residential and commercial lawns, parks, some sports complexes, and as a pasture grass (Busey 2003; Sauer 1972). Stenotaphrum has long been considered the primary host of the SCB (Reinert & Kerr 1973; Reinert et al. 1995; Vittum et al. 1999). The SCB has been identified on 9 other grass hosts (Cherry & Nagata 1997; Slater 1976). Resistant cultivars Floratam, Floralawn, FX-10, and Captiva have been developed and deployed to help manage this pest (Busey 1993; Cherry & Nagata 1997; Dudeck et al. 1986; Horn, et al. 1973; Reinert & Dudeck 1974). Recently, populations of SCB have been identied that have overcome the resistance in each of these cultivars (Busey & Center 1987; Cherry & Nagata 1997; Reinert 2008). This study was established to characterize the reproductive potential and development of the SCB on 24 cultivars of turfgrass from 7 genera in 8 turfgrasses used across the Southern U.S.A. M ATERIALS AND M ETHODS This study was conducted under greenhouse conditions during Jul-Sep 2008 at the Texas AgriLife Research and Extension Center at Dallas, TX, U.S.A. A total of 24 cultivars (Table 1) including St. Augustinegrass, (5) zoysiagrass ( Zoysia spp.) (5), bermudagrass ( Cynodon spp.) (5), buffalograss ( Buchlo dactyloides (Nutt.) Engelm.) (2), centipedegrass ( Eremochloa ophiuroides (Munro) Hack.) (1), seashore paspalum ( Paspalum vaginatum Swartz) (2), bahiagrass ( Paspalum notatum Flugge) (2), and tall fescue ( Festuca arundinacea Schreb.) (2) were evaluated for their susceptibility to SCB infestation and development. Plugs of grass grown either in the eld or greenhouse were divided and planted into 18-cell tra ys and allowed to grow to cover the whole cell. Cells measured 7.5 7.5 cm and 4 cm deep. Plants were fertilized bi-monthly during establishment with Mirac le-Gro All Purpose fertilizer (24-8-16 + B (200 ppm), Cu (700 ppm), Fe (1500 ppm), Mn (500 ppm), Mo (5 ppm), Zn (600 ppm)) (Scotts, 14111 Scottslawn Road, Marysville, Ohio) at ~8.25 kg of N ha -1 month -1 Once sufcient growth was achieved to provide near complete coverage of the entire cell (ca. 14 weeks), plugs from 4 cells of each cultivar were repotted into 15-cm diam plastic pots and allowed to establish for 2 weeks. Each pot was lled with soil within 2.5 cm of the top. Potted plants were then tted with a cylindrical plastic cage (a modication of Starks & Burton 1977) to exclude extraneous insects and to conne the SCB. Cages were made of Lexan 8010 Film (0.2 mm thickness) (General Electric Plastics, 4600 AC Bergen op Zoom, The Netherlands) and measured 32.5 cm tall and 2.5 cm in diam and were vented on opposite sides with two, 8-cm diameter ventilation holes to allow air circulation within the cage. Ventilation holes and the top end of the cage were covered with Voile 118 Decorator Fabric in White # 235-004-81 (Hancock Fabrics, Plano, Texas, hancockfabrics.com) cut 15 mm larger than the holes and secured with glue. On 6-8 Jul 2008, 10 adults (5 male and 5 female) were introduced into each cage. Before bugs were introduced, each pot was lled to the top with ne topdressing sand. When each cage was inserted over the plant in the pot the area between the cage and the wall of the pot was backlled with additional sand to form an escapeproof barrier to the conned insects. Pots were maintained in the greenhouse in a randomized complete block design with 4 replicates and held on full size aluminum sheet pans that were 45 cm 65 cm 18 gauge (WINCO Industries Co ., Lodi, New Jersey). Potted plants were provided sub-surface irrigation by lling the pans with 1.5-2.0 cm of water as needed to avoid wilting of the test grasses. Watering was done every 3-4 d. After the pots were allowed to soak-up water for about 2 h, the excess water was drained to avoid causing deterioration of the root system. Cages had to be opened about every 2 weeks so the grass could be clipped, since there was not enough room for the continued plant growth. The clipping process was done over one of the aluminum sheet pans so that any SCB adults or nymphs that were removed with the clippings or that tried to escape could be collected with a hand aspirator and returned to the grass when the cage was put back in the pot. SCB for this experiment were collected by vacuum sampling the bugs from a residential lawn of S. secundatum in the Houston, Texas area. A modication of the procedure for vacuuming (Nagata & Cherry 2007; and personal communication) was used. An Echo Shred N Vac model ES210 (Echo Inc., Lake Zurich, Illinois) leaf blower/ vacuum was modied by cutting-off the distal 15cm end of the vacuum tube. This unit has an 87.5 cm long intake tube (11.25 cm diam) and produces 225.31 km/h (140 mph) of vacuum. A 20-cm long piece of French drain pipe (10.3 cm outside diameter) that t loosely within the intake tube was shimmed to t the inside diam of the vacuum tube by wrapping it with duct tape, close to each end, so it would t snugly inside to reattach the 2 pieces of the intake vacuum tube. When the 2 pieces of tube were re-joined, a 20-cm diameter piece of polyester Tricot interlocking netting (mesh size ca. 9.6 8 per cm, 24 20 per inch) cut

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160 Florida Entomologist 94(2) June 2011 from the material of a BioQuip superior aerial net (Cat. No. 7215NA, BioQuip Products, Rancho Dominguez, CA), material was inserted at the outer end of the French drain insert to form a 15cm deep collecting basin to catch insects that were dislodged as the grass was vacuumed. The potted plants were maintained in the greenhouse until the week of 22 Sep 2008, when the total number of bugs produced on each plant was assayed. Plants in the experiment (1 replicate at a time) were individually submerged in 18.9-liter plastic buckets of water (the plant was weighted with a stone so it would stay submerged) and all SCB nymphs and adults that oated to the surface within 30 min were removed, identied by instars, and counted. After a plant had been submerged for 5 min and again at 15 min, the canopy of the plant was agitated by T ABLE 1.R ATE OF REPRODUCTION AND DEVELOPMENT OF SOUTHERN CHINCH BUG ON CULTIVARS AND GENERA OF TURFGRASS Genera of grasses Cultivars Nymphs Adults a, b (A) Total a, b (N + A) 1st a, b 5th a, b Total nymphs a, b (N) Stenotaphrum secundatum (St Augustinegrass) Raleigh 34.3 a*45.8 a*163.0 a*17.8 a*180.8 a* A** TX Common 25.3 a30.0 b117.5 ab4.3 b121.8 b A Captiva 30.7 a18.5 b93.3 b4.3 bc97.6 b A FX-10 1.0 bc0.0 c1.3 d0.0 d1.3 d B Floratam 0.8 bc0.3 c1.1 d0.0 d1.1 d B Zoysia spp. (Zoysiagrass) P alisades 5.8 b0.5 c16.5 c2.5 bcd19.0 c A Emerald 1.3 bc1.0 c8.8 cd1.0 cd9.8 cd AB Zorro 1.3 bc2.3 c8.0 cd0.0 d8.0 cd AB Empire 0.8 bc0.8 c4.3 cd1.3 bcd5.6 cd AB Cavalier 0.0 c0.0 c0.3 d0.8 d1.1 d B Buchlo dactyloides (Buffalograss) 609 3.7 bc0.3 c7.5 cd2.5 bcd10.0 cd ns Prairie 0.8 bc0.0 c1.0 cd0.8 d1.8 d Festuca arundinacea (Tall Fescue) Rebel 2.0 bc1.0 c5.0 cd1.0 cd6.0 cd ns Paladin 2.8 bc0.0 c3.3 cd0.8 d4.1 cd Cynodon spp. (Bermudagrass) Tifton 10 1.3 bc0.5 c2.8 cd0.3 d3.1 cd ns Tifway 0.0 c1.3 c1.3 cd0.0 d1.3 d Texturf 10 0.3 c0.0 c0.5 d0.0 d0.5 d TifSport 0.0 c0.0 c0.0 d0.0 d0.0 d Common 0.0 c0.0 c0.0 d0.0 d0.0 d Paspalum notatum (Bahiagrass) Argintine 1.8 bc0.0 c2.0 cd0.0 d2.0 d ns Pensacola 0.0 c0.0 c0.0 d0.0 d0.0 d Paspalum vaginatum (Seashore Paspalum) Seadw arf 0.3 c0.0 c1.5 cd0.5 d2.0 cd ns AZ-1 0.0 c0.0 c0.0 d0.0 d0.0 d Eremochloa ophiuroides (Centipedegrass) Tifblaire 0.0 c0.3 c 0.3 d0.3 d0.6 d a Mean number of 1st, 5th instars, total nymphs, adults, and total population on each turfgrass cultivar after an 11-week development period. b Data in each column was transformed as ( n + 0.001) for analysis; untransformed means are reported. *Means in a column followed by the same lower case letter are not signicantly different by F ishers protected LSD ( P = 0.05) (Analysis among all turf groups). **Means in the total column for eac h grass followed by the same upper case letter are not signicantly different by Fishers protected LSD ( P = 0.05) or by Students t -test. (Analysis within a turf group only).

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Reinert et al.: Susceptibility of Turfgrasses to Southern Chinch Bug161 hand to dislodge any bugs that had failed to let loose and oat to the surface. This procedure was a modication of the otation method that has been used widely to accurately assay eld populations of SCB for chemical efcacy tests (Reinert 1974, 1982). Data Analysis and Statistics Data for the number of SCB for each growth stage for each cultivar were analyzed by Analysis of Variance (ANOVA) (PROC GLM) for a randomized complete block design with 4 replications to test for differences in the number of progeny that had developed on each cultivar. Data for cultivars were also grouped by species of grass ( Zoysia and Cynodon each contained 2 species, but were analyzed by genus only) and analyzed to determine suitability among the 8 types of turfgrass tested. The transformations (n + 0.001) was used on eac h data set to achieve normality and homogeneity of variance before analysis (Kuehl 2000) but untransformed means are presented. Means were compared at the 5% level of signicance with Fishers least-signicant difference (LSD) multiple range test. For the total population column, means were also compared within each grass genera by Students t -test (SAS Institute 2009). R ESULTS AND D ISCUSSION This method of caging the SCB on potted grass plants in a no-choice experiment worked well to assay the reproductive and developmental potential on each cultivar. Caging was necessary to conne the bugs on the grasses, but the main problem with this type and size of cage was that cages were not large enough to accommodate the growth potential of several of the grasses, and they had to be opened during the experiment to clip and remove leaf material from many of the cultivars. The modied Echo blower/vacuum also worked well for collecting large numbers of SCB specimens for this type of study. Stenotaphrum (St. Augustinegrass) Stenotaphrum as expected, served as the best host among the 8 turfgrass groups The highest population was produced on Raleigh with all 5 instars and adults present in substantial numbers for a mean of 163.0 nymphs, 17.8 adults, and a total population of 180.8 bugs per plant after the 11-week test period (Table 1). Texas Common was the second best host (117.5 nymphs, 121.8 total), followed by Captiva with 93.3 nymphs and 97.5 total bugs produced on it. Analysis conducted across all 8 groups of turfgrass showed that Raleigh produced signicantly more SCB than either Texas Common or Captiva, but all 3 cultivars serve as good reproductive hosts and all 3 mean populations far exceeded the accepted damage threshold level of 20 to 30 bugs per 0.1 m 2 (Reinert 1972; Buss & Unruh 2006). The highest individual SCB population on any of the replicate plants of Raleigh, Texas Common, and Captiva was 311, 252, and 155, respectively. Neither FX-10 nor Floratam served as an acceptable host with this population of SCB and they yielded only an average of 1.3 and 1.1 total bugs, respectively. Moreover, all of the bugs on these 2 cultivars had developed on only 1 replicate plant. Additionally, they were all rst instars, except for 1 third instar on 1 of the FX-10 replicate plants and 1 fth instar on 1 Floratam replicate plant. Analysis conducted only on the 5 cultivars of Stenotaphrum showed that population levels on FX-10 and Floratam were not signicantly different, and they were signicantly lower than those on the 3 susceptible cultivars (Raleigh, Texas Common, and Captiva). In a related study in a lab no-choice experiment with the same population of SCB, adult survival was high on Raleigh, Texas Common, and Captiva (72-78%), but survival on Floratam and FX-10 was only 48 and 58%, respectively, after 7 d of connement (Reinert unpublished data). However, when Floratam and FX-10 were rst released (Horn et al. 1973; Busey 1993), both cultivars consistently provided >80% antibiosis within 7 d for populations of SCB adults that were collected from lawns in Florida (Reinert & Dudeck 1974; Reinert 1978). More recently, however, Cherry & Nagata (1997) showed that oviposition of eggs was high and survival on Floratam, Seville, Bitterblue, and FX-10 cultivars was 88.6 to 75.6% for populations of SCB collected from Florida lawns. Zoysia (Zoysiagrass) Among the 5 Zoysia cultivars, Palisades served as the best host for SCB with the developing population consisting of all 5 instars and adults A mean of 16.5 nymphs and 19.0 total bugs had developed on this cultivar during the 11-week test period. When the Zoysia cultivars were analyzed either among the total cultivars or separately for the genus the same statistical separations were recorded (Table 1). Palisades produced a signicantly higher number of SCB than Cavalier (mean total of only 1 SCB), but the population on Palisades was not signicantly higher than either Emerald, Zorro, or Empire. Although Zoysia is not normally considered a primary host of the SCB (Reinert et al. 1995), this study shows certain cultivars, Palisades along with Emerald, Zorro, and Empire can serve as acceptable reproductive hosts with mean development of 5.5 total bugs during this study. This would be an equivalent of 31 bugs per 0.1 m2, which is within the considered threshold of damage on Stenotaphrum. Three of the replicate

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162 Florida Entomologist 94(2)June 2011plants of Palisades had total populations >23 SCB. Also, 1 replicate plant each of Zorro, Emerald, and Empire had total populations of 27, 26, and 14 SCB, respectively. Population development on Cavalier in the present study with B. insularis was the lowest among the Zoysia cultivars tested with an average of 1.1 bugs per replicate plant. Studies with a related Blissus species, the western chinch bug ( B occiduus Barber), showed that Zoysia and particularly the cultivars Zenith, Meyer, and Crowne, serve as acceptable hosts for that species as well (Eickhoff et al. 2006, 2007). Populations of B. occiduus preferred both Buchlo and Zoysia. Cavalier along with Emerald and Zorro produced the lowest number of B. occiduus in their greenhouse study and these cultivars were listed as moderately resistant (Eickhoff et al. 2007). Cavalier expresses good resistance to both species of Blissus.Buchlo (Buffalograss)For the 2 cultivars of Buchlo only served as a good host for SCB with 7.5 nymphs and a total of 10.0 bugs per plant (Table 1). This would be equivalent to 56.5 bugs 0.1 m2 which is within the threshold of damage on Stenotaphrum. Although there was a large difference between the mean number of SCB that developed on the 2 cultivars, there was no signicant difference due to a large amount of variance among the replicates.Other Turfgrass GeneraSurprisingly, both cultivars of Festuca, Rebel, and Paladin, did support low development of SCB with total mean numbers of 6 and 4.1 bugs per replicate plant, respectively (Table 1). The development on the 5 Cynodon cultivars was very low. Poor development of SCB has also been reported on Cynodon (no cultivar identied) by Cherry & Nagata (1997). Slater (1976) in his study of host relationships of Blissinae described Cynodon as a breeding host for the SCB. However, Kelsheimer & Kerr (1957) reported Cynodon to be rarely attacked by the SCB. One of the authors has received numerous reports of chinch bug feeding on and damage to Cynodon in Florida, Texas, and in island nations throughout the Caribbean, but most likely these populations and their damage were caused by another Blissus species. Cynodon has been reported as a host of B. leucopterus leucopterus Say (Lynch et al. 1987). The 2 cultivars of P. notatum, 2 cultivars of P. vaginatum, and 1 cultivar of Eremochloa did not support much SCB development (<2.0 bugs per plant) in this study. Also, only 1 adult developed on 1 of the Eremochloa cv. Tifblaire replicate plants. Kelsheimer & Kerr (1957) reported Eremochloa to be an occasional host for the SCB. Additionally, Kerr (1966) reported that SCB will attack other lawn grasses ( P. notatum, Cynodon, Eremochloa, and Zoysia) but mostly it is a problem on Stenotaphrum. Other common hosts include crabgrass ( Digitaria spp.), torpedograss ( Panicum repens L.), and pangolagrass (Digitaria eriantha Steud) (Slater & Baranowski 1990). This study conrms the high suitability of Stenotaphrum as a developmental host for the SCB. We also show that 4 cultivars of Zoysia and the cultivar 609 Buchlo serve as good breeding hosts and may have potential for damage by SCB. ACKNOWLEDGMENTSThis study was supported in part by grants from the Texas Turfgrass Research, Extension, and Education Endowment. Appreciation is extended to J. E. McCoy for his technical assistance.REFERENCES CITEDBRANDENBURG, R. L., AND VILLANI, M. G. 1995. Handbook of Turfgrass Insect Pests. Entomol. Soc. America, Lanham, Maryland. BUSEY, P. 1993. Registration of FX-10 St. Augustinegrass. Crop Sci. 33: 214-215. BUSEY, P. 2003. St. Augustinegrass, Stenotaphrum secundatum (Walt.) Kuntze, Ch. 20, pp. 309-330 In M. D. Casler and R. R. Duncan [eds.], Turfgrass Biology, Genetics, and Breeding. John Wiley & Sons, Inc., Hoboken, New Jersey, 367 pp. BUSEY, P., AND CENTER, B. 1987. Southern chinch bug (Hemiptera: Heteroptera: Lygaeidae) overcomes resistance in St. Augustinegrass. J. Econ. Entomol. 80: 608-611. BUSS, E. A., AND UNRUH, J. B. 2006. Insect management in your Florida lawn. Univ. Florida IFAS Ext. CIR427, 12 pp. (available at http://edis.ifas.u.edu/pdfles/LH/LH03400.pdf). CHERRY, R. H., AND NAGATA, R. T. 1997. Ovipositional preference and survival of southern chinch bugs (Blissus insularis Barber) on different grasses. Intl. Turfgrass Soc. Res. J. 8: 981-986. DUDECK, A. E., REINERT, J. A., AND BUSEY, P. 1986. Registration of Floralawn St. Augustinegrass. Crop Sci. 26: 1083. EICHHOFF, T. E., BAXENDALE, F. P., AND HENG-MOSS, T. M. 2006. Host preference of the chinch bug, Blissus occiduus J. Insect Sci. 7: 1-6. EICHHOFF, T. E., HENG-MOSS, T. M., AND BAXENDALE, F. P. 2007. Evaluation of warm-season turfgrasses for resistance to the chinch bug, Blissus occiduus HortScience 42(3): 718-720. HENRY, T. J., AND FROESCHNER, R. C. [Eds.]. 1988. Catalog of the Heteroptera or True Bugs of Canada and the Continental United States. E. J. Brill, Leiden. HORN, G. C., DUDECK, A. E., AND TOLER, R. W. 1973. Floratam St Augustinegrass: A fast growing new variety of ornamental turf resistant to St. Augustine decline and chinch bugs. Florida Agr. Exp. Stn. Circ. S-224.

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Reinert et al.: Susceptibility of Turfgrasses to Southern Chinch Bug163KELSHEIMER, E. G., AND KERR, S. H. 1957. Insects and Other Pests of Lawns and Turf. Florida Agr. Exp Stn. Circ. S-93. KERR, S. H. 1966. Biology of the lawn chinch bug, Blissus insularis Florida Entomol. 49(1): 9-18. KUEHL, R. O. 2000. Design of Experiments: Statistical Principles of Research Design and Analysis. Duxbury Press, Albany, New York. LYNCH, R. E., SOME, S., DICKO, I., WELLS, H. D., ANDMONSON, W. G. 1987. Chinch bug damage to bermudagrass. J. Entomol Sci. 22: 153-158. MORTORELL, L. F. 1976. Annotated Food Plant Catalog of the Insects of Puerto Rico. Agr. Exp Stn., Univ. Puerto Rico 303 pp. NAGATA, R., AND CHERRY, R. 2007. Resistance to 2 classes of insecticides in southern chinch bugs (Hemiptera: Lygaeidae). Florida Entomol. 90: 431-434. PAINTER, R. H. 1928. Notes on the injury to plant cells by chinch bug feeding. Ann. Entomol. Soc. America 21: 232-242. REINERT, J. A. 1972. Control of the southern chinch bug, Blissus insular in south Florida. Florida Entomol. 55: 231-235. REINERT, J. A. 1974. Tropical sod webworm and southern chinch bug control in Florida. Florida Entomol. 57: 275-279. REINERT, J. A. 1978. Antibiosis to the southern chinch bug by St. Augustinegrass accessions. J. Econ. Entomol. 71: 21-24. REINERT, J. A. 1982. Carbamate and synthetic pyrethroid insecticides for control of organophosphate resistant southern chinch bugs. J. Econ. Entomol. 75(4): 716-718 REINERT, J. A. 2008. Southern chinch bugs on St. Augustinegrass Out of control in Texas. Texas Turfgrass. Winter 2008: 10-11. REINERT, J. A., AND DUDECK, A. E. 1974. Southern chinch bug resistance in St. Augustinegrass. J. Econ. Entomol. 67: 275-277. REINERT, J. A., HELLER, P. R., AND CROCKER, R. L. 1995. Chinch bugs, pp. 38-42 In R. L. Brandenburg and M. G. Villani [eds.], Handbook Turfgrass Insect Pests. Entomol. Soc. America, Publ. Dept., Lanham, Maryland. 140 pp. REINERT, J. A., AND KERR, S. H. 1973. Bionomics and control of lawn chinch bugs. Bull. Entomol. Soc. America 19: 91-92. SAS INSTITUTE. 2009. SAS system for Windows, release 9.1. SAS Institute, Cary, North Carolina. SAUER, J. D. 1972. Revision of Stenotaphrum (Graminease: Paniceae) with attention to its historical geography. Brittonia 24: 202-222. SLATER, J. A. 1976. Monocots and chinch bugs: A study of host plant relationships in the Lygaeid subfamily Blissinae (Hemiptera: Lygaeidae). Biotropica 8(3): 143-165. SLATER, J. A., AND BARANOWSKI, R. M. 1990. Lygaeidae of Florida (Hemiptera: Heteroptera). Vol. 14. Arthropods of Florida and Neighboring Land Areas. Florida Dept. Agr. Cons. Serv., Gainesville, Florida. STARKS, K. J., AND BURTON, R. L. 1977. Greenbugs: Determining biotypes, culturing, and screening for plant resistance. U.S.D.A.-A.R.S. Tech. Bull. No. 1556: 12 pp. SWEET, M. H. 2000. Seed and chinch bug (Lygaeoidae), pp. 143-264 In C. W. Schaefer and A. R. Paninzzi [eds.], Heteroptera of Economic Importance. CRC Press LLC, Boca Raton, Florida. VITTUM, P. J., VILLANI, M. G., AND TASHIRO, H. 1999. Turfgrass Insects of the United States and Canada. Cornell Univ. Press, 422 pp.

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164 Florida Entomologist 94(2) June 2011 TOMARUS SUBTROPICUS (COLEOPTERA: SCARABAEIDAE) LARV AL FEEDING HABITS O LGA S. K OSTROMYTSKA 1 AND E ILEEN A. B USS 2 1 Entomology Department, Rutgers, The State University of New Jersey, 93 Lipman Drive, New Brunswick, NJ 08901 2 Entomology and Nematology Department, University of Florida, P.O. Box 110620, Gainesville, FL 32611 A BSTRACT The importance of soil organic matter for Tomarus subtropicus Blatchley larval development and survival, the amount of damage larvae could cause on turfgrasses, and potential larval host range were investigated in greenhouse experiments. First instars were reared individually in seedling trays containing sand or peat, with or without St. Augustinegrass. Survival, developmental stage, and nal weight were recorded 1 month after introduction. First instars died in the pots with peat but no grass, so it appears that grass roots were critical for larval growth and development. Soil organic matter did not signicantly affect grub weight gain and development, but more root loss occurred with grass grown in sand. In host range tests (2005 and 2006), rst and third instars were reared on 6 species of warm season grasses and ryegrass. Grub weight gain, development, survival and grass root reduction were determined 2 months after introduction. Larval survival ranged from 62-93% if grubs were reared on warm season grasses to only 40% if reared on ryegrass. Grubs reared on warm season grasses gained weight and successfully developed into third instars, indicating that all of the tested warm season turfgrasses were suitable for larval T. subtropicus growth and development. Grub feeding caused signicant root reduction of all grasses in our study, which ranged from 36 to 87% and differed among grass species. As result, quality ratings and clipping yields decreased for most of the turfgrasses after 5 weeks of infestation, but bahiagrass and seashore paspalum were less affected by T. subtropicus root feeding, compared to the other grass species Key Words: sugarcane grub, host range, bermudagrass, sugarcane, St. Augustinegrass, ryegrass, root reduction, soil organic matter, grub weight gain and development R ESUMEN La importancia de materia orgnica en el suelo para el desarrollo y sobrevivencia de larvas de Tomarus subtropicus Blatchley, la cantidad de dao que las larvas puedan causar en el csped y el rango potencial de hospederos por las larvas fueron investigados en experimentos realizados en invernaderos Se criaron los primeros instares individualmente en bandejas usadas para plantillas con arena o turba y con o sin el csped San Augustin. El sobrevivencia, el estadio de desarrollo y el peso nal fueron anotados 1 mes despus de la introduccin. Los primeros instares murieron en las macetas con turbo y sin grama, esto parece indicar que las raices de grama son bsicas para el crecimiento y desarrollo de las larvas. La materia orgnica del suelo no afecto el aumento en el peso y el desarrollo de las larvas, pero hubo una mayor prdida de las raices de la grama sembrada en arena. En pruebas del rango de los hospederos (2005 y 2006), se criaron los primeros y terceros instares sobre 6 especies de grama de la estacin clida y sobre centeno. Se determinaron el aumento en el peso de las larvas, el desarrollo, la sobrevivencia reduccin en las raices de la grama 2 meses despues de la introduccin. El sobrevivencia de las larvas fue entre 62-93% en las larvas criadas sobre grama de la estacin clida y solo 40% en larvas criadas sobre centeno. Las larvas criadas sobre grama de la estacin clida aumentaron en peso y se desarrollaron exitosamente en instares de tercer estadio, que indica que todas las clases de grama de la estacin clida probadas fueron apropiadas para el crecimiento y desarrollo de larvas de T. subtropicus. En nuestro estudio la alimentacin de las larvas caus una reduccin signicativa de las raices de 36 a 87% y varan entre las especies de grama. Como un resulto, el indice de la calidad y el rendimiento de las cortadas de grama diminuy para la mayora de las clases de grama despus de 5 semanas de infestacin, pero el csped Bahia y el paspalum costero fueron menos afectados por la alimentacin de T. subtropicus sobre las raices, comparados con las otras especies de grama. Tomarus subtropicus Blatchley is a destructive turfgrass pest along Floridas Gulf and Atlantic Coasts, but is also distributed along coastal Alabama, Georgia, South Carolina, and North Caro-

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Kostromytska & Buss: Tomarus subtropicus Host Range 165 lina (Cartwright 1959). As with other grub species, T. subtropicus grubs directly damage turf by their root-feeding and tunneling behaviors (Ritcher 1966; Tashiro 1987; Braman & Pendley 1993). This pest is univoltine, with eggs present from late Jun to early Aug, first instars from Jul to Aug, second instars from Aug to Sep, and mostly third instars from Oct to Feb in Florida (Kostromytska & Buss 2008). Tomarus subtropicus attacks the roots of sugarcane ( Saccharum spp.) (Gordon & Anderson 1981), St. Augustinegrass ( Stentaphrum secundatum (Walt. Kuntze)) (Kostromytska & Buss 2008), and bermudagrass ( Cynodon dactylon (L.)) (Summers 1974; Reinert 1979; Prewitt & Summers 1981), resulting in crop yield loss and large patches of dead turfgrass. Its potential to feed on and injure other warm season turfgrasses has not been assessed. Tomarus subtropicus grubs also feed on plant roots in ornamental plant beds, and cause plant dieback (E. Buss, personal observation). White grub feeding habits vary depending on the species Some species (e.g., Cotinis nitida L.) obtain nutrients from soils high in organic matter (Brandhorst-Hubbard et al. 2001), while others need live plant roots (e.g., Popillia japonica Newman, Rhizotrogus majalis (Razumowsky), Cyclocephala spp., Phyllophaga spp.). Some scarab species consume soil organic matter as rst instars then switch to live roots in later instars (Litsinger et al. 2002). The larval feeding habits of T. subtropicus have not been clearly described. Tomarus subtropicus females oviposit and their offspring develop in soil with a high organic matter content (e .g., muck soil in sugarcane elds) (Cherry & Coale 1994), but they also survive and develop in sandy soils with low organic matter in residential environments (Kostromytska & Buss 2008). Because the urban landscape is a complex system with many plant species, understanding the feeding preference of key pests helps to explain pest distribution, abundance, and damage related to feeding, and can affect the management strategies used against them. We conducted no-choice tests to assess the effect of soil organic matter on T. subtropicus survival and development, and to determine whic h other warm season turfgrasses could be hosts for T. subtropicus grubs. M ATERIALS AND M ETHODS Effect of 2 Soil Types on First Instars A no-choice test with a nutrient-poor soil (sand) and an organic soil (Black Velvet peat (Black Gold Compost Co., Oxford, Florida)) was conducted from Jul to Aug 2005 to evaluate T. subtropicus larval growth and survival. The roots of Palmetto St. Augustinegrass plugs were washed, and grass plugs were replanted into the seedling tray cells (8 8 8 cm) with either sand or peat (48 cells for eac h growing media). Another 24 cells were lled with peat, but no grass. Grass was maintained in the greenhouse for 2 weeks before the experiment. Cells were arranged in a randomized complete block design. Adult T. subtropicus were collected from infested St. Augustinegrass lawns in Punta Gorda (Charlotte County) and Fort Myers (Lee County), Florida, and held in the laboratory to obtain eggs and young larvae (Kostromytska 2007). Grubs (26 d old; mean weight: 0.028 0.002 g) were randomly assigned to the following treatments: peat only, peat and grass, or sand and grass (24 replicates or cells for each). Cells of grass planted in sand or peat (24 cells of each) were uninfested controls. Individual rst instars were placed in a depression (2.5 cm deep, 0.7 cm in diameter) made in the center of each cell and covered with soil. Each cell was provided 30 mL of water daily. After 1 month, cells were visually inspected, and surviving grubs were weighed. Grass roots were washed with a #10 sieve, oven-dried in paper bags for 48 h at 55C, and root dry weights were recorded. Data were analyzed by an ANCOVA (SAS Institute 2004) with soil type as a factor, posttreatment grub weight as a dependent variable and initial grub weight as a covariate. A two-way ANOVA was also conducted with soil type and grub presence as factors and dry root weight as a dependent variable. Tukeys HSD test was conducted for mean separation. Survival and Growth of Third Instar T. subtropicus Reared on Different Turfgrass Species Six warm season and 1 cool season turfgrasses were evaluated as possible hosts for T. subtropicus grubs. Tested grasses included Palmetto St. Augustinegrass Tifway bermudagrass ( C. dactylon transvaalensis Burtt-Davy), Empire zoysiagrass ( Z. japonica Steud.), common centipedegrass ( Erimochloa ophiuroides (Munro) Hack), P ensacola bahiagrass ( Paspalum notatum Flugge), Sea Dwarf seashore paspalum ( Paspalum vaginatum Swartz), and Gulf annual ryegrass ( Lolium multiorum Lam.). Thirty plugs (15 cm diameter) of eac h warm season grass species were obtained from the University of Florida Plant Science Unit in Citra (Marion County), Florida, in Aug 2005. Soil was washed off the roots and the grass plugs were planted with FaFard mix #2 (Conrad Fafard, Inc., Agawam, Massachusetts) in plastic pots (15 cm diameter). Annual ryegrass was seeded at a rate of 0.05 kg/m 2 Grass was watered daily and fertilized monthly with 24.4 kg of N per ha during 2 months of establishment. Pots were arranged in a randomized complete block design in the greenhouse. Initial grub weights were obtained, then 1 recently molted (<7 d old) third instar was put in a shallow depression on the soil of each of 15 pots

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166 Florida Entomologist 94(2) June 2011 for each turfgrass species. Grubs that failed to dig into the soil within 10 min were replaced. Larval survival, weight, and weight gain were determined after 8 weeks. Daylight was supplemented with lights to provide a photoperiod of 16:8 h (L:D) and the average ambient greenhouse temperature was ~23.4C. Pots were watered with 150 mL of tap water every other day. Each turfgrass species was maintained at its recommended height (Turgeon 2002): 1.3 cm for seashore paspalum, 2.5 cm for bermudagrass, 5.1 cm for centipedegrass and zoysiagrass, and 7.6 cm for annual ryegrass, St. Augustinegrass and bahiagrass. To assess the amount of feeding damage, grass clippings were collected weekly and fresh weights were taken within 2 h. Clippings were then oven-dried for 48 h at 55C, and weighed. After 8 weeks of infestation, grass roots were cut within 2 mm of the plant crown, washed with a #20 sieve, oven-dried for 48 h (55C), and dry root weights were recorded. Tomarus subtropicus Neonate Survival, Growth and Development on 6 Warm Season Grasses The warm season turfgrass species noted in the previous section were tested as potential hosts for T. subtropicus larvae in a greenhouse experiment in 2006. Grass plugs were planted into 10-cm plastic pots with native soil (94% sand, 4% clay, 2% silt and 1.6% organic matter), and allowed 2 months to establish. First instars (1-3 d old) were weighed immediately before being individually placed in a hole (8 mm diameter, 5 cm deep) in the soil of each pot, and were covered with soil. Grass was watered daily as needed and fertilized weekly (0.6 kg of N per ha, Miracle Gro Scotts Miracle-Gro Products Inc., Marysville, OH). Four replicates per grass species were arranged and each replicate included 6 infested and 6 uninfested control pots. Grubs were removed from the pots after 8 weeks, and larval survival, weight, and head capsule width were determined. Grass clippings were collected and processed weekly, as previously described. Grass color and density were visually assessed on a scale from 1 (yellow, sparse) to 9 (dark green, very dense) and total grass quality was calculated by averaging the two scores. After the grubs were removed, grass roots were cut to within 2 mm of the plant crown, washed with a #20 sieve, and placed in paper bags. The remaining plant parts were collectively placed into paper bags, and ovendried for 48 h (55C). Dry root weight and dry total plant yield were recorded. Statistical Analysis The correlation between initial and nal grub weights was tested before analysis. If the 2 variables were signicantly correlated, the ANCOVA GLM procedure (SAS Institute 2004) was used to analyze the effect of turf species on grub nal weight with a correction for initial weight for all experiments. Percent of root reduction was calculated as averaged root weights of controls minus root weights of infested plants and divided by averaged weights in controls. Analysis of variance (GLM procedure, SAS Institute 2004) was used to determine the effect of turf species on the percentage of grub survival and development, and effect of grub presence on dry root weight. Proportion data were arcsine square root transformed before analysis. Clipping weights and grass quality ratings were analyzed by a repeated measure analysis (SAS Institute 2004) with time as a repeated within-group factor and grub presence as a between-group factor. Means were separated by Tukeys HSD. R ESULTS AND D ISCUSSION Effect of 2 Soil Types on First Instars Tomarus subtropicus grubs fed on live St. Augustinegrass roots, regardless of soil type, in this test. Two grubs (8%) that were reared on peat without grass survived, but they remained first instars, and all other grubs in this treatment died. All grubs provided with St. Augustinegrass roots, regardless of soil type, were second instars when the test was evaluated. Grub survival to the second instar when reared on peat with grass was 83%, and 75% when reared on sand with grass. Tomarus subtropicus body weights were statistically similar when grubs were reared on grass grown in peat (0.87 0.07 g) or grass grown in sand (0.74 0.08 g). Grub feeding signicantly reduced St. Augustinegrass dry root weight compared to uninfested cells, regardless of soil type ( F = 156.66; df = 1, 85; P < 0.0001). Uninfested pots had statistically similar dry St. Augustinegrass root weights (1.24 0.06 g in peat; 1.17 0.06 g in sand). The nal dry root weight of infested grass grown in sand (0.31 0.05 g) was signicantly lower than in infested grass grown in peat (0.59 0.05 g) ( F = 4.59; df = 1, 85; P = 0.03), indicating that more root herbivory ma y have occurred in the pots with sand. Peat, like muck soil, is an organic soil, consisting of poorly decomposed animal and plant remnants (Brown 2009) with organic matter content ranging from 40 to 80% (Andriesse 1988; Litaor et al. 2005; Kechavarzi et al. 2010) which can provide nutrients (e.g., carbon, nitrogen, sulfur, and phosphorous) to plants and soil fauna (Andriesse 1988; Killham 1994). Although the nutrient content of the soils or turfgrass were not measured in our test (fertilization and irrigation were consistent across all treatments), it is possible that the peat provided additional nutrients to the grass, which could lead to either more efcient grub

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Kostromytska & Buss: Tomarus subtropicus Host Range 167 feeding or increased compensatory plant growth (Radcliffe 1970; Brown and Gauge 1990; Steinger & Mller-Schrer 1992; Sparling et al. 2006). In addition, grubs may, while feeding on grass roots, acquire additional nutrients when ingesting soil with greater organic matter content (Seastedt 1985; Brown & Gange 1990), and thus cause less root damage. The tendency for insects to consume more of a nutritionally inferior food to overcome the lack of needed nutrients has been documented for many taxa (King 1977; Yang & Joern 1994; Obermaier & Zwolfer 1999; Berner et al. 2005). Survival and Growth of Third Instar T. subtropicus Reared on Different Turfgrass Species Third instar initial weights (1.97 0.08 g) were statistically similar among treatments ( F = 0.71; df = 6, 76; P = 0.64). However, initial and nal grub weights were signicantly correlated (P earsons r = 0.37, P = 0.001) and were included as covariates in the analysis Final grub weight differed statistically among grasses (Table 1). Third instar weights when reared on ryegrass (1.96 0.2 g) and bermudagrass (2.1 0.2 g) were significantly lower than grub weights on any of the other grasses tested ( F = 8.51; df = 6, 76; P < 0.0001). However, 66.7% of the grubs survived on bermudagrass and only 40% survived on ryegrass. Analysis of grass dry root weight with and without grubs indicated that there was a signicant root reduction in all pots with warm season grasses, but not in the pots with ryegrass (Fig. 1). These data suggest that ryegrass was a poor larval host for T. subtropicus Annual ryegrass w as the only C 3 grass included in the test. C 3 grasses typically are more benecial nutritionally to herbivores than C 4 grasses (Barbehenn & Bernays 1992), however physical properties could be a key factor for herbivore host selection (Scheirs et al. 2001). Suitability of annual ryegrass as a host for T. subtropicus larvae may be affected by the physical structure of the grass root system. Numerous ryegrass ne roots created a dense mesh through the entire pot, which may have reduced grub movement. At evaluation, live grubs in ryegrass pots were in soil chambers that were located near pot walls and not more than 5 cm deep. In contrast, the other grasses had thick main roots from which roots branched closer to the pot walls and bottom, and grubs were often found in the center of the pots at different depths. Grass leaf growth changed differently among grass species over time (summarized statistics are in Table 2). For St. Augustinegrass, the main effect of grub presence and the interaction of grub feeding with time were signicant. On T ABLE 1. L ARVAL T. SUBTROPICUS SURVIVAL AND GROWTH ON DIFFERENT GRASS SPECIES AND ASSOCIATED ROOT REDUCTION IN 2005 AND 2006. Grass species % Grub survival Initial grub weight (g SEM) 1 Final grub weight (g SEM) 2 Proportional weight gain 3 Root reduction (% SEM) 4 2005 Bahiagrass 93.3 6.71.8 0.2a3.05 0.2b1.8 0.1a27.4 8.8bc Bermudagrass 66.7 6.71.7 0.2a2.10 0.2a1.1 0.3ab64.9 3.2a Centipedegrass 66.7 24.01.9 0.3a3.43 0.2b2.1 0.3a60.5 4.9aa Ryegrass 40.0 0.02.2 0.7a1.96 0.2a1.0 0.1b17.2 4.9ca Seashore paspalum86.7 6.72.1 0.2a3.57 0.2b1.8 0.1a49.1 9.1ab St. Augustinegrass66.7 6.72.2 0.2a3.18 0.1b1.4 0.1a52.2 7.7ab Zoysiagrass 86.7 6.72.0 0.1a3.04 0.1b1.8 0.2a55.3 6.4ab 2006 Bahiagrass 79.3 7.90.028 0.002a2.62 0.2abc103.7 11.2abc48.1 4.6bc Bermudagrass 66.8 6.70.034 0.003a2.20 0.2c 69.6 8.3c87.5 4.6a Centipedegrass 91.5 4.90.030 0.002a2.43 0.2bc 87.6 0.7bc36.3 4.2c Seashore paspalum62.5 10.50.031 0.003a3.35 0.2a126.2 15.6a60.8 4.6ab St. Augustinegrass87.3 4.30.033 0.002a2.62 0.2abc 92.2 7.9abc65.0 6.8ab Zoysiagrass 70.8 7.90.030 0.002a3.13 0.1ab114.3 8.7ab80.9 2.8a1Initial grub weight was not significantly different among treatments at = 0.05 (2005: F = 1.64; df = 6, 69; P = 0.15 and 2006: F = 1.84; df = 5, 143; P = 0.11).2Means within columns with different letters are statistically different at = 0.05 (2005: F = 8.51; df = 6, 76; P < 0.0001 and 2006: F = 5.04; df = 5, 109; P = 0.0004). 3Proportions were calculated by dividing the final weights by initial weights. Means within columns with different letters are statistically different at = 0.05 (2005: F = 3.89; df = 6, 76; P = 0.002 and 2006: F = 3.95; df = 5, 109; P = 0.0026).4Means within columns with different letters are statistically different at = 0.05 (2005: F = 6.32; df = 6, 105; P < 0.0001 and 2006: F = 16.52; df = 5, 111; P < 0.0001).

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168 Florida Entomologist 94(2)June 2011average, clippings collected from the infested pots weighed less than clippings from uninfested control pots, and this difference increased over time. For bahiagrass, bermudagrass, and centipedegrass the main effect of grub presence was signicant, whereas the interaction between grub presence and time was not signicant. Clipping yield of these grasses changed signicantly over time in all pots, but uninfested pots on average yielded more clippings. For zoysiagrass, the main effect of grub presence was not signicant although the main effects of time and the interaction of time and grub presence were signicant. Thus, decrease in clipping weight over time was more pronounced in the pots with grubs. Only the main effect of time was signicant for ryegrass and seashore paspalum, so grass growth changed over time, but variation of clipping yield was not related to grub presence.Tomarus subtropicus Neonate Survival, Growth, and Development on 6 Warm Season GrassesOn average, 76.3% of grubs survived across treatments and 70.8% of all (94% of survivors) grubs reached the third instar (Table 1). Percentage survival, percent of grubs that reached the third instar, and head capsule width did not differ among the grasses (F = 1.64; df = 5, 23; P = 0.20; F = 2.17; df = 5, 23; P = 0.10; and F = 1.73; df = 5, 109; P = 0.13). Mean initial rst instar weight (0.031 0.01 g) did not correlate with mean grub nal weight (2.75 0.86 g) ( r = 0.11, P = 0.22), and was not included in the analysis as a covariate. Final grub weight and proportional weight gain differed among grasses. Similar to the result obtained in the 2005-experiment, grubs feeding on bermudagrass gained less weight (weight gain about 70 times initial weight) when compared to grubs reared on seashore paspalum (weight gain about 126 times initial weight) and zoysiagrass (weight gain about 114 times) (Table 1). Similar to the 2005-experiment, bermudagrass appeared to be a poorer host for T. subtropicus compared to the other warm season grasses. However, T. subtropicus larvae are reported to damage bermudagrass (Reinert 1979), so despite the slower grub growth, this grass may still be an acceptable host. Reduced grub growth may be inuenced by a smaller root mass in bermudagrass (grubs consumed on average 87.5% of root mass). Fig. 1. Reduction of root mass caused by third instar T. subtropicus feeding, 2005. Means marked with different letter are different at a = 0.05 (F = 39.51; df = 1, 130; P < 0.0001) TABLE 2.STATISTICS SHOWING EFFECTS OF TIME, GRUB FEEDING, AND THEIR INTERACTION ON CLIPPING YIELD DURING AN 8-WEEK PERIOD IN 2005 AND 2006. Grass species Grub feeding Time Time*Grub F df PF df PF df P 2005 Bahiagrass 8.501, 27<0.012.487, 210.051.057, 210.43 Bermudagrass11.221, 23<0.0144.497, 17<0.011.967, 170.12 Centipedegrass12.801, 23<0.013.207, 170.021.67, 170.20 Seashore paspalum3.071, 190.1020.127, 13<0.012.577, 130.07 St. Augustinegrass0.091, 270.762.927, 210.030.677, 210.74 Zoysiagrass 9.471, 23<0.011.997, 170.112.697, 170.05 2006 Bahiagrass 1.561, 410.225.437, 35<0.011.997, 350.08 Bermudagrass8.101, 38<0.015.387, 32<0.012.477, 320.04 Centipedegrass4.661, 440.046.097, 38<0.010.367, 380.92 Seashore paspalum0.301, 390.596.887, 33<0.010.687, 330.69 St. Augustinegrass1.491, 440.015.387, 32<0.012.477, 320.04 Zoysiagrass 4.611, 460.049.487, 34<0.011.297, 340.28

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Kostromytska & Buss: Tomarus subtropicus Host Range 169Grub movement was also limited, so grubs could not migrate in search of food after the previous source had been exhausted. Grub feeding caused signicant root reduction of all grasses in our study ( F = 70.61; df = 6, 287; P < 0.0001) (Fig. 2). The percent of root reduction ranged from 36 to 87% and differed among grasses (F = 16.52; df = 5, 111; P < 0.01) (Table 1). However, the total plant yield was reduced only for bahiagrass and bermudagrass (F = 22.81; df = 11, 287; P = 0.001) (Fig. 3). Root loss does not necessarily reduce aboveground plant growth, and in some cases, minor root damage can lead to increased or compensatory foliage growth (Humphries 1958; Seastedt et al. 1988; Brown & Gange 1990; Bardgett et al. 1999; Blossey & Hunt-Joshi 2003). Measurements of grass yield over time demonstrated that tested grasses responded differently to T. subtropicus herbivory (statistics are summarized in Table 2). Centipede and zoysiagrass tended to yield fewer leaf clippings if infested with grubs, and clipping weights varied over time (the main effects of infestation and time on clipping yield were signicant), but effect of grub feeding was not signicantly stronger with time (interaction was not signicant). The interaction of the 2 factors was signicant for bermudagrass and St. Augustinegrass, so grub feeding decreased clipping yield beginning week 5. Grub feeding did not affect clipping yield in pots of bahiagrass and seashore paspalum. Grub feeding reduced the quality ratings for St. Augustinegrass, bermudagrass, zoysiagrass and centipedegrass, but not for bahiagrass and seashore paspalum (statistics are summarized in Table 3). Differences in grass quality were apparent 4 weeks (St. Augustinegrass, bermudagrass), 6 weeks (zoysiagrass), and 8 weeks (centipedegrass) after grubs were introduced. Grass crowns could be pulled easily from the pots with grubs, but grass remained green in all pots. Our study demonstrated that T. subtropicus can successfully survive and develop on bahiagrass, bermudagrass, centipedegrass, zoysiagrass and seashore paspalum, and conrmed that St. Augustinegrass was an adequate host (Reinert 1979), regardless of soil organic content. Quality ratings and clipping yields decreased for most of the turfgrasses after 5 weeks of infestation, but bahiagrass and seashore paspalum were less affected by T. subtropicus root feeding, compared to the other grass species. It was previously reported that 3 grubs per 0.1 m2 could severely damage bermudagrass (Reinert 1979), but the grasses in our study could tolerate approximately 5 and 12 third instar grubs per 0.1 m2 in 2005 and 2006, respectively, despite >50% root reduction. Environmental conditions (temperature, photoperiod, herbivore aboveground grazing or mowing, fertilization, and irrigation practices) can signicantly affect plant tolerance to root herbivory in addition to plants characteristics and insect density (Ladd & Buriff 1979; Seastedt et al. 1988; Brown & Gange 1990; Potter et al. 1992; Crutcheld et al. 1995; Crutcheld & Potter 1995; Braman & Raymer 2006). For instance, 4 Japanese beetle (Popillia japonica Newman) grubs per 15cm pot (~20 grubs per 0.1 m2) signicantly reduced Poa pratensis L. clipping yield in a greenhouse study (Ladd & Buriff 1979), but clipping yield from other eld and greenhouse tests was unaffected by 60-90% root reduction from 40-60 grubs per 0.1 m2 and 24-30 grubs per 0.1 m2, respectively (Potter et al. 1992; Crutcheld & Potter 1995). During our experiment, grass was regularly irrigated, and although the roots were dramatically reduced and crowns could be easily removed from infested pots, the foliage remained green. Most third instar-feeding in the eld occurs during the Fig. 2. Reduction of root mass caused by larval T. s ubtropicus feeding, 2006. Means marked with different letters are different at = 0.05 (F = 70.61; df = 1, 287; P < 0.0001) Fig. 3. Effect of grub feeding on total plant yield by warm season grasses, 2006. Means marked with different letter are different at a = 0.05 ( F = 5.51; df = 1, 253; P = 0.02)

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170 Florida Entomologist 94(2)June 2011fall (Kostromytska 2007), which coincides with reduced rainfall and slower warm season turfgrass growth. Thus, drought and/or other environmental stresses during this time, in addition to root damage by grubs, may more quickly overwhelm the grass. ACKNOWLEDGMENTSWe are grateful for the collection sites, assistance, and cooperation provided by E. McDowell (Tonys Pest Control), P. Quartuccio (All-Service Pest Management), Pest Solutions Plus, N. Palmer (Master Gardener), Greig Henry, and Trish Wood. REFERENCES CITEDANDRIESSE, J. P.1988. Nature and management of tropical peat soils. FAO Soils Bulletin 59. Rome, Italy. BARDGETT, R. D., COOK, R., YEATES, G. W., AND DENTON, C. S. 1999. The influence of nematodes on below-ground processes in grassland ecosystems. Plant Soil. 212: 23-33. BARBEHENN, R. V., AND BERNAYS, E. A. 1992. Relative nutritional quality of C3 and C4 grasses for a graminivorous lepidopteran, Paratrytone melane (Hesperiidae). Oecologia 92: 97-103. BERNER, D., BLANCKENHORN, W. U., AND KOERNER, C. 2005. Grasshoppers cope with low host quality by compensatory feeding and food selection N limitation challenged. Oikos 111: 525-533. BLOSSEY, B., AND HUNT-JOSHI, T. R. 2003. Belowground herbivory by insects: inuence on plants and aboveground herbivores. Annu. Rev. Entomol. 48: 521-547. BRAMAN, S. K., AND PENDLEY, A. F. 1993. Growth, survival, and damage relationships of white grubs in bermudagrass vs. tall fescue, pp. 370-372 In Research and Technical Papers, 7th Intl. Turfgrass Soc. Res. Conf., 18-24 July, Palm Beach, Florida. BRAMAN, S. K., AND RAYMER, P. L. 2006. Impact of Japanese beetle (Coleoptera: Scarabaeidae) feeding on seashore paspalum. J. Econ. Entomol. 99: 16991704. BRANDHORST-HUBBARD, J. L., FLANDERS, K. L., AND APPEL, A. G. 2001. Oviposition site and food preference of the green June beetle (Coleoptera: Scarabaeidae). J. Econ. Entomol. 94: 628-633. BROWN, R. B. 2009. Soil texture. http://edis.ifas.u.edu/ ss169. (Last accessed July 20, 2010). BROWN, V. K., AND GANGE, A. C. 1990. Insect herbivory below ground. In M. Begon, A. H. Fitter and A. Macfadyen (eds.), Advances in Ecological Research 20: 158. Academic Press Inc., San Diego, California. CARTWRIGHT, O. L. 1959. Scarab beetles of the genus Bothynus in the United States (Coleoptera: Scarabaeidae). Proc. United States Natl. Mus. 108: 515540. CHERRY, R. H., AND COALE, F. J. 1994. Oviposition of the sugarcane grub, Ligyrus subtropicus (Coleoptera: Scarabaeidae) in different soils. J. Agr. Entomol. 11: 345-348. CRUTCHFIELD, B. A., POTTER, D. A., AND POWELL, J. 1995. Irrigation and nitrogen fertilization effects on white grub injury to Kentucky bluegrass and tall fescue turf. Crop Sci. 35: 1122-1126. CRUTCHFIELD, B. A., AND POTTER, D. A. 1995. Tolerance of cool-season turfgrasses to feeding by Japanese beetle and southern masked chafer (Coleoptera: Scarabaeidae) grubs. J. Econ. Entomol. 88: 13801387. GORDON, R., AND ANDERSON, D. 1981. The species of Scarabaeidae (Coleoptera) associated with sugarcane in south Florida. Florida Entomol. 64: 119-138. HUMPHRIES, E. C. 1958. Effect of removal of a part of the root system on the subsequent growth of the root and shoot. Ann. Bot. 22: 251-257. KECHAVARZI, C., DAWSON, Q., AND LEEDS-HARRISON, P. B. 2010. Physical properties of low-lying agricultural peat soils in England. Geoderma 154:196-202. KILLHAM, K. 1994. Soil Ecology. Cambridge University Press. Cambridge, UK. KING, P. D. 1977. Effect of plant species and organic matter on feeding behaviour and weight gain of larval black beetle Heteronychus arator (Coleoptera: Scarabaeidae). New Zealand J. Zool. 4: 445-448. KOSTROMYTSKA, O. 2007. Seasonal Phenology, Host Range, and Management of Tomarus Subtropicus (Coleoptera: Scarabaeidae) in Turfgrass. M.S. thesis, University of Florida, Gainesville, Florida. KOSTROMYTSKA, O. S., AND BUSS, E. A. 2008. Seasonal phenology and management of Tomarus subtropicus (Coleoptera: Scarabaeidae) in St. Augustinegrass. J. Econ. Entomol. 101: 1847-1855. LADD, JR., T. L., AND BURIFF, C. R. 1979. Japanese beetle: influence of larval feeding on bluegrass yields at two levels of soil moisture. J. Econ. Entomol. 72: 311314. LITAOR, M. I., REICHMANN, O., HAIM, A., AUERSWALD, K., AND SHENKER, M. 2005. Sorption characteristics TABLE 3.STATISTICS SHOWING EFFECT OF TIME, GRUB FEEDING, AND THEIR INTERACTION ON TURFGRASS QUALITY IN2006. Grub feedingTimeTime*Grub Grass species F df PF df PF df P Bahiagrass 0.61, 440.443.907, 400.150.957, 400.43 Bermudagrass 9.081, 44<0.0117.077, 40<0.015.547, 40<0.01 Centipedegrass 5.711, 440.0213.517, 40<0.018.667, 40<0.01 Seashore paspalum0.041, 440.834.617, 400.010.377, 400.70 St. Augustinegrass9.081, 44<0.0117.077, 40<0.015.547, 40<0.01 Zoysiagrass 32.231, 44<0.0114.087, 40<0.019.827, 40<0.01

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Kostromytska & Buss: Tomarus subtropicus Host Range 171of phosphorus in peat soils of a semi-arid altered wetland. Soil Sci. Soc. America J. 69:1658-1665. LITSINGER, J. A., LIBETARIO, E. M., AND BARRION, A. T. 2002. Population dynamics of white grubs in the upland rice and maize environment of Northern Mindanao, Philippines. Int. J. Pest Manag. 48: 239-260. OBERMAIER, F., AND ZWOLFER, H. 1999. Plant quality or quantity? Host exploitation strategies in three Chrysomelidae species associated with Asteraceae host plants. Entomol. Exp. Appl. 92: 165-177. POTTER, D. A., PATTERSON, C. G., AND REDMOND, C. T. 1992. Inuence of turfgrass species and tall fescue endophyte on feeding ecology of Japanese beetle and southern masked chafer grubs (Coleoptera: Scarabaeidae). J. Econ. Entomol. 85: 900-909. PREWITT, J. C., AND SUMMERS, T. E. 1981. White grubs of sugarcane in South Florida, pp. 49-50 In Proc. 2nd Inter-American Sugar Cane Seminar, October 1981, Miami, Florida. Inter-American Transport Equipment Co., Miami, Florida. RADCLIFFE, J. E. 1970. Some effects of grass grub ( Costelytra zealandica (White)) larvae on pasture plants. New Zealand J. Agric. Res. 13: 87-104. REINERT, J. A. 1979. Response of white grubs infesting bermudagrass to insecticides. J. Econ. Entomol. 72: 546-548. RITCHER, P. O. 1966. White Grubs and Their Allies, A Study of North American Scarabaeoid Larvae. Oregon State University Monograph Series 4: 1-219. SAS INSTITUTE. 2004. SAS 9.1.2 Qualication Tools Users Guide. SAS Institute, Cary, North Carolina. SCHEIRS, J., DE BRUYN, L., AND VERNAGEN, R. 2001. A test of the C3-C4 hypothesis with two grass miners. Ecol. 82: 410-421. SEASTEDT, T. R. 1985. Maximization of primary and secondary productivity by grazers. American Nat. 126: 559-564. SEASTEDT, T. R., RAMUNDO, R. A., AND HAYES, D. C. 1988. Maximization of densities of soil animals by foliage herbivory: empirical evidence, graphical and conceptual models. Oikos 51: 243-248. SPARLING, G. P., WHEELER, D., VESELY, E. T., ANDSCHIPPER, A. 2006. What is soil organic matter worth? J. Environ. Qual. 35: 548-557. STEINGER, T., AND MLLER-SCHRER, H. 1992. Physiological and growth responses of Centaurea maculosa (Asteraceae) to root herbivory under varying levels of interspecic plant competition and soil nitrogen availability. Oecologia 91: 141-149. SUMMERS, T. 1974. Florida sugarcane attacked by white grubs. Proc. American Soc. Sugar Cane Technol. 3: 124. TASHIRO, H. 1987. Turfgrass Insects of the United States and Canada. Cornell University Press, Ithaca, New York. TURGEON, A. J. 2002. Turfgrass Management. Sixth edition. Prentice Hall, Upper Saddle River, New Jersey. YANG, Y., AND JOERN, A. 1994. Compensatory feeding in response to variable food quality by Melanoplus differentialis Physiol. Entomol. 19: 75-82.

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172 Florida Entomologist 94(2) June 2011 VAGILITY AS A LIABILITY: RISK ASSESSMENT OF THE LEAF-BLOTCHING BUG EUCEROCORIS SUSPECTUS (HEMIPTERA: MIRIDAE), A PROSPECTIVE BIOLOGICAL CONTROL AGENT OF THE AUSTRALIAN TREE MELALEUCA QUINQUENERVIA G ARY R. B UCKINGHAM 1,* S USAN A. W INERITER 1 J ASON D. S TANLEY 2 P AUL D. P RATT 3 AND T ED D. C ENTER 3 1 USDA-ARS Invasive Plants Research Laboratory, P.O. Box 147100, Gainesville, FL 32614-7100 2 Division of Plant Industry, Florida Department of Agriculture and Consumer Services, P.O. Box 147100, Gainesville, FL 32614-7100 3 USDA-ARS Invasive Plant Research Laboratory, 3225 College Ave., Fort Lauderdale, FL 33314 *Retired A BSTRACT Melaleuca quinquenervia (Cav.) S.T. Blake (Myrtales: Myrtaceae) forms dense monocultures that displace native vegetation in wetlands of southern Florida, USA. Faunal studies in the trees native Australian range revealed several prospective biological control agents, including the leaf-blotching bug, Eucerocoris suspectus Distant (Hemiptera: Miridae). This herbivore was imported into quarantine to assess risk to Florida native and ornamental species after preliminary Australian studies had indicated that it might be useful. Ornamental Melaleuca spp. suffered heavy feeding in no-choice adult feeding trials, with moderate feeding on some native Myrtaceae. Native species sustained light to heavy feeding in multichoice adult feeding trials and in a no-choice nymphal feeding trial. Feeding increased on native species in a large enclosure after M. quinquenervia was cut, allowed to dry, and then removed. Nymphs completed development only on M. quinquenervia and ornamental bottlebrushes, Melaleuca spp. However, inability to fully develop on non-target species is of limited importance as a criterion for release of insects with highly mobile immature stages as compared to less vagile species. Local movement from the host to other plant species could result in unacceptable non-target damage despite seemingly adequate developmental specificity. This insect would clearly harm native and ornamental Myrtaceae and should therefore not be released. K ey Words: Biological control, Hemiptera, Eucerocoris suspectus host range, Melaleuca quinquenervia Miridae, Myrtaceae, risk assessment, weed control R ESUMEN Melaleuca quinquenervia (Cav.) S.T. Blake (Myrtales: Myrtaceae) forma monoculturas densas que desplazen la vegetacin nativa en las tierras humedas en el sur de la Florida, EEUU. Estudios faunsticas realizados en el rango nativo Australiano del rbol revelan varios agentes de control biolgico prospectivos, incluyendo un chinche que mancha las hojas, Eucerocoris suspectus Distant (Hemiptera: Miridae). Este herbvoro fue importado al laboratorio de cuarentena para evaluar su riesgo hacia las especies nativas de la Florida y ornamentales despus de que estudios preliminares en Australia indicaron que esta especie puede ser til. Especies ornamentales de Melaleuca sufrieron niveles fuertes de alimentacin en pruebas sin opcin de los adultos con alimentacin moderada en plantas nativas de la familia Myrtaceae. Las especies nativas sostuvieron alimentacin leve y fuerte en pruebas de opciones multiples de alimentos para los adultos y en pruebas sin opcin de alimentos para las ninfas. La alimentacin aument sobre las especies nativas en un cercado grande despus que la M. quinquenervia fue cortada, puesta a secar y quitada. Las ninfas completaron su desarrollo solamente sobre M. quinquenervia y especies de Melaleuca ornamentales. Sin embargo, la incapacidad para desarrollar completamente sobre especies que no son el enfoque es de importancia limitada como un criterio para la liberacin de insectos con estadios de immaduros altamente moviles comparado con especies menos moviles El movimento local de un hospedero a otras especies de plantas puede resultar en dao no aceptable en plantas que son el enfoque a pesar de que la especicidad del desarrollo parece adecuada. Este insecto claramente daara las Myrtaceae nativas y ornamentales y por ello no debe ser liberado.

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Buckingham et al.: Risk Assessment for Eucerocoris suspectus 173 Melaleuca quinquenervia (Cav.) S. T. Blake is a large tree of Australian origin and one of numerous invasive plants threatening the Florida Everglades. This introduced tree forms expansive monocultures and spreads rapidly from prolific seed production. Its presence and rapid spread hinders restoration of many south Florida ecosystems including sawgrass prairies, hardwood hammocks, and even pine uplands (Bodle 1998; Turner et al. 1998). Invaded habitats are transformed into nearly pure stands of M. quinquenervia trees, thereby altering the function and structure of these systems. A biological control program began in 1986 to curtail the M. quinquenervia invasion by inhibiting its reproduction. Eucerocoris suspectus Distant (Hemiptera: Miridae) seemed an excellent candidate based upon the injury it caused to young shoots (Fig. 1) in Australia (Burrows & Balciunas 1999). It was introduced into quarantine during 1996 to complete host range evaluations by focusing on native and cultivated Myrtaceae. Herein, we report results of host range studies that led us to reject this species. Burrows & Balciunas (1999) described the biology and life history of E. suspectus as follows. F emales insert eggs into the young shoots. Nymphal development progresses through 5 instars and requires about 17 d but is inuenced by plant quality. A single female produces up to 163 progeny and adults live up to 72 d. Adults and nymphs feed on the sap of young leaves and shoots causing distinctive brown blotches on the foliage. Their dispersive capacity is unknown but both nymphs and adults are very active, making them difcult to contain, and are readily able to disperse onto nearby vegetation. M ATERIALS AND M ETHODS Laboratory Cultures Dr. Charles Turner and staff of the USDA-ARS Australian Biological Control of Weeds Laboratory collected E. suspectus adults near Brisbane, Australia, during Jul and Aug 1996. A shipment arrived in quarantine at Gainesville, Florida on Fig. 1. A female Eucerocoris suspectus and feeding scars on Melaleuca quinquenervia

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174 Florida Entomologist 94(2) June 2011 12 Jul with 17 of 38 adults alive (5 males and 12 females). A second shipment arrived on 31 Aug, which contained 4 of 75 adults alive (not sexed). Adults were placed on seed-grown M. quinquenervia saplings of varying sizes, usually less than 2 m tall. The saplings were sleeved with ne-mesh netting (100 holes/cm 2 ) and held in air-conditioned quarantine greenhouses. Plants were fertilized with an encapsulated fertilizer, watered regularly, and occasionally sprayed with a soap-vegetable oil mixture to control insect pests. They were not sprayed after the bugs were added. During rearing, adults and/or nymphs, which preferred new growth, were removed and placed on new saplings, depending upon the amount of damage and the amount of remaining leaf material. They were reared continuously from Jul 1996 to Jun 1997, during which time 5 host range studies, designated I-V, were conducted. Each study consisted of 1 or more separate trials designated A-L. No-choice Adult Feeding and Oviposition Trials (Study I) Potted test plants (Table 1) 1-2 m tall bearing new growth on the stem tips (hereafter referred to as shoots) were individually caged in quarantine greenhouses in sleeves of nylon netting. Groups of 5 pairs of adults were randomly assigned to the cages. Most plant species were set up within 2 d of the commencement of the study but 3 species were set up 11-13 d later. Each group of plants ( n = 3 groups) was assigned a separate control plant of M. quinquenervia to comprise trials A, B, and C (T able 1, Test ID). Adults and nymphs were removed at 7-11-d intervals and placed separately on new plants. Each exposed plant was held to assess further nymphal emergence. Adults were transferred to a new plant of the same species as many as 4 times (Table 1). Adult survival was recorded at each plant change. Nymphs were removed, counted, and placed together on a new plant of the same species. The number of shoots bearing damaged leaves and the number of leaves with feeding blotches were recorded for most species. Damage results were categorized by a threetiered intensity scale: + = not damaged; ++ = moderately damaged; and +++ = heavily damaged. The presence of eggs was noted at the end of the test. Water-lled vials with bouquets of exT ABLE 1. R ESULTS OF THE NO CHOICE ADULT FEEDING AND OVIPOSITION TRIALS WITH POTTED MYRTACEAE AND LYTHRACEAE EXPOSED TO E UCEROCORIS SUSPECTUS (S TUDY I). Test species a Trial ID b, c No. Plants Tested Time to 100% Mortality (d) Feeding Intensity d Nymphs Produced (no.) e Melaleuca citrina (Curtis) Dum. Cours A4 27+++ 91 Melaleuca citrina (broad-leaved) B4>42+++184 Melaleuca viminalis ( Sol. ex Gaertn.) ByrnesA4 27+++181 Melaleuca viminalis Little John C4 32+++ 51 Calyptranthes pallens Griseb.* C1 9+ 0 Calyptranthes pallens *B 1 1 0 + 0 Calyptranthes zuzygium (L.) Sw.* C3 32++ 0 Eugenia axillaris (Sw.) Willd.* C1 9++ 0 Eugenia confusa DC.* B1 10+ 0 Eugenia foetida Pers.* C1 9++ 0 Eugenia foetida *B 1 1 0 + + 0 Eugenia uniora L. B2>10+++ 0 Lagerstroemia indica L. (Lythraceae) A1>25+ 0 Leptospermum scoparium J. R. Forst. & G. Forst. B2 20+ 0 Melaleuca quinquenervia (Cav.) Blake C4>42+++18 f Melaleuca quinquenervi a B4>25+++401 Melaleuca quinquenervia A4>25+++257 g Myrcianthes fragrans (Sw.) McVaugh* B1 10++ 0 Psidium friedrichsthalianum (O. Berg.) Nied.C2 17+++ 0 Psidium cattleianum Sabine C3 32+++ 0 Syzygium paniculatum Gaertn. A1 10++ 0 a Florida natives are indicated with *. b Plants with the same letter had the same Melaleuca quinquenervia control plant. c Five pairs of adults on a potted plant covered with mesh sleeve, adults moved weekly to a new plant, old plant was held for nymphal emergence, total plants tested with those adults. d Subjective feeding estimate, compared with feeding on M. quinquenervia ; at first plant change M. quinquenervia reps. had 129168 leaves with >10 feeding spots. e Total nymphs produced on all plants exposed to that cohort of adults. f Progeny of one female, four of the 5 females were trapped and died in the release vial. g The test ended when all adults were dead on the 3 companion plants. Two females were still alive on M. quinquenervia

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Buckingham et al.: Risk Assessment for Eucerocoris suspectus 175 cised shoots were tethered to Melaleuca citrina (Curtis) Dum.Cours and Melaleuca viminalis (Sol. ex Gaertn.) Byrnes at the third and fourth plant changes to provide supplemental food as the original plant material was insufcient following extensive feeding.No-choice Nymphal Feeding Trials (Study II)Techniques were similar to those for the previous adult test except that 10 rst instars (1.3 to 1.7 mm in length) were placed on the plants instead of adults. Supplemental bouquets of excised shoots in water-lled vials were tethered to test plants that had too few suitable ushing shoots to support the insects after the rst week. Four native Eugenia spp. were included with M. quinquenervia in trial D, and 2 non-target species of Melaleuca along with M. quinquenervia in trial E (Table 2). Insect survival, the number of adults produced, and the number of leaves attacked were recorded weekly for 28 d. Adults that developed during the test were retained on the plants with the remaining nymphs.Multi-choice Adult Feeding and Oviposition Trials without M. quinquenervia (Study III)In trial F, 2 bouquets of shoots in water-lled vials of each of 10 test plant species (Table 3) were placed into a glass-topped wooden cage (44.5 x 44.5 44.5 cm, l w h) in a greenhouse with daily mean temperature 22-24C (range 19-33C), and 78-81% RH (range 45-97%). Natural lighting was supplemented with uorescent lights to maintain a 16L:8D photoperiod. One bouquet of each species was randomized to one of 10 positions in each half of the cage. Ten females and 2 males were released in the cage. Feeding was subjectively estimated on a scale of 0 to 5 from light to heavy after 2 d when the bouquets were replaced and again after 3 d when trial F ended. The plants were examined for eggs at the end of the trial. In trial G, 1 potted plant of each of 4 species was placed in a cloth screen cage (0.6 0.6 1.2 m, l w h) in a greenhouse. The test species included 3 Myrtaceae, pineapple guava (Feijoa sellowiana (O. Berg) O. Berg), bay rum tree ( Pimenta racemosa (Mill.) J. W. Moore), and java plum (Syzygium cumini (L.) Skeels), and 1 Rutaceae, lemon (Citrus limon (L.) Burm.f. (pro. Sp.) ( medica aurantifolia)). Three pairs of adults were released in the cage. Two plants with extensive feeding were removed during the second d and the other 2 plants were left until d 11. The plants were checked for eggs and nymphs when the test ended.Large Enclosure Multi-choice Adult Feeding Trials with and without M. quinquenervia (Study IV)Potted plants, 1-1.5 m tall, were exposed to adults in a large walk-in screen enclosure (1.8 x 1.5 1.8 m, l w h) in a greenhouse. Plants were randomized to 1 of 9 positions in 3 rows of 3 each, in trials H and I. A M. quinquenervia plant was placed next to the test plant in the center of the enclosure at the start of each trial. The 15 test plant species are listed in Table 4. All were Myrtaceae except Morella cerifera (L.) Small (Myricales: Myricaceae), which was considered at riskTABLE 2.NO-CHOICE NYMPHAL FEEDING AND DEVELOPMENT TRIALS ON NATIVE EUGENIA SPP. AND EXOTIC MELALEUCA SPP. (STUDY II). Test Plant Survival (%)a after: No. AdultsbLeaves attacked (cumulative no.)c7 d28 d 7 d 28 d Trial D Eugenia axillaris 00020 Eugenia confusa 0006 Eugenia foetida 00028 Eugenia rhombea (Berg) Krug & Urb.300 1 34 76 Melaleuca quinquenervia 6030 5 50+ 344+ Trial E Melaleuca citrina 1000 7 50 284+ Melaleuca citrina (broad-leaved) 100 0 20 20 Melaleuca viminalis 8040 4 50+ 347+ Melaleuca viminalis Little John10070 8 50 459 Melaleuca quinquenervia 8050 6 50+ 446+aTen small nymphs per plant, new adults were left on the plant with the remaining nymphs.bSome adults died before 28 d when trapped in the folds of the mesh sleeve.cPlants with a + were difficult to count accurately so the count is a minimum.

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176 Florida Entomologist 94(2)June 2011because of limited use by other Melaleuca herbivores (Wheeler 2005; Pratt et al. 2009). Two specimens of Eugenia DC. were placed together at the same position in trial H because they had fewer shoots than the others. Ten pairs of adults were released on the M. quinquenervia plant. Damaged leaves were counted daily except for trial H, which was not assessed until the third d. The few damaged leaves on the test plants were removed daily to avoid duplicate counting. Damaged leaves were not removed from M. quinquenervia because this would have resulted in total defoliation of the tree. The resultant cumulative counts were therefore underestimates for M. quinquenervia inasmuch as most leaves would have been subjected to repeated feeding. The M. quinquenervia was cut on the fth day and the pieces were tied to the trunk to allow the leaves to dry and the bugs to disperse. The dried M. quinquenervia was removed on the seventh day and the trial terminated on the tenth day. The plants in trial I were examined for eggs when the test ended. Melaleuca viminalis and the 2 forms of M. citrina were randomized to 9 positions, 3 each, in trial J. Due to a limitation of standard M. viminalis plants, 1 M. viminalis cultivated variety Little John was also incorporated into the trial. We placed 2 pots together at one position for M. citrina and one for M. viminalis because they had fewer shoots than the other plants. Eighteen males were released on the ceiling in the center of the cage. Three survivors were removed on the fth day, but 2 more were found on the seventh day when damaged leaves were counted. Males were used to avoid oviposition in order to preserve the plants for other uses.No-choice Starvation Trial with Nymphs and Adults (Study V)Three potted sugarcane plants, Saccharum ofcinarum L. (Cyperales: Poaceae), and 2 potted lemon plants were individually caged in nylon sleeves in the greenhouse (trial K). Three females, 2 males, and 6 medium-sized nymphs were released in each cage where they remained until they died. The number of leaves with feeding spots was counted after all bugs were dead. An additional trial (trial L) was conducted to determine if discoloration observed on the plants in trial K was due to feeding damage. A leaf of a sugarcane plant and a lemon plant was covered with a small net sleeve. One pair of adults and 2 nymphs were released in the small sleeve. Each plant was enclosed in a larger sleeve. All plants in trials K and L were examined for eggs when the test ended. RESULTSNo-choice Adult Feeding and Oviposition Trials (Study I)Feeding was moderate to heavy on nearly all plants tested (Table 1). The 2 non-target Melaleuca spp., the 2 Psidium spp., and Eugenia uniora L. were most heavily attacked. Native Myrtaceae were noticeably damaged but, with the exception of Calyptranthes zuzygium (L.) Sw. on which they survived for 32 d, the adult bugs died within 10 d. Nymphs developed and eggs were found only on non-target Melaleuca spp. and M. quinquenervia Lagerstroemia indica L. suffered only light damage, although some nymphs livedTABLE 3. MULTI-CHOICE ADULT FEEDING AND OVIPOSITION TRIAL ON MYRTACEAE WITHOUT MELALEUCA QUINQUENERVIA(STUDY III). Test species (Trial F) Feeding intensityaEggs Day 2bDay 5cBouquet 1Bouquet 2Bouquet 1Bouquet 2 Calyptranthes pallens 14450 Calyptranthes zuzygium 00000 Eugenia axillaris 21546 Eugenia confusa 11310 Eugenia foetida 00112 Eugenia uniora 23220 Leptospermum scoparium 00000 Myrcianthes fragrans 11310 Psidium friedrichsthalianum 23350 Psidium cattleianum 31541aTen and 2 released in the cage. Feeding estimate: 1 = Light, scattered feeding, no large blotches; 2 = Light-medium; 3 = Medium, noticeable feeding, on multiple leaves; 4 = Medium-heavy; 5 = Heavy, some leaves blackened, some abscinded.bBouquets were randomized in each half of cage and were replaced in d 2.cTest terminated on d 5 when eggs were counted.

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Buckingham et al.: Risk Assessment for Eucerocoris suspectus 177at least 25 d on it. Damage was usually distributed throughout the plant with over 50% of the shoots attacked on 7 of 10 species. In general, survival on most test plants was quite long (Table 1).No-choice Nymphal Feeding Trials (Study II)Small nymphs were dead by d 7 on 3 native Eugenia spp. in trial D, but 30% survived on E. rhombea Ridl. (Table 2). One became an adult, but it and the remaining nymphs were dead by d 28. Only the newest leaves on E. rhombea were attacked but they were heavily damaged and abscinded. Similar feeding and damage was observed on the other Eugenia spp., with few leaves attacked due to low nymphal survival but heavy damage levels (Table 2). Older leaves were also attacked on E. axillaris (Sw.) Willd. but with little damage. On M. quinquenervia, 60% of nymphs survived to d 7 and 50% became adults. Two died after being entrapped in the folds of the cloth sleeve, so survivorship would probably have been somewhat greater. Survival and feeding on non-target Melaleuca spp. in trial E were similar to those on M. quinquenervia except on the broad-leaved M. citrina. All nymphs were alive on d 7 on M. citrina and M. viminalis Little John and 80% were alive on M. viminalis and on M. quinquenervia. All were dead on M. citrina by d 28, but 40% and 70% were alive on the 2 M. viminalis plants and 50% on M. quinquenervia Most surviving nymphs developed to adults on both nontarget Melaleuca spp. and M. quinquenervia and some adults died before 28 d. Feeding on 2 non-target Melaleuca spp. was comparable to that experienced by M. quinquenervia (Table 2). Feeding on the test plants was usually distributed throughout the canopy, with most foliage damaged at shoot tips.TABLE 4. WALK-IN ENCLOSURE MULTI-CHOICE ADULT FEEDING TRIALS ON MYRTACEAE WITH AND WITHOUT MELALEUCA QUINQUENERVIA (STUDY IV). Test Species Leaves with feeding blotches, cumulative tally on day 3 5 7 10Eggs Trial H. Ten adult pairs, potted plants at 9 positions, M. quinquenervia next to central plant Calyptranthes pallens 1151 1 Calyptranthes zuzygium 0000 Eugenia axillaris 1 3 11 16 Eugenia confusa 0 1 14 22 Eugenia foetida 8 8 25 36 Eugenia rhombea 0044 Melaleuca quinquenervia 164293DryingaRemovedb Myrcianthes fragrans 2 2 30 36 Morella cerifera (L.) Small (Myricaceae) 0111 Psidium longipes (Berg) McVaugh 0 0 22 22 Trial I. Set up same as Trial H but eggs were assessed at the end of evaluation Melaleuca citrina Melaleuca citrina (broad-leaved) 2 3 13 21no Melaleuca viminalis 3 28104254yes Eucalyptus camaldulensis Dehnh. 0011n o Eucalyptus camaldulensis 4c11c12c23 cyes Leptospermum scoparium 0000n o Melaleuca quinquenervia 258328DryingRemoved Psidium friedrichsthalianum 0070n o Psidium cattleianum 0000n o Trial J. Eighteen potted plants at 9 positions, 3 of each species Melaleuca citrina 23d Melaleuca citrina (broad-leaved) 38 Melaleuca viminalis 127 aMelaleuca quinquenervia was cut on d 5 and left in cage to dry slowly.bMelaleuca quinquenervia was removed from the cage on d 7.cNumber of stems fed upon. Feeding was on stems not on leaves.dTotal of the 3 positions, 122 leaves attacked on C. viminalis were all on 1 plant.

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178 Florida Entomologist 94(2)June 2011Multi-choice Adult Feeding and Oviposition Trials without M. quinquenervia (Study III)All species in trial F except C. zuzygium and Leptospermum scoparium J. R Forst. & G. Forst. sustained damage, but Eugenia foetida Pers. was not damaged until after d 2 (Table 3). Most species had light to medium damage by d 2. Four species showed medium to heavy feeding on one bouquet on d 5 (Calyptranthes pallens Griseb., E. axillaris Psidium friedrichsthalianum (O. Berg.) Nied., and P. cattleianum Sabine). Eggs were found on 3 species: the natives E. axillaris and E. confusa and the cultivated P. cattleianum. Two of the 4 species in trial G were attacked on d 1. Five of 7 leaves on P. racemosa and 4 of 5 leaves on S. cumini had more than 5 feeding blotches. The trial ended on d 11, with no feeding on lemon but with 14 leaves damaged on A. sellowiana, half of which had more than 5 feeding blotches. No live insects were recovered at the end of trial G. Nymphs only emerged from eggs in the stems of A. sellowiana None emerged from stems of P. racemosa or S. cumini .Large Enclosure Multi-choice Adult Feeding Trials With and Without M. quinquenervia (Study IV)With M. quinquenervia present in trials H and I, there was little feeding on test plants by d 5 except on one of 2 plants of M. citrina and 1 plant of M. viminalis (Table 4). However, the feeding on both non-target plants was much less than that on M. quinquenervia Feeding increased on most plants after M. quinquenervia was cut, but was still minor except on the 2 plants already mentioned. Feeding on M. viminalis on d 10 was similar to that on M. quinquenervia on d 3. Eggs were found only on M. citrina M. viminalis and M. quinquenervia in trial I. There was little feeding on the broad-leaved M. citrina in this trial, although it was heavily attacked by adults in nochoice trials. Feeding on plants of other non-target species was not particularly damaging, but was noticeable. Little feeding occurred on either variety of M. citrina in trial J. Almost all feeding on M. viminalis 96%, and all on M. citrina occurred at one of the 3 positions within the cage. Also, the attacked leaves were on relatively few shoot tips, not widely distributed over the plant (M. citrina 3 of 25 tips had feeding M. citrina (broad-leaved), 5 of 10, and M. viminalis 9 of 100).No-choice Starvation Trial with Nymphs and Adults (Study V)All insects died on sugarcane plants ( n = 3) by the eighth d without feeding in trial K. On lemon plants (n = 2) all insects were dead by the sixth d, but 7 and 8 leaves were damaged on 2 of the plants. This damage was slight, perhaps due to test probing, and all damaged leaves had less than 5 feeding blotches. Brown streaks were observed along the veins on some sugarcane leaves, but when additional insects were conned on new leaves (trial L) to determine if this streaking was symptomatic of feeding damage, none resulted. No eggs were found on any of the test plants. DISCUSSIONThe host range of the Melaleuca leaf-blotching bug, E. suspectus may be considered acceptable based on the fact that the insect was able to complete development only on M. quinquenervia other exotic Melaleuca spp. and once on E. rhombea. However, the vagile nature of this insect would enable it to feed on a wide variety of plants that are not developmental hosts. Feeding damage proved especially troublesome because of the frequency that this insect probed or test fed on non-target plants. Thus, even though E. suspectus is stenophagous, it presents a risk to rare endemic plant species such as E. rhombea that are sympatric with M. quinquenervia in southern Florida. The exact nature of this risk cannot be known without further study but the precautionary principal, which dictates conservative actions, would disqualify release of this insect. We did not test unusually high numbers of insects per cage, for instance, but unacceptably high levels of collateral damage were observed on nontarget species in large cage trials. In contrast, and contrary to cage tests, Burrows & Balciunas (1999) observed that adults released on test plants in a shade-house fed and reproduced only on M. quinquenervia Our concerns, however, were conrmed through eld observations by personnel at the Brisbane laboratory: damage was observed on mixed Myrtaceae in a garden plot, all stages of E. suspectus were found on bottlebrush, Melaleuca spp., and damage to guava was severe (Purcell et al. 2000). Bottlebrushes are relatively common ornamentals in Florida and some other states. These were originally placed in the genus Callistemon but have since been synonymized with Melaleuca (Craven 2006). These results matched those of Burrows & Balciunas (1999) quite closely in terms of common genera tested in cage tests. They reported noticeable feeding on Melaleuca (=Callistemon), Psidium, and Syzygium, as did we. Nymphal survival to adult was 47% on M. viminalis in their tests as compared to 50% herein. Damage from an equal amount of feeding on Calyptranthes, Eucalyptus, Eugenia, and Psidium was greater than that on Melaleuca. The damaged young leaves and young stems dried and abscinded on those genera, as they often did on Melaleuca, but there were fewer leaves on nontarget plants than on the longer, foliose shoots of

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Buckingham et al.: Risk Assessment for Eucerocoris suspectus 179the target weed. Thus, an equal number of attacked leaves among test plants resulted in a disproportionate level of damage on non-target hosts as compared to Melaleuca. However, this level of non-target damage was limited to a few test species as many more leaves were usually attacked on Melaleuca. The present data also have relevance to experimental protocols for host range testing. Evaluation of an herbivores host range is an effort to maximize predictive precision within the bounds of practicality. Multiple replicated experiments provide insight to variation in an herbivores host preferences, but limited nancial resources necessitate abandoning continued testing (replication) at early stages of evaluation for herbivores that demonstrate broad host ranges. Although development of E. suspectus appears to be conned to Melaleuca, non-host feeding by the highly mobile nymphs and adults is too broad and too damaging for this bug to be used for biological control. Therefore, many tests were terminated with few replicates when it became apparent that this insect was not a suitable candidate for release. Continuation of testing for the sake of additional replicates would have wasted time and resources, so testing was curtailed in favor of more suitable candidates. ACKNOWLEDGMENTSWe thank Mayana Roberg Anderson for assistance with plant maintenance. We further acknowledge D. W. Burrows, J. K. Balciunas, M. F. Purcell, K. E. Galway, J. A. Goolsby, J. R. Makinson, D. Mira, and the late C. E. Turner for signicant contributions towards the study of Eucerocoris suspectus Mention of trade names or commercial products in this publication is solely for the purpose of providing specic information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. This research was supported, in part, by grants from the South Florida Water Management District and the Florida Department of Environmental Protection Bureau of Invasive Plant Management.REFERENCES CITEDBODLE, M. 1998. Dial M for melaleuca. Wildland Weeds. 1: 9-10. BURROWS, D. W., AND BALCIUNAS, J. K. 1999. Hostrange and distribution of Eucerocoris suspectus (Hemiptera: Miridae), a potential biological control agent for the paperbark tree Melaleuca quinquenervia (Myrtaceae). Environ. Entomol. 28: 290-299. CRAVEN, L. A. 2006. New combinations in Melaleuca for Australian species of Callistemon Myrtaceae). Novon 16: 468-475. CRAVEN, L. A. 2009. Melaleuca (Myrtaceae) from Australia. Novon 19: 444-453. PRATT, P. D., RAYAMAJHI, M. B., CENTER, T. D., TIPPING, P. W. AND WHEELER, G. S. 2009. The ecological host range of an intentionally introduced herbivore: a comparison of predicted versus actual host use. Biol. Control 49: 146-153. PURCELL, M. F., GALWAY, K. E., GOOLSBY, J. A., MAKINSON, J. R., AND MIRA, D. 2000. Field plot experiments, a method of assessing the host range of biological control agents for Melaleuca quinquenervia in its native range, pp. 684-685 In N. R. Spencer [ed.] Proc. X Inter. Symp. Biol. Control Weeds. Montana State University, Bozeman Montana, USA. 1030 pp. TURNER, C. E., CENTER, T. D., BURROWS, D. W. ANDBUCKINGHAM, G. R. 1998. Ecology and management of Melaleuca quinquenervia an invader of wetlands in Florida, U.S.A. Wetlands Ecol. Manage. 5: 165178. WHEELER, G. S. 2005. Maintenance of a narrow host range by Oxyops vitiosa; a biological control agent of Melaleuca quinquenervia Biochem. Syst. Ecol. 33: 365-383.

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180 Florida Entomologist 94(2) June 2011 COMPARISON OF SYNTHETIC FOOD-BASED LURES AND LIQUID PROTEIN BAITS FOR CAPTURE OF ANASTREPHA SUSPENSA (DIPTERA: TEPHRITIDAE) ADULTS N ANCY D. E PSKY P AUL E. K ENDRA J ORGE P EA 1 AND R OBERT R. H EATH Subtropical Horticultural Research Station, United States Department of Agriculture, Agricultural Research Service, 13601 Old Cutler Road, Miami, FL 33158, U.S.A. 1 University of Florida, Tropical Research and Education Center, 18905 SW 280th Street, Homestead, FL 33031, U.S.A. A BSTRACT Field tests were conducted in south Florida to compare capture of the Caribbean fruit y, Anastrepha suspensa (Loew), in Multilure traps baited with either of the liquid protein baits torula yeast/borax or Nulure/borax, or with food-based synthetic lures including two-component Biolure (ammonium acetate, putrescine) and three-component Biolure (ammonium acetate, putrescine, trimethylamine). The highest relative proportion of females captured was in traps baited with the two-component Biolure (44-61%), intermediate capture was in traps baited with the three-component Biolure (14-24%) or torula yeast/borax (8-25%), and the lowest capture tended to be in traps baited with Nulure/borax (0-19%). Similar results were obtained for capture of males. Tests of the unipak two-component Biolure, which has a reduced ammonium acetate release rate and is a single package with both ammonium acetate and putrescine sections, captured similar numbers of both females and males as Biolure formulated in 2 individual packages. Traps baited with unipak Biolure combined with the addition of a trimethylamine lure captured fewer females than the unipak alone, but this was greater than capture in traps baited with torula yeast/borax. Our studies conrmed that the best lure for A. suspensa is ammonium acetate and putrescine. However, C. capitata -targeted traps baited with three-component Biolure should be as effective for A. suspensa detection and monitoring as traps baited with torula yeast/borax. The unipak two-component Biolure will provide the improved handling that has been requested by users. Key Words: Caribbean fruit y, Biolure, ammonium acetate, unipak, torula yeast R ESUMEN En pruebas de campo realizadas en el sur de Florida, se compararon resultados de captura de la mosca de la fruta del Caribe Anastrepha suspensa (Loew), capturadas en trampas Multilure que habian sido cebadas con cebos de proteina liquida de levdura torula/borax o con Nulure/borax, o con atrayentes alimenticios sinteticos los cuales incluian Biolure con dos componentes (acetato de ammonia, putrescina) y Biolure con 3 componentes (acetato de ammonia, putrescina, trimethylamina). El mas alto porcentajge de captura de hembras ocurrio en trampas cebadas con Biolure de dos componentes (44-61%), un nivel de captura intermedio ocurrio en trampas cebadas con Biolure de 3 componentes (14-24%) o con los cebos de levadura torula /borax (8-25%), mientras que la captura mas baja fue en aquellas trampas cebadas con Nulure/borax (0-19%). Se obtuvieron resultados similares en la captura de machos. Pruebas unipak del Biolure de dos componentes, el cual da una tasa de liberacion reducida de acetato de ammonio y el cual es un atrayente individual con secciones de acetato de ammonio y putrescina, no mostro diferencias en captura de machos o hembras entre el Biolure de dos componentes formulado como atrayentes individuales o como unipak. La captura intermedia de las hembras fue obtenida en trampas cebadas con unipak Biolure combinado con un atrayente individual de trimethylamina, pero esta captura fue mayor que la obtenida cuando se usaron trampas cebadas con levadura torula /borax. Nuestros estudios conrmaron que el mejor atrayente para A suspensa es el acetato de ammonio y putrescina. Sin embargo trampas destinadas a atrapar C. capitata y cebadas con acetato de amonio, putrescina y trimethylamina deben ser tan efectivas como aquellas trampas cebadas con levadura torula /borax para la deteccion y monitoreo de A. suspensa El unipak con Biolure de dos componentes es igualmente efectivo y dara una mejor facilidad para manipulacion, la cual ha sido pedida por los usuarios. Translation provided by the authors.

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Epsky et al.: Standard Baits for Anastrepha suspensa 181 Tephritid fruit ies are among the most important pests of fruits and vegetables in the world, and use of traps and lures are important components of fruit y pest management programs. Standard trapping protocols have been developed for fruit y detection and monitoring and, depending on target species, different female-targeted baits may be used (IAEA 2003). Selection of trap and lure depends on the purpose of trapping, availability of materials and cost. Although use of a single trap type that would capture the highest number of females and males of multiple species would have many advantages, species-specic traps that use lures specic to the target fruit y and environmental conditions are the best systems available currently (Daz-Fleischer et al. 2009). Among the numerous liquid protein baits, agencies primarily use torula yeast/borax solutions for detecting and monitoring Anastrepha spp. and NuLure/borax solutions for the Mediterranean fruit y Ceratits capitata Weidemann (Epsky et al. 1993; Heath et al. 1993, 1994). Food-based synthetic attractants have been developed based on volatile chemicals released from liquid protein baits. A two-component Biolure attractant comprised of ammonium acetate and putrescine is used in traps that target Anastrepha spp. (Heath et al. 1995; Epsky et al. 1995, Thomas et al. 2001). Addition of trimethylamine is used in traps that target C. capitata (Heath et al. 1997), and McPhail-type traps baited with the Biolure three-component attractant are equal to or better than liquid protein-baited traps for capture of C. capitata females (Epsky et al. 1999). Capture of Anastrepha spp. ies by traps baited with either the two-component or three-component attractant, however, is more variable (IAEA 2007). Initial studies found no difference in capture of the Mexican fruit y, Anastrepha ludens (Loew), in traps baited with Biolure ammonium acetate and putrescine with or without the third Biolure component, trimethylamine (Heath et al. 1997). However, Holler et al. (2006) found that addition of trimethylamine reduced capture of the Caribbean fruit y, Anastrepha suspensa (Loew). Regulatory agencies deploy McPhail-type traps baited with the three-component Biolure (ammonium acetate putrescine, and trimethylamine; Suterra LLC, Bend, OR), which is commercially available, primarily to monitor for new infestations of C. capitata in areas currently yfree and questions remain on the effectiveness of this trapping system for detecting and/or monitoring A. suspensa Due to problems with the deployment of the synthetic attractants as individual lures several versions of the components combined in a single lure have been developed and tested (Jang et al. 2007; Navarro-Llopis et al. 2008) including two-component and three-component unipak versions of Biolure (Holler et al. 2009). We report results of eld tests that were conducted in south Florida to compare capture of A. suspensa in traps baited with either of the liquid protein baits or with the Biolure twoand three-component food-based synthetic attractant (both individual lures and unipak lure) to deter mine relative effectiveness of these standard trapping systems. M ATERIALS AND M ETHODS Traps and Lures MultiLure traps (Better World Manufacturing Inc., Fresno, CA, USA) were used in all experiments. Liquid protein baits included aqueous solutions of torula yeast/borax (three 5-g pellets, 2.25:2.75 yeast:borax, in 300 mL water) (ERA International, Freeport, NY) and NuLure (Miller Chemical & Fertilizer Co., Hanover, PA) as a 300 mL aqueous solution of 9% NuLure (vol:vol) and 3% borax (wt:vol; sodium tetraborate decahydrate). Synthetic attractants included individual component Biolure formulations of ammonium acetate, putrescine, and trimethylamine, and the unipak two-component Biolure formulation of ammonium acetate and putrescine (Suterra LLC, Bend, OR). The membrane-release area of the ammonium acetate lure in the two-component unipak has been reduced from ~35 mm diam. on individual component lure to ~23 mm diam. on unipak lure. This lowers the release rate of ammonium acetate, which improves capture of the Mexican fruit y, Anastrepha ludens (Loew) but has no effect on capture of A. suspensa (Thomas et al. 2008). Traps baited with synthetic lures contained 300 mL 10% polypropylene glycol (vol:vol; LowTox, Prestone, Danbury, CT, USA) aqueous solution to retain captured ies. Field Tests Field tests were conducted at the Tropical Research and Education Center (TREC), University of Florida, Homestead, FL. Experiment 1 compared capture of ies in traps baited with (1) NuLure/borax, (2) torula yeast/borax, (3) two-component Biolure (individual ammonium acetate and putrescine lures), and (4) three-component Biolure (individual ammonium acetate, putrescine, and trimethylamine lures). Experiment 2 compared capture of ies in traps baited with (1) torula yeast/borax, (2) two-component Biolure, (3) unipak two-component Biolure, and (4) unipak two-component Biolure plus trimethylamine individual Biolure. Tests were conducted in two hosts, Surinam cherry, Eugenia uniora L., and guava, Psidium guajava L., for experiment 1; and in one host, guava, for experiment 2. For the tests in Surinam cherry, all 4 treatments were placed around the periphery of a large tree in fruit. There were 3 blocks (replicates) of traps, with 2 m

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182 Florida Entomologist 94(2) June 2011 between traps within a block and 10 m between blocks. For the tests in guava, there was 1 trap per tree with the traps placed in 3 rows (blocks/ replicates) of trees. There were at least 10 m between rows and 10 m between traps within a row. For both experiments, traps were sampled every 7 d, and numbers of male and female ies were recorded. Traps were sampled for 4 wk per sampling period, for a total of 4 sampling periods in experiment 1 (sample periods 1-2 in Surinam cherry, sample periods 3-4 in guava), and 1 sampling period in experiment 2. The protein bait solutions were replaced every 7 d, but the synthetic lures were not replaced during a sampling period. A complete randomized design was used for trap placement at the start of each sampling period. Traps were rotated sequentially to the next position within a block at time of sampling, so that all treatments were in all positions within each sampling period. Trees were in fruit throughout both experiments, but the tests in Surinam cherry were conducted toward the end of the fruiting season (18 Jun to 13 Aug, 2008) and the experiments in guava were conducted in the beginning of the fruiting season (experiment 1 Jun 25 to Aug 20, 2008; experiment 2 Aug 27 to Sep 17, 2009). Fruiting in guava trees began earlier in 2008 than in 2009 because the trees were trimmed in spring 2009. Statistical Analysis Effect of treatment and either sample period (experiment 1) or sample week (experiment 2) were analyzed by two-way ANOVA in a factorial model with interaction (Proc GLM, SAS Institute 2000) followed by LSD mean separation ( P = 0.05) for signicant factors When necessary, data were transformed prior to analysis to satisfy conditions of equal variance (Box et al. 1978). Numbers of total ies per trap per day and percentage females for ies captured in traps baited with the Biolure two-component attractant were analyzed as assessments of population level. Summary statistics are presented as average standard deviation. R ESULTS Experiment 1 Number of total A. suspensa captured per trap per da y in traps baited with the two-component Biolure was affected by sampling period ( P = 9.08, df = 3, 44; P < 0.0001; log ( x + 1) transformed data). Numbers of A. suspensa in traps in Surinam c herry decreased from 4.0 4.4 ies per trap per day in sampling period 1 to 2.0 2.3 ies per trap per day in sampling period 2. Numbers in traps in guava increased from 0.4 0.2 in sampling period 3 to 5.8 5.0 in sample period 4. Percentage of females captured in these traps was also affected by sampling period ( P = 3.27, df = 3, 48; P = 0.0321; square-root ( x + 0.5) transformed data). All captures were female biased, and percentage of females captured during sampling periods 1, 2, 3 and 4 were 86.6 10.6, 80.8 14.5, 60.8 .0, and 76.1 16.7, respectively. Numbers of female and male ies per trap per block were converted to relative trapping efciency to facilitate comparisons among the range of population levels tested during the different sampling periods (Epsky et al. 1999). There was a signicant interaction between sampling period and treatment for female ( F = 3.17; df = 9, 32; P = 0.0075) but not for male ( F = 0.97; df = 9, 32; P = 0.4825) relative trapping efciencies Therefore, one-way analyses were used to test effect of treatment within each sampling period for females and over all sampling periods for males. Traps baited with the two-component Biolure had the highest relative trapping efciency for females for all sampling periods (Table 1). The next highest relative trapping efciencies were for traps baited with the three-component Biolure and with torula yeast/borax solution. Relative trapping efciency in traps baited with NuLure/borax was signicantly less than capture in traps with the three-component attractant for the sampling periods in which the population was the lowest (sample periods 2 and 3), Treatment also had an effect on capture of males ( F = 24.24; df = 3, 44; P < 0.0001). The highest relative trapping efciency of males w as 64.5 20.2% in traps baited with the two-component Biolure, and this was higher than traps baited with the three-component Biolure, NuLure/borax or torula yeast/borax (12.4 10.9, 12.2 12.7, and 10.9 10.5, respectively). Experiment 2 Number of total A. suspensa captured per trap per da y in traps baited with the two-component Biolure was affected by sample week ( P = 23.83, df = 3, 8; P = 0.0003; log ( x + 1) transformed data). Numbers increased from 6.5 0.6 in week 1 to 29.0 6.2 in week 4. Percentage of females captured in these traps was not affected by sampling period ( P = 1.62, df = 3, 8; P = 0.2591; square-root ( x + 0.5) transformed data). Overall, captures were female biased, but the percentage of females captured decreased slightly from 74.7 8.9 in week 1 to 65.9 5.1 in week 4. As in experiment 1, numbers of female and male ies per trap per block were converted to relative trapping efciency for subsequent analysis. There was no interaction between treatment and sample week, so data from all sample weeks were pooled and effect of treatment was analyzed with one-way ANOVA. Both individual and unipak two-component Biolure formulations captured more females than traps baited with torula yeast/

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Epsky et al.: Standard Baits for Anastrepha suspensa 183 borax (Fig. 1 solid bars; F = 7.73; df = 3, 44; P = 0.0003), with intermediate capture with unipak two-component Biolure plus trimethylamine. There was no difference in number of males captured in traps baited with two-component Biolure or unipak two-component Biolure (Fig. 1 shaded bars; F = 3.37; df = 3, 44; P = 0.0268), and both treatments captured more males than traps baited with either with unipak Biolure plus trimethylamine or torula yeast/borax. D ISCUSSION Glass McPhail traps baited with torula yeast/ borax solution have been the standard trapping system for Anastrepha spp. since early studies found that torula yeast performed better than a number of other yeast formulations (Lopez et al. 1971), and most studies have evaluated liquid protein baits in these traps. Torula yeast/borax captured more A. suspensa than NuLure/borax in tests with glass McPhail traps (Epsky et al. 1993). Plastic McPhail traps baited with two-component Biolure captured more ies than glass McPhail traps baited with either torula yeast/borax or two-component Biolure in tests in Florida in Surinam cherry, loquat ( Eriobotrya japonica [Thunb.] Lindl.), and guava (Thomas et al. 2001; Hall et al. 2005). Tests in grapefruit, Citrus paradisi Macfady, in Florida found that captures in Multilure traps baited with either two-component Biolure or torula yeast/borax were higher than in traps baited with NuLure/borax (Thomas et al. 2008). Tests in sapodilla, Manilkara zapota Van Royen, and mamey sapote, Pouteria sapota (Jacq.), in Puerto Rico, however, found more A. suspensa were captured in Multilure traps baited with torula yeast/borax than with two-component Biolure (Pingel et al. 2006). In the only previous test of three-component Biolure, Holler et al. (2006) found that the highest capture was in plastic McPhail traps baited with two-component Biolure, and that there was no difference between capture in plastic McPhail traps baited with three-component Biolure or glass McPhail traps baited with torula yeast/borax. Results from that study, in which traps were placed in scattered wild guava trees, were the same as the results from our study. Unipak versions of two-component and threecomponent Biolure have been shown to be equal T ABLE 1.R ELATIVE TRAPPING EFFICIENCY (%) FOR CAPTURE OF FEMALE A NASTREPHA SUSPENSA IN FIELD TESTS CONDUCTED IN HOMESTEAD, FL. MULTILURE TRAPS WERE USED FOR ALL BAITS AND EACH SAMPLE PERIOD WAS4 WEEKS. SURINAM CHERRY WAS AT THE END OF THE FRUITING SEASON IN SAMPLE PERIOD 2 AND GUAVA WAS AT THE BEGINNING OF THE FRUITING SEASON IN SAMPLE PERIOD 3. Bait* Surinam cherry** Guava Sample period 1Sample period 2Sample period 3Sample period 4 Two-component BioLure 44.1 4.5 a60.2 10.2 a60.9 12.8 a60.9 20.5 a Three-component BioLure 19.4 8.5 b24.2 13.9 b14.3 9.4 b23.7 15.8 b Torula yeast/borax 17.3 7.2 b10.1 4.3 bc24.8 12.6 b7.5 2.2 b NuLure/borax 19.2 12.8 b5.5 2.9 c0 c 8.0 4.4 b F 4.22 21.56 27.17 11.06 df 3,8 3, 8 3,8 3,8 P 0.0459 0.0003 0.0002 0.0032*Ammonium acetate and putrescine alone (two-component Biolure) or with trimethylamine (three-component Biolure) in traps with 300 mL 10% propylene glycol solution, 3 torula yeast/borax pellets in 300 mL water, 9% NuLure and 3% borax in 300 mL water **Means within a column followed by the same letter are not signicantly different (LSD mean separation test on square root (x + 0.5)-transformed data, non-transformed mean standard deviation presented). Fig. 1. Relative trapping efciency (%) for capture o f female (solid bars) and male (shaded bars) Anastrepha s uspensa in eld tests conducted in Homestead, FL. Multilure traps were used for all baits and traps were sampled for 4 weeks. Treatments included two-component Biolure as individual ammonium acetate and putrescine lures (Standard AP), unipak two-component Biolure (Unipak AP), unipak two-component Biolure plus trimethylamine individual Biolure (Unipak AP + T) in traps with 300 mL 10% propylene glycol solution, and 3 torula yeast/borax pellets (TY/borax) in 300 mL water. Bars headed by the same lowercase (females) or uppercase (males) letter are not signicantly different (LSD mean separation test on square root ( x + 0.5)transformed data, non-transformed means presented).

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184 Florida Entomologist 94(2)June 2011to the same components formulated in single lure formulations for capture of sterile C. capitata released in Florida and for capture of A. suspensa in traps placed in backyard plantings of host fruit trees in Sarasota/Bradenton and in Ft. Pierce (Holler et al. 2009). We recorded similar results for A. suspensa in our tests in south Florida. Additionally, we found that the unipak two-component in combination with trimethylamine may be better than torula yeast/borax for A. suspensa monitoring. This may be due to the lower release rate of ammonia from the unipak two-component formulation. The greater amount of ammonia from the individual lure formulation used in experiment 1, when combined with the amines from the trimethylamine lure, may have been repellant to A. suspensa Our studies conrmed that the best lure for A suspensa is two-component Biolure ammonium acetate and putrescine, however, C. capitata -targeted traps baited with three-component Biolure ammonium acetate, putrescine and trimethylamine should be as effective as traps baited with torula yeast/borax for A. suspensa detection and monitoring. The unipak two-component Biolure is equally effective and will provide the improved handling that has been requested by users. ACKNOWLEDGMENTSThe authors thank M. Gill (USDA-ARS, Miami, FL) for coordinating the eld tests, and D. Long, J. Sanchez, W. Montgomery, C. Allen, I. Filpo, and J. Tefel (USDAARS, Miami, FL) for technical assistance; Joan Fisher (Suterra LLC, Bend, OR) for supplying unipak two-component Biolures; Jerome Niogret (USDA-ARS, Miami, FL), David Jenkins (USDA-ARS, Mayaguez, PR) and Donald Thomas (USDA-ARS, Weslaco, TX) for reviewing an earlier version of this manuscript. This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or recommendation by the USDA. REFERENCES CITEDBOX, G. E. P., HUNTER, W. G., AND HUNTER, J. S. 1978. Statistics for Experimenters. An Introduction to Design, Data Analysis, and Model Building. J. Wiley & Sons, New York, NY. DAZ-FLEISCHER, F., ARRENDONDO, J., FLORES, S., MONTOYA, P., AND ALUJA, M. 2009. There is no magic fruit y trap: multiple biological factors inuence the response of adult Anastrepha ludens and Anastrepha obliqua (Diptera: Tephritidae) individuals to MultiLure traps baited with BioLure or nulure. J. Econ. Entomol. 102: 86-94. EPSKY, N. D., HEATH, R. R., SIVINSKI, J. M., CALKINS, C. O., BARANOWSKI, R. M., AND FRITZ, A. H. 1993. Evaluation of protein bait formulations for the Caribbean fruit y (Diptera: Tephritidae). Florida Entomol. 76: 626-635. EPSKY, N. D., HEATH, R. R., GUZMAN, A., AND MEYER, W. L. 1995. Visual cue and chemical cue interactions in a dry trap with food-based synthetic attractant for Ceratitis capitata and Anastrepha ludens (Diptera: Tephritidae). Environ. Entomol. 24: 1387-1395. EPSKY, N. D., HENDRICHS, J., KATSOYANNOS, B. I., VSQUEZ, L. A., ROS, J. P., ZMREOGLU, A., PEREIRA, R., BAKRI, A., SEEWOORUTHUN, S. I., AND HEATH, R. R. 1999. Field evaluation of female-targeted trapping systems for Ceratitis capitata (Diptera: Tephritidae) in seven countries. J. Econ. Entomol. 92: 156-164. HALL, D. G., BURNS, R. E., JENKINS, C. C., HIBBARD, K. L., HARRIS, D. L., SIVINSKI, J. M., AND NIGG, H. N. 2005. Field comparison of chemical attractants and traps for Caribbean fruit y (Diptera: Tephritidae) in Florida citrus. J. Econ. Entomol. 98: 1641-1647. HEATH, R. R., EPSKY, N. D., LANDOLT, P. J., AND SIVINSKI, J. 1993. Development of attractants for monitoring Caribbean fruit ies (Diptera: Tephritidae). Florida Entomol. 76: 233-244. HEATH, R. R., EPSKY, N. D., BLOEM, S., BLOEM, K., ACAJABON, F., GUZMAN, A., AND CHAMBERS, D. 1994. pH effect on the attractiveness of a corn hydrolysate to the Mediterranean fruit y and several Anastrepha species (Diptera: Tephritidae). J. Econ. Entomol. 87: 1008-1013. HEATH, R. R., EPSKY, N. D., GUZMAN, A., DUEBEN, B. D., MANUKIAN, A., AND MEYER, W. L. 1995. Development of a dry plastic insect trap with food-based synthetic attractant for the Mediterranean and Mexican fruit ies (Diptera: Tephritidae). J. Econ. Entomol. 88: 1307-1315. HEATH, R. R., EPSKY, N. D., DEUBEN, B. D., RIZZO, J.,AND JERONIMO. F. 1997. Adding methyl-substituted ammonia derivatives to a food-based synthetic attractant on capture of the Mediterranean and Mexican fruit ies (Diptera: Tephritidae). J. Econ. Entomol. 90: 1584-1589. HOLLER, T., SIVINSKI, J., JENKINS, C., AND FRASER, S. 2006. A comparison of yeast hydrolysate and synthetic food attractants for capture of Anastrepha suspensa (Diptera: Tephritidae). Florida Entomol. 89: 419-420. HOLLER, T., PEEBLES, M., YOUNG, A., WHITEMAN, L., OLSON, S., AND SIVINSKI, J. 2009. Efcacy of the Suterra biolure individual female fruit y attractant packages vs. the unipak version. Florida Entomol. 92: 667-669. JANG, E. B., HOLLER, T. C., MOSES, A. L., SALVATO, M. H., AND FRASER, S. 2007. Evaluation of a single-matrix food attractant tephritid fruit y bait dispenser for use in federal trap detection programs. Proc. Hawaiian Entomol. Soc. 39: 1-8. IAEA (INTERNATIONAL ATOMIC ENERGY AGENCY). 2003. Trapping Guidelines for Area-wide Fruit Fly Programmes. International Atomic Energy Agency, Vienna, Austria, 47 pp. IAEA (INTERNATIONAL ATOMIC ENERGY AGENCY). 2007. Development of Improved Attractants and Their Integration into Fruit Fly SIT, IAEA-TECDOC-1574, International Atomic Energy Agency, Vienna, Austria, 230 pp. LOPEZ, F., STEINER, L. F., AND HOLBROOK, F. R. 1971. A new yeast hydrolysate-borax bait for trapping the Caribbean fruit y. J. Econ. Entomol. 64: 15411543. NAVARRO-LLOPIS, V., ALFARO, F., DOMNGUEZ, J., SANCHIS, J. AND PRIMO, J. 2008. Evaluation of traps and

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Epsky et al.: Standard Baits for Anastrepha suspensa 185lures for mass trapping of Mediterranean fruit y in citrus groves. J. Econ. Entomol. 101: 126-131. PINGEL, R. L., EPSKY, N. D., AND GOENAGA, R. 2006. Field trials to attract fruit ies (Diptera: Tephritidae) in commercial sapodilla, mamey sapote, and carambola orchards in Puerto Rico. J. Agric.Univ. Puerto Rico 90:109-113. SAS INSTITUTE. 2000. SAS system for Windows release 8.01. SAS Institute, Cary, NC. THOMAS, D. B., HOLLER, T. C., HEATH, R. R., SALINAS, E. J, AND MOSES, A. L. 2001. Trap-lure combinations for surveillance of Anastrepha fruit ies (Diptera: Tephritidae). Florida Entomol. 84: 344-351. THOMAS, D. B., EPSKY, N. D., SERRA, C. A., HALL, D. G., KENDRA, P. E., AND HEATH, R. R. 2008. Ammonia formulations and capture of Anastrepha fruit ies (Diptera: Tephritidae). J. Entomol. Sci. 43: 76-85.

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186 Florida Entomologist 94(2) June 2011 FOOD-BASED LURE PERFORMANCE IN THREE LOCATIONS IN PUERTO RICO: ATTRACTIVENESS TO ANASTREPHA SUSPENSA AND A. OBLIQUA (DIPTERA: TEPHRITIDAE) D AVID A. J ENKINS 1 N ANCY D. E PSKY 2 P AUL E. K ENDRA 2 R OBERT R. H EATH 2 AND R ICARDO G OENAGA 1 1 USDA-ARS, Tropical Agriculture Research Station, 2200 Ave. P.A. Campos, Mayaguez, PR 00680-5470 2 USDA-ARS-Subtropical Horticulture Research Station, 13601 Old Cutler Road, Miami, FL 33158 A BSTRACT Lures based on odors released by hydrolyzed protein were assessed for their attractiveness to Anastrepha obliqua and A. suspensa at 3 locations in Puerto Rico in Aug through Oct 2009. Lures compared inc luded ammonium acetate combined with putrescine, hydrolyzed corn protein (Nulure) with borax, freeze-dried Nulure, freeze-dried Nulure in combination with ammonium acetate, freeze-dried Nulure in combination with ammonium acetate and putrescine, and the Unipak lure, a single lure containing ammonium acetate and putrescine. Where the distribution of trapped ies departed signicantly from what would be expected given an equal attraction of the baits, Nulure and freeze-dried Nulure always attracted fewer ies than the other baits tested, regardless of species, sex, or location. Although all of the baits or bait combinations containing ammonium acetate attracted more ies than the Nulure or freeze-dried Nulure baits, there was a distinct trend of ammonium acetate and putrescine and the Unipak lures to attract more ies after the 4th week of the study and for the freeze-dried Nulure with ammonium acetate or in combination with ammonium acetate and putrescine to attract more ies in the 1st 4 weeks of the study. This trial is unique in that it was conducted in orchards of carambola, Averrrhoa carambola (Oxalidaceae), a poor host for both y species Our results are compared with other studies on lures of A. obliqua and A. suspensa and the implications for monitoring/detecting pest Tephritidae are discussed. K ey Words: ammonium acetate, putrescine, Nulure, McPhail trap R ESUMEN Trampas que trabajan a base de olores liberados por protena hidrolizada se evaluaron como atrayentes de las moscas Anastrepha obliqua y A. suspensa en tres localidades en Puerto Rico durante agosto a octubre de 2009. Las trampas utilizadas en el estudio incluyeron acetato de amonio en combinacin con putrescina, protena hidrolizada de maz (NuLure) con brax, NuLure liolizado en combinacin con acetato de amonio y putrescina, y la trampa Unipak la cual contiene acetato de amonio y putrescina en una sola mezcla. Las trampas NuLure y NuLure liolizada atrajeron menos moscas que el resto de las trampas irrespectivamente de la especie, sexo, o localidad. Aunque todas las trampas o combinaciones de estas con acetato de amonio atrajeron ms moscas que las trampas NuLure o NuLure liolizada, hubo una clara tendencia de las trampa de acetato de amonio y putrescina y la trampa Unipak a atraer ms moscas despus de la cuarta semana a partir de comenzado el estudio y de las trampas NuLure liolizadas con acetato de amonio o en combinacin con acetato de amonio y putrescina a atraer ms moscas en las primeras cuatro semanas del estudio. Este estudio es nico en que se llev a cabo en huertos de carambola Averrrhoa carambola (Oxalidcea), un cultivo que es un pobre hospedero de ambas especies de moscas. Nuestros resultados se comparan con otros estudios con trampas de A. obliqua y A. suspensa y las implicaciones para el monitoreo y deteccin de plagas Tephritidae son discutidos. Translation provided by the authors. Although less than 10% of the 199 described species of Anastrepha are considered economically important (White & Elson-Harris 1992; Aluja 1994; Norrbom 2004), the occurrence of any of these economically important species in a region has a negative impact on growers. Growers may be restricted from exporting their produce to certain markets, or may have to subject their fruit to expensive post-harvest sterilization measures (Simpson 1993). The island of Puerto Rico contains populations of 2 economically important species; the Caribbean fruit y, A. suspensa (Loew) and the West Indian fruit y A. obliqua (Macquart) (Jenkins & Goenaga 2008). Although there are populations of A. suspensa in Florida, there are no populations of A. obliqua there, making it risky to transport some Puerto Rican produce to Florida. Establishment of A. obliqua in Florida could jeopardize mango and other subtropical fruit crops

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Jenkins et al.: Food-based Anastrepha spp. Lures in Puerto Rico187 Regulatory agencies spend considerable effort and expense monitoring large areas for these and other potentially invasive Anastrepha spp. (Anonymous 2010). The need for effective monitoring/ detection devices has resulted in a long history of studies on attractants for Anastrepha spp. (Heath et al. 1993). Females of all frugivorous species of Tephritidae that have been studied, including Anastrepha spp., are anautogenous, i.e., they need to consume protein as adults for ovary development (Drew & Yuval 2000). Exploiting this need for protein, a variety of potential lures based on odors released from hydrolyzed proteins have shown some degree of attractiveness, including ammonia (released from ammonium acetate, ammonium bicarbonate and urine) (Bateman & Morton 1981; Burditt et al. 1983), and hydrolyzed torula yeast (Lopez et al. 1971; Burditt 1982). For many years hydrolyzed torula yeast in a liquid suspension, along with borax to reduce cadaver decay, was used in 1 piece glass McPhail traps to monitor and detect populations of Anastrepha spp. (Anonymous 1989; Heath et al. 1993). A series of modications to the trap and the lures ha ve improved the utility and effectiveness of the trap (Epsky et al. 1993; Heath et al. 1995; Thomas et al. 2001). Heath et al. (1995) identied some common volatiles from baits and decomposing fruit that were attractive to A. suspensa These inc luded ammonia, acetic acid (both released from ammonium acetate) and putrescine. Although not attractive when deployed alone (Heath et al. 2004), putrescine has been shown to be a potent synergist to ammonium acetate for capture of both A. ludens and A. supensa (Kendra et al. 2008). Thomas et al. (2001) pointed out that the design of the 1-piece glass McPhail trap was difcult to service, especially with the new lures, and prone to damage. A 2piece plastic version of the McPhail trap has since been widely adopted by regulatory agencies. However, despite many studies, no single bait has been identied to satisfy the needs of regulatory agencies. Ideally, a bait would be easy to apply, long-lasting, attractive to target species (often multiple target species; regulatory agencies in Florida are currently monitoring for A. obliqua and the Mediterranean fruit y Ceratitis capitata Wied., among many others) combined with low non-target attractiveness APHIS-PPQ in Puerto Rico currently deploys a battery of traps and lures to detect and monitor pest Tephritidae; trimethylamine and ammonium acetate plus putrescine are used in Multilure traps (2-piece plastic McPhail traps) to detect C. capitata ; methyl eugenol is used in Jackson traps (tent-shaped stic ky cards) to detect Oriental fruit ies, Bactrocera dorsalis (Hendel) and carambola fruit ies B. carambolae Drew & Hancock, and Cuelure is used in J ackson traps to detect melon fruit y, B. cucurbitae (Coquillett), and Queensland fruit ies B. tryoni (Froggatt) (Anonymous 2010). In addition, torula yeast is still used at some trap sites (Saez, personal communication). Current lures for detecting/monitoring pest Anastrepha spp. include Nulure (Miller Chemical & F ertilizer, Hanover, PA.), a hydrolyzed corn protein lure (Gilbert et al. 1984), a freeze-dried preparation of Nulure (Heath et al. unpublished), ammonium acetate combined with putrescine (Biolure, Suterra LLC, Bend, Oregon), and the Unipak (Suterra LLC), a single bait dispenser containing ammonium acetate and putrescine (Holler et al. 2009). Our objective in this study was to compare these lures, as well as certain combinations (freeze-dried Nulure combined with ammonium acetate, or combined with both ammonium acetate and putrescine) for relative attractiveness to populations of A. suspensa and A. obliqua in Puerto Rico We chose to conduct these trials in carambola, Averrhoa carambola (Oxalidaceae), because orc hards of this fruit were available to the researchers in 3 different regions of Puerto Rico. Additionally, this is a poor host of both A. obliqua and A. suspensa ; collections of thousands of carambola fruit yielded no pupae of A. supensa and relatively few pupae of A. obliqua principally when preferred hosts such as mango, were not available (Jenkins & Goenaga 2008). Most lure trials are conducted in orchards of preferred hosts; by conducting these trials in a poor host environment we evaluated efcacy of lures for detection of pest Anastrepha at low population levels. M ATERIALS AND M ETHODS Study Sites Field trials were conducted in Sep and Oct of 2009, a time we had determined to be peak season for both y species (Jenkins, unpublished). All trap blocks were set in experimental orchards of carambola located at the USDA-ARS Tropical Agriculture Research Experimental Station in Isabela, PR, and at the University of Puerto Rico Agricultural Experiment Stations in Corozal and Juana Diaz, PR. All orchards were planted in 1999 and were composed of 10 rows, each row containing 22 trees. Trees were planted in a quincunx system 3.7 m apart with 5.5 m between rows. All of the 6 internal rows had 9 varieties of tree planted randomly throughout the row. The varieties, grafted onto Goldenstar rootstock, were Arkin, B-10, B-16, B-17, Kajang, Kari, Lara, SriKembangan, and Thai Knight. The 2 rows of trees on either side of these 6 internal rows were composed entirely of Arkin grafted onto Goldenstar rootstock. The rst 2 trees and the last 2 trees of each row were also Arkin grafted onto Goldenstar rootstock. We have never recovered A. suspensa from thousands of carambola fruit and relatively low numbers of A. obliqua have been recovered

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188 Florida Entomologist 94(2) June 2011 from carambola fruit (Jenkins & Goenaga 2008); nonetheless past experience has demonstrated that both species can be trapped in relative abundance from orchards of this fruit with no demonstrable effects of fruit variety on trap catch (Jenkins, unpublished). Traps and Lures All baits were tested using plastic 2-piece Multilure traps (Better World Manufacturing, Inc., Fresno, CA). Commercial lures (Suterra, LLC, Bend, OR) consisted of ammonium acetate and putrescine (Biolure MFF), and the newly formulated Unipak. A total of 6 lures or lure combinations were tested in each block as follows: 1.Ammonium acetate + putrescine (=AAPt) 2.Nulure with borax 3.Freeze-dried Nulure 7 (=FDN7) 4.Freeze-dried Nulure 7 + ammonium acetate (=FDN7 + AA) 5.Freeze-dried Nulure 7 + ammonium acetate + putrescine (=FDN7 + AAPt) 6.Unipak For all treatments except the 9% Nulure with borax, the trap uid consisted of 200 mL of a 10% solution of propylene glycol (Qualichem Technologies, GA) and water. The trap uid for the 9% Nulure lure (18 mL) consisted of 3% Borax (6 g) mixed with 182 mL of water. The Nulure and freeze-dried Nulure (9 g) were dissolved in the respective trap uid. Nulure and freeze-dried Nulure baits were changed every 2 weeks (3 times during the study). The ammonium acetate and putrescine lures were changed every 4 weeks (once during the study). Trap Block Design Traps were deployed in 3 rows of each orchard. Rows with traps were at least 2 rows from the orchard border and separated by at least 1 trap-less row from another row with traps. All 6 treatments were represented in each of the 3 rows. Trees with traps were separated from other trees with traps by at least 1 tree. Traps were rotated to subsequent positions within rows each time they were checked. Traps at all sites were checked for fruit ies on Monday and Friday of each week between 24 Aug 2009 and 16 Oct 2009. All fruit ies were returned to the laboratory for identication and stored in 95% EtOH. Statistical Analyses The large number of independent variables we were comparing combined with the low number of replicates we were forced to use made the use of an ANOVA unsuitable for our purposes. Chisquare analyses were used to compare the observed number of ies of each species and sex trapped in each treatment to the expected number of ies under the assumption that ies would be equally distributed among the treatments if there was no difference in attraction. These analyses were conducted for each sex of each species for each week of the study (a total of 8 weeks) and for the total number of ies trapped throughout the study for each site. Chi-square probabilities exceeding 0.05 were labeled as insignicant. When the total number of ies trapped during a given week or at a given site was less than 30, i.e., an expected distribution among the treatments would be less than 5 (30 ies divided by 6 treatments = 5), the result was regarded as too weak to make inferences, even if the analyses indicated signicant departure from the null hypothesis (= there was no difference in the distribution of ies among the treatments). R ESULTS Too few A. suspensa were caught in traps at the Isabela site (9 females and 1 male total) to merit analysis. A total of 294 A. obliqua ies were caught in Isabela, 167 of which were females (58.0%) and 127 of which were males (42.0%). Throughout the 8 weeks of the trial, the percentage of females trapped averaged 57.9% 4.7 (SEM). Of the 294 A. obliqua trapped at the Isabela site during the experiment, 23% were in traps baited with freeze-dried Nulure and ammonium acetate and putrescine, 19% were in traps baited with freeze-dried Nulure and ammonium acetate, 19% in traps baited with UniPak lures, 17% were in traps baited with ammonium acetate plus putrescine, 14% were in traps baited with freeze-dried Nulure, and 7% in traps baited with Nulure. Chi-square analyses indicated that the distribution of A. obliqua (combined sexes) among the treatments departed signicantly from the null hypothesis although this was not true for every week of the study (Table 1). A total of 231 Anastrepha spp. individuals were trapped at the Corozal site One hundred and fty nine (69%) of these were identied as A. obliqua of which 109 (69%) were females and 50 (31%) were males Of the 72 A. suspensa identied from traps at Corozal, 54 (75%) were female and 18 (25%) were male. Throughout the 8 weeks of the trial, the percentage of female A. obliqua trapped averaged 70.2% 3.0 (SEM) and the percentage of female A. suspensa trapped averaged 75.3% 3.8 (SEM). Of the 159 A. obliqua trapped at the Corozal site during the experiment, 30% were in traps baited with ammonium acetate and putrescine, 28% were in traps baited with freeze-dried Nu-

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Jenkins et al.: Food-based Anastrepha spp. Lures in Puerto Rico189 T ABLE 1. N UMBER OF FLIES CAPTURED BY WEEK AND BY BAIT AT THE I SABELA SITE C HI SQUARE ANALYSES WERE PERFORMED ON COMBINED SEXES WITHIN A SPECIES Number of ies AAPtNulureFDN7FDN7 + AAFDN7 + AAtUniPakTotal ies WeekSexMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemale 2 value 2 prob 1 A. obliqua 013513732202131612.60.0280 A. suspensa 00010000000001NANA 2 A. obliqua 333138414111643284531.8<0.0001 A. suspensa 00000000000000NANA 3 A. obliqua 412041339863281618.70.0020 A. suspensa 00000000000000NANA 4 A. obliqua 25224361203419154.10.5320 A. suspensa 00000001000001NANA 5 A. obliqua 010001132100369.00.1090 A. suspensa 01000001020004NANA 6 A. obliqua 3110233126510162119.30.0020 A. suspensa 00000011000011NANA 7 A. obliqua 1420012413096219.20.1000 A. suspensa 00000001010002NANA 8 A. obliqua 9130224012215142728.3<0.0001 A. suspensa 00000000000000NANA Total A. obliqua 22291310162426303138193612716725.4<0.0001 A. suspensa 01010014030019NANA

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190 Florida Entomologist 94(2)June 2011lure and ammonium acetate and putrescine, 13% were in traps baited with UniPak lures, 12% were in traps baited with freeze-dried Nulure and ammonium acetate, 11% were in traps baited with freeze-dried Nulure, and 6% were in traps baited with Nulure. Of the 72 A. suspensa trapped at the Corozal site during the experiment, 33% were in traps baited with ammonium acetate and putrescine, 26% were in traps baited with UniPak lures, 25% were in traps baited with freeze-dried Nulure and ammonium acetate and putrescine, 8% were in traps baited with freeze-dried Nulure and ammonium acetate, 6% were in traps baited with freeze-dried Nulure, and 1% were in traps baited with Nulure. As at the Isabela site, ammonium acetate and putrescine, freeze-dried Nulure combined with ammonium acetate or combined with ammonium acetate and putrescine, and the Unipak trapped more ies than the Nulure or the freeze-dried Nulure (Table 2). This was true for both A. obliqua and A. suspensa A total of 157 Anastrepha spp. individuals were trapped at the Juana Diaz site. Ninety four (59.9%) of these were identied as A. suspensa of which 78 (83.0%) were female and 16 (17%) were male. A total of 63 (40.1%) A. obliqua were trapped at the Juana Diaz site, of which 49 (77.8%) were female and 14 (22.2%) were male. Throughout the 8 weeks of the trial, the percentage of female A. obliqua trapped averaged 80.0% 4.2 (SEM) and the percentage of female A. suspensa trapped averaged 75.3% + 7.3 (SEM). Of the 63 A. obliqua trapped at the Juana Diaz site during the experiment, 24% were in traps baited with freeze-dried Nulure combined with ammonium acetate and putrescine, 21% were in traps baited with freeze-dried Nulure, 17% were in traps baited with UniPak lures, 16% were in traps baited with freeze-dried Nulure and ammonium acetate and putrescine, 13% were in traps baited with ammonium acetate and putrescine, and 10% were in traps baited with Nulure. Of the 94 A. suspensa trapped at the Juana Diaz site during the experiment, 27% were in traps baited with freeze-dried Nulure and ammonium acetate, 26% were in traps baited with ammonium acetate and putrescine, 21% were in traps baited with freeze-dried Nulure and ammonium acetate and putrescine, 16% were in traps baited with UniPak lures, 6% were in traps baited with Nulure, and 4% were in traps baited with freeze-dried Nulure. Generally, too few ies were captured of either species to make a condent analysis except when captures were summed for the duration of the experiment (Table 3). No signicant departures from the null hypothesis were detected in the distribution of A. obliqua ies among the different baits. At all 3 sites there was a consistent temporal pattern in capture; freeze-dried Nulure combined with ammonium acetate or combined with ammonium acetate and putrescine caught more ies in the rst 4 weeks, whereas ammonium acetate plus putrescine or the Unipak lures caught more ies after the fourth week (Tables 1-3). The only exception occurred at the Juana Diaz site when the ammonium acetate and putrescine combination caught more A. suspensa than expected in the rst week (Table 3). DISCUSSIONFor all locations, species and sexes, where a Chi-square analysis detected signicant departure from equal distribution among the different baits (and at least 30 ies were captured) Nulure or freeze-dried Nulure consistently attracted the fewest ies. This would suggest that the higher attractiveness of the Unipak, ammonium acetate and putrescine lures and the freeze-dried Nulure in combination with either ammonium acetate or ammonium acetate and putrescine is attributable to the common factor of these lures, namely, the presence of ammonium acetate in all of these lures. However, bait attractiveness was not constant over time, with a general trend of freezedried Nulure in combination with ammonium acetate or in combination with ammonium acetate and putrescine attracting more ies in the rst 4 weeks of the study and ammonium acetate and putrescine or Unipak lures attracting more ies in the fourth week and later. This appears to be consistent with the anecdotal reporting that freshly opened ammonium acetate lures are less attractive than those that have been out a week or more (Thomas et al. 2008). This is potentially due to the dosage of ammonia released; Thomas et al. (2008) demonstrated that higher doses of the ammonia signicantly reduced capture of A. suspensa and A. ludens compared to lower doses. Also, Kendra et al. (2005) demonstrated that increased doses of ammonia decreased the capture of female A. suspensa with undeveloped ovaries. However, fresh ammonium acetate and putrescine lures were placed in the eld on the 5th week of this study, approximately when they began to catch more ies. Also, the freeze-dried Nulure combined with ammonium acetate or ammonium acetate and putrescine caught more ies early in the study, suggesting that the freshly opened ammonium acetate packages are not too strong, or that combined with the freeze-dried Nulure, the ammonium acetate packages are attractive at higher doses. Many studies have been conducted on the attractiveness of certain baits, but comparing these studies in a meaningful manner is difcult and subject to speculation. This is because these studies are often conducted in different regions, trap different species of ies, different strains of ies (wild versus lab-reared), test different combina-

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Jenkins et al.: Food-based Anastrepha spp. Lures in Puerto Rico191TABLE 2. NUMBER OF FLIES CAPTURED BY WEEK AND BY BAIT AT THE COROZAL SITE. CHI-SQUARE ANALYSES WERE PERFORMED ON COMBINED SEXES WITHIN A SPECIES. Number of ies AAPtNulureFDN7FDN7 + AAFDN7 + AAtUniPakTotal ies WeekSexMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemale 2 value 2 prob 1 A. obliqua 01012016060031415.10.0100 A. suspensa 000000001102138.00.1560 2 A. obliqua 012000030410385.90.3150 A. suspensa 0000001200001215.00.0100 3 A. obliqua 3421063571600153251.5<0.0001 A. suspensa 0100100105011810.30.0660 4 A. obliqua 69020000330391737.2<.0001 A. suspensa 1400010022124927.3<0.0001 5 A. obliqua 030012013102495.00.4160 A. suspensa 0400010111031105.90.3150 6 A. obliqua 2100020000042717.00.0050 A. suspensa 2200010002002511.00.0510 7 A. obliqua 3301000000154942.7<.0001 A. suspensa 33100000022561017.80.0030 8 A. obliqua 480023001032101327.9<.0001 A. suspensa 130000010112279.00.1090 Total A. obliqua 18304551341514305165010948.3<0.0001 A. suspensa 717101315414415185437.5<0.0001

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192 Florida Entomologist 94(2)June 2011TABLE 3. NUMBER OF FLIES CAPTURED BY WEEK AND BY BAIT AT THE JUANA DIAZ SITE. CHI-SQUARE ANALYSES WERE PERFORMED ON COMBINED SEXES WITHIN A SPECIES. Number of ies AAPtNulureFDN7FDN7 + AAFDN7 + AAtUniPakTotal ies WeekSexMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemale 2 value 2 prob 1 A. obliqua 0301031213002124.90.4332 A. suspensa 19230004161152313.60.0190 2 A. obliqua 000010000200127.00.2206 A. suspensa 13000017070221917.00.0050 3 A. obliqua 0101221312125113.50.6234 A. suspensa 0401020802212189.40.0940 4 A. obliqua 011000110510379.20.1013 A. suspensa 000011130103288.00.1560 5 A. obliqua 0000011000051616.40.0057 A. suspensa 1100000000122310.60.0600 6 A. obliqua 010000000100024.00.5494 A. suspensa 010000000201045.00.4160 7 A. obliqua 0012000000021410.60.0599 A. suspensa 010000000010114.00.5490 8 A. obliqua 0200130000001514.00.0156 A. suspensa 110000100100225.00.4160 Total A. obliqua 082449462132914495.10.4038 A. suspensa 4202413322119510167825.9<0.0001

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Jenkins et al.: Food-based Anastrepha spp. Lures in Puerto Rico193tions of lures, are conducted in orchards of different crop species, and at different times of the year, all of which can impact the outcome. Nonetheless, it is useful to summarize these studies because baits will be used in a variety of conditions/locations/seasons to monitor/detect a variety of pest Tephritidae. Regionally, the experiments most similar to the present study are those of Pingel et al. (2006) comparing the attractiveness of ammonium acetate plus putrescine with torula yeast plus borax in commercial orchards of 3 crop species in southern Puerto Rico, coinciding climactically and geographically with our Juana Diaz site. Conducted in Apr to May of 2002, their study found that the torula yeast outperformed the ammonium acetate and putrescine combination in orchards of mamey sapote and sapodilla, but the ammonium acetate and putrescine lure attracted more ies than the torula yeast in carambola orchards. The difference between the effectiveness of the 2 lures in the different orchards is striking and they point out the preponderance of A. obliqua in the carambola orchard (94% trapped ies in the carambola orchard were A. obliqua) whereas the other orchards had higher relative populations of A. suspensa Anastrepha suspensa was more abundant in the carambola orchard at the Juana Diaz site during our study. In a Colombian mango orchard A. obliqua was caught in traps baited with Nulure and borax more frequently than in traps baited with ammonium acetate with putrescine, torula yeast, or ammonium bicarbonate with putrescine (Epsky et al. 2003). However, in another study in a Mexican Pouteria sapota (Sapotaceae) orchard, traps baited with ammonium acetate with putrescine caught more A. obliqua than traps with the other baits, and in a Mexican mango orchard Nulure and ammonium acetate with putrescine both caught more A. obliqua than traps baited with other lures (Epsky et al. 2003). Furthermore, traps baited with torula yeast in Costa Rica and Honduras caught more A. obliqua than traps baited with the other lures. Anastrepha suspensa does not occur in any of the locations of the cited trial and so no comparison can be made with the A. suspensa results of our study. A similar study indicated that ammonium acetate was the best lure for detection of A. suspensa in Florida but that Nulure or torula yeast were the best lures for A. obliqua in the Dominican Republic, based on traps in mango orchards (Thomas et al. 2008). In a study conducted in cages in Mexico, DazFleischer et al. (2009) found ammonium acetate and putrescine were more attractive to A. obliqua than Nulure, but the ammonium acetate and putrescine was not as attractive to A. ludens Anastrepha ludens is an economic pest which regulatory agencies in the United States would like to be able to detect. They also found that attractiveness varied according to whether the ies tested were wild or reared in the laboratory for several generations. There is strong evidence that the attractiveness of a particular lure to a given y is based on that ys physiological state, usually the stage of ovary development and a possible explanation for our results may be that populations varied physiologically over the duration of the experiment. Electroantennagram studies on A. suspensa indicated that immature females (females with little ovary development) were more responsive to ammonia and to ammonium bicarbonate lures, while females with mature ovaries were more responsive to putrescine and to carbon dioxide (Kendra et al. 2005; Kendra et al. 2009). Diaz-Fleischer et al. (2009) found that diet of the target y did inuence subsequent capture in traps, with more protein-starved individuals being captured by protein baits. Nulure, followed by freeze-dried Nulure, consistently attracted the fewest ies in our study, regardless of species, sex, or location. This contrasts with the results obtained by Thomas et al. (2008) in mango orchards in Dominican Republic, where A. obliqua was most attracted to Nulure and torula yeast baits. It is conceivable that Nulure would attract more ies in different seasons. Liquid lures, including Nulure and torula yeast, have been shown to be more attractive in the dry season than in the wet season (Heath et al. 1997) and the Thomas et al. study was conducted at the beginning of the wet season. Diaz-Fleischer et al. (2009) recently concluded that there is no magic fruit y trap, based on the complex interactions of y species, physiological state and bait preference, aggravated by low trap efciency. One particular short-coming was the number of ies that entered a trap and successfully escaped from it. It is certainly true that these interactions are complex and that no single bait or trap will sufce for all target species in all regions. It has long been known that different tephritid species respond differently to hydrolyzed protein from different sources; A. ludens was more attracted to hydrolyzed cottonseed oil than to hydrolyzed corn protein (Lopez and Becerril 1967). Anastrepha striata, A. serpentina, A. obliqua and A. balloui preferred baits of hydrolyzed soy protein to torula yeast hydrolysate (Jiron and Soto-Manitiu 1989). Despite all of the improvements to the traps themselves and the lures, estimates of percent capture have not changed over more than 20 years; Calkins et al. (1984) and Diaz-Fleischer et al. (2009) came up with estimates of about 10% of the available population. Kendra et al. (2010) were able to recapture up to 35% of released A. suspensa though. The results of this study conrm what has been suspected; that trap baits will have to be tailored based on regional and seasonal use.

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194 Florida Entomologist 94(2)June 2011ACKNOWLEDGMENTSWe thank Elkin Vargas and Rosemarie Boyle (USDA-ARS, Mayaguez, Puerto Rico) for technical assistance; Amy Roda (USDA-APHIS, Miami, Florida) and Paul Robbins (USDA-ARS, Fort Pierce, Florida) for helpful suggestions with the manuscript. This report presents the results of research only; mention of a proprietary product does not constitute an endorsement by the USDA.REFERENCES CITEDALUJA, M. 1994. Bionomics and management of Anastrepha. Annu. Rev. Entomol. 39: 155-173. ANONYMOUS. 1989. Florida Fruit Fly Detection Manual. USDA, APHIS, PPQ and Florida DACS, DPI, Gainesville, Florida. ANONYMOUS. 2010. Puerto Rico and USVI Fruit Fly Trapping Protocol. USDA-APHIS-PPQ. 312 pp. BATEMAN, M. A., AND MORTON, T. C. 1981. The importance of ammonia in proteinaceous attractants for fruit ies (Diptera: Tephritidae). Australian J. Agric. Res. 32: 883-903. BURDITT, JR., A. K. 1982. Anastrepha suspensa (Loew) (Diptera: Tephritidae) McPhail traps for survey and detection. Florida Entomol. 65: 367-373. BURDITT, JR., A. K., MCGOVERN, T. P., AND GREANY, P. D. 1983. Anastrepha suspensa (Loew) (Diptera: Tephritidae) response to chemical attractants in the eld. Proc. Florida State Hort. Soc. 96: 222-226. CALKINS, C. O., SCHROEDER, W. A., AND CHAMBERS, D. L. 1984. The probability of detecting the Caribbean fruit y, Anastrepha suspensa (Loew), (Diptera: Tephritidae) with various densities of McPhail traps. J. Econ. Entomol. 77: 198-201. DAZ-FLEISCHER, F., ARREDONDO, J., FLORES, S., MONTOYA, P., AND ALUJA, M. 2009. There is no magic fruit y trap: multiple biological factors inuence the response of adult Anastrepha ludens and Anastrepha obliqua (Diptera: Tephritidae) individuals to multilure traps baited with Biolure or Nulure. J. Econ. Entomol. 102: 86-94. DREW, R. A. I., AND YUVAL, B. 2000. The evolution of fruit y feeding behavior, pp. 737-740 In M. Aluja and A. L. Norrbom [eds.], Fruit Flies (Tephritidae): Phylogeny and Evolution of Behavior. CRC Press, Boca Raton, FL. EPSKY, N. D., HEATH, R. R., SIVINSKI, J. M., CALKINS, C. O., BARANOWSKI, R. M., AND FRITZ, A. H. 1993. Evaluation of protein bait formulations for the Caribbean fruit y (Diptera: Tephritidae). Florida Entomol. 76: 626-634. EPSKY, N. D., KENDRA, P. E., AND HEATH, R. R. 2003. Development of lures for detection and delimitation of invasive Anastrepha fruit ies. Proc. Caribbean Food Crops Soc. 39: 84-89. GILBERT, A. J., BINGHAM, R. R., NICOLAS, M. A., ANDCLARK, R. A. 1984. Insect trapping guide. Pest Detection/ Emergency Projects, State of California Department of Food and Agriculture, Sacramento, CA. HEATH, R. R., EPSKY, N. D., LANDOLT, P. J., AND SIVINSKI, J. M. 1993. Development of attractants for monitoring Caribbean fruit ies (Diptera: Tephritidae). Florida Entomol. 76: 233-244. HEATH, R. R., EPSKY, N. D., GUZMAN, A., DUEBEN, B. D., MANUKIAN, A., AND MEYER, W. L. 1995. Development of a dry plastic insect trap with food-based synthetic attractant for the Mediterranean and Mexican fruit ies (Diptera: Tephritidae). J. Econ. Entomol. 88: 1307-1315. HEATH, R. R, EPSKY, N. D., MIDGARDEN, D., AND KATSOYANNOS, B. I. 2004. Efcacy of 1,4-diaminobutane (putrescine) in food-based synthetic attractant for capture of Mediterranean and Mexican fruit ies (Diptera: Tephritidae) J. Econ. Entomol. 97: 1126-1131. HOLLER, T. C., PEEBLES, M., YOUNG, A., WHITEMAN, L., OLSON, S., AND SIVINSKI, J. 2009. Efcacy of the Suterra Biolure individual female fruit y attractant packages vs. the Unipak version. Florida Entomologist 92: 667-669. JENKINS, D. A., AND GOENAGA, R. 2008. Host breadth and parasitoids of fruit ies ( Anastrepha spp.) (Diptera: Tephritidae) in Puerto Rico. Environ. Entomol. 37: 110-120. JIRON, L. F., AND SOTO-MANITIU, J. 1989. Field evaluation of attractants for the capture of Anastrepha spp. (Diptera: Tephritidae), pests of fruits in tropical America. III. Hydrolyzed protein and torula yeast. Revista Brasileira de Entomolgia. 33: 353-356. KENDRA, P. E., MONTGOMERY, W. S., MATEO, D. M., PUCHE, H., EPSKY N. D., AND HEATH, R. R. 2005. Effect of age on EAG response and attraction of female Anastrepha suspensa (Diptera: Tephritidae) to ammonia and carbon dioxide. Environ. Entomol. 34: 584-590. KENDRA, P. E., EPSKY, N. D., MONTGOMERY, W. S., ANDHEATH, R. R. 2008. Response of Anastrepha suspensa (Diptera: Tephritidae) to terminal diamines in a food-based synthetic attractant. Environ. Entomol. 37:1119-1125. KENDRA, P. E., MONTGOMERY, W. S., EPSKY N. D., ANDHEATH, R. R. 2009. Electroantennagram and behavioral responses of Anastrepha suspensa (Diptera: Tephritidae) to putrescine and ammonium bicarbonate lures. Environ. Entomol. 38: 1259-1266. KENDRA, P. E., EPSKY, N. D., AND HEATH, R. R. 2010. Effective sampling range of food-based attractants for sterile Anastrepha suspensa (Diptera: Tephritidae) J. Econ. Entomol. 103: 533-540. LOPEZ, F., AND BECERRIL, O. H. 1967. Sodium borate inhibits decomposition of the protein hydrolysates attractive to the Mexican fruit y. J. Econ. Entomol. 60: 137-140. LOPEZ, F., STEINEER, L. F., AND HOLDBROOK, F. R. 1971. A new yeast hydrolysate-borax bait for trapping the Caribbean fruit y. J. Econ. Entomol. 64: 1541-1543. NORRBOM, A. L. 2004. The Diptera Site http:// www.sel.barc.usda.gov/Diptera/tephriti/Anastrep/ Anastrep.htm PINGEL, R. L., EPSKY, N. D., AND GOENAGA, R. 2006. Field trials to attract fruit ies (Diptera: Tephritidae) in commercial sapodilla, mamey sapote, and carambola orchards in Puerto Rico. J. Agric. Univ. Puerto Rico 90: 109-113. SIMPSON, S. E. 1993. Development of the Caribbean fruit-y free zone certication protocol in Florida. Florida Entomol. 76: 228-233. THOMAS, D. B., EPSKY, N. D., SERRA, C., HALL, D. G., KENDRA, P. E., AND HEATH, R. R. 2008. Ammonia formulations and capture of Anastrepha fruit ies (Diptera: Tephritidae). J. Entomol. Sci. 43: 76-85. WHITE, I. M., AND ELSON-HARRIS, M. M. 1992. Fruit ies of economic signicance: their identication and bionomics. CAB International Wallingford, xii + 601 pp.

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Ovruski et al.: Host Preference By Diachasmimorpha longicaudata 195 HOST PREFERENCE BY DIACHASMIMORPHA LONGICAUDATA (HYMNEOPTERA: BRACONIDAE) REARED ON LARVAE OF ANASTREPHA FRATERCULUS AND CERATITIS CAPITATA (DIPTERA: TEPHRITIDAE) S ERGIO M. O VRUSKI L AURA P. B EZDJIAN G UIDO A. V AN N IEUWENHOVE P ATRICIA A LBORNOZ -M EDINA AND P ABLO S CHLISERMAN Laboratorio de Investigaciones Ecoetolgicas de Moscas de la Fruta y sus Enemigos Naturales (LIEMEN). Planta Piloto de Procesos Industriales Microbiolgicos y Biotecnologa (PROIMI) CCT Tucumn CONICET. Avda. Belgrano y Pje. Caseros, (T4001MVB) San Miguel de Tucumn, Tucumn, Argentina A BSTRACT The preferences of Diachasmimorpha longicaudata (Ashmead) for larvae of Anastrepha fraterculus (Wiedemann) and Ceratitis capitata (Wiedemann) were evaluated under laboratory conditions in no-choice and dual-choice tests, based on percent parasitism, proportion of emerged parasitoids, proportion of female offspring, and number of parasitoid female visits to and ovipositor probes on the articial oviposition device as different measures of host preference. In no-choice tests D. longicaudata females did not demonstrate a signicant preference between C. capitata and A. fraterculus larvae. Nevertheless, D. longicaudata females showed a strong preference for A. fraterculus larvae in dual-choice test. Although female biased parasitoid progeny resulted in all assays, signicantly more D. longicaudata female offspring emerged from A. fraterculus pupae than from C. capitata pupae. Thus, this study conrmed that both C. capitata and A. fraterculus are appropriate host for rearing D. longicaudata but also provided evidence that female parasitoid progeny yield can be substantially improved by using A. fraterculus larvae as the host instead of C. capitata larvae. Key Words: fruit ies, parasitoids, host preference, biological control, Argentina R ESUMEN Se evalu la preferencia de Diachasmimorpha longicaudata (Ashmead) por larvas de Anastrepha fraterculus (Wiedemann) y Ceratitis capitata (Wiedemann) bajo condiciones de laboratorio en situaciones de eleccin y no-eleccin. Las variables consideradas para el anlisis fueron el porcentaje de parasitismo, la proporcin de parasitoides emergidos, la proporcin de descendientes hembras, el nmero de hembras que visitaron la unidad articial de oviposicin y el nmero de hembras que realizaron pruebas con el ovipositor en la unidad. Los resultados de los ensayos de no-eleccin mostraron que las hembras de D. longicaudata no tienen una signicativa preferencia por las larvas de una u otra especie de tefrtido. No obstante, en el ensayo de eleccin, las hembras del parasitoide manifestaron una signicativa preferencia por las larvas de A. fraterculus En todos los ensayos realizados, la proporcin de descendientes hembras de D. longicaudata obtenida fue superior a la de los machos, aunque signicativamente ms hembras del parasitoide se obtuvieron de puparios de A. fraterculus El presente estudio conrma que tanto las larvas de C. capitata como las de A. fraterculus son adecuadas para criar D. longicaudata en laboratorio, aunque tambin seala que el empleo de larvas de A. fraterculus mejoran sustancialmente la produccin de descendientes hembras del parasitoide. Translation provided by the authors. The South American fruit y, Anastrepha fraterculus (Wiedemann), and the Mediterranean fruit y, Ceratitis capitata (Wiedemann) are 2 of the major pests currently affecting fruit crops in Argentina (Guilln & Snchez 2007). Early biological control attempts to suppress both tephritid pest species resulted in the use of exotic parasitoids (Ovruski et al. 2000). Diachasmimorpha longicaudata (Asmead) is 1 of 5 exotic parasitoids introduced into Argentina from Costa Rica and Mxico (Ovruski et al. 2003). It was originally collected in the Malaysia-Philippine region and is a solitary, koinobiont, larval-prepupal endoparasitoid of several tephritid species (Montoya et al. 2000). At present, D. longicaudata is considered 1 of the most signicant biological control agents for augmentative releases against economically important fruit y species in several Latin American countries (Montoya et al. 2007; Paranhos et al. 2008; Lpez et al. 2009). Although small scale releases of D. longicaudata were made in the Citrus-growing areas of northern Argentina during the 1960s (Ovruski et al. 2000), the permanent establishment of this

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196 Florida Entomologist 94(2)June 2011 opiine parasitoid on A. fraterculus has been veried as a direct result of early classical biological control programs (Oroo & Ovruski 2007). Currently, the suitability for successfully rearing D. longicaudata on larvae of either C. capitata or A. fraterculus is being studied in the PROIMI insectary in San Miguel de TucumnArgentina, as part of an augmentative release program against both tephritid fruit y species. Therefore, the study here presented was conducted to evaluate the effects of both C. capitata and A. fraterculus on parasitism, parasitoid emergence, and sexual ratio of offspring in D. longicaudata under laboratory conditions. Furthermore, both the number of visiting and oviposition events was documented to assess the parasitoid female preference for 1 or the other host tephritid species. M ATERIALS AND M ETHODS The study was performed at the Biological Control Division of Planta Piloto de Procesos Industriales Microbiolgicos y Biotecnologa (PROIMI) located in San Miguel de Tucumn, Argentina. The colony of D. longicaudata was originally established in 1999 with individuals imported from Mxico (Ovruski et al. 2003), where this colony had been reared in the laboratory on Anastrepha ludens (Loew) larvae (Montoya et al. 2000). First, D. longicaudata was successfully reared at the PROIMI laboratory on late-third instars of C. capitata Then, in 2005 a second colony of D. longicaudata was established on late-third instars of A. fraterculus Parasitoid colonies were held in cubical Plexiglas cages (30 cm) covered by organdy screen on both lateral sides, at a capacity of 300 pairs per cage at 25 1C; 75 5% RH, and 12:12 (L:D) h photoperiod. The parasitoid rearing cage was provided with water and honey every other day. The general C. capitata and A. fraterculus rearing procedures were carried out as described by Ovruski et al. (2003) and by Vera et al. (2007), respectively. Both A. fraterculus and C. capitata puparia were selected from different samples and weighed for host quality evaluation. Each species of fruit y was exposed to 10 mated D. longicaudata females in cubical Plexiglas cages (30 cm) under both dual-choice and nochoice assays. In the choice assay, an oviposition unit (an organdy screen-covered petri dish, 8 cm diameter, 0.8 cm deep) containing 300 laboratoryreared third-instars of A. fraterculus (11 d old) was placed on the oor of the test cage along with another oviposition unit containing 300 laboratory-reared third-instars of C. capitata (6 d old). Larvae of both fruit y species were placed in the units with articial diet (brewer yeast + wheat germ + sugar + water). Oviposition units were positioned in the central part of the test cage; each unit was placed 1 cm from the side wall and separated by 10 cm from the other unit. In the nochoice assays, an identical oviposition unit containing 300 third-instars of A. fraterculus (or 300 third-instars of C. capitata ) was placed on the oor of the central part of the test cage away from the walls. All female parasitoids used in experiments were 7-8 d old and deprived of any host larvae before testing. The females used in no-choice tests came either from parasitized puparia of A. fraterculus or from parasitized puparia of C. capitata In the choice assay, 5 females stemming from parasitized puparia of A. fraterculus and 5 females stemming from parasitized puparia of C. capitata were used jointly This combination of parasitoids from different origins was used so as to ameliorate a possible conditioned response by the previous experience with the host on which it was reared (Godfray 1994). Two control tests (no parasitoids) were made to determine both natural A. fraterculus and C. capitata mortality and emergence rates. Each test, including control treatments, was replicated 22 times. Each replicate lasted 24 h. All assays were conducted in the laboratory under the environmental conditions described previously. Behavioral observations can be used to provide evidence of host preference for solitary parasitoids (Manseld & Mills 2004). For this reason, upon release of parasitoids into each test cage, the number of female visits to and ovipositor probes in the oviposition units was recorded. Odor concentrations of host fruit (Messing & Jang 1992) or ovipositiondeterring pheromone of tephritid y (Prokopy & Webster 1978) were not considered in the assays because oviposition units with articial diet were used. The female parasitoids were observed once every 15 min during the rst 3 h and each observation lasted 30 s (Duan & Messing 2000a). A visit was recorded each time a female arrived on the oviposition unit after release. An ovipositor probe was conrmed each time a female parasitoid inserted its ovipositor through the top organdy screen of the oviposition dish. After the 3-h observations, all oviposition units remained exposed to female parasitoids for 21 h to nish a 24-h period (Duan & Messing 2000a). Then, all oviposition dishes were removed from the cages, and y larvae were directly transferred into plastic cups (7 cm diameter, 6.7 cm deep) containing a 2 cm-vermiculite layer on the bottom as pupation medium. Later, each cup was tightly covered with a piece of organdy cloth on the top. Thus, y pupae were held within plastic cups with moist, sterilized vermiculite until eclosion. After that, the number and sex of the emerged parasitoids, the number of emerged ies, and the number of uneclosed puparia were checked. Uneclosed puparia were dissected 2 weeks after emergence of the last adult parasitoid in each cup to check for the presence or absence of recognizable immature parasitoid stages (larvae, prepupae, or pupae) and/or fully developed pherate-adult parasitoids.

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Ovruski et al.: Host Preference By Diachasmimorpha longicaudata 197 Both the parasitism percentage and the number and sex ratio of emerged parasitoid progeny were used as 3 suitable variables to measure host preference, in addition to the behavioral observations (Manseld & Mills 2004). Parasitism percentage was calculated by dividing the total number of emerged and unemerged parasitoids into the total number of larvae exposed in the oviposition unit. The proportion of emerged parasitoids was calculated as the total number of emerged offspring divided by the total number of recovered pupae. The proportion of emerged ies was computed as the total number of retrieved adult ies divided by the total number of recovered pupae. The proportion of dead pupae was determined as the total number of pupae that did not yield ies or parasitoids divided into the sum of eclosed and uneclosed puparia. Data on parasitism, parasitoid and y emergences, sexual ratio of parasitoid offspring (as proportion of females), pupal mortality, and the number of female visits to and ovipositor probes on the articial oviposition device were analyzed by a 2-sample unpaired t -test ( P = 0.05) in nochoice assays, and by a paired t -test ( P = 0.05) in the choice assay. Moreover, the numbers of emerged adults and dead pupae recorded from each fruit y species per assay were statistically compared with control treatments by means of one-way analyses of variance ( P < 0.05). Means were separated with a Tukey honest signicant difference test (HSD) ( P = 0.05). The proportion data were transformed to arcsine square root before analysis. All untransformed means ( SEM) were presented in the text. Pupal weight difference between A. fraterculus and C. capitata was analyzed by a Mann-Whitney Rank Sum test ( P = 0.05). R ESULTS From the dual-choice test, signicantly higher parasitism and emerged adult parasitoid percentages were recorded from A. fraterculus than from C. capitata (Table 1). When these 2 fruit y species were analyzed in the no-choice tests, there was no signicant difference for either of these 2 measures of host preference (Table 1). Sex ratios were female biased when D. longicaudata was reared from either host fruit y species. However, the proportion of female offspring was always signicantly higher when the parasitoid was reared on A. fraterculus than on C. capitata (Table 1). The proportion of emerged A. fraterculus and C. capitata adults was signicantly different between dual-choice, no-choice, and no-exposure control tests ( F (2, 63) = 260.0, P < 0.0001 for A. fraterculus ; F(2, 63) = 311.8, P < 0.0001 for C. capitata Table 2). A signicantly higher proportion of A. fraterculus adults were recovered from the no-choice test than from dual-choice test (Table 2). In contrast, significantlyTABLE 1.MEAN (SEM) PERCENTAGE PARASITISM BY DIACHASMIMORPHA LONGICAUDATA, AND PROPORTION OF ADULT PARASITOIDS AND FEMALE PROGENY EMERGED FROMCERATITIS CAPITATA AND ANASTREPHA FRATERCULUS FOR BOTH DUAL-CHOICE AND NO-CHOICE TESTS. Fly species Dual-choice test No-choice test % Parasitism % emerged adult parasitoids % parasitoid female progeny % Parasitism % emerged adult parasitoids % parasitoid female progeny C. capitata 17.4 1.5 a 13.3 1.1 a 50.7 2.8 a 37.6 2.0 a 32.1 1.5 a 55.0 1.1 a A. fraterculus 35.5 2.1 b 25.3 2.3 b 82.4 1.5 b 43.2 2.2 a 36.3 1.8 a 79.5 1.6 b paired-t = 7.60paired-t = 5.86 paired-t = 5.86unpaired-t = 1.90unpaired-t = 1.95 unpaired-t = 12.5 df = 21.0 df = 21.0 df = 21.0 df = 42.0 df = 42.0 df = 42.0 P < 0.0001 P < 0.0001 P < 0.0001 P = 0.0643 P = 0.0585 P < 0.0001Values in the same column with the same latter are not signicantly different (paired and unpaired t-test, P = 0.05).

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198 Florida Entomologist 94(2)June 20112.5-times greater proportion of C. capitata adults emerged from the dual-choice test than from nochoice test (Table 2). The signicantly lowest proportion of dead y pupae was recorded from no-exposure control tests (F(2, 63) = 28.6, P < 0.0001 for A. fraterculus ; F(2, 63) = 38.9, P < 0.0001 for C. capitata Table 2). Signicantly greater proportion of dead C. capitata pupae was recorded from no-choice tests than from dual-choice tests (Table 2). Under both dualand no-choice conditions, the mean numbers of D. longicaudata female visits to the oviposition units containing A. fraterculus larvae were signicantly similar to those of parasitoid visits to the oviposition units containing C. capitata larvae (paired-t = 1.89, df = 21.0, P = 0.0732 for dual-choice test; unpairedt = 0.47, df = 42.0, P = 0.6435 for no-choice test; Fig. 1 A). Similarly, in the no-choice assays, there were no signicant differences in the mean numbers of parasitoid females probing the oviposition articial devices (unpaired-t = 0.58, df = 42.0, P = 0.5631; Fig. 1 B). In contrast, in the dual-choice test, a signicantly greater number of D. longicaudata females were observed probing the oviposition unit containing A. fraterculus larvae that the device containing C. capitata larvae (paired-t = 5.54, df = 21.0, P < 0.0001; Fig. 1 B). DISCUSSIONWhile D. longicaudata attacked both C. capitata and A. fraterculus larvae at similar rates when only 1 of the species was present, they preferred A. fraterculus when provided a choice. This divergence may be suggestive of the relative host size differences. For example, A. fraterculus larvae used as host in this study were twice as large as C. capitata larvae (T = 60100.0, P < 0.0001, n = 200). Previous studies conducted by Messing et al. (1993), Cancino et al. (2002) and Lpez et al. (2009), found that D longicaudata females prefer large hosts. Eben et al. (2000) also pointed to the progeny sex ratio as a measure of host larva preference in D. longicaudata. These authors found that D. longicaudata reared from a larger species, A. ludens (Loew), in mango (Mangifera indica L.) had a much higher proportion of female progeny than those parasitoids that had developed in a smaller species, A. obliqua (Macquart), infesting the same fruit. Behavioral observations provided further evidence for a preference for A. fraterculus over C. capitata larvae. In dual-choice tests D. longicaudata females are more likely to exhibit oviposition behaviors on devices containing A. fraterculus. However, Silva et al. (2007) found that D. longicaudata females did not discriminate between the volatiles produced by C. capitata or A. fraterculus larvae. In contrast to the present study, the larvae exposed by Silva et al. (2007) were feeding inside infested guava fruits ( Psidium guajava L.). It has been repeatedly demonstrated that D. longicaudata females respond to fruit volatiles, especially from rotting fruits (Greany et al. 1977; Leyva et Fig. 1 (A and B). Mean ( SEM) (A) number of D. long icaudata female visits to, (B) and ovipositor probes on the oviposition units containing articial diet plus third-instars of A. fraterculus or C. capitata recorded in no-choice and dual-choice tests. Bars in each graph followed by the same letter indicate no signicant differences [unpaired t-test (P = 0.05) in the no-choice tests, and paired t-test (P = 0.05) in the dual-choice test] TABLE 2.MEAN (SEM) PROPORTION OF EMERGED ADULTS AND DEAD PUPAE FROM CERATITIS CAPITATA AND ANASTREPHA FRATERCULUS RECORDED IN CHOICE, NO-CHOICE, AND CONTROL TESTS. Tests % emerged A. fraterculus adults % emerged C. capitata adults % dead A. fraterculus pupae % dead C. capitata pupae Dual-choice17.3 1.8 a63.2 1.7 a34.2 1.4 a25.5 1.1 a No-choice24.6 2.6 b25.1 1.9 b32.3 2.0 a37.4 2.5 b Control82.4 1.3 c85.3 1.6 c17.6 1.3 b14.7 1.6 cValues in the same column with the same latter are not signicantly different (Tukey`s test, P < 0.05).

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Ovruski et al.: Host Preference By Diachasmimorpha longicaudata 199al. 1991; Messing & Jang 1992; Purcell et al. 1994; Eben et al. 2000; Carrasco et al. 2005). Chemical cues derived from fermentation of the articial rearing medium can be exploited for host searching by D. longicaudata (Duan & Messing 2000b). However, it is possible that differences in host larval substrates might have inuenced D. longicaudata s host detection ability. Duan & Messing (2000b) found that C. capitata larvae outside of the substrate on which they fed generated vibration and chemical cues that stimulated oviposition in Diachasmimorpha tryoni Cameron, another generalist opiine fruit y larval parasitoid (Wharton 1989). In the case of D. longicaudata, chemical cues produced by C. capitata larvae had little inuence on probing behavior (Duan & Messing 2000b). However, it is possible that D. longicaudata females may respond more positively to chemical cues of A. fraterculus larvae than to those from C. capitata larvae. In addition to larval frass, other parts of the host larva such as hemolymph, alimentary canal, fat bodies, labial glands, and mandibular glands may be the source of 1 or more kairomones that stimulate oviposition movements in larval parasitoid species (Arthur 1981). Therefore, additional research should be performed to further dene specicity of D. longicaudata female responses to chemical cues from both A. fraterculus and C. capitata larvae. Based on this requirement, we plan to conduct a second series of future experiments with D. longicaudata and 2 neotropical opiine fruit y larval parasitoids. Although dual-choice test results obtained in the present study provide reliable information on host rank order preferences for D. longicaudata, the ecological considerations on preference cannot be conjectured from this data. Therefore, we are currently verifying the host preference by D. longicaudata in eld-cage tests using different host fruit species which are commonly infested by C. capitata and/or A. fraterculus larvae in the eld. Finally, this study conrmed previous data indicating that both C. capitata (Ovruski et al. 2003; Viscarret et al. 2006) and A. fraterculus (Ovruski et al. 2007) are suitable hosts for laboratory rearing of D. longicaudata in Argentina. It also provided evidence that female parasitoid progeny yield can be highly improved by using A. fraterculus larvae as host instead of C. capitata larvae. ACKNOWLEDGMENTSI express my gratefulness to Arnaldo Mangeaud (UNCo, Crdoba, Argentina) for the statistical help and to Carolina Chiappini, Natalia Salinas, and Josena Buonocore (PROIMI, Tucumn, Argentina) for technical assistance. Special thanks to Jorge Cancino-Diaz and Pablo Montoya (Mexican MOSCAMED Program, Metapa de Domnguez, Chiapas, Mxico), and Pablo Liedo (ECOSUR, Tapachula, Chiapas, Mxico) for allowing me to introduce D. longicaudata specimens to Argentina from Mxico, and to 2 anonymous referees for helping us produce a better paper. I thank Teresa Vera and Eduardo Willink (EEAOC, Tucumn, Argentina) for providing me with the rst A. fraterculus reared specimens. This study was supported by Consejo Nacional de Investigaciones Cientcas y Tcnicas de Argentina (CONICET) (grant PIP/2005 No. 5129) and by Agencia Nacional de Promocin Cientca y Tecnolgica de Argentina through Fondo Nacional de Ciencia y Tecnologa (FONCyT) (grant PICT/2006 No. 2402). REFERENCES CITEDARTHUR, A. P. 1981. Host acceptance by parasitoids, pp. 97-120 In D. A. Nordlund, R. L. Jones, and W. J. Lewis [eds.], Semiochemicals. Their Role in Pest Control. John Wiley & Sons, Inc., New York, USA. CANCINO, J., VILLALOBOS, P., AND DE LA TORRE, S. 2002. Changes in the rearing process to improve the quality of mass production of the fruit y parasitoid Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae), pp. 74-82 In N. C. Leppla, K. A. Bloem, and R. F. Luck [eds.], Quality Control for Mass-reared Arthropods. Proc. 8th and 9th workshop of the IOBC, Florida, USA. CARRASCO, M., MONTOYA, P., CRUZ-LOPEZ, L., AND ROJAS, J. C. 2005. Response of the fruit y parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae) to mango fruit volatiles. Environ. Entomol. 34: 576-583. DUAN, J. J., AND MESSING, R. H. 2000a. Host Specicity Tests of Dichasmimorpha kraussii (Hymenoptera: Braconidae), a Newly Introduced Opiine Fruit Fly Parasitoid with Four Nontarget Tephritids in Hawaii. Biol. Control 19: 28-34. DUAN, J. J., AND MESSING, R. H. 2000b. Effects of Host Substrate and Vibration Cues on Ovipositor-Probing Behavior in Two Larval Parasitoids of Tephritid Fruit Flies. J. Insect Behav. 13(2): 175-186. EBEN, A., BENREY, B., SIVINSKI, J., AND ALUJA, M. 2000. Host Species and Host Plant Effects on Performance of Diachasmimorpha longicaudata (Hymenoptera: Braconidae). Environ. Entomol. 29(1): 87-94. GODFRAY, H. C. J. 1994. Parasitoids Behavioral and Evolutionary Ecology, Princeton University Press, Princeton, USA, 473 pp. GREANY, P. D., TUMLINSON, J. H., CHAMBERS, D. L., ANDBOUSH, G. M. 1977. Chemical mediated host nding by Biosteres (Opius) longicaudatus a parasitoid of tephritid fruit y larvae. J. Chem. Ecol. 3: 189-195. GUILLN, D., AND SNCHEZ, R. 2007. Expansion of the national fruit y control programme in Argentina, pp. 653-660 In M. J. B. Vreysen, A. S. Robinson, and J. Hendrichs [eds.], Area-Wide Control of Insect Pests: from Research to Field Implementation. Springer, The Netherlands. LEYVA, J. L., BROWNING, H. W., AND GILSTRAP, F. E. 1991. 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200 Florida Entomologist 94(2)June 2011Diachasmimorpha longicaudata (Hymenoptera: Braconidae)?. Florida Entomol. 92(3): 441-449. MANSFIELD, S. AND MILLS, N. J. 2004. A comparison of methodologies for the assessment of host preference of the gregarious egg parasitoid Trichogramma platneri Biol. Control 29: 332-340. MESSING, R. H., AND JANG, E. B. 1992. Response of the fruit y parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae) to host fruit stimuli. Environ. Entomol. 21: 1189-1195. MESSING, R. H., KLUNGNESS, L. M., PURCELL, M., ANDWONG, T. T. Y. 1993. Quality control parameters of mass-reared opine parasitoids used in augmentative biological control of tephritid fruit ies in Hawaii. Biol. Control 3: 140-147. MONTOYA. P., LIEDO, P., BENREY, B., CANCINO, J., BARRERA, J. F., SIVINSKI, J., AND ALUJA, M. 2000. Biological Control of Anastrepha spp. (Diptera: Tephritidae) in mango orchards through augmentative releases of Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae). Biol. Control 18: 216-224. MONTOYA, P., CANCIO, J., ZENIL, M., SANTIAGO, G., ANDGUTIERREZ, J. M. 2007. The augmentative biological control component in the Mexican National Campaign against Anastrepha spp. fruit ies, pp. 661-670 In M. J. B. Vreysen, A. S. Robinson, and J. Hendrichs [eds.], Area-Wide Control of Insect Pests: from Research to Field Implementation. Springer, The Netherlands. OROO, L. E., AND S. M. OVRUSKI. 2007. Presence of Diachasmimorpha longicaudata (Hymenoptera: Braconidae) in a guild of parasitoids attacking Anastrepha fraterculus (Diptera: Tephritidae) in northwestern Argentina. Florida Entomol. 90(2): 410-412. OVRUSKI, S. M., ALUJA, M., SIVINSKI, J., AND WHARTON, R. A. 2000. Hymenopteran parasitoids on fruit infesting tephritidae (Diptera) in Latin America and the southern United States: diversity, distribution, taxonomic status and their use in fruit y biological control. Intl. Pest Management Reviews 5: 81-107. OVRUSKI, S. M., COLIN, C., SORIA, A., OROO, L., ANDSCHLISERMAN, P. 2003. Introduccin y establecimiento en laboratorio de Diachasmimorpha tryoni y Diachasmimorpha longicaudata (Hymenoptera: Braconidae, Opiinae) para el control biolgico de Ceratitis capitata (Diptera: Tephritidae, Dacinae) en la Argentina. Rev. Soc. Entomol. Argentina 62: 4959. OVRUSKI, S. M., OROO, L. E., SCHLISERMAN, P., ANDNUEZ CAMPERO, S. 2007. The effect of four fruit species on the parasitization rate of Anastrepha fraterculus (Diptera: Tephritidae, Trypetinae) by Diachasmimorphalongicaudata (Hymenoptera: Braconidae, Opiinae) under laboratory rearing conditions. Biocontrol Sci.Technol. 17: 1079-1085. PARANHOS, B. J., COSTA, M. L. Z., OVRUSKI, S. M., ALVES, R. M., LUMMER, L. B., AND WALDER, J. M. M. 2008. Offspring in response to parental female densities in the fruit y parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae: Opiinae). Florida Entomol. 91(4): 628-635. PROKOPY, R. J., AND WEBSTER, R. P. 1978. Oviposition deterring pheromone of Rhagoletis pomonella, a kairomone for its parasitoid Opius lectus J. Chem. Ecol. 4: 481-494. PURCELL, M. F., JACKSON, C. G., LONG, J. P., ANDBATCHLOR, M. A. 1994. Inuence of guava ripening on parasitism levels of the oriental fruit y, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), by Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) and other parasitoids. Biol. Control 4: 396-403. SILVA, J. W. P., BENTO, J. M. S., AND ZUCCHI, A. R. 2007. Olfactory response of three parasitoid species (Hymenoptera: Braconidae) to volatiles of guavas infested or not with fruit y larvae (Diptera: Tephritidae). Biol. Control 41: 304-311. VERA, M. T., ABRAHAM, S., OVIEDO, A., AND WILLINK, E. 2007. Demographic and quality control parameters of Anastrepha fraterculus (Diptera: Tephritidae) maintained under articial rearing. Florida Entomol. 90: 53-57. VISCARRET, M. M., LA ROSSA, R., SEGURA, D. F., OVRUSKI, S. M., AND CLADERA, J. L. 2006. Evaluation of the parasitoid Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) reared on a genetic sexing strain of Ceratitis capitata (Wied.) (Diptera: Tephritidae). Biol. Control 36: 147-153. WHARTON, R. A. 1989. Classical biological control of fruit infesting Tephritidae, pp. 303-313 In A. S. Robinson and G. Hooper, G. [eds.], World Crop Pests: Fruit Flies, Their Biology, Natural Enemies, and Control. Vol. 3b. Elsevier Amsterdam, The Netherlands.

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Lehnert et al.: Color Quantication201 A NEW METHOD FOR QUANTIFYING COLOR OF INSECTS M ATTHEW S. L EHNERT 1,2 M URAT O. B ALABAN 3 AND T HOMAS C. E MMEL 2 1 Department of Entomology and Nematology, University of Florida, Bldg. 970 Natural Area Drive, P.O. Box 110620, Gainesville, FL 32611-0620 2 McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida, S.W. 34th Street and Hull Road, P.O. Box 112710, Gainesville, FL 32611-2710 3 Fishery Industrial Technology Center, University of Alaska Fairbanks, 118 Trident Way, Kodiak, AK 99615 A BSTRACT We describe a method to quantify color in complex patterns on insects, using a combination of standardized illumination and image analysis techniques. Two color comparisons were investigated: (1) the percentage of blue in the submarginal band of the hindwing in yellow and dark morph females of Papilio glaucus L., and (2) the percentage of orange hues in the wings of 2 putative subspecies of Eastern Tiger Swallowtail, P. g. glaucus L. and P. g. maynardi Gauthier. Live specimens were photographed in a light-box with standardized lighting and a color standard. Digital images were processed in LensEye software to determine the percentage of selected colors. No signicant differences were found in the percentage of blue between yellow and dark morph females, but the percentage of orange hues between P. g. glaucus and P. g. maynardi differed signicantly. Color quantication can be a useful tool in studies that require color analysis. Key Words: color analysis, color quantication, buttery comparison, digital image, Papilio glaucus R ESUMEN Se describe un mtodo para cuanticar el color en los patrones complejos de los insectos, utilizando una combinacin de iluminacin estandarizada y de la tcnica de anlisis de imagen. Se investigaron dos comparaciones de color: (1) el porcentaje de azul en la banda submarginal de las alas posteriores en las hembras de forma amarilla y de forma oscura de Papilio glaucus L. y (2) el porcentaje de tonos de color anaranjado en las alas de dos subespecies putativos de Papilio glaucus, P. g. glaucus L. y P. g. maynardi Gauthier. Se tomaron fotos de especimenes vivos en una caja de luz con iluminacin estandarizada y un estndar de color. Las imgenes digitales fueron procesadas usando el programa LensEye para determinar el porcentaje de los colores seleccionados. No se encontraron diferencias signicativas en el porcentaje de color azul en las hembras de forma amarilla y de forma oscura, pero el porcentaje de tonos anaranjados entre P. g. glaucus y P. g. maynardi diferan signicativamente. Cuanticacin del color puede ser una herramienta til en los estudios que requieren de un anlisis de color. Color and color patterns have been used to study a wide range of ecological and evolutionary topics, including sexual selection (Punzalan et al. 2008), aposematism (Brower 1958), industrial melanism (Kettlewell 1961), and mimicry (Jiggins et al. 2001; Saito 2002). Color is used in the classication of organisms to verify species and population properties, and subspecies (Brower 1959). The color of buttery life stages and wings is used to understand evolutionary-developmental patterns and phenotypic plasticity (Starnecker & Hazel 1999; Nice & Fordyce 2006; Otaki 2008). However, most of these studies are hindered in their ability to quantify color. When reporting quantied colors, RGB (red, green, blue) and L*, a*, and b* values (L* = lightness, scale: 0-100; a* = green to red, scale: -120120; and b* values = blue to yellow, scale: -120120) are typically used. RGB are digitally represented by 256 values each, meaning a total of more than 16 million possible color combinations (Balaban 2008), but the colors produced by these values are typically non-uniform and do not correlate well to human vision (Pedreschi et al. 2006). However, L*, a*, and b* values are combined together to represent a color that can be used in a comparative context to other similar colors (Pedreschi et al. 2006), and do account for the way humans perceive color. Existing methods for quantifying color include simple visual estimates, with or without the use of a book of color standards for reference such as Munsells (1976), spectrophotometry (Stevens et al. 2007), color software with RGB applications

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202 Florida Entomologist 94(2)June 2011 (Villafuerte & Negro 1998), and colorimetery (Yagiz et al. 2009). Human vision is color biased (Wyszecki & Stiles 1982); factors such as lighting condition, illumination, and color are context-dependent (Endler 1990; Zuk & Decruyenaere 1994), and make color difcult to quantify. Specimens need to be nearly homogenous in color and have an almost at surface to be accurately represented with colorimetry (Balaban 2008; Yagiz et al. 2009), and common image software such as Adobe Photoshop has limitations when standardizing or calibrating a digital image and when quantifying the color patterns of complex images with large color variation. Our objective was to introduce the use of image analysis with the LensEye software as a tool to quantify the color of insects. LensEye software was developed specically for color quantication purposes, which makes it more user-friendly than other general color analysis programs such as Adobe Photoshop. LensEye has been used in food and agricultural sciences (Balaban 2008; Yagiz et al. 2009), but its application to entomological studies is novel. To illustrate this process, the wing colors of male and female Eastern Tiger Swallowtail butteries, Papilio glaucus L., were analyzed in 2 comparisons: (1) the percentage of blue on the hindwing between yellow and dark morph females of P. glaucus and (2) the percentage of orange hues between males of the 2 subspecies P. g. glaucus L. and P. g. maynardi Gauthier. In the rst comparison, we predicted that the percentage of blue on the hindwing would be similar in yellow and dark morph females, because to our knowledge no previous reports have suggested a larger amount of blue in either morph. In the second comparison, we expected that males of P. g. maynardi would have a signicantly larger percentage of the wings represented by high a* and b* values when compared with P. g. glaucus as a combination of these values (reds and yellows) likely produces the orange hues that are diagnostic for this subspecies. To our knowledge, this is the rst report of color quantication of tiger swallowtail butteries. M ATERIALS AND M ETHODS Study Species and Specimen Preparation The Eastern Tiger Swallowtail, Papilio glaucus L. (Lepidoptera: Papilionidae), is a large multi-colored buttery found throughout the eastern half of the USA (Scriber 1996). Females are polymorphic and are either yellow with black stripes or melanic (Clarke & Sheppard 1962; Scriber 1996; Scriber et al. 1996); both forms have blue scales along the submarginal region of the dorsal side of the hindwings. Currently, 2 putative subspecies are recognized, P. g. glaucus and P. g. maynardi ; the latter has a unique orange background color rather than the yellow found on the glaucus subspecies (Maynard 1891; Scriber 1986). Papilio g. maynardi is primarily found in Florida, but occasionally is found in other southeastern states (Maynard 1891; Brower 1959; Scriber 1986; Lindroth 1991). Ten yellow females and 10 dark morph females of P. glaucus were captured from Cedar Key and Lake Placid, Florida to compare the percentage of blue in the hindwings between these morphs. To compare the percentage of orange hues between the 2 subspecies, 10 males were collected from La Fayette, Georgia, and 10 males from Lake Placid, Florida, to represent the P. g. glaucus and P. g. maynardi subspecies, respectively. All specimens were captured during Apr-Jun, 2008, representing what is likely the spring brood of P. glaucus in these regions. All butteries were captured with a buttery net and placed into glassine envelopes for transport. The live adults of P. glaucus were cooled in a walk-in refrigerator at 4C, removed from the glassine envelopes, and their wings spread at 4C on white Styrofoam to expose the dorsal side of the wings, positioned as if prepared for a professional insect collection. Spreading was facilitated with insect pins placed near the costal and A1 veins of the forewing and the anal vein and distal portion of M3 vein of the hindwing proximal to the tail. No pins were inserted into the body. Once a buttery was spread, it was removed from the walk-in refrigerator and walked to the equipment for color analysis. Protocol for Color Analysis Each buttery was placed individually in a light-box with D65 standardized lighting (Luzuriaga et al. 1997), and a Labsphere (North Sutton, NH) yellow color standard was placed next to the buttery. Inside the light-box, a Nikon D200 digital camera was fastened to a stand approximately 0.3 m tall so that the camera faced down, and was xed at a specic height and connected to a computer by a USB cable (camera specications listed in Table 1). The light-box door was closed and a photograph was taken of the buttery. Once in the light-box, it took less than 30 sec to process an individual buttery. The computer used Camera ControlPro software (Nikon, Tokyo, Japan) to control the act of taking a photograph with the camera; therefore, a photograph could be taken from the computer while the camera was enclosed within the light-box, and the picture would upload onto the computer. Two types of software were used for color analysis: Adobe Photoshop 6.0 (Adobe Systems Inc, San Jose, California) used for image adjustments, modications, and edits, and LensEye (Engineering and CyberSolutions, Gainesville, Florida), used for color quantication and analysis).

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Lehnert et al.: Color Quantication203 The digital photographs (JPEG) (Fig. 1a) were cleaned in Adobe Photoshop 6.0 to isolate the images necessary for color analysis. The eraser tool was used to remove insect pins, feces, and additional artifacts created during photographing. The image of the Labsphere color standard was cleaned by selecting the elliptical marquee tool that was used to highlight a yellow circular area within the color standard, which was moved with the move tool to the left of the buttery, and the remainder of the color standard was erased. This process created 2 nal images: a buttery and a yellow circle. The image resolution was adjusted to 700 pixels wide by selecting Image in the main toolbar, then resize and image size, and saved as a 24 bit BMP image (Fig. 1b). Females of P. glaucus were cleaned with the use of the eraser tool until only one hindwing remained. Males of P. g. glaucus and P. g. maynardi were cleaned so the entire buttery (minus antennae) remained. Cleaned images were opened and analyzed in LensEye software. In Lenseye, the objects of interest were separated from the background by designating the background color to consist of any pixel with RGB colors between 220 and 255, and the colors per axis (4096 color blocks) option was selected. This color information was displayed as the % of total object area. Objects smaller than a user-selected threshold of 100 pixels were ignored, ensuring only the buttery and color standard would be analyzed. In the color calibration option, the L*, a*, and b* values of the color standard were entered (L*, a*, and b* value of 90.17, -3.27, and 74.30, respectively), and the image was calibrated by selecting the Process Image tab. The software then calculated the average L*, a*, and b* values of the color standard from the uncalibrated image, and adjusted the color of each pixel in the image so that the average color of the standard in the image would equal that of the given reference values; this process calibrated all objects in the image (Fig. 1c). A spreadsheet was produced listing the percentage of each color (color ID#) and the average and standard deviation of the L*, a*, and b* values based on each pixel in the object. Each color ID # has a unique L*, a*, and b* value (Table 2), and the inT ABLE 1. C AMERA SPECIFICATION USED FOR COLOR ANALYSIS OF L EPIDOPTERAN WINGS Image Quality Compressed RAW (12-bit) Image Size Large (3872 2592) Lens VR 18-200 mm F/3.5-5.6 G Focal Length 35 mm Sensitivity ISO 100 Optimize Image Custom High ISO NR Off Exposure Mode Manual Metering Mode Multi-Pattern Shutter Speed 1/3 sec F/11 Exposure Comp.: (in Camera) 0 EV Focus Mode AF-S Exposure Comp.: (by Capture NX) 0 EV Sharpening Auto Tone Comp. Auto Color Mode Model Saturation Normal Hue Adjustment 0 White Balance Direct Sunlight T ABLE 2.S ELECTED COLOR ANALYSIS RESULTS FROM L ENSEYE SOFTWARE OF L EPIDOPTERAN WINGS Color ID# 1 Color StandardButtery 2 347201.534 348802.076 374408.74 374501.805 376200.271 Lab L*90.1771.61 StdDev L*0.372.84 Lab a*-3.2714.18 StdDev a*0.711.72 Lab b*74.378.42 StdDev b*3.126.93 NBS namebrilliant yellowstrong orange yellow 1 Each Color ID# represents a specific color (available in the software) with a unique L*, a*, and b* value. 2 The numbers represent the percentage of each color (Color ID#) in the image. Percentages do not equal 100, because this is only a selected portion of the entire spreadsheet from the analysis. The numbers that correspond to the Lab L*, Lab a*, and Lab b* represent the average L*, a*, and b* value of the image. The NBS name represents the name of the color using the average L* a* and b* values.

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204 Florida Entomologist 94(2) June 2011 formation for each color was provided in the color block information in the software. Both comparisons required the use of the color contours option in LensEye software. For the rst comparison, the most abundant colors of blue (color ID #) were selected from the spreadsheet and the L*, a*, and b* values of these colors were searched for in the color block information option. To analyze the calibrated image, it had to be reopened and reprocessed in LensEye. The show contours option was selected revealing a table with options for selecting thresholds, where the blue L*, a*, and b* values were entered. On the image, the L*, a*, and b* contour settings were manipulated by interactively adjusting them and evaluating the quantity of blue pixels that were highlighted in the image to nd the range of blue color values that encompassed the entire blue area on the buttery. After 2 images of both yellow and dark morph females were manipulated, the following settings were deemed best suited for the task: L* contour greater than 20, a* contour less than 19, b* contour less than 25. These threshold values were entered for each of the 20 images and the software selected all the pixels that met the above criteria (all blue areas were highlighted in red, Fig. 2A). The percentages of blue colors of the total wing area were recorded for each image by selecting the report contour option. For the second comparison, we used a male of P. g. maynardi from Lake Placid, Florida, to determine color composition to represent the maynardi subspecies. The image of this male was calibrated to receive the spreadsheet with the color ID # information, and the color moderate-orangeyellow (L*, a*, and b* values equal to 70, 9, and 60, respectively) was chosen to represent the threshold to distinguish P. g. maynardi from P. g. glaucus This color was chosen because it was the lightest orange hue represented by the specimen in the image, and we also wanted to include darker hues of orange in our analysis, as these colors also may be present on the wings of P. g. maynardi Calibrated images of the males were reopened in LensEye and reprocessed. The show contours option was selected and the L*, a*, and b* contour values were entered into the threshold space. All values greater than the chosen threshold values were highlighted, because these values (higher a* and b* values) would represent darker orange colors in the buttery wings than the moderate-orange-yellow color (Fig. 2B). The report contour option was chosen to record the percentage of wing area highlighted. Statistical Analysis We used a Welchs t test (two-tailed; P = 0.05) to evaluate differences in the percentage of blue between yellow and dark morph females, and the percentage of orange on the wings of males of the 2 subspecies. Fig. 1. Example of sequential images produced during color analysis. The raw image of the buttery and color standard (a) is saved as a JPEG and opened in A dobe Photoshop where it is cleaned and saved as a bitmap image 700 pixels wide (b). The cleaned image is opened in Lenseye and calibrated and the colors quantied (c).

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Lehnert et al.: Color Quantication205 Fig. 2. Example of images used to determine the percentage of blue on hindwings of females of P. glaucus (A), and to study color differences between subspecies of P. glaucus (B), with designated L*, a*, and b* color values. In image A, the dark morph female (a) has more blue extending proximally from the submarginal band compared with the yellow morph female (b). The regions of blue interpreted by Lenseye using specied values are highlighted in red by the software. The percentage of blue in image (a) and (b) is 21.4% and 12.8%, respectively. In image B, the same threshold for colors with a higher L*, a*, and b* value than moderate-orange-yellow were used for all males of P. glaucus Papilio g. glaucus (a) has less orange than P. g. maynardi (b), as indicated by the red. Image (a) and (b) have 5.0% and 20.6% of the wings at or above the designated threshold. Both sets of images (A and B) display the calibrated image on the left and the analyzed image on the right.

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206 Florida Entomologist 94(2) June 2011 R ESULTS The dark morph and yellow females did not differ signicantly in the percentage of blue on the hindwing (mean SE) (16.98 percent 3.10 and 14.2 percent 1.4, respectively) ( t = 1.5858; df = 18; P = 0.1411). However, the pattern of blue differed between the morphs (Fig. 2A). All yellow females had blue scales restricted to the submarginal area of the hindwing, resulting in less than 20% of blue color on the hindwing, which was similar to some dark females, but other dark females had blue that continued proximally and became more random and scattered, resulting in a larger variation of blue color in these morphs. Four of the dark morph females had over 20% of blue scales on the hindwing, synonymous with the scattered blue scale phenotype, but the large variation in this morph led to an average quantity of blue not signicantly different from that of the yellow morph. Males of Papilio glaucus maynardi from Lake Placid, Florida, had signicantly more orange than the butteries from La Fayette, Georgia (9.97 percent 2.18 and 0.52 percent 0.90, respectively) ( t = 4.007; df = 18; P = 0.0021), 80% of the analyzed P. glaucus from La Fayette had 0% of the wings at or above the designated L*, a*, and b* threshold used to represent moderate-orange-yellow. Although P. g. maynardi from Lake Placid, Florida, was visually distinct from the northern subspecies, the range of orange hues on the wings would have been difcult to quantify without a computer vision system and image analysis software. Lenseye highlighted only the areas of the wings we were interested in analyzing. Even small patches of blue in the hindwing were highlighted, verifying the softwares sensitivity to interpreting specied colors in an intricate color pattern. D ISCUSSION The application of image analysis software and our methods open a new avenue for quantifying color that could inuence understanding of color components in ecological and evolutionary systems. For instance, color associated with the effects of temperature or host plant (phenotypic plasticity) (Price 2006), range distributions of hybrid zones (Blum 2002; Gay et al. 2008), oral color changes in response to insect pollination (Paige & Whitham 1985), and seasonal polyphenisms (Hazel 2002) can be quantied. This study also provides a means to analyze color of live specimens, which could have important implications to studies of endangered species. In this study, the butteries seemed unaffected by the method, and were capable of ight, copulation, and oviposition after the study, veried by additional studies (M.S.L., unpublished data). Our methods also provide a protocol to quantify museum specimens, for instance, in studying how color dynamics of populations have shifted over time. Our method allows the use of thresholds to study colors of interest and to determine their percentage compared with the rest of the image. For example, the blue scales scattered over the hindwing of a dark morph female were quantied, even though these small blue spots were on a black background. Additionally, similar, but different, colors (yellow-orange) were quantied to distinguish 2 entities. Papilio glaucus maynardi is relatively unstudied, and there are conicting reports concerning its distribution (Forbes 1960; Harris 1972; Howe 1975; Mather & Mather 1985; Scriber 1986; Lindroth et al. 1988). Our method could provide a means to determine its distribution. Other aspects of its evolutionary history could be addressed, such as determining if the subspecies represent a color cline or a rapid shift in color, suggesting similar dynamics of a narrow hybrid zone where one phenotype rapidly shifts to the other. The primary limitation of our method, and other color quantication methods, is that standardized lighting is necessary; therefore, these methods would not be reliable in all situations, such as comparing the color of buttery wings from photographs taken outdoors under different lighting conditions. We addressed this issue by using a light-box with standardized lighting. Other source and processing errors may have occurred, such as instrumental inaccuracies of the light-box, camera, and software; however, to minimize these errors we used the same camera and light specications for each individual. In addition, there may be a source error in that populations of P. glaucus may experience a seasonal polyphenism, which could alter our interpretations of the data sets. We addressed this issue by collecting the individuals from the various locations during a similar time period. ACKNOWLEDGMENTSWe thank Jonathan Doyle and Matthew Standridge (both at McGuire Center for Lepidoptera and Biodiversity, University of Florida, Gainesville) for technical assistance in cleaning up photographs for analysis and for testing the protocol, and Alberto De Azeredo (Food Science and Human Nutrition Department, University of Florida, Gainesville) for camera, software, and photograph assistance. Jonathan Doyle assisted in collecting the P. glaucus. We thank Peter Adler (Department of Entomology, Soils, and Plant Sciences, Clemson University, Clemson), and Richard Lehnert, and 3 anonymous reviewers for editorial comments on the manuscript.REFERENCES CITEDBALABAN, M. O. 2008. 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ZUK, M., AND DECRUYENAERE, J. G. 1994. Measuring individual variation in colour: a comparison of two techniques. Biol. J. Linn. Soc. 53: 165-173.

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208 Florida Entomologist 94(2) June 2011 THE LARGE DECAPITATING FLY PSEUDACTEON LITORALIS (DIPTERA: PHORIDAE): SUCCESSFULLY ESTABLISHED ON FIRE ANT POPULATIONS IN ALABAMA S ANFORD D. P ORTER 1 L. C. F UDD G RAHAM 2 S ETH J. J OHNSON 3 L ARRY G. T HEAD 4 AND J UAN A. B RIANO 5 1 Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive, Gainesville, FL 32608 2 Department of Entomology and Plant Pathology, Auburn University, 301 Funchess Hall, Auburn, AL 36849-5413 3 Department of Entomology, 400 Life Sciences Building, Louisiana State University Agricultural Center, Baton Rouge, LA 70803 4 Biological Control of Pests Research Unit, USDA-ARS, P.O. Box 67, Stoneville, MS 38776 Current Address: 10303 Wildcat Road, Collinsville, MS 39325 5 South American Biological Control Laboratory, USDA-ARS, Bolivar 1559 (1686) Hurlingham, Buenos Aires, Argentina A BSTRACT The large re ant decapitating y, Pseudacteon litoralis Borgmeier, from northeastern Argentina w as successfully released as a self-sustaining biocontrol agent of imported re ants in south central Alabama in 2005. Five years later, this y is rmly established at the original release site and has expanded outward at least 18 km. Nevertheless, populations remain very low considering P. litoralis is one of the most abundant re ant decapitating ies in large areas of its range in South America. The reasons for low densities and why we were only able to establish this y at 1 of 9 release sites in 4 states (2003-2006) are unknown, but problems with host-matching, release procedures, weather conditions, and competition with previously released decapitating ies are discussed as possible factors. Key Words: Solenopsis invicta biological control, low population density R ESUMEN La mosca grande del norte y centro-este de Argentina, Pseudacteon litoralis Borgmeier, decapitadora de la hormiga de fuego (hormiga bra va), fue liberada exitosamente como agente de control biolgico de la hormiga de fuego importada en el sur-centro de Alabama en 2005. Cinco aos despus, esta mosca se encuentra rmemente establecida en ese sitio y se ha expandido al menos 18 km; sin embargo, las poblaciones permanecen muy bajas considerando que P. litoralis es una de las moscas decapitadoras de hormiga de fuego ms abundante en su rea de Amrica del Sur. Se desconocen las razones de las bajas densidades y el por qu del establecimiento de esta mosca en slo uno de los nueve sitios de liberacin en cuatro estados (2003-2006), pero se discuten como posibles factores los problemas de correspondencia de hospederos, procedimientos de liberacin, condiciones climticas y competencia con moscas decapitadoras liberadas previamente. Translation provided by the authors. The decapitating y Pseudacteon litoralis Borgmeier (Fig. 1) is a parasitoid of the red imported re ant, Solenopsis invicta Buren, the blac k imported re ant, Solenopsis richteri Forel, and 3 other species of saevissima complex re ants in southern Brazil, Paraguay, and northern Argentina (Patrock et al. 2009). Pseudacteon litoralis is the largest of the common Pseudacteon species that attack re ants and specializes in parasitizing the largest sizes of re ant workers (Morrison et al. 1997). It is active throughout the daylight hours, but prefers dawn and especially dusk (Pesquero et al. 1996). As with several other Pseudacteon phorids (e.g., P. tricuspis and P. nocens ), sex is probably determined environmentally primarily by the size of the host, rather than genetically like most other insects (Morrison et al. 1999). Males of P. litoralis are not attracted to re ant mounds like P. tricuspis and P. obtusus (Porter & P esquero 2001; Calcaterra et al. 2005). In the lab, mating appeared to occur on and around black objects in the top of the large attack boxes (SDP, unpubl. obs.). This y is one of the most abundant re ant decapitating ies throughout much of its range in South America both numerically and spatially (Calcaterra et al. 2005; Patrock et al. 2009, personal observations, SDP). Like other species in the genus, P. litoralis is

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Porter et al.: Fire Ant Decapitating Fly Established in Alabama209 highly host-specic (Porter & Gilbert 2004; Weissog et al. 2008) probably because these ies use re ant alarm pheromones to nd their hosts (Vander Meer & Porter 2002) and also because of their highly specialized life history of decapitating re ant workers and then pupating inside their empty head capsules (Porter et al. 1995). The characteristics discussed above made P. litoralis an attractive target for release as a selfsustaining or c lassical re ant biological control agent. The objectives of this paper are to document the release and establishment of P. litoralis in south central Alabama and to describe the fate of 8 additional eld releases conducted in Florida, Mississippi, and Louisiana from the spring of 2003 to the summer of 2006. M ATERIALS AND M ETHODS The original source population for the P. litoralis ies discussed in this paper was from several sites just off Route 11 about 6 kilometers south of San J usto, Santa Fe, Argentina (30.550S, 60.607W). About 1,800 re ant workers parasitized with P. litoralis were brought bac k to Gainesville, FL in Apr 2001. The re ants at the collection sites were S. invicta although probably not the same biotype as that found in the United States (Ross & Trager 1991; Caldera et al. 2008). By the summer of 2001 the newly established P. litoralis laboratory colony had dropped to about 1000 individuals (about 20-30 pupae per da y, assuming a 40-d life cycle) and remained at this level through the end of 2001, after which numbers began to gradually increase. In the winter of 2002, 100 or so males were added to the San Justo colony from a collection site on the Paraguay River near Herradura, Formosa, Argentina (26.514S, 58.284W). The S. invicta ants at this site were probably more similar to the U .S. biotype, but still not quite the same. By the time releases had begun in the spring of 2003 the colony was producing about 500 pupae per day. Maximum production was about 1,000 pupae per day in Jan 2006. Releases were conducted at sites where re ants were abundant (Table 1). We selected sites with a large percentage of monogyne colonies because monogyne or single-queen re ant colonies have a higher percentage of the larger workers preferred by P. litoralis females (Morrison et al. 1997). Most sites were near water sources and had patches of tall grass or shrubbery that was assumed to help protect y pupae from being killed in the sun. All of the sites were pastures except the Florida Ironwood Golf Course (Table 1) which was a mixture of fairways, lake edges, and service roads along drainage canals. The Alabama release site (Table 1) was drenched by Hurricane Dennis just before the nal groups of parasitized ants were released in Jul 2005. Fig. 1. Female Pseudacteon litoralis y preparing to oviposit in the thorax of a re ant worker. T ABLE 1. F IELD RELEASE DATA FOR THE FIRE ANT DECAPITATING FLY P SEUDACTEON LITORALIS Site County, StateStart DateDuration (days)Number ReleasedFate Mickle FarmAlachua, FLMay 2003~3~150 a Failed Morrill Farm Alachua, FL15 May 200312 2,400 a Failed Whitehurst FarmA f Marion, FL15 Sep 200321 4,500 b Failed c Knox Site d,f Clay, MS4 Aug 2004206,400 b Failed Whitehurst FarmB f Levy, FL25 Apr 200527 5,200 b Failed Ironwood Golf Course f Alachua, FL10 May 2005324,800 b Failed c Biddle Farm Wilcox, AL21 Jun 200518 4,600 b Established Idelwilde Res. StationE. Feliciana, LA15 May 200620 5,200 b Failed Morrill Farm f Alachua, FL18 May 200644 17,200 e Failed a Adult ies released over disturbed mounds. b Estimated parasitized re ant workers. c First-generation adult ies recovered at release site. d Fire ants at this site were primarily hybrids (black red); ants at all the other sites were the red imported re ant, S. invicta e Adult ies emerged from pupae in shaded emergence box in eld. f Sites where P. curvatus ies were established prior to the release of P. litoralis P. tricuspis was previously established at all sites except the Mississippi site

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210 Florida Entomologist 94(2) June 2011 Competing P. tricuspis ies were present at all of the P. litoralis release sites except the Mississippi site where P. tricuspis had been unable to establish on the hybrid re ants (T able 1). At the time P. litoralis was released, P. curvatus ies were not present at the Mic kle and Morrill release sites in Florida, the Louisiana site, or the Alabama site (until 2007). The P. litoralis ies were released at the rst 2 sites (T able 1) as adult ies over disturbed re ant mounds as was the procedure for P. tricuspis (Porter et al. 2004). However, only a few of the females were observed to hover over and attempt to oviposit in the disturbed workers. The next 6 releases (Table 1) were conducted by releasing workers parasitized in the laboratory back into their mother colonies as described for P. curvatus (Vazquez et al. 2006). The hope was that emerging females would naturally mate with nearby males and then be attracted to attac k re ant workers. At the nal site (Table 1), pupae on moist plaster trays were placed inside a large emergence box (61 by 41 by 51 cm; height, width, depth) in the eld. This was done several days before the pupae were due to emerge. The box was shaded to prevent overheating and placed on a stand coated with Fluon to limit access for ants and other arthropods. Upon emergence, the ies ew to the light and exited through window screen that protected the pupae from access of larger organisms. Average emergence rates of adult ies from pupae in this box was 84%, a value comparable to that achieved with good rearing procedures in the laboratory. Initial surveys to determine whether the ies had established were usually conducted in the late afternoon or early evening by disturbing several mounds at or near the release site and aspirating all ies that were attracted to the mounds (Porter et al. 2004; Vazquez et al. 2006). Beginning in 2006, most surveying in Florida was accomplished with sticky traps (baited with live ants) supplemented by aspiration (Puckett et al. 2007; Porter 2010). Sticky traps baited with either live ants or freeze killed ants were also tried in Alabama in 2008. We did not conduct prerelease surveys to detect the presence of P. litoralis at our release sites because none of the 20 or so South American Pseudacteon species that attack red imported re ants ha ve ever been found in North America (unless they were intentionally released) despite extensive collections and observations over many years (Porter et al. 2004; Patrock et al. 2009; Porter 2010; Plowes et al. 2011). R ESULTS The decapitating y P. litoralis only became established at the release site in Alabama (Table 1). This site was a series of small weedy pastures encircled by trees and shrubbery (~7 ha). Releases were conducted in overgrown areas near the tree lines of the pastures. The rst P. litoralis y was recovered at this site on 20 J un 2006. This collection occurred a year after the release even though sampling had been conducted several times previously in both 2005 and 2006. The next ies were detected a year later on 23 Jul (2 ies) and 31 Jul 2007 (7 ies). In 2008 (Jun and Jul) 3 years after the release, P. litoralis ies were collected with aspirators at 5 sites: the release site (1 y), 6 km south (1), 11 km south (2), 6 km west (1), and 18 km west (1). In the summer 2008 (Jun and Jul), sticky traps were placed every half mile along road right-of-ways for 10 miles in each of the 4 cardinal directions (80 total traps) for the sole purpose of monitoring P. litoralis expansion. This w as repeated 3 times. Many P. curvatus and P. tricuspis ies were found on the traps, but no P. litoralis ies. In Jun 2009, single ies were collected 2, 6, and 14 km north of the release site. In Jul and Aug 2010, a total of 7 ies were collected on 3 different occasions at the release site. Throughout this period, abundance of P. litoralis was always low; P. litoralis was not collected at most of the sites surveyed, and they were generally found in only a small fraction of disturbed mounds inspected. However, 113 ies were aspirated at the release site in the early morning on 16 Sep 2010, an abundance that is equivalent to high densities of this species in South America. To date, all P. litoralis in Alabama have been collected with aspirators First generation, eld-reared P. litoralis females were found about 6 weeks after 2 of the 6 Florida releases (T able 1). Unfortunately, repeated monitoring (2003-2010) failed to detect any additional ies, including in the fall of 2010 when 4 sites near each of the 3 major release areas were checked twice for P. litoralis ies (Sep and Oct, 74 total mounds). The Louisiana site was rst sampled 4 months after the release (Sep 2006). This release site was rechecked twice in 2009 (Apr and Sep) and twice in 2010 (Apr and Sep) without nding P. litoralis Five other sites were sampled near the release site (1.6-5.2 km away) in 2009 (Apr and Sep) and again in 2010 (Apr and Sep). Ten mounds were inspected at each of the Louisiana sample sites, but no P. litoralis ies were collected even though both P. curvatus and P. tricuspis ies were collected. Flies also were not detected at the Mississippi site which was checked 11 times after the release (Sept-Nov, 2004) and once in Jul 2005, almost a year after the release. Three locations near the Mississippi site were checked in Sep 2010, but only a few dozen P. curvatus ies were found. DISCUSSIONThe large decapitating y, P. litoralis is rmly established on red imported re ants in south

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Porter et al.: Fire Ant Decapitating Fly Established in Alabama211central Alabama. Populations of this species are generally low, but they have survived through 5 winters and they have expanded at least 18 km from the release site. This makes P. litoralis the third decapitating y species released and successfully established on imported re ant populations in the United States. The rst 2 Pseudacteon species, P. tricuspis and P. curvatus were released at numerous sites across the Southeast and currently cover about 65% and 90% of the imported re ant range in the United States, respectively, (Callcott et al. 2011). A fourth Pseudacteon species, P. obtusus, has been established in Texas and Florida (Gilbert et al. 2008; SDP) and a fth very small species, P. cultellatus, is currently being released in Florida (SDP). In addition to the ies mentioned above, several other parasitic arthropods (Williams et al. 2003), 2 species of mermithid nematodes (Poinar et al. 2007), 2 species of microsporidian pathogens, and at least 3 kinds of viruses, are being investigated as potential re ant biocontrol agents (Oi & Valles 2009). The expansion rate of P. litoralis from the release site in Alabama has proven difcult to monitor because low densities make this y difcult to detect at sample sites. Despite low densities, the rate of expansion for P. litoralis in Alabama is similar to expansion rates reported for P. tricuspis in Texas and Louisiana, but probably less than the very abundant P. curvatus in Florida and Mississippi (Henne et al. 2007; Porter 2010). The low densities of P. litoralis at sites in Alabama is curious because P. litoralis is consistently one of the most abundant decapitating ies across most of its range in South America both numerically and spatially (Calcaterra et al. 2005; Patrock et al. 2009). The large number of ies recently collected (Sep 2010) from the release site is encouraging, but it is unknown whether this represents a new trend or is just a temporal quirk. The apparent failure to establish P. litoralis at the other 8 sites was disappointing. We made releases at sites with a variety of habitats and climates in hopes that variety would increase the probability of success. The Mississippi site was chosen in hopes that the ies might do better on the S. invicta x S. richteri hybrid re ants found at that site. It is possible that populations have been established at some sites listed in Table 1, but densities are still too low to be easily detected, as has occurred on several occasions with P. curvatus (Graham et al. 2003; Vazquez et al. 2006). Nevertheless, this possibility seems unlikely at the Florida, Louisiana, and probably Mississippi sites considering the frequency and duration of the sampling efforts in those areas. Repeated failures to establish P. litoralis in the eld is reminiscent of failures to establish P. curvatus collected from black re ants in South America on red re ants in the United States (Graham et al. 2003; Callcott et al. 2011). Perhaps a biotype of P. litoralis better adapted to the biotype of red imported re ants found in the United States would have been more successful. However, we tried twice to establish additional laboratory colonies of P. litoralis from ies collected along the Parana River near Herradura, Formosa, Argentina (Apr 2003, 314 ies; Dec 2005, 1400 ies). Unfortunately, both attempts failed as did other attempts to culture P. litoralis ies collected in Sa Paulo State, Brazil (1997) and the Corrientes area of Argentina (2004-2006). Exactly why we were able to culture the ies collected from San Justo, but not the P. litoralis ies collected elsewhere is unknown, although it may be related to problems with mating since the adult females seemed to be attracted normally to the re ant workers we provided to them in the laboratory attack boxes. While poor host matching may have been a problem, other factors may also have been important in the failure of P. litoralis to establish at some of release sites, especially since they did establish in Alabama and thus should have been able to be established elsewhere on S. invicta re ants. Competition with previously released species is one likely explanation. Our colleagues in Texas provide strong evidence that the presence of P. curvatus at their release sites greatly diminished the success rate of establishing P. obtusus (Plowes et al. 2011). Similarly in Florida, competition between P. curvatus, P. tricuspis, and the recently released P. obtusus appears to be greatly reducing P. tricuspis populations (SDP and Lu, unpublished). However, competition with P. curvatus was not a problem with the rst 2 releases in Florida or with the releases in Alabama and Louisiana because P litoralis was released at these sites before P. curvatus was present. Poor weather conditions may have been another factor at some of the failed sites. Examination of release records for P. tricuspis (Callcott et al. 2011) indicates that summer releases were about half as successful as releases in the spring or fall. Five of the 9 P. litoralis releases, including the successful one in Alabama (Table 1), were at least partly carried out during hot summer months (although rain and clouds from Hurricane Dennis likely reduced negative impacts of summer heat for the Alabama release). Another possible problem is that U.S. re ant populations may not have enough major workers to sustain large numbers of P. litoralis but intercontinental comparisons of worker polymorphism have not been done to see if this is a real concern. Certainly, U.S. re ant colonies do have many workers in the size range which P. litoralis prefers to parasitize (Porter & Tschinkel 1985; Morrison et al. 1997; Morrison et al. 1999). Poor release technique is another explanation. This would certainly seem to be true for the rst 2 releases, because the adult

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212 Florida Entomologist 94(2)June 2011ies did not show much interest in the disturbed re ant mounds and very few ies were used at the rst site. The large release box used in the last release was an effort to try something different than what had previously been done. The lack of any rst-generation eld-reared ies at this release site was disappointing considering the number of ies released and the extended period of the release. In the fall of 2006, we made the decision to focus on other biocontrol agents with higher probabilities of success. Nevertheless, P. litoralis is rmly established in Alabama and will presumably expand into other states. While P. litoralis was locally abundant on one occasion in 2010, it failed at most of the release sites and remained rare in Alabama over most of the last 5 years, a curious situation considering P. litoralis is one of the most abundant species of re ant decapitating ies throughout most of its range in South America (Calcaterra et al. 2005; Patrock et al. 2009). ACKNOWLEDGMENTSVicky Bertagnolli, Kelly Ridley, Mel Leap, and Jennifer Reese assisted with eld releases and collections in Alabama. Lloyd Davis, Darrell Hall, David Milne, and Roberto Pereira assisted with eld releases in Florida. Don Henne assisted with releases in Louisiana. Evita Gourley, Mary Vowell and Dan Harsh assisted with releases in Mississippi. Luis Calcaterra is thanked for assistance with logistics in Argentina and eld work near Herradura.REFERENCES CITEDCALCATERRA, L. A., PORTER, S. D., AND BRIANO, J. A. 2005. Distribution and abundance of re ant decapitating ies (Diptera: Phoridae: Pseudacteon ) in three regions of southern South America. Ann. Entomol. Soc. America 98: 85-95. CALDERA, E. J., ROSS, K. G., DEHEER, C., AND SHOEMAKER, D. D. 2008. Putative native source of the invasive re ant Solenopsis invicta in the USA. Biol. Invasions 10: 1457-1479. CALLCOTT, A.-M. A., PORTER, S. D., WEEKS, R. D., JR., GRAHAM, L. C., JOHNSON, S. J., AND GILBERT, L. E. 2011. Fire ant decapitating y cooperative release programs (1994-2008): Two Pseudacteon species, P. tricuspis and P. curvatus rapidly expand across imported re ant populations in the southeastern United States. J. Insect Sci. 11: 19: insectscience.org/11.19 GILBERT, L. E., BARR, C. L., CALIXTO, A. A., COOK, J. L., DREES, B. M., LEBRUN, E. G., PATROCK, R. J. W., PLOWES, R. M., PORTER, S. D., AND PUCKETT, R. T. 2008. Introducing phorid y parasitoids of red imported re ant workers from South America to Texas: outcomes vary by region and by Pseudacteon species released. Southwestern Entomol. 33: 15-29. GRAHAM, L. C., PORTER, S. D., PEREIRA, R. M., DOROUGH, H. D., AND KELLEY, A. T. 2003. Field releases of the decapitating y Pseudateon curvatus (Diptera: Phoridae) for control of imported re ants (Hymenoptera: Formicidae) in Alabama, Florida, and Tennessee. Florida Entomol. 86: 334-339. HENNE, D. C., JOHNSON, S. J., AND CRONIN, J. T. 2007. Population spread of the introduced red-imported re ant parasitoid, Pseudacteon tricuspis Borgmeier (Diptera: Phoridae), in Louisiana. Biol. Control 42: 97-104. MORRISON, L. W., DALLAGILO-HOLVORCEM, C. G., ANDGILBERT, L. E. 1997. Oviposition behavior and development of Pseudacteon ies (Diptera: Phoridae), parasitoids of Solenopsis re ants (Hymenoptera: Formicidae). Environ. Entomol. 26: 716-724. MORRISON, L. W., PORTER, S. D., AND GILBERT, L. E. 1999. Sex ratio variation as a function of host size in Pseudacteon ies (Diptera: Phoridae), parasitoids of Solenopsis re ants (Hymenoptera: Formicidae). Biol. J. Linn. Soc. 66: 257-267. OI, D. H., AND VALLES, S. M. 2009. Fire ant control with entomopathogens in the USA, pp. 237-257 In A. E. Hajek, T. R. Glare and M. OCallaghan [eds.], Use of Microbes for Control and Eradication of Invasive Arthropods. Springer Science+Business Media B.V. PATROCK, R. J. W., PORTER, S. D., GILBERT, L. E., ANDFOLGARAIT, P. J. 2009. Distributional patterns of Pseudacteon associated with the Solenopsis saevissima complex in South America. J. Insect Sci. 9:60, 17 pp. (available online: insectscience.org/9.60). PESQUERO, M. A., CAMPIOLO, S., FOWLER, H. G., ANDPORTER, S. D. 1996. Diurnal patterns of ovipositional activity in two Pseudacteon y parasitoids (Diptera: Phoridae) of Solenopsis re ants (Hymenoptera: Formicidae). Florida Entomol. 79: 455457. PLOWES, R. M., LEBRUN, E. G., AND GILBERT, L. E. 2011. Introduction of the re ant decapitating y Pseudacteon obtusus in the United States: factors inuencing establishment in Texas. BioControl (in press). POINAR, G. O., JR., PORTER, S. D., TANG, S., AND HYMAN, B. C. 2007. Allomermis solenopsii n. sp. (Nematoda: Mermithidae) parasitizing the re ant Solenopsis invicta Buren (Hymenoptera: Formicidae) in Argentina. Syst. Parasitol. 68: 115-128. PORTER, S. D. 2010. Distribution of the Formosa strain of the re ant decapitating y Pseudacteon curvatus (Diptera: Phoridae) three and a half years after releases in North Florida. Florida Entomol. 93: 107-112. PORTER, S. D., AND TSCHINKEL, W. R. 1985. Fire ant polymorphism: the ergonomics of brood production. Behav. Ecol. Sociobiol. 16: 323-336. PORTER, S. D., AND PESQUERO, M. A. 2001. Illustrated key to Pseudacteon decapitating ies (Diptera: Phoridae) that attack Solenopsis saevissima complex re ants in South America. Florida Entomol. 84: 691-699. PORTER, S. D., AND GILBERT, L. E. 2004. Assessing host specicity and eld release potential of re ant decapitating ies (Phoridae: Pseudacteon ), pp. 152-176 In R. G. Van Driesche and R. Reardon [eds.], Assessing Host Ranges for Parasitoids and Predators Used for Classical Biological Control: A Guide to Best Practice. FHTET-2004-03, USDA Forest Service, Morgantown, West Virginia. PORTER, S. D., NOGUEIRA DE S, L. A., AND MORRISON, L. W. 2004. Establishment and dispersal of the re ant decapitating y Pseudacteon tricuspis in North Florida. Biol. Control 29: 179-188. PORTER, S. D., PESQUERO, M. A., CAMPIOLO, S., ANDFOWLER, H. G. 1995. Growth and development of Pseudacteon phorid y maggots (Diptera: Phoridae) in the heads of Solenopsis re ant workers (Hy-

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Porter et al.: Fire Ant Decapitating Fly Established in Alabama213menoptera: Formicidae). Environ. Entomol. 24: 475479. PUCKETT, R. T., CALIXTO, A., BARR, C. L., AND HARRIS, M. 2007. Sticky traps for monitoring Pseudacteon parasitoids of Solenopsis re ants. Environ Entomol 36: 584-8. ROSS, K. G., AND TRAGER, J. C. 1991. Systematics and population genetics of re ants ( Solenopsis saevissima complex) from Argentina. Evolution 44: 21132134. VANDER MEER, R. K., AND PORTER, S. D. 2002. Fire ant, Solenopsis invicta worker alarm pheromones attract Pseudacteon phorid ies, Proc. Imported Fire Ant Conference, 77-80. The Georgia Center for Continuing Education, Athens, GA. VAZQUEZ, R. J., PORTER, S. D., AND BRIANO, J. A. 2006. Field release and establishment of the decapitating y Pseudacteon curvatus on red imported re ants in Florida. BioControl (Dordrecht) 51: 207216. WEISSFLOG, A., MASCHWITZ, U., SEEBAUER, S., DISNEY, R. H. L., SEIFERT, B., AND WITTE, V. 2008. Studies on European ant decapitating ies (Diptera: Phoridae): II. observations that contradict the reported catholicity of host choice by Pseudaction formicarum Sociobiology 51: 87-94. WILLIAMS, D. F., OI, D. H., PORTER, S. D., PEREIRA, R. M., AND BRIANO, J. A. 2003. Biological control of imported re ants (Hymenoptera: Formicidae). American Entomol. 49: 150-163.

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214 Florida Entomologist 94(2) June 2011 HOST SPECIFICITY OF ANTHONOMUS TENEBROSUS (COLEOPTERA: CURCULIONIDAE), A POTENTIAL BIOLOGICAL CONTROL AGENT OF TROPICAL SODA APPLE (SOLANACEAE) IN FLORIDA J. M EDAL 1 N. B USTAMANTE 1 E. B REDOW 2 H. P EDROSA 2 W. O VERHOLT 1 R. D AZ 1 AND J. C UDA 1 1 University of Florida, Department of Entomology and Nematology, FL. 32611 2 Universidade Federal do Paran, Curitiba, Brazil A BSTRACT Multiple-choice and no-choice tests were conducted at the Florida Department of Agriculture quarantine facility to determine the host specicity of the South American ower bud weevil, Anthonomus tenebrosus Boheman, intended for biological control of the exotic weed tropical soda apple (TSA), Solanum viarum Dunal in Florida, USA. Ninety-one plant species in 21 families were inc luded in multiple-choice feeding and oviposition experiments, including the target weed and the 6 major cultivated Solanaceae: bell pepper ( Capsicum annuum L.), chili pepper ( C. frutescens L.), tomato ( Lycopersicon esculentum Mill.), tobacco ( Nicotiana tabacum L.), eggplant ( Solanum melongena L.), and potato ( Solanum tuberosum L.). Plant bouquets with ower -buds of 8 to 10 randomly selected plant species, always including TSA ( S. viarum ) were exposed to 10-20 A. tenebrosus adults for 1 to 2 weeks. Oviposition and feeding were observed twice a week. No-choice host-specicity tests were also conducted with A. tenebrosus adults using potted owering plants. Ten adults were exposed to 29 plant species individually tested for 1 to 2 weeks Plant species in each test were replicated 3 or 4 times. All tests showed that A. tenebrosus fed and laid eggs only on the target weed. No eggs were deposited on any of the other of the 91 plant species tested. Host-specicity tests indicated that a host range expansion of A. tenebrosus to include any of the crops, and native Solanaceae and non-solanaceous plants tested is highly unlikely. A petition for eld release in the USA was submitted to the Technical Advisory Group for Biological Control Agents of Weeds (TAG) in Oct 2007 Key Words: host-specicity tests, weed biological control, Solanum viarum Solanaceae R ESUMEN Pruebas de ovoposicin y alimentacin (con y sin eleccin), se realizaron para evaluar la especicidad del picudo del botn oral, de origen suramericano, Anthonomus tenebrosus Boheman, como agente potencial para control biolgico de bola de gato, Solanum viarum Dunal en los Estados Unidos Las pruebas se efectuaron en la cuarentena del Departamento de Agricultura de la Florida. Noventa y una especies de plantas, en 21 familias, fueron incluidas en las pruebas de especicidad de mltiples eleccin, incluyendo la maleza objetivo y las seis plantas cultivadas pertenecientes a la familia Solanaceae ms importantes: chile dulce ( Capsicum annuum L.), chile ( Capsicum frutescens L.), tomate ( Lycopersicon esculentum Mill.), tabaco ( Nicotiana tabacum L.), berenjena ( Solanum melongena L.), y papa ( Solanum tuberosum L.). En cada prueba se utilizaron racimos orales de ocho a diez plantas escogidas al azar inc luyendo siempre la planta objetivo las cuales fueron expuestas a 10-20 adultos de A. tenebrosus por una a dos semanas. Registros de alimentacin y ovoposicin fueron realizados dos veces por semana. Pruebas de alimentacin/ovoposicin sin eleccin fueron tambin realizadas usando plantas en oracin. Diez adultos fueron expuestos a 29 especies de plantas en forma individual por una a dos semanas. Cada prueba tuvo tres o cuatro repeticiones. Las pruebas mostraron que A. tenebrosus se aliment y coloc posturas solo en bola de gato Ninguna postura fu depositada en las otras 90 especies de plantas evaluadas. Las pruebas indicaron que la posibilidad de A. tenebrosus de llegar a ser una plaga de las Solanaceae cultivadas es muy remota. La solicitud al comit TAG para liberar el picudo en los Estados Unidos fue presentada en octubre 2007. Tropical soda apple (TSA), Solanum viarum Dunal (Solanaceae), is an invasive weed native to southeastern Brazil, northeastern Argentina, Paraguay, and Uruguay that has invaded Florida grasslands and natural ecosystems. In 1988, TSA was rst reported in the USA in Glades County, Florida (Coile 1993; Mullahey & Colvin 1993); the introduction pathway is unknown. In 1993, a survey of beef cattle operations in south Florida estimated 157,145 ha of infested pasture land, twice the infestation present in 1992 (Mullahey et al. 1994). The infested area increased to more than

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Medal et al.: Host Specicity of Anthonomus tenebrosus 215 303,000 ha in 1995-96 (Mullahey et al. 1998). Currently, more than 404,000 ha are believed to be infested in Florida (Medal et al. 2010b). Due, at least in part, to favorable environmental conditions, the lack of natural enemies (herbivores and pathogens), and seed dispersal by wildlife and cattle feeding on the fruits. TSA has been spreading rapidly and has been observed in the majority of the counties in Florida and also in Alabama, Georgia, Louisiana, Mississippi, North Carolina, Pennsylvania, South Carolina, Tennessee, Texas, and Puerto Rico (Bryson & Byrd Jr. 1996; Dowler 1996; Mullahey et al. 1993, 1998; Medal et al. 2003, 2010a). Although TSA has been reported in Pennsylvania and Tennessee, it is highly probable that does not overwinter in these states. Patterson (1996) studied the effects of temperatures and photoperiods on TSA in controlled environmental chambers and speculated that the range of TSA could expand northward into the midwestern US. S. viarum was placed on the Florida and Federal Noxious Weed Lists in 1995. TSA typically invades improved pastures, where it reduces livestock carrying capacity. Foliage and stems are unpalatable to cattle; dense stands of the prickly shrub prevent access of cattle to shaded areas, which results in summer heat stress (Mullahey et al. 1998). TSA control costs for Florida ranchers were estimated at $6.5 to 16 million annually (Thomas 2007), and economic losses from cattle heat stress alone were estimated at $2 million (Mullahey et al. 1998). TSA is a reservoir for at least 6 crop viruses (potato leafroll virus, potato virus Y, tomato mosaic virus, tomato mottle virus, tobacco etch virus, and cucumber mosaic virus) and the early blight of potato and tomato fungus, Alternaria solani Sorauer (McGovern et al. 1994a, 1994b; McGovern et al. 1996). In addition, major insect pests utilize TSA as an alternate host; including Colorado potato beetle, Leptinotarsa decemlineata ( Say); tomato hornworm Manduca quinquemaculata (Haworth); tobacco hornworm, M. sexta (L.); tobacco budworm, Helicoverpa virescens (Fabricius); tomato pinworm, Keiferia lycopersicella (Walsingham); green peach aphid, Myzuz persicae (Sulzer); silverleaf whitey biotype B of Bemisia tabaci (Gennadius); soybean looper, Pseudoplusia includens (Walker); and the southern green stink bug Nezara viridula (L.) (Habeck et al. 1996; Medal et al. 1999; Sudbrink et al. 2000). TSA also reduces biodiversity in natural areas, ditch banks, and roadsides by displacing native vegetation (Langeland & Burks 1998). TSA interferes with restoration efforts in Florida by invading areas that are reclaimed following phosphate mining operations (Albin 1994). TSA Management practices in Florida pastures primarily involve herbicide applications and mowing (Sturgis & Colvin 1996; Mislevy et al. 1996, 1997; Akanda et al. 1997). Herbicides or mowing provide temporary weed suppression at an estimated cost of $61 and $47 per ha, respectively (Thomas 2007). However, application of these control methods is not always feasible in rough terrain or inaccessible areas. In June 1994, the rst exploration for TSA natural enemies in South America was conducted by University of Florida and Brazilian researchers (Medal et al. 1996). Sixteen species of insects were found attacking the weed during this 2week survey. Host specicity tests were initiated in 1997 by J. Medal (University of Florida) in collaboration with the Universidade Estadual Paulista, Jaboticabal campus, Brazil, and the USDA Biological Control Laboratory in Hurlingham, Buenos Aires province, Argentina, and in Stoneville, MS. The South American leaf-feeder Gratiana boliviana (Chrysomelidae) was approved for eld release in Florida in summer 2003. In total, at least 230,000 beetles have been released in 39 Florida counties since the summer 2003. The beetles established at almost all the release sites in central/south Florida and they are having extensive defoliations and reducing the weed fruit production on TSA plants (Medal & Cuda 2010; Medal et al. 2010a; Overholt et al. 2009, 2010). A second potential TSA biocontrol agent is the ower-bud weevil Anthonomus tenebrosus Boheman (Coleoptera: Curculionidae). This insect was collected on TSA in Rio Grande do Sul, Brazil (29.66465S, 50.80171W) by the late Daniel Gandolfo and Julio Medal in April 2000. The identity of A. tenebrosus was conrmed by Drs. Wayne Clark (Auburn University AL) and Germano Rosado Neto (Universidade Federal do Paran in Curitiba, Brazil). Voucher specimens of A. tenebrosus are deposited at Auburn University, Alabama, at the Universidade Federal do Paran Curitiba campus, Brazil, and at the Florida State Collection of Arthropods, Division of Plant Industry in Gainesville, Florida. This species does not have a common name in South America. The only known A. tenebrosus host plants in South America are S. viarum and S. acculeatisimum The biology of A. tenebrosus was studied by Da vis (2007) at the quarantine facility in Gainesville, Florida. Eggs are inserted individually into TSA ower-buds, and hatch in 3-5 days. Larvae are cream-colored with a yellowish brown head capsule. They feed on the contents of the owerbud, and this feeding prevents the ower-bud from opening. There is typically 1 larva, but occasionally 2 larvae in a single ower-bud. As larval feeding progresses, the ower-bud senesces and drop from the plant. Three larval stadia are completed in 7-13 days. The pupal stage is completed in 3-7 days inside the fallen ower bud. Pupae resemble the adult in form; they are cream-colored but darken shortly before eclosion. Emerging adults chew their way out of the ower-bud. De-

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216 Florida Entomologist 94(2) June 2011 velopment from egg to adult stage lasts 11-69 days. Longer developmental times are apparently not associated with seasonal differences as they occurred throughout the year. Adults can live up to 210 days under laboratory conditions. Adult size appears to be related to food abundance during development rather than beetle sex. Copulation has been observed a few hours after adult emergence and throughout the oviposition period. At least 7-8 generations per year can occur under laboratory conditions (temperature 24 3C, relative humidity 50-70%) conditions. M ATERIALS AND M ETHODS Host Specicity Tests Laboratory host specicity tests with A. tenebrosus adults were conducted from May 2000 to January 2003 at the Florida Department of Agriculture and Consumer Services-Division of Plant Industry quarantine facility in Gainesville, Florida. Open eld host-specicity tests were conducted at the Universidade Federal do Paran Agricultural Experiment Station in Paran state, Brazil from Oct 2005 to Mar 2007. For Florida tests, A. tenebrosus adults were collected from TSA plants in Rio Grande do Sul, Brazil and introduced onto caged plants of TSA plants growing in 1-gallon pots to establish a laboratory colony in quarantine. In this article we report the results of various host-specicity tests with the ower-bud weevil A. tenebrosus, to assess its possible use as biological control agent of the non-native weed tropical soda apple Multiple-Choice Feeding and Oviposition Tests Ninety-one plant species in 21 families were included in the feeding oviposition preference tests at the Gainesville quarantine (Table 1). Tested plants included 53 species in the family of the target weed (Solanaceae), 26 of which were from the genus Solanum and 27 from 14 other genera that inc lude plants of agricultural or ecological importance. Ten species represented 5 families (Boraginaceae, Convolvulaceae, Ehretiaceae, Nolanaceae, Polemoniaceae) very close related to Solanaceae within the order Polemoniales (Heywood 1993) were also included. Twentyeight plant species representing 15 families, most of them with economic and/or environment value in North America, were also tested. The target weed ( S. viarum ), and other 9 plant species in Solanaceae were tested at least 3 times (T able 1). These included the natives Solanum donianum Walpers, listed as a threatened plant in Florida (Coile 1998), and S. americanum Mill, 2 non native-weeds ( S. tampicense Dunal, S. torvum Sw.), and the 5 major cultivated Solanaceae (bell pepper Capsicum annuum L., tomato, Lycopersicon esculentum Mill., tobacco, Nicotiana tabacum L., eggplant, Solanum melongena L., and potato, Solanum tuberosum L.). Bouquets of leaves and ower -buds of 8 to 10 plant species, always including TSA were simultaneously exposed to 1020 A. tenebrosus adults (approximately 50% males and 50% females) in c lear plastic round containers (26 cm diameter by 9 cm height, with four 4-7 cm diameter vents drilled along the sides of the container to allow for air circulation). At the beginning of each test, the insects were placed at the bottom center of each container to allow them to choose any tested plants. Plant species in each test were replicated 3-4 times (1 replication of tested plants in each separate container). Bouquets were exposed to A. tenebrosus adults for 1 to 2 weeks Observations of oviposition and feeding were made twice a week, and consumed bouquets were replaced as needed. Flower-buds were checked for oviposition and eggs were counted weekly. On the last day of each experiment, ower-buds were scored for feeding damage, and eggs laid on them were counted. Leaf and ower bud area consumed was visually estimated using a scale from 0 to 5 (0 = no feeding, 1 = probing or <5% of area consumed, 2= light feeding or 5-20% of the area, 3 = moderate feeding or 21-40%, 4 = heavy feeding or 41-60%, and 5 = intense feeding or >60% of the area consumed). No-Choice Adult Feeding and Oviposition Tests No-choice host specicity tests were also conducted with A. tenebrosus adults at the Gainesville-quarantine facility using potted plants (2060 cm height) in cages Cages were made of clearplastic cylinders (15 cm diam, 50-60 cm height), with a mesh screen at the top and covering 6 circular holes (6 cm diam) located in pairs at the bottom, middle, and upper part of the cylinder to allow for air circulation. A. tenebrosus adults were exposed to 29 plant species in 3 families including the native S. donianum and all major cultivated Solanaceae (T able 2). Five to 7 plant species with ower-buds were individually tested each time due to limited cage numbers. Plants were exposed to 10 A tenebrosus adults (5 males, 5 females) for 1 to 2 weeks; each test plant was replicated 3 or 4 times. Adults were F 2 or F 3 progeny from adults originally collected in southern Brazil and reared in quarantine on TSA. Adults had either recently eclosed from pupae or were still young less than 1 week old. Plants were replaced as needed. At the end of the testing periods, feeding and oviposition were recorded. First Field Experiment in Brazil A multiple-choice, open field experiment was conducted at the Universidade Federal do Paran, Agriculture Experimental Farm Canguiri. A.

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Medal et al.: Host Specicity of Anthonomus tenebrosus 217 T ABLE 1. A NTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE MULTIPLE-CHOICE TESTS. Plant Family Species Common Names *indicates native species No. of Plants No. of Insects Feeding Score1Eggs Laid per Female Category 1. Genetic types of the target weed species found in North America SOLANACEAE Tribe Solaneae Genus Solanum Subgenus Leptostemonum Section Acantophora Solanum viarum Dunal Tropical soda apple 40 430 3-5 5-9 Category 2. Species in the same genus as the target weed, divided by subgenera (if applicable). Tribe Solaneae Genus Solanum Subgenus Leptostemonum Section Acantophora Solanum capsicoides All. Red soda apple 12 160 0 0 Section Lasiocarpum Solanum quitoense Lam. Naranjilla 4 40 0 0 Solanum pseudolulo Heise Falso lulo 4 40 0 0 Solanum sessiliorum Dunal Cocona nightshade 4 40 0 0 Section Micracantha Solanum jamaicense Mill. Jamaican nightshade 3 30 0 0 Solanum tampicense Dunal Wetland nightshade 10 110 1 0 Section Melongena Subsection Lathyrocarpum Solanum carolinense L. Horse nettle*44 000 Solanum dimidiatum Raf. Western horsenettle*33 000 Solanum elaeagnifolium Cav. Silverleaf nightshade*44 000 Subsection Melongena Solanum melongena L. Eggplant Cv. Black Beauty 77 000 Cv. Classic 44 000 Cv. Market 77 000 Cv. Asian Long Purple 3 35 0-1 0 Section Persicariae*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Solanaceous taxonomic categories were taken from the Radboud University of Nijmegen, Netherland website (www.bagard.sci.kun.nl). Most of the plant common names are from: http:/www.plants.usda.gov.

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218 Florida Entomologist 94(2)June 2011Subgenus Leptostemonum Solanum bahamense Bahama nightshade 7 75 0 0 Section Torva Solanum torvum Sw. Turkey berry 12 140 0 0 Solanum verbascifolium L. Mullein nightshade*33 000 Subgenus Solanum Solanum americanum Mill. American nightshade*10 100 0 0 Solanum diphyllum L. 2-leaf nightshade*33 000 Solanum erianthum Don. Potato tree*33 000 Solanum jasminoides Paxt. White potato vine 7 75 1 0 Solanum mauritianum Scop. Earleaf nightshade 4 40 0 0 Solanum nigrescesns Mart. & Gal Divine nightshade*33 000 Solanum nigrum L. Black nightshade*45 000 Solanum pumillum Dunal Rock outcrop Solanum*33 000 Solanum seaforthianum Scop. Brazilian nightshade 3 30 0 0 Solanum tuberosum L. Potato 8 95 0 0 Category 3. Species in other genera in the same family as the target weed, divided by subfamily (if applicable). Genus Acnistus Acnistus australe (Griseb.) Griseb. Acnistus 3 30 0 0 Genus Iochroma Iochroma sp. Iochroma 3 30 0 0 Genus Physalis Physalis angulata L. Cutleaf groundcherry 3 30 0 0 Physalis arenicola Kearney Cypresshead*33 000 Physalis crassifolia Benth Yellow groundcherry*33 000 Physalis gigantea L. Strawberry groundcherry 3 30 0 0 Physalis ixocarpa Brot. Tomatillo 3 30 0 0 Physalis pubescens L Husk tomato*33 000 Physalis walteri Nutt. Walters groundcherry*33 000 Tribe Daturae Genus Brugmansia Brugmansia sanguinea (Ruiz & Pav.) Don Angels trumpet 3 30 0 0 Genus Datura TABLE 1. (CONTINUED) ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE MULTIPLE-CHOICE TESTS. Plant Family Species Common Names *indicates native species No. of Plants No. of Insects Feeding Score1Eggs Laid per Female*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Solanaceous taxonomic categories were taken from the Radboud University of Nijmegen, Netherland website (www.bagard.sci.kun.nl). Most of the plant common names are from: http:/www.plants.usda.gov.

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Medal et al.: Host Specicity of Anthonomus tenebrosus 219Datura discolor Bernh Desert thorn-apple*33 000 Datura metel L. Devils trumpet 3 30 0 0 Datura meteloides D. Devils weed*33 000 Datura stramonium L. Jimson weed*33 000 Tribe Lycieae Genus Lycium Lycium carolinianum Walter Carolina desert-thorn*33 000 Lycium fremontii Gray. Fremont desert thorn*33 000 Genus Lycopersicon Lycopersicon esculentum Mill. Tomato 12 130 0 0 Tribe: Nicandreae Genus: Nicandra Nicandra physaloides (L.) Gaertn. Apple of Peru 3 30 0 0 Tribe Nicotianae Genus Nicotiana Nicotiana tabacum L. Tobacco 8 100 0 0 Nicotiana rustica L. Aztec tobacco 7 75 0 0 Nicotiana sylvestris Speg. & Comes Woodland tobacco 3 30 0 0 Genus Nierembergia Nierembergia scoparia Sendtri Broom cupower 3 30 0 0 Tribe Salpiglossidae Genus Salpiglossis Salpiglossis sinuata Ruiz & Pav Painted tongue 3 30 0 0 Genus Schizanthus Schizanthus spp. Buttery ower 3 30 0 0 Tribe Solandeae Genus Solandra Solandra glandiora Swartz Showy chalicevine 3 30 0 0 Category 4. Threatened and endangered species in the same family as the target weed divided by subgenus, genus, and subfamily. Section Torva Solanum donianum Walpers Mullein nightshade*99 000 Category 5. Species in other families in the same order that have some phylogenetic, morphological, or biochemical similarities to the target weed. BORAGINACEAE TABLE 1. (CONTINUED) ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE MULTIPLE-CHOICE TESTS. Plant Family Species Common Names *indicates native species No. of Plants No. of Insects Feeding Score1Eggs Laid per Female*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Solanaceous taxonomic categories were taken from the Radboud University of Nijmegen, Netherland website (www.bagard.sci.kun.nl). Most of the plant common names are from: http:/www.plants.usda.gov.

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220 Florida Entomologist 94(2)June 2011Heliotrope sp. Heliotrope 3 30 0 0 Myosotis alpestris Schmidt Forget-Me-Not*33 000 Convolvulus purpurea L. Morning-glory*33 000 Ipomoea batata (L.) Lam. Sweet-potato 3 30 0 0 Evolvulus muttallianus Shaggy dwarf morning-glory*33 000 EHRETIACEAE Cordia sebestena L. Largeleaf geigertree*33 000 NOLANACEAE Nolana paradoxa Lindl. Chilean bellower 3 30 0 0 POLEMONIACEAE Cobaea scandens Cav. Catedral bells 3 30 0 0 Gilia tricolor Benth Birds-eye gilia*33 000 Phlox panuculata L. Fall phlox*33 000 Category 6. Species in other orders that have some morphological or biochemical similarities to the target weed or that share the same habitat. ANACARDIACEAE Mangifera indica L. Mango 3 30 0 0 Pistacia vera L. Cultivated pistachio 3 30 0 0 APIACEAE Daucus carota L. Carrot 3 30 0 0 ASTERACEAE Helianthus annuus L. Common sunower*33 000 Lactuca sativa L. Lettuce 3 30 0 0 CAMPANULACEAE Campanula persicifolia L Peachleaf bellower*33 000 CRUCIFERAE Brassica oleracea L. var. botrytis Broccoli 3 30 0 0 CUCURBITACEAE Citrullus lanatus (Thumb) Watermelon 3 30 0 0 Cucurbita sativus L. Cucumber* 33 000 ERICACEAE Vaccinium ashei Rende Rabbiteye blueberry*33 000 FABACEAE Glycine max (L.) Merrill Soybean 3 30 0 0 TABLE 1. (CONTINUED) ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE MULTIPLE-CHOICE TESTS. Plant Family Species Common Names *indicates native species No. of Plants No. of Insects Feeding Score1Eggs Laid per Female*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Solanaceous taxonomic categories were taken from the Radboud University of Nijmegen, Netherland website (www.bagard.sci.kun.nl). Most of the plant common names are from: http:/www.plants.usda.gov.

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Medal et al.: Host Specicity of Anthonomus tenebrosus 221Phaseolus vulgaris L. Kidney bean 3 30 0 0 LOBELIACEAE Lobelia cardinalis L. Cardinalower* 3 30 0 0 LOGANIACEAE Buddleia davidii Franch Buttery bush 3 30 0 0 POACEAE Oryza sativa L. Rice 3 30 0 0 Saccharum ofcinarum L. Sugarcane 3 30 0 0 Zea mays L. Corn* 3 30 0 0 ROSACEAE Fragariax ananassa Duchesne Garden strawberry 3 30 0 0 Malus pumilla Mill. Paradise apple*3 30 0 0 Rosa sp. Miniature rose 3 30 0 0 Rubus betulifolius Small Blackberry*33 000 RUTACEAE Citrus sinensis (L.) Osbeck Sweet orange 3 30 0 0 Citrus limon (L.) Burm. Lemon 3 30 0 0 Citrus paradise Mcfady Grapefruit 3 30 0 0 Murraya paniculata (L.) Jacq. Orange Jasmine 6 60 0 0 SCROPHULARIACEAE Antirrhinum majus L. Garden snapdragon 3 30 0 0 Nemensia strumosa Benth Capejewels 3 30 0 0 Category 7. Any plant on which close relatives of the biological control agent (within the same genus) have been found or recorded to feed/ or reproduce. MALVACEAE Gossypium hirsutum L. Cotton 10 100 0 0 SOLANACEAE Genus Capsicum Capsicum annuum L. Bell pepper 8 80 0 0 Capsicum frutescens L. Chili pepper 4 40 0 0 Genus Solanum Solanum sisymbriifolium Lam. Sticky nightshade 3 30 1 0 TABLE 1. (CONTINUED) ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE MULTIPLE-CHOICE TESTS. Plant Family Species Common Names *indicates native species No. of Plants No. of Insects Feeding Score1Eggs Laid per Female*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Solanaceous taxonomic categories were taken from the Radboud University of Nijmegen, Netherland website (www.bagard.sci.kun.nl). Most of the plant common names are from: http:/www.plants.usda.gov.

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222 Florida Entomologist 94(2)June 2011tenebrosus adults were collected in Rio Grande do Sul state in Dec 2005. These insects were reared in screened cages (0.6 0.6 0.9 m) at the Neotropical Biological Control Laboratory in Curitiba on TSA plants growing in 1-2 gallon pots to provide progeny weevils for the experiment. One hundred A. tenebrosus adults recently emerged from pupae were released in the field (30 20m2) with 5 plant species (TSA, eggplant cv. Black Beauty, bell-pepper, potato, and tomato). Seven plants of each species tested (35 plants/plot, 1m between plants, 35m2/plot, 4 plots, 10m between plots) were randomly assigned in each of the experimental plots following a Complete Block Randomized Experimental Design with 4 replications. Test plants (n = 140) were transplanted in Oct 2005, and insects were released when plants were flowering during the last week of Dec 2005 on the ground approximately 1m from any plant. All plants were thoroughly examined weekly from 22 Dec 2005 to 31 Mar 2006, and number of adults, feeding, and number of egg on the plants were recorded.Second Field Experiment in BrazilAnother multiple-choice, open eld experiment exposing A. tenebrosus adults to owering eggplant cv. Black Beauty, tomato, potato, andTABLE 2.ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE NO-CHOICE TESTS. Plant family Species Common names (*indicates native Species) No. of Plants No. of Insects Feeding Score*Eggs/female SOLANACEAE Capsicum annuum Bell pepper99000 Capsicum frutescens Chili pepper77000 Lycopersicon esculentum Tomato99000 Nicotiana tabacum Tobacco77000 Nierembergia scoparia Broom cupower33000 Physalis crassifolia Yellow groundcherry*33000 Solanum americanum American nightshade*33000 Solanum capsicoides Red soda apple33000 Solanum carolinense Horse nettle*33000 Solanum citrullifolium Watermelon nightshade*33000 Solanum dimidiatum Western horsenettle*33000 Solanum diphillum 2-leaf nightshade33000 Solanum donianum Mullein nightshade77000 Solanum elaeagnifolium Silverleaf nightshade*33010 Solanum heterodoxum Melonleaf nightshade*33000 Solanum jamaicense Jamaican nightshade33000 Solanum jasminoides White potato vine*33000 Solanum melongena Eggplant cv. Black Beauty33000 cv. Classic33000 cv. Market33000 cv. Asian Long Purple99000 Solanum nigrescens Divine nightshade*33000 Solanum pumilum Rock-outcrop Solanum*33000 Solanum ptycanthum Wonder berry*33000 Solanum retroexum Sunberry*33000 Solanum scabrum Garden huckleberry*33000 Solanum tampicense Wetland nightshade77010 Solanum torvum Turkeyberry77010 Solanum tuberosum Potato99000 Solanum viarumTropical soda apple9903-54-11 MALVACEAE Gossypium hirsutum L.Cotton99000 CONVOLVULACEAE Ipomoea batata (L.) Lam.Sweet-potato99000*0=No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Most of the plant common names are from: http:/www.plants.usda.gov.

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Medal et al.: Host Specicity of Anthonomus tenebrosus 223bell pepper, but not TSA, was conducted at the Universidade Federal do Paran, Agriculture Experimental Farm Canguiri, Brazil from Dec 2006 to Mar 2007. Control plots with owering TSA plants alone, were also established at the Neotropical Biological Control Laboratory in Curitiba located approximately 45 km from the cultivated crop plots to prevent plant species interference. Distance between plants was similar to the rst experiment. Field collected A. tenebrosus weevils were released into crop and TSA plots (80 and 76 adults, respectively). Beetles were randomly released in groups (6-10) on the ground but not on any test plant. Evaluations (visual estimation of number of insects and feeding) were made weekly checking thoroughly each of the plants tested. RESULTS AND DISCUSSIONMultiple-Choice Feeding-Oviposition TestsIn the quarantine multiple-choice tests, A. tenebrosus adults fed moderately to intensively (>20% of the area offered) on S. viarum, the target weed (Table 1). Weevils did some probing or exploratory feeding (<5% of the area offered) on S. tampicense Dunal (an exotic weed of Mexico-Central America-Caribbean origin and established and expanding in central-south Florida), on S. sisymbriifolium Lam., and S. jasminoides Paxt. (weeds of South-American origin also present in Florida), and on eggplant cv. Asian Long Purple (crop of economic importance). No feeding was observed on any of the other 86 plant species in 21 families that were tested. A. tenebrosus adults lay from 5 to 9 eggs inside TSA ower-buds during the 1-2 week period of the test (Table 1). No eggs were deposited on any of the other 90 plant species tested, including the threatened S. donianum. Although minor A. tenebrosus feeding occurred on eggplant in quarantine, this insect has never been recorded attacking eggplant in South America. Expanded host ranges of weedbiocontrol insects under conned laboratory conditions have been reported by South African researchers (Neser et al. 1988; Hill & Hulley 1995; Olckers et al. 1995; Hill & Hulley 1996; Olckers 1996). They indicated that almost all agents tested for biocontrol of Solanum weeds have fed on closely related plant species that are never attacked under natural conditions. For example, Gratiana spadicea (Klug) (Coleoptera: Chrysomelidae) screened as a potential biocontrol agent of S. sisymbrifolium in South-Africa (Hill & Hulley 1995), fed and completed development on eggplant in the laboratory. In 1994, this insect was released in South-Africa based mainly on the lack of records as an eggplant pest in South America. Gratiana spadicea is established on S sisymbriifolium with no reports of attacks in South African eggplant elds. Multiple choice tests conducted at the USDA-South American Biological Control Laboratory in Hurlingham, Argentina with Anthonomus sisymbrii Hustache by late Daniel Gandolfo, showed this weevil fed and lay eggs on eggplant and potato, although the number of eggs lay on these crops was signicantly lower than on TSA. He also reported that 75% of the eggs on potato and 25% on eggplant were abnormally oviposited outside the ower-buds. Gandolfo indicated that A. sisymbrii could use eggplant, and possibly other Solanum, at least for feeding purposes that may result in an economic impact (Gandolfo et al. 2004). The only known natural hosts of A. sisymbrii are S. sisymbrifolium, S. viarum, and S. aculeatissimum (Medal unpublished data). To corroborate the specicity and safety of A. tenebrosus a weevil related to A. sisymbrii, 2 open eld experiments (discussed later) were conducted in Brazil, which indicated that A. tenebrosus did not represent a threat to eggplant and other economic crops tested under natural conditions and it is safe as a biological control agent of TSA.No-Choice Adult Feeding TestsStarvation (no-choice) tests with A. tenebrosus adults exposed to individual potted plants (29 species in 3 families) in quarantine cages indicated that this insect fed and laid eggs (range: 411; average, 8 eggs per female) only on TSA (Table 2). Feeding on TSA was moderate to intense (>21% of the area offered) compared to a probing or exploratory feeding (<5%) observed on S. elaeagnifolium, S. tampicense and on S. torvum. No eggs were laid on any of the 28 non-target plant species tested including 4 eggplant cultivars (Black Beauty, Classic, Market, Asian Long Purple).First Field Experiments in BrazilIn the open-eld planted with TSA, bell-pepper, tomato, potato, and eggplant, A. tenebrosus adults (100) fed and laid eggs, and larvae developed only on TSA. A total of 83 eggs, 21 larvae, and 51 adults of A. tenebrosus were recorded on TSA plants. Feeding on TSA ower-buds was moderate to heavy (21 to 50% of the area). No feeding was observed on any of the Solanaceous crops tested. This eld test conrms that A. tenebrosus feeds and develops only on TSA and does not represent a threat to eggplant, tomato, potato, or bell-pepper.Second Field Experiment in BrazilThe eld test exposing A. tenebrosus adults to eggplant, tomato, potato, and bell-pepper, showed that no adults or immature stages were found on these crops tested when TSA plants were not

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224 Florida Entomologist 94(2)June 2011present. In a separate plot, A. tenebrosus feeding on TSA ower buds was moderate (21-30%), contrary to no-feeding on the crops tested. A total of 124 eggs, 72 larvae, and 45 adults of A. tenebrosus were recorded on TSA plants. This test showed that A. tenebrosus adults fed and laid eggs, and larvae developed only on TSA, with no utilization of eggplant, potato, tomato, and bell pepper when TSA is not present. The laboratory and open-eld experiments indicated that no A. tenebrosus feeding damage and reproduction on the native solanaceous plants and crops tested are likely to occur. It is expected that this weevil will complement the TSA damage by G. boliviana in south and central Florida. ACKNOWLEDGMENTSWe thank Howard Frank (University of Florida), and Julieta Brambila (United States Department of Agriculture, Animal and Plant Health Inspection Service) and three anonymous reviewers for reviewing the manuscript. We also thank Wayne Clark (Auburn University), and Germano Rosado Neto (Universidade Federal do Paran Curitiba, Brazil) for the identication of Anthonomus tenebrosus This research was funded by USDA-APHIS.REFERENCES CITEDAKANDA, R. A., MULLAHEY, J. J., AND SHILLING, D. G. 1997. Tropical soda apple (Solanum viarum) and bahiagrass (Paspalum notatum) response to selected PPI, PRE, and POST herbicides, p. 35 In Abstracts of the Weed Science Society of America Meeting. Orlando, Florida. WSSA Abstracts Vol. 37. ALBIN, C. L. 1994. Non-indigenous plant species nd a home in mined lands, pp. 252-253 In D. C. Schmitz and T. C. Brown [eds.], An Assessment of Invasive Non-Indigenous Species in Floridas Public Lands. Technical Report TSA-94-100. Department of Environmental Protection, Tallahassee, Florida, United States. BRYSON, C. T., AND BYRD, JR., J. D. 1996. Tropical soda apple in Mississippi, pp. 55-60 In J. J. Mullahey [ed.], Proc. Tropical Soda Apple Symp. Bartow, Florida. University of Florida, IFAS. CHANDRA, V., AND SRIVASTAVA, S. N. 1978. Solanum viarum Dunal syn. Solanum khasianum Clarke, a crop for production of Solasadine. Indian Drugs 16: 53-60. COILE, N. C. 1993. Tropical soda apple, Solanum viarum Dunal: The plant from hell. Florida Department of Agriculture and Consumer Services, Division of Plant Industry. Botany Circular No. 27, 4 pp. COILE, N. C. 1998. Notes on Floridas endangered and threatened plants. Florida Department of Agriculture & Consumer Services, Bureau of Entomology, Nematology and Plant Pathology. Botany Section Contribution No. 38, 2nd edition, 119 pp. DAVIS, B. J. 2007. Evaluation of articial diets for rearing Anthonomus tenebrosus (Coleoptera: Curculionidae), a potential biological control agent of tropical soda apple, Solanum viarum. Thesis, University of Florida, 104 pp. DOWLER, C. C. 1996. Some potential management approaches to tropical soda apple in Georgia, pp. 41-54 In J. J. Mullahey [ed.], Proc. Tropical Soda Apple Symp. Bartow, Florida. University of Florida, IFAS. GANDOLFO, D., MACKAY, F., AND CAGNOTTI. 1994. Tropical soda apple, pp. 46-55 In Annual Report, South American Biological Control Laboratory, USDAARS. Hurlingham, Argentina. HABECK, D. H., MEDAL, J. C., AND CUDA, J. P. 1996. Biological control of tropical soda apple, pp. 73-78 In J. J. Mullahey [ed.], Proc. Tropical Soda Apple Symp. Bartow, Florida. University of Florida, IFAS. HEYWOOD, V. H. [Ed.]. 1993. Flowering Plants of the World. Oxford University Press, 335 pp. HILL, M. P., AND HULLEY, P. E. 1995. Biology and host range of Gratiana spadicea (Klug, 1829) (Coleoptera: Chrysomelidae: Cassidinae), a potential biological control agent for the weed Solanum sisymbrifolium Lamarck (Solanaceae) in South Africa. Biol. Control 5: 345-352. HILL, M. P., AND HULLEY, P. E. 1996. Suitability of Metriona elatior (Klug) (Coleoptera: Chrysomelidae: Cassidinae) as a biological control agent for Solanum sisymbrifolium Lam. (Solanaceae). African Entomol. 4: 117-123. LANGELAND, K. A., AND BURKS, K. C. [Eds.], 1998. Identication & biology of non-native plants in Floridas natural areas. University of Florida. Gainesville, FL., 165 pp. MCGOVERN, R. J., POLSTON, J. E., DANYLUK, G. M., HEIBERT, E., ABOUZID, A. M., AND STANSLY, P. A. 1994a. Identication of a natural weed host of tomato mottle geminivirus in Florida. Plant Dis. 78: 11021106. MCGOVERN, R. J., POLSTON, J. E., AND MULLAHEY, J. J. 1994b. Solanum viarum: weed reservoir of plant viruses in Florida. Int. J. Pest Management 40: 270273. MCGOVERN, R. J., POLSTON, J. E., AND MULLAHEY, J. J. 1996. Tropical soda apple ( Solanum viarum Dunal): Host of tomato, pepper, and tobacco viruses in Florida, pp. 31-34 In J. J. Mullahey [ed.], Proc. Tropical Soda Apple Symp. Bartow, Florida. University of Florida, IFAS. MEDAL, J. C., CHARUDATAN, R., MULLAHEY, J. J., ANDPITELLI, R. A. 1996. An exploratory insect survey of tropical soda apple in Brazil and Paraguay. Florida Entomol. 79(1): 70-73. MEDAL, J. C., PITELLI, R. A., SANTANA, A., GANDOLFO, D., GRAVENA, R., AND HABECK, D. H.1999. Host specicity of Metriona elatior a potential biological control agent of tropical soda apple, Solanum viarum Dunal, in the USA. BioControl 44: 1-16. MEDAL, J. C., SUDBRINK, D., GANDOLOFO, D., OHASHI, D., AND CUDA, J. P. 2002. Gratiana boliviana, a potential biocontrol agent of Solanum viarum: Quarantine host-specicity testing in Florida and eld surveys in South America. BioControl 47: 445-461. MEDAL, J. C., GANDOLFO, D., AND CUDA, J. P. 2003. Biology of Gratiana boliviana the rst biocontrol agent released to control tropical soda apple in the USA. University of Florida-IFAS Extension Circular ENY, 3p. MEDAL, J., OVERHOLT, W., STANSLY, P., RODA, A., OSBORNE, L., HIBBARD, K., GASKALLA, R., BURNS, E., CHONG, J., SELLERS, B., HIGHT, S., CUDA, J. P., VITORINO, M., BREDOW, E., PEDROSA-MACEDO, J., ANDWIKLER, C. 2008. Establishment, spread, and initial

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Medal et al.: Host Specicity of Anthonomus tenebrosus 225impacts of Gratiana boliviana (Chrysomelidae) on Solanum viarum in Florida, pp. 591-596 In R. Sforza, M. C. Bon, H. C. Evans, P. E. Hatcher, H. Z. Hinz, B. G. Rector [eds.]. Proc. XII International Symp. Biological Control of Weeds. La Grande Motte, France. MEDAL, J., BUSTAMANTE, N., OVERHOLT, W., DIAZ, R., STANSLY, P., RODA, A., AMALIN, D., HIBBARD, K., GASKALLA, R., SELLERS, B., HIGHT, S., AND CUDA, J. 2010a. Biological control of tropical soda apple (Solanaceae) in Florida: Post-release evaluation. Florida Entomol. 93: 130-132 MEDAL, J., BUSTAMANTE, N., VITORINO, M., BEAL, L., OVERHOLT, W., DIAZ, R., AND CUDA, J. 2010b. Host specicity tests of Gratiana graminea (Coleoptera: Chrysomelidae), a potential biological control agent of tropical soda apple (Solanaceae). Florida Entomol. 93: 231-242. MEDAL, J., AND CUDA, J. 2010. Establishment and initial impact of the leaf-beetle Gratiana boliviana (Chrysomelidae), rst biocontrol agent released against tropical soda apple in Florida. Florida Entomol. 93 (4): 493-500. MISLEVY, P, MULLAHEY, J. J., AND COLVIN, D. L. 1996. Management practices for tropical soda apple control: Update, pp. 61-67 In J. J. Mullahey [ed.], Proc. Tropical Soda Apple Symp. Bartow, Florida. University of Florida, IFAS. MISLEVY, P., MULLAHEY, J. J., AND MARTIN, F. G. 1997. Tropical soda apple control as inuenced by clipping frequency and herbicide rate, In Abstracts of Weed Sci. Soc. of America Meeting, Orlando, Florida. WSSA Vol. 37. MULLAHEY, J. J., AND COLVIN, D. L. 1993. Tropical soda apple: A new noxious weed in Florida. Univ. of Florida, Florida Cooperative Extension Service, Fact Sheet WRS, 7p. MULLAHEY, J. J., NEE, M., WUNDERLIN, R. P., ANDDELANEY, K. R. 1993. Tropical soda apple ( Solanum viarum): a new weed threat in subtropical regions. Weed Technology 7: 783-786. MULLAHEY, J. J., HOGUE, P., HILL, K., SUMNER, S., ANDNIFONG, S. 1994. Tropical soda apple census. Florida Cattleman Magazine 58:3. MULLAHEY, J. J., SHILLING, D. G., MISLEVY, P., ANDAKANDA, R. A. 1998. Invasion of tropical soda apple (Solanum viarum) into the U.S.: Lessons learned. Weed Technology 12: 733-736. NESER, S., ZIMMERMANN, H. G., ERB, H. E., AND HOFFMANN, J. H. 1988. Progress and prospects for the biological control of 2 Solanum weeds in South Africa, pp. 371-381 In E.S. Delfose, [ed.], Proc. VII International Symp. Biological Control of Weeds, Rome, Italy, (Instituto Sperimentale per la Patologia Vegetale Ministerio dell Agriculture e delle Foreste, Rome. OLCKERS, T., ZIMMERMANN, H. G., AND HOFFMANN, J. H. 1995. Interpreting ambiguous results of hostspecicity tests in biological control of weeds: assessment of 2 Leptinotarsa species (Chrysomelidae) for the control of Solanum elaeagnifolium (Solanaceae) in South Africa. Biol. Control 5: 336-344. OLCKERS, T. 1996. Improved prospects for biological control of three Solanum weeds in South Africa, pp. 307312 In V. C. Moran and J. H. Hoffmann [eds.], Proc. IX International Symp. Biological Control of Weeds, Stellenbosch, South Africa. University of Cape Town, South Africa. OVERHOLT, W. A., DIAZ, R., HIBBARD, K. L., RODA, A. L., AMALIN, D., FOX, A. J., HIGHT, S. D., MEDAL, J. C., STANSLY, P. A., CARLISLE, B., WALTERS, J. H., HOGUE, P. J., GARY, L. A., WIGGINS, L. F., KIRBY, C. L., AND CRAWFORD, S. C. 2009. Releases, distribution and abundance of Gratiana boliviana (Coleoptera: Chrysomelidae), a biological control agent of tropical soda apple (Solanum viarum, Solanaceae) in Florida. Florida Entomol. 92: 450-457. OVERHOLT, W. A., DIAZ, R., MARKLE, L., AND MEDAL, J. C. 2010. The effect of Gratiana boliviana (Coleoptera: Chrysomelidae) herbivory on growth and population density of tropical soda apple (Solanum viarum) in Florida. Biocontrol Sci. Technol. 20: 791807. PATTERSON, D. T. 1996. Effects of temperature and photoperiod on tropical soda apple ( Solanum viarum Dunal) and its potential range in the United States, pp. 29-30 In J. J. Mullahey [ed.], Proc. Tropical Soda Apple Symp. Bartow, Florida. University of Florida, IFAS. SUDBRINK, JR. D. L., SNODGRASS, G. L., BRYSON, C. T., MEDAL, J. C., CUDA, J. P., AND GANDOLFO, D. 2000. Arthropods associated with tropical soda apple, Solanum viarum in the Southeastern USA, p.154 In Program Abstracts, X International Symp. Biol. Control of Weeds, 4-9 July 1999. Bozeman, MT. USDA-ARS/Montana State University, Bozeman. STURGIS, A. K., AND COLVIN, D. L. 1996. Controlling tropical soda apple in pastures, p. 79 In J. J. Mullahey [ed.], Proc. Tropical Soda Apple Symp. Bartow, Florida. University of Florida, IFAS. THOMAS, M., 2007. Impact of tropical soda apple on Floridas grazing land. The Florida Cattleman and Livestock Journal 71: 37-38.

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226 Florida Entomologist 94(2) June 2011 EFFECT OF ORGANIC MULCHES ON SOIL SURFACE INSECTS AND OTHER ARTHROPODS H ARSIMRAN K. G ILL 1 R OBERT M C S ORLEY 1 AND M ARC B RANHAM 1 1 Entomology and Nematology Department, University of Florida, Gainesville, Florida 32611-0620, USA A BSTRACT Four different types of organic mulches were evaluated for their effects on soil surface insects and related arthropods. Field experiments were conducted in fall 2007 and 2008 near Citra, Florida. In both the years, ve treatments were compared: cowpea ( Vigna unguiculata (L.) Walp.) mulch, sunn hemp ( Crotalaria juncea L.) mulch, sorghum-sudangrass ( Sorghum bicolor Moench S. sudanense ((Piper)] Stapf) mulch, pine bark nuggets, and unmulched control. Data were collected on insects and other arthropods using pitfall traps. Results indicate that organic mulches can affect a wide range of different insects. Diptera, dominated by Asyndetus spp. (Dolichopodidae), were most dense in pine bark plots in both years. Populations of small plant-feeding insects suc h as Aphididae, Thripidae, and Aleyrodidae were most dense in cowpea and unmulched control plots in one season. It is possible that these insects were affected by weed growth in cowpea and control plots. Ants, which tend or feed on small plant feeders, were fairly abundant in these plots as well, as were predatory beetles. Some groups, such as Collembola (mainly Isotomidae), spiders, and Orthoptera (Acrididae and Gryllidae) were unaffected by mulches. Key Words: cover crop residue, organic mulch, insect community, pine bark R ESUMEN Se evaluaron cuatro diferentes tipos de coberturas orgnicas por sus efectos sobre los insectos y artrpodos relacionados de la supercie del suelo. Se realizaron los experimentos de campo en el otoo del 2007 y 2008 cerca de Citra, Florida. En ambos aos, se compararon cinco tratamientos: el mantillo de caup ( Vigna unguiculata (L.) Walp.), el mantillo de c- amo sunn ( Crotalaria juncea L.), el mantillo de sorgo-pasto de Sudn ( Sorghum bicolor Moenc h S. sudanense (Piper) Stapf), pedazos de la corteza de pino, y sin cobertura (control). Se utilizaron trampas de cada para obtener los datos de los insectos y de los otros artrpodos Los resultados indican que el mantillo orgnico puede afectar a una amplia gama de diferentes insectos. Los Diptera, dominado por las especies de Asyndetus (Dolichopodidae), fueron ms densas en parcelas de corteza de pino en ambos aos Las poblaciones de insectos que se alimentan de plantas pequeas, tales como Aphididae, Thripidae y Aleyrodidae eran ms densas en caup y parcelas sin cobertura (control) en una temporada. Es posible que estos insectos fueron afectados por el crecimiento de malezas en las parcelas de caup y del control. Las hormigas, que atienden o se alimentan de insectos que se alimentan de plantas pequeas, fueron bastante abundantes en estas parcelas, al igual que los escarabajos depredadores. Algunos grupos, como los colmbolos (principalmente Isotomidae), araas, y ortpteros (Acrididae y Gryllidae) no fueron afectados por las coberturas. Use of cover crop residues as organic mulches has a number of advantages to farming systems such as reducing soil erosion, conserving soil moisture, moderating soil temperature, improving inltration of water, and providing a slow-release source of nutrients (Gruda 2008; Hatwig & Ammon 2002; Hatwig & Hoffman 1975; Powers & McSorley 2000; Snapp et al. 2005; Westerman & Bicudo 2005). Plant mulches can be an effective way to provide shelter for predatory insects (Johnson et al. 2004) and to control weeds (Reeleder et al. 2004; Teasdale et al. 2004). Mulches can help to maintain soil moisture required for plant vigor and to promote plant tolerance to the attack of insect pests (Johnson et al. 2004). Cover crops and intercrops have been used as living mulches for managing some insect pests. Alfalfa ( Medicago sativa L.) and kura clover ( Trifolium ambiguum M. Bieb.) mulches increased predator populations to manage European corn borer ( Ostrinia nubilalis Hbner) (Prasifka et al. 2006). Eggs and larval densities of pest caterpillars were higher in broccoli ( Brassica oleracea L. var. botrytis ) monoculture when compared to broccoli with undersown mulc hes like strawberry clover ( Tribolium fragiferum L.), white clover ( Tribolium repens L.), and yellow sweet clover ( Melilotus ofcinalis L.) (Hooks & Johnson 2004). Alfalfa living mulc h increased predators to manage outbreaks of the invasive soybean aphid, Aphis glycines Matsumura (Schmidt et al. 2007). While these examples suggest that living mulc hes may offer resources to support preda-

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Gill et al.: Effect of Organic Mulches on Soil Surface Arthropods227 tors, non-living mulches derived from killed cover crops, hay from cover crops, or composted waste products may offer benets as well. In sweetpotato ( Ipomoea batatas (L.) Lam.), higher numbers of re ants rove beetles, and carabid beetles were captured using pitfall traps in plots covered with killed-cover crop (Jackson & Harrison 2008). Also, the injury level from soil insect pests to roots of sweetpotato was lower in killed-cover crop plots than in conventional plots. In an apple ( Malus domestica Borkh.) orchard, the dominance of several carabid species depended on different factors inc luding sampling dates and different types of ground cover including plastic mulch and straw mulch (Miarro & Dapena 2003). Predation of beet armyworm, Spodoptera exigua (Hbner), pupae w as 33% greater in cover crop mulch as compared with conventional production plots (Pullaro et al. 2006). Mulch from sunn hemp ( Crotalaria juncea L.) hay was effective in reducing incidence of lesser cornstalk borer Elasmopalpus lignosellus (Zeller) on bean ( Phaseolus vulgaris L.) (Gill et al. 2010). Changes in cropping systems affect insect pests and their natural enemies (Hummel et al. 2002). Organic mulches might provide hiding places to harbor populations of natural enemies. Different types of cover crops harbor distinctive complexes of benecial insects, pest arthropods, and their diverse trophic relationships (Bugg & Waddington 1994). Many previous studies that used mulches for the management of insect pests focused especially on ying insects moving into mulched areas (Brown & Tworkoski 2004; Gill et al. 2010; Hooks & Johnson 2004; Prasifka et al. 2006; Pullaro et al. 2006; Reeleder et al. 2004; Schmidt et al. 2007; Tremelling et al. 2002). The effects of mulches on insects and other soil arthropods living on the soil surface is a relatively less explored area. More information is needed on arthropods that are active on the soil surface where the mulches occur, and how different materials on the soil surface affect these arthropods. To answer these questions, the present study was designed with main objective to determine the impact of mulches on the community of arthropods that live and move on the soil surface. The purpose was to obtain an overview of various arthropod groups that were active on the soil surface, rather than focusing on selected key species. M ATERIALS AND M ETHODS Field experiments were conducted in fall 2007 and 2008 at the University of Florida Plant Science Research and Education Unit (29N, 82W), Citra, Florida. The soil at the experimental site was Arredondo sand (95% sand, 2% silt, 3% clay) with 1.5% organic matter (Thomas et al. 1979). Fall 2007 The experimental eld was sprayed with glyphosate (Roundup, Monsanto, St. Louis, Missouri) to kill weeds on Sep 26 followed by rototilling on Oct 3. Average soil moisture measured gravimetrically before planting was 6.1%. Five treatments compared were: cowpea ( Vigna unguiculata (L.) Walp.) mulch; sunn hemp mulch; sorghum-sudangrass ( Sorghum bicolor Moench S. sudanense (Piper) Stapf) mulch; pine bark nuggets as mulc h (HTC Hood Timber Co., Adel, GA); and unmulched control. Treatments were arranged in a randomized complete block design with ve replications (total of 25 plots). Individual plots for each treatment were 3.0 m long and 2.4 m wide and the distance between plots was 3.0 m. All plots were planted with Roma II bush beans ( Phaseolus vulgaris L.) on Oct 4. Seeds were spaced 10 cm apart at a rate of 30 seeds per row in two rows per plot. The mulches used were readily available or easily supplied by cover crop residues. Cover crop mulches were obtained from crops of Iron and Clay cowpea, Tropic Sun sunn hemp, and Growers Choice sorghum-sudangrass planted in early Jul. Mulches were obtained from these cover crops (prior to owering) planted near the experimental site. To obtain mulches, these cover crops were harvested on Oct 11 by clipping plants at the base, removing above-ground biomass, and applying it to the plots The resulting mulches (3-5 cm deep) were a composite of leaves and stems and were applied by hand over the entire plot, next to the rows of bean plants. Therefore, except for the plant rows, the entire plot was covered with mulch. Mulches were applied only once at the start of experiment on Oct 11, using the following amounts of material: cowpea (18.1 kg fresh wt/ plot), sunn hemp (15.9 kg fresh wt/ plot), and sorghum-sudangrass (17.7 kg fresh wt/plot). The pine bark nuggets (29.8 kg fresh wt/plot) were not obtained from cover crops, but were purchased locally. Plots were irrigated as needed using drip irrigation, and no insecticides were applied during the course of the experiment. Fall 2008 The experiment was repeated at the same site in the fall 2008, with the same treatments. Experimental procedures remained the same with a few minor changes. The experimental eld was sprayed with glyphosate to kill weeds in the rst week of Sep followed by rototilling on Sep 16. Average soil moisture measured gravimetrically at planting was 6.9%. Beans were planted on Oct 7. Cowpea (12.7 kg fresh wt/plot), sunn hemp (15.9 kg fresh wt/plot), sorghum-sudangrass (13.6 kg fresh wt/plot), and pine bark nuggets (29.8 kg fresh wt/plot) were applied on Oct 9. Early frost in

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228 Florida Entomologist 94(2) June 2011 each season caused severe damage to the bean plants, so that crop harvests were not possible. Data collection Insects were collected on several sampling dates in both seasons (Oct 24, Nov 6, Nov 20, Dec 3, and Dec 17 in 2007; Oct 13, Oct 28, Nov 9, and Nov 24 in 2008). Pitfall traps were used for capturing insects that run or move on the soil surface (Borror et al. 1989). A plastic sandwich container (14 cm 14 cm 4 cm) was used as a pitfall trap. One pitfall trap w as placed in the middle of each plot, and buried so that the upper edge was ush with the soil surface. The traps were lled three quarters with water, along with 3 to 4 drops of dish detergent (Ultra Joy, Procter and Gamble, Cincinnati, Ohio) to break surface tension, ensuring that the insects would remain in the trap. Pitfall traps were set out in the morning (9:00 am) and collected at approximately the same time (9:00 am) the next day (which was recorded as the sampling date). The traps were brought to the laboratory, kept in a cold room at 10C, and contents transferred and stored in 70% ethanol in vials. Insects and related arthropods were identied to order and family levels using a dissecting microscope. Data analysis All statistical analyses were performed using the Statistical Analysis System (SAS) package (version 9.1; SAS Institute, Cary, North Carolina). Data for each dependent variable (insect groups) were analyzed across all sampling dates in each year using repeated measures (PROC MIXED procedure of SAS) to examine the effects of treatment, sampling date, and interactions between treatments and sampling dates. Since no interactions were found, data were pooled across sampling dates for calculations of means and standard errors of the means. When treatment effects were signicant ( P 0.05), least square means (LS) values were computed to compare means of mulc h treatments. R ESULTS Fall 2007 Diptera were affected ( P 0.05) by mulches, and were more common in pine bark mulc h than in sunn hemp and sorghum-sudangrass (Table 1). Diptera consisted mainly of Dolichopodidae (43.9%, Asyndetus spp.) followed by Mycetophilidae (fungus gnats) and other micro-dipterans (37.1%) and other Diptera (19.0%). Cicadellidae and small plant-feeding insects were not signicantly ( P 0.05) affected by treatment, but Cicadellidae showed some an interesting trend ( P T ABLE 1. E FFECT OF MULCH TREATMENTS ON ARTHROPOD TAXA ( NUMBERS / PITFALL TRAP ) THROUGHOUT THE SEASON IN C ITRA F LORIDA 2007. Treat 1 Hymenoptera 2 Collembola 2 Homoptera 2 Diptera 2 Orthoptera 2 AraneaeColeoptera 2 Others 2 CP 6.48 1.51 a58.52 8.76 a1.48 0.29 a5.56 0.71 ab1.28 0.37 a0.52 0.13 a2.16 0.50 a1.64 0.34 a SH5.08 1.42 a138.20 42.39 a1.04 0.30 a4.12 0.67 b0.60 0.16 a0.76 0.18 a1.48 0.32 a0.72 0.20 a SO6.96 3.22 a 70.00 11.53 a1.32 0.30 a4.32 0.68 b0.84 0.21 a0.68 0.15 a1.80 0.33 a0.92 0.36 a PB3.00 0.64 a84.36 30.78 a0.88 0.22 a7.32 0.99 a1.24 0.25 a1.08 0.57 a0.96 0.20 a1.04 0.26 a C2.52 0.51 a86.80 27.82 a2.80 1.03 a5.28 0.86 ab1.00 .45 a0.48 0.13 a1.52 0.37 a1.44 0.35 a P > F0.26380.41280.08990.01110.49280.51360.19180.2272 F value1.421.042.344.320.880.841.691.55 1 Treatments CP = cowpea, SH = sunn hemp, SO = sorghum-sudangrass, PB = pine bark, C = unmulched control 2 Hymenoptera = Formicidae; Collembola = Isotomidae and Sminthuridae; Homoptera = Cicadellidae; Diptera = Dolichopodidae, Mycetophilidae and micro-dipterans; Orthoptera = Acrididae and Gryllidae; Coleoptera = Staphylinidae, Carabidae, Elateridae, and Chrysomelidae; others = Aphididae, Aleyrodidae, and Thripidae Data are means standard error of 25 replications (data pooled across 5 sampling dates). Means in columns for each sampling date followed by the same letters do not differ signicantly based on least square means (P 0.05).

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Gill et al.: Effect of Organic Mulches on Soil Surface Arthropods229 0.10) toward greater abundance in unmulched control plots. Small plant-feeding insects consisted of aphids (72.7%, Aphididae), whiteies (24.3%, Aleyrodidae), and thrips (3.0%, Thripidae). The numbers of Formicidae (mixture of Pheidole spp., and Dorymyrmex spp.), Collembola (Isotomidae with a few Sminthuridae), Orthoptera (mixture of Melanoplus spp., Dichromorpha spp., and Gryllus spp.), Araneae, and Coleoptera (Staphylinidae Carabidae, Elateridae, and Chrysomelidae) did not differ among treatments (Table 1). In addition, the few micro-Hymenoptera (mainly small parasitoid wasps) collected were also not affected by treatments (data not shown). Beetles collected were from the families Staphylinidae (23.4%), Carabidae (12.2%, Anisodactylus spp.), Elateridae (14.2%, Conoderus spp.), and Chrysomelidae (48.4%, Altica spp.), but none of these individual families were signicantly ( P 0.05) affected by treatments. A few specimens of other plant-feeding insects were occasionally re covered at low levels in pitfall traps including cutworms (Noctuidae), plant hoppers (Fulgoridae), spittlebugs (Cercopidae), and stink bugs (Pentatomidae), but none were affected by treatments ( P 0.10). Fall 2008 In this season, Diptera were more common in pink bark mulch than in sunn hemp and unmulched control plots (Table 2). Diptera consisted mainly of Dolichopodidae (81.1%) followed by fungus gnats (Mycetophilidae) and other microdipterans (3.7%) and other Diptera (15.2%). Formicidae were affected by mulches, and were greatest in cowpea plots. Total numbers of beetles were greater in unmulched control and cowpea than in sorghum-sudangrass. Beetles collected were from the families Staphylinidae (71.7%), Carabidae (21.6%), Elateridae (3.2%), and Chrysomelidae (3.4%), but none of these individual families were signicantly ( P 0.05) affected by treatments Small plant-feeding insects were most abundant in cowpea and unmulched control plots. Small plant-feeding insects consisted of aphids (73.0%, Aphididae), whiteies (26.0%, Aleyrodidae), and thrips (0.9%, Thripidae). The numbers of Cicadellidae, Araneae, Collembola (mostly Isotomidae), and Orthoptera (Acrididae and Gryllidae) collected did not differ among treatments (Table 2). D ISCUSSION The arthropods recovered during this study encompassed a variety of trophic groups and feeding habits (Table 3). Effects of treatments on different insect groups varied, but some interesting patterns were evident. Several insect groups, including ants, beetles, and small plant feeding inT ABLE 2. E FFECT OF MULCH TREATMENTS ON ARTHROPOD TAXA (NUMBERS/PITFALL TRAP) THROUGHOUT THE SEASON IN CITRA, FLORIDA, 2008. Treat1Hymenoptera2 Collembola2Homoptera2Diptera2Orthoptera2AraneaeColeoptera2Others2CP 12.00 1.80 a22.20 2.87 a3.90 0.99 a9.85 1.94 ab2.95 0.85 a0.45 0.15 a2.25 0.70 ab6.00 1.27 a SH4.49 0.96 b27.00 6.29 a2.05 0.52 a6.60 1.23 b1.70 0.41 a1.00 0.74 a1.20 0.28 bc1.70 0.52 bc SO3.75 0.95 b28.00 4.66 a3.10 0.63 a10.10 1.92 ab1.65 0.47 a1.15 0.46 a1.15 0.29 c3.00 0.62 bc PB3.80 0.72 b28.60 3.68 a1.75 0.34 a14.30 2.08 a1.50 0.48 a0.40 0.15 a1.30 0.42 bc1.10 0.23 c C5.10 0.84 b38.70 8.59 a3.90 1.00 a7.65 1.35 b2.10 0.56 a0.25 0.10 a2.50 0.55 a4.15 0.88 ab P > F <0.0001 0.4777 0.2364 0.0050 0.1500 0.5105 0.0439 <0.0001 F value 14.76 0.91 1.51 5.17 1.90 0.85 2.99 12.881Treatments CP = cowpea, SH = sunn hemp, SO = sorghum-sudangrass, PB = pine bark, C = unmulched control2Hymenoptera = Formicidae; Collembola = Isotomidae; Homoptera = Cicadellidae; Diptera = Dolichopodidae, Mycetophilidae and micro-dipterans; Orthoptera = Acrididae and Gryllidae; Coleoptera = Staphylinidae, Carabidae, Elateridae, and Chrysomelidae; others = Aphididae, Aleyrodidae, and Thripidae. Data are means standard error of 20 replications (data pooled across 4 sampling dates). Means in columns for each sampling date followed by the same letters do not differ signicantly based on least square means (P 0.05).

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230 Florida Entomologist 94(2)June 2011TABLE 3. ARTHROPODS AND THEIR FEEDING HABITS IN THE NATURAL ENVIRONMENT. Arthropods Feeding habitsReferences Hymenoptera (Formicidae: Pheidole, Dorymyrmex) Mainly predators of small invertebrates Wilson 2005 Some ants feed on plant sap, nectar, honeydew or fungi Triplehorn & Johnson 2005 Collembola (Isotomidae and Sminthuridae) Usually fungi associated with decaying vegetation Coleman et al. 2004 Homoptera (Cicadellidae) Mainly herbivores feeds on plant sap Redak et al. 2003 Diptera (Dolichopodidae: Asyndetus and Mycetophilidae)Dolichopodidae mainly predators of small invertebrates Ulrich & Schmelz 2001 Mycetophilidae feed on fungus Triplehorn & Johnson 2005 Orthoptera (Gryllidae: Gryllus and Acrididae: Melanoplus Dichromorpha) Generalist herbivores, feed on most kinds of vegetation including weeds Capinera 1993 Araneae Generalist predators of small-sized arthropods Riechert & Lockley 1984 Coleoptera: (Staphylinidae), Most Staphylinidae are facultative predators Frank & Thomas 2010 Carabidae: (Anisodactylus spp.), Anisodactylus spp. are typically predators but granivory has been recently reported Sasakawa 2009 Elateridae: (Conoderus spp.), Conoderus spp. eat seeds, and feed on stem and roots of seedlings and lead to weak plant stand Mossler 1993, and Chrysomelidae: (Altica spp.) Altica spp. generally feed on different kinds of plants Jenkins et al. 2009 Others (Aphididae, Aleyrodidae, and Thripidae) Plant feeders Triplehorn & Johnson 2005

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Gill et al.: Effect of Organic Mulches on Soil Surface Arthropods231sects (aphid, whiteies, and thrips), were highest in unmulched control or cowpea plots in one season. It is possible that weeds (including nutsedges, grasses, and broadleaf) in unmulched control and cowpea plots may have led to the higher numbers of small plant-feeding insects in these plots. Cowpea mulch degraded quickly and allowed the emergence of weeds after 3-4 weeks. At this time, broadleaf weeds covered about 10% of the surface area in unmulched control and cowpea plots, but <5% in other plots. Broadleaf weeds consisted of Florida pusley ( Richardia scabra L.), eveningprimrose (Oenothera laciniata Hill.), and cudweed (Gnaphalium spp.). Beetles are the largest and most diverse group of insects, and varied in their response to treatment over the two seasons, reaching highest numbers in cowpea plots in 2008. The majority of these were Staphylinidae and Carabidae, which are predators, and the increased abundance of potential prey insects (Aphididae, Cicadellidae etc.) in unmulched control plots may have stimulated these predatory beetles as well (Table 3). Ants have been observed to feed on or tend sucking insects such as aphids and whiteies (Borror et al. 1989), so their increased numbers may be related to the other insects in unmulched control and cowpea plots. This effect was observed by Pullaro et al. (2006) who recorded a greater number of re ants in plots with killed-cover crop mulch compared with conventional plots. Flies were most common in pine bark plots in both years, possibly because pine bark was the only mulch that did not degrade as fast as others (C:N ratio = 208:1), and may have served as cover for these insects and their larvae. This mulch may have provided favorable habitat for long-legged ies (Dolichopodidae) that typically inhabit organic debris and feed on small invertebrates on the soil surface (Borror et al. 1989; Triplehorn & Johnson 2005; Ulrich & Schmelz 2001). Collembola were unaffected by treatments, with similar levels in mulched and unmulched plots. This was unexpected since the degradation of mulch could provide a continuous supply of organic matter. Generally, Collembola are cryptozoic and feed on fungi associated decaying organic matter (Coleman et al. 1996; Powers & McSorley 2000). We were surprised to nd a number of aphids, whiteies, and thrips in pitfall traps. The pitfall trap is the one of the most commonly used methods to sample insects and other arthropods on the soil surface (Southwood & Henderson 2000). On the other hand, small plant feeders such as aphids, whiteies, and thrips are typically sampled by other methods such as sticky cards rather than pitfall traps (Southwood & Henderson 2000). However, small numbers of them will fall from vegetation into pitfall traps as well (Tremelling et al. 2002). Future studies should anticipate presence of some small plant feeders in pitfall traps, which could probably be better explained by concurrent sampling of above-ground vegetation by other methods. CONCLUSIONSThe present study suggests that insects varied in their responses to different mulches. During both years, ies (mainly Dolichopodidae) were found in highest numbers in pine bark plots throughout the season. Several other groups were affected indirectly due to the effects of mulches on weed growth. Weed growth in unmulched control and cowpea plots may have led to increased populations of small plant feeders such as aphids, thrips, and whiteies. Ants that tend or feed on small plant feeders were more abundant in these plots as well, as were predatory beetles in 2008. Some groups, such as Collembola, spiders, and parasitoid wasps, were unaffected by mulches, while others such as leafhoppers showed only minimal trends. ACKNOWLEDGMENTSThis paper is submitted in partial fulllment of the requirements for the PhD degree of the senior author. The authors also thank Heidi HansPetersen, Namgay Om, and Romy Krueger for their assistance in the eld, and Buck Nelson and the staff of PSREU for management of eld plots. Lyle Buss of the Entomology and Nematology Department at the University of Florida was very helpful with identication of some of the insect genera. The authors also thank Danielle Treadwell, Gaurav Goyal, and Susan Webb of the University of Florida for reviewing and improving an earlier version of the manuscript. 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232 Florida Entomologist 94(2)June 2011GILL, H. K., MCSORLEY, R., GOYAL, G., AND WEBB, S. E. 2010. Mulch as a potential management strategy for lesser cornstalk borer, Elasmopalpus lignosellus (Insecta: Lepidoptera: Pyralidae), in bush bean (Phaseolus vulgaris ). Florida Entomol. 93: 183-190. GRUDA, N. 2008. The effect of wood fiber mulch on water retention, soil temperature and growth of vegetable plants. J. Sustain. Agric. 32: 629-643. HATWIG, N. L., AND AMMON, H. 2002. Cover crops and living mulches. Weed Sci. 50: 688-699. HARTWIG, N. L., AND HOFFMAN, L. D. 1975. Suppression of perennial legume and grass cover crops for notillage corn. Proc. Northeast. Weed Sci. Soc. 29: 8288. HOOKS, C. R., AND JOHNSON, M. W. 2004. Using undersown clovers as living mulches: effects on yields, lepidopterous pest infestation, and spider densities in a Hawaiian broccoli agroecosystem. Int. J. Pest Manag. 50: 115-120. HUMMEL, R. L., WALGENBACH, J. F., HOYT, G. D., ANDKENNEDY, G. G. 2002. 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Bioresource Technol. 96: 215-221. WILSON, E. O. 2005. Oribatid mite predation by small ants of the genus Pheidole Insectes Sociaux 52:263-265.

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Xu et al.: Dryinidae from the Nanling National Nature Reserve233 TWO NEW SPECIES OF DRYINIDAE (HYMENOPTERA: CHRYSIDOIDEA FROM NANLING NATIONAL NATURE RESERVE, CHINA Z AIFU X U 1 M ASSIMO O LMI 2 AND J UNHUA H E 3 1 College of Natural Resources and Environment, South China Agricultural University Guangzhou, Guangdong 510642, P. R. China 2 Corresponding author. Department of Plant Protection, University of Tuscia, Via San Camillo de Lellis, I-01100 Viterbo, Italy 3 Institute of Insect Sciences, Zhejiang University, Hangzhou 310029, P. R. China A BSTRACT Anteon nanlingense sp. nov and Anteon longum sp. nov. are described from Nanling National Nature Reserve (Guangdong P.R. China). A check-list of Dryinidae from Nanling National Nature Reserve is presented. Key Words: Dryinidae, Anteon nanlingense Anteon longum new species, Nanling Nature Reserve China R ESUMEN Se describen por primera vez a Anteon nanlingense sp. nov. y Anteon longum sp. nov. ambos colectados en la Reserva Natural Nanling (Guangdong P.R. China); asimismo, se realiza un listado de los Dryinidae presentes en dicha reserva. Translation provided by the authors. Dryinidae (Hymenoptera: Chrysidoidea) are parasitoids of Hemiptera: Auchenorrhyncha (Guglielmino & Olmi 1997, 2006, 2007). The species of Dryinidae inhabiting China have been studied in the last 10 years mainly by He & Xu (2002), Xu, He & Olmi (2001) and Xu, Olmi & He (2006a, 2006b, 2006c, 2007, 2008, 2009a, 2009b, 2009c, 2010, 2011). With approximately 126 described species, Anteon Jurine, 1807, is 1 of the largest genera of the Oriental region. Two additional new species of Anteon are described herein. They were collected in 1 of the most interesting protected areas of P .R. China, i.e., Nanling National Nature Reserve. This large park includes the highest mountain of Guangdong Province, Mt. Shikengkong (1902 m). This paper presents a revised check-list of Dryinidae inhabiting Nanling National Nature Reserve. M ATERIALS AND M ETHODS The descriptions follow the terminology used by He & Xu (2002) and Olmi (1984, 1994, 1999). The measurements reported are relative, except for the total length (head to abdominal tip, without the antennae), which is expressed in mm. In the descriptions, POL is the distance between the inner edges of the lateral ocelli; OL is the distance between the inner edge of a lateral ocellus and the median ocellus; OOL is the distance from the outer edge of a lateral ocellus to the compound eye; OPL is the distance from the posterior edge of a lateral ocellus to the occipital carina; TL is the distance from the posterior edge of an eye to the occipital carina. The material studied in this paper is deposited in the Hymenoptera Collection of South China Agricultural University, Department of Entomology, Guangzhou, Guangdong, P. R. China (SCAU). S YSTEMATIC A CCOUNTS Anteon nanlingense sp. nov. (Fig. 1) Material examined: Holotype: Female, P.R. CHINA, Guangdong Prov., Nanling National Nature Reserve, 4-5.X.2004, Zaifu Xu (SCAU). Description. Holotype female; Macropterous; length 2.4 mm; head black, except mandibles, clypeus and anterior half of face are testaceous; ventral side of head black, except a median testaceous stripe; antenna testaceous; prothorax testaceous; rest of mesosoma black; petiole black; gaster testaceous, except some brown areas on dorsal side; legs testaceous-whitish. Antenna clavate; antennal segments in following proportions: 10:4:10:7:6.5:7:7:6:6:8.5. Head shiny, smooth, punctate, without sculpture among punc-

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234 Florida Entomologist 94(2) June 2011 tures; anterior half of face rugose; frontal line complete; face with 2 lateral longitudinal keels around orbits and directed towards antennal toruli; POL = 5; OL = 4; OOL = 5; OPL = 3.5; TL = 3; greatest breadth of posterior ocellus much shorter than OPL (2:3.5); occipital carina complete. Pronotum shiny, smooth, with anterior surface weakly rugose; posterior surface smooth, nely punctate, without sculpture among punctures; posterior surface shorter than scutum (8:12.5), more than twice as broad as long (18:8); pronotal tubercles reaching tegulae. Scutum shiny, nely punctate, without sculpture among punctures. Notauli incomplete, reaching approximately 0.9 length of scutum. Scutellum and metanotum shiny, without sculpture. Propodeum reticulate rugose, with a strong transverse keel between dorsal and posterior surface; posterior surface with 2 longitudinal keels and median area shiny, as rugose as lateral areas, with some smooth areas. Forewing hyaline, without dark transverse bands; distal part of stigmal vein much shorter than proximal part (5:9). Fore tarsal segments in following proportions: 8:2.5:2.5:4:16; fore tarsal segment 2 curved into a hook. Segment 5 of fore tarsus (Fig. 1) with basal part slightly longer than distal part (10:7). Enlarged claw (Fig. 1) with a proximal prominence bearing a long bristle. Segment 5 of fore tarsus (Fig. 1) with 2 rows of 3 + 24 lamellae; distal apex with a group of 8 lamellae. Male. Unknown. Hosts. Unknown. Etymology. This species is named after its occurrence in Nanling National Nature Reserve, China. Remarks. Anteon nanlingense resembles A. xuexini Xu, He & Olmi, 2001, from P.R. China, Zhejiang Prov However, in A. nanlingense the prothorax is testaceous the notauli reach approximately 0.9 length of scutum and the anterior half of the face is dull and rugose, whereas in A. xuexini the prothorax is black, the notauli reach 0.60.7 length of scutum and the anterior half of the face is smooth and punctate Following the above description of A. nanlingense the key to the females of Oriental Anteon presented by Xu, He & Olmi (2001) can be modied by replacing couplet 11 as follows: 11Scutellum testaceous-reddish . . . . . . . . . . . . . . . . . . . . . . . . .A subdignum Olmi Scutellum black . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 11Anterior half of face dull, rugose, posterior half punctate, without sculpture among punctures; prothorax testaceous; notauli reaching approximately 0.9 length of scutum . . . . . . . . . A. nanlingense sp. nov. Face completely nely punctate, smooth; prothorax black; notauli reaching approximately 0.60.7 length of scutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A xuexini Xu, He & Olmi Anteon longum sp. nov. (Fig. 2) Material examined: Holotype: female, P.R. CHINA, Guangdong Prov., Nanling National Nature Reserve, 4-5.X.2004, Zaifu Xu (SCAU). Description. Holotype female, Macropterous, length 3.1 mm; head black, except mandibles testaceous; antenna testaceous; mesosoma black; gaster brown; legs testaceous. Antenna clavate; antennal segments in following proportions: 12:6:10:8:7:8:7:7:7:10. Head shiny; face rugose, mainly on lateral regions, with a large area in front of anterior ocellus smooth, punctate and without sculpture among punctures; vertex weakly rugose behind posterior ocelli and on temples; frontal line complete; face with 2 lateral keels near orbits directed towards antennal toruli; anterior third of face and clypeus densely hairy; rest of head almost hairless; POL = 5; OL = 4; OOL = 4; OPL = 5; TL = 5; greatest breadth of posterior ocellus shorter than OPL (3:5); occipital carina complete. Pronotum shiny, with anterior surface rugose; posterior surface shiny, punctate, without sculpture among punctures, shorter than scutum (9:16), more than twice as broad as long (22:9); pronotal tubercles reaching tegulae. Scutum, scutellum and metanotum shiny, smooth, nely puncFigs. 1 and 2. Chelae of holotypes of Anteon nanling ense sp. nov. (1) and Anteon longum sp. nov. (2). Scale bars 0.10 mm for 1 and 0.11 mm for 2.

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Xu et al.: Dryinidae from the Nanling National Nature Reserve235 tate, without sculpture among punctures. Notauli incomplete, reaching approximately 0.7 length of scutum. Propodeum with a strong transverse keel between dorsal and posterior surface; dorsal surface reticulate rugose; posterior surface with 2 complete longitudinal keels and median area as rugose as lateral areas. Forewing hyaline, without dark transverse bands; distal part of stigmal vein much shorter than proximal part (5:11). Fore tarsal segments in following proportions: 8:2.5:2.5:6:19. Enlarged claw (Fig. 2) with a proximal prominence bearing a long bristle. Segment 5 of fore tarsus (Fig. 2) with basal part slightly longer than distal part (11:8), with 2 rows of approximately 6 + 27 lamellae; distal apex with a group of about 7 lamellae. Male. Unknown. Hosts. Unknown. Etymology. This species is named after the conspicuous length of the holotype. Remarks. Anteon longum resembles A. acre Olmi, 1991, from Vietnam and Taiwan. However, in A. longum the posterior surface of pronotum is longer than half of scutum and OPL is longer than OOL, whereas in A. acre the posterior surface of pronotum is shorter than half of scutum and OPL is muc h shorter than OOL. Following the above description of Anteon longum the key to the females of Oriental Anteon presented by Xu, He & Olmi (2001) can be modied by replacing couplet 56 as follows: 56Segment 4 of fore tarsus as long as segment 1 . . . . . . . . . . . . . . . . . . . .A insertum Olmi Segment 4 of fore tarsus shorter than segment 1 . . . . . . . . . . . . . . . . . . . . . . . .56 56Posterior surface of pronotum shorter than half of scutum; head with OPL much shorter than OOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A acre Olmi Posterior surface of pronotum longer than half of scutum; head with OPL longer than OOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. longum sp. nov. C HECK LIST OF D RYINIDAE OF N ANLING N ATIONAL N ATURE R ESERVE This check-list is the result of many years of research by 1 of the authors (Prof. Zaifu Xu ) in Nanling National Nature Reserve The following 28 species were found: Aphelopinae Aphelopus maculiceps Bergman, 1957 Aphelopus nepalensis Olmi, 1984 Aphelopus taiwanensis Olmi, 1991 Aphelopus zhaoi Xu, He & Olmi, 1998 Conganteoninae Fiorianteon rugosum Olmi, 1991 Anteoninae Anteon bauense Olmi, 1984 Anteon borneanum Olmi, 1984 Anteon chaoi Xu & He, 1997 Anteon dum Olmi, 1991 Anteon hirashimai Olmi, 1993 Anteon insertum Olmi, 1991 Anteon lankanum Olmi, 1984 Anteon lini Olmi, 1996 Anteon longum new species Anteon nanlingense new species Anteon songyangense Xu, He & Olmi, 1998 Anteon thai Olmi, 1984 Anteon wengae Xu, Olmi & He, 2006b Anteon yasumatsui Olmi, 1984 Dryininae Dryinus adgressor Xu, Olmi & He, 2006c Dryinus chenae Xu, Olmi & He, 2007 Dryinus indianus (Olmi, 1984) Dryinus irregularis Olmi, 1984 Dryinus punctulatus Xu, Olmi & He, 2008 Dryinus pyrillivorus Olmi, 1986 Dryinus sinicus Olmi, 1987 Dryinus stantoni Ashmead, 1904 Gonatopodinae Neodryinus grandis Xu, Olmi & He, 2011 CONCLUSIONSNanling National Nature Reserve is a large mainly mountainous area covered with dense forests. This range, separating Guangdong and Hunan Provinces, hosts populations of temperate and tropical species. This environment explains why the above check-list is composed mainly of 3 genera of Dryinidae: Aphelopus Dalman, 1823, Anteon Jurine, 1807, and Dryinus Latreille, 1804. Notably These genera include species with macropterous females that parasitize mainly forest leafhoppers and planthoppers. Cicadellidae: Typhlocybinae are parasitized by Aphelopus; Cicadellidae: Deltocephalinae, Eurymelinae, Iassinae, Idiocerinae, Ledrinae, Macropsinae and Tartessinae are parasitized by Anteon; many families of Fulgoromorpha (Acanaloniidae, Cixiidae, Flatidae, Fulgoridae, Issidae, Lophopidae, Ricaniidae and Tropiduchidae) are parasitized by Dryinus (Guglielmino & Olmi 1997, 2006, 2007). The subfamily Gonatopodinae, characterized mainly

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236 Florida Entomologist 94(2)June 2011by apterous females, parasitizes Hemiptera feeding on herbaceous plants, so that the species usually do not live in forests and prefer grasslands. Among the few genera of Gonatopodinae with macropterous females, Neodryinus Perkins, is better adapted to live in forests, because the species parasitize Flatidae, Nogodinidae and Ricaniidae, which feed both on herbaceous plants and on shrubs and trees. Currently Nanling National Nature Reserve is known to host 28 of the 193 dryinid species listed in China by He & Xu (2002). ACKNOWLEDGMENTSWe thank Mr. Zhongrun Zhang, Mr. Desong Ruan, Mr. Jingxian Liu, Mr. Jujian Chen, Mr. Bin Xiao, Mr. Hanjian Huang, Mr. Wuqing Fan, Mr. Huayan Chen, Mr. Bo Qiu, Miss Liqiong Weng, Miss Jieming Yao, Miss Juanjuan Ma, Miss Zeng Jie, Miss Chundan Hong, Miss Yali Cai, from South China Agricultural University for their assistance with eldwork.REFERENCES CITEDASHMEAD, W. H. 1904. Descriptions of new genera and species of Hymenoptera from the Philippine Islands. Proc. U.S. National Museum 28(1387): 127-158. BERGMAN, B. H. H. 1957. A new Dryinid Parasite of Leafhoppers in Java. Entomol. Ber. 17: 9-12. DALMAN, J. W. 1823. Analecta entomologica. Typis Lyndhianis, Holmiae: 104 pp. GUGLIELMINO, A., AND OLMI, M. 1997. A host-parasite catalog of world Dryinidae (Hymenoptera: Chrysidoidea). Contrib. Entomology, International 2(2): 165-298. GUGLIELMINO, A., AND OLMI, M. 2006. A host-parasite catalog of world Dryinidae (Hymenoptera: Chrysidoidea): rst supplement. Zootaxa 1139: 35-62. GUGLIELMINO, A., AND OLMI, M. 2007. A host-parasite catalog of world Dryinidae (Hymenoptera: Chrysidoidea): second supplement. Boll. Zool. Agr. Bachic., Ser. ii, 39: 121-129. HE, J., AND XU, Z. 2002. Hymenoptera Dryinidae. Fauna Sinica 29. Science Press, Beijing: 464 pp. JURINE, L. 1807. Nouvelle mthode de classer les Hymnoptres et les Diptres, 1. Hymnoptres. Paschoud, Genve: 320 pp. LATREILLE, P. A. 1804. Nouvelle dictionnaire dHistoire naturelle 24. F. Dufart, Paris: 104 pp. OLMI, M. 1984. A revision of the Dryinidae (Hymenoptera). Mem. Am. Entomol. Inst. 37: 1-1913. OLMI, M. 1986. New species and genera of Dryinidae (Hymenoptera Chrysidoidea). Frustula Entomol. (1986), N.S., 7-8 (20-21): 63-105. OLMI, M. 1987. New species of Dryinidae (Hymenoptera, Chrysidoidea). Fragmenta Entomol. 19: 371-456. OLMI, M. 1991. Supplement to the revision of the world Dryinidae (Hymenoptera: Chrysidoidea). Frustula Entomol. (1989), N.S., 12(25): 109-395. OLMI, M. 1993. A new generic classication for Thaumatodryininae, Dryininae and Gonatopodinae, with descriptions of new species (Hymenoptera Dryinidae). Boll. Zool. Agr. Bachic., Ser. ii, 25: 57-89. OLMI, M. 1994. The Dryinidae and Embolemidae (Hymenoptera: Chrysidoidea) of Fennoscandia and Denmark. Fauna Entomologica Scandinavica 30. E. J. Brill, Leiden: 100 pp. OLMI, M. 1996. New Anteoninae from Taiwan (Hymenoptera: Dryinidae). Oriental Insects 30: 171180. OLMI, M. 1998. New Embolemidae and Dryinidae (Hymenoptera Chrysidoidea). Frustula Entomol. (1997), N. S., 20(33): 30-118. OLMI, M. 1999. Hymenoptera DryinidaeEmbolemidae. Fauna dItalia 37. Edizioni Calderini, Bologna: 425 pp. PERKINS, R. 1905. Leafhoppers and their natural enemies (Pt. I. Dryinidae). Rep. Work Exp. Station Haw. Sugar Planters Assoc., Division of Entomology, Bull. No. 1(I): 1-69. XU, Z., AND HE, J. 1997. Four new species of the genus Anteon Jurine from Guizhou, China (Hymenopter: Dryinidae). Wuyi Sci. J. 13: 106-110. XU, Z., HE, J., AND OLMI, M. 1998. New species of Dryinidae from China (Hymenoptera, Chrysidoidea). Phytophaga 8: 21-37. XU, Z., HE, J., AND OLMI, M. 2001. Descriptions of new species of Dryinidae from China (Hymenoptera: Chrysidoidea). Frustula Entomol. (2000), N.S., 23(36): 1-22. XU, Z., OLMI, M., AND HE, J. 2006a. Description of a new species of Anteon Jurine from the Peoples Republic of China and of the male of Anteon dum Olmi (Hymenoptera: Dryinidae). Zootaxa 1164: 57-61. XU, Z., OLMI, M., AND HE, J. 2006b. Descriptions of ve new species of Anteon Jurine from China (Hymenoptera: Chrysidoidea: Dryinidae). J. Kansas Entomol. Soc. 79(2): 92-99. XU, Z., OLMI, M., AND HE, J. 2006c. A contribution to the knowledge of Dryininae and Gonatopodinae of China (Hymenoptera: Dryinidae). Oriental Insects 40: 91-96. XU, Z., OLMI, M., AND HE, J. 2007. Two new species of Dryinus Latreille (Hymenoptera: Dryinidae) from China. Florida Entomol. 90(3): 453-456. XU, Z., OLMI, M., AND HE, J. 2008. Descriptions of two new species of Dryinus Latreille from China (Dryinidae). J. Kansas Entomol. Soc. 81(1): 8-11. XU, Z., OLMI, M., AND HE, J. 2009a. Two new species of Dryinidae (Hymenoptera: Chrysidoidea) from China. Florida Entomologist 92(2): 217-220. XU, Z., OLMI, M., AND HE, J. 2009b. A taxonomic revision of the Oriental species of Thaumatodryinus Perkins, with descriptions of two new species from P. R. China (Hymenoptera: Dryinidae). Zootaxa 2175: 19-28. XU, Z., OLMI, M., AND HE, J. 2009c. Description of a new species of Lonchodryinus (Hymenoptera: Dryinidae) from China. Oriental Insects 43: 11-14. XU, Z., OLMI, M., AND HE, J. 2010. Two new species of Anteon (Hymenoptera: Dryinidae) from China. Florida Entomol. 93(3); 403-406. XU, Z., OLMI, M. AND HE, J. 2011. Revision of the Oriental species of the genus Neodryinus Perkins, 1905 (Hymenoptera, Dryinidae, Gonatopodinae), with description of a new species from P.R. China. Zootaxa 2790: 1-22.

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Tufts et al.: Viral Pellets as Biological Control for S. invicta 237 DELIVERY SYSTEM USING SODIUM ALGINATE VIRUS LOADED PELLETS TO RED IMPORTED FIRE ANTS ( SOLENOPSIS INVICTA HYMENOPTERA: FORMICIDAE) D ANIELLE M. T UFTS 1,3 K YLE S PENCER 1 W AYNE B. H UNTER 2 AND B LAKE B EXTINE 1 1 University of TexasTyler, Biology Department, 3900 University Blvd., Tyler, TX 75799 2 ARS, USDA, U.S. Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945 3 Current Afliation: School of Biological Sciences, University of Nebraska-Lincoln, 323 Manter Hall, Lincoln, NE 68588 A BSTRACT Microencapsulation as a delivery mechanism of SINV-1 and other molecules such as dsRNA, offers an approach to Solenopsis invicta Buren management that is target specic and ts current approac hes to baiting ants with toxins and/or RNA-interference. The delivery method presented here targets ground dwelling, foraging ants with an ant-infecting virus which is specic to the genus, Solenopsis Endemic ant-infecting viruses, like S. invicta viruses (SINV -1, SINV-2, and SINV-3) are being evaluated for efcacy in S. invicta population suppression. In this study, SINV-1 (TX5 strain) was extracted from S. invicta colonies and microencapsulated in sodium alginate pellets Pellets containing extracted whole virions were offered to conrmed non-infected S. invicta colonies. Colonies were sampled every 5 d and tested by reverse transcription polymerase c hain reaction (RT-PCR) for presence of viral RNA. The longevity of control and viral pellets were also evaluated. Within 30 d, post-feeding of virus, 35% of S. invicta colonies acquired SINV-1 infection ( P = 0.03). Thus, microencapsulation as a delivery mec hanism was successful to deliver SINV-1 to S. invicta colonies. Future incorporation of this economically affordable method can be implemented to deliver biological agents for specic ant species and to augment current approac hes that bait ants. While a virus was used to demonstrate delivery, an adequate and affordable virus production system still needs to be developed before a viral strategy can be adopted as a tool for biological control of re ants. Key Words: ant management, anti-infecting virus, Solenopsis invicta virus (SINV), microencapsulation, mortality, biological control R ESUMEN Microencapsulacin como un mecanismo para entregar el virus SINV-1 y otras molculas como dsARN, ofrece una aproximacin al manejo de Solenopsis invicta Buren que es un objetivo especco y se ajusta a los enfoques actuales de cebos para hormigas txicos y / o interferencia de ARN. El mtodo de entrega presentado aqui se enfoca sobre hormigas que hbitan el suelo o que forrajean y utiliza un virus que infecta especcamente a hormigas del gnero Solenopsis Los virus endmicos que infectan las hormigas, como los virus de S. invicta (SINV-1, SINV-2 y SINV-3) estan siendo evaluados para su ecacia en suprimir poblaciones de S. invicta En este estudio, el virus SINV-1 (cepa TX5) fue extraido de colonias de S. invicta y microencapsulado en granulos de alginato de sodio. Se ofrecieron granulos con los virus encapsulados a las colonias de S. invicta que fueron conrmadas de no estan infectadas Las colonias fueron examinadas cada cinco dias y probadas usando la reaccin reversa de la transcripcin de la cadena polimerasa (RT-RCP) para la presencia de ARN viral. La longevidad de los granulos virales y su control fueron evaluados. En un perodo 30 das, posterior a la ingestin de la virus, el 35% de las colonias de S. invicta adquireron una infeccin de SINV (P = 0,03). Por lo tanto, la microencapsulacin como un mecanismo para entregar el SINV-1 a las colonias de S. invicta fue exitosa. La incorporacin de este mtodo economicamente asequible puede ser implimentado en el futuro para entregar agentes biolgicos para especies especcas de hormigas y para aumentar los mtodos actuales que usan cebo para controlar las hormigas Aunque el virus fue usado para demonstrar la entrega, todavia se necesita desarollar un sistema de produccin adecuada y asequible antes que una estrategia viral pueda ser adoptada como una herramienta de control biolgico para las hormigas de fuego. The red imported re ant ( Solenopsis invicta Buren) invaded North America in the 1930s and since then has become a serious threat to humans and devastated many endemic wildlife species

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238 Florida Entomologist 94(2) June 2011 (Taber 2000). Solenopsis invicta colonies were quic kly established in the USA because of their aggressive behavior, large colony size, and lack of natural predators (Taber 2000). Therefore, a safe and species specic method for reducing ant colony size and population density of S. invicta is desperately needed. A positive single-stranded RNA virus in the Family Dicistroviridae was reported to only infect ants in the S olenopsis genus (Valles et al. 2007). The S. invicta viruses (SINV-1, SINV-2, and SINV -3) and genotypes (SINV-1A and SINV-1 TX5) infect all stages of development and caste members (Valles et al. 2004; Valles & Strong 2005; Valles et al. 2007; Valles & Hashimoto 2009; Tufts et al. 2010). Rapid replication of SINV-1 (TX5) can quickly produce an infected re ant colony. Although acute mortality observed within eld populations of ants under natural viral infection has not been signicant, when the entire colony becomes infected under controlled conditions the colony dies (Valles et al. 2004). Other examples of insects which became infected with single stranded RNA (ssRNA) viruses related to SINV-1 also showed increased mortality and colony collapse, e.g., leafhoppers (Hunter et al. 2006; Hunnicutt et al. 2006) and honey bees ( Apis mellifera L.) (Cox-Foster et al. 2007). These studies suggest that the effect of high virus titers or multiple virus infections ma y be required to cause colony collapse. A virus closely related to SINV-1, the Israeli acute paralysis virus (IAPV), infects honey bees in approximately 90% of apiaries (Johnson 2010). However, only in conjunction with other pathogens (i.e. other viruses or parasites) did it produce colony collapse (Cox-Foster et al. 2007; VanEngelsdorp et al. 2009). Fire ants, which are also hymenopterans, are under increased stress from the implementation of several biological control agents, e.g., phorid ies ( Pseudacteon spp.; Graham et al. 2003), fungi ( Beauveria bassiana ; Baird et al. 2007), and microsporidian protozoan ( Thelohania solenopsae ; Oi & Williams 2002; Oi & Valles 2008). These natural enemies continue to be investigated with limited success with respect to their effective use in reducing re ant populations; therefore, the addition of multiple viral pathogens may increase the efcacy of these currently used approaches. Costly, chemical control programs have been successful, but often displace re ant colonies to surrounding regions, with re-colonization occurring as the chemicals break down and re ant populations increase. The entomopathogenic fungus, B. bassiana w as microencapsulated in sodium alginate pellets and tested as a potential biological control method with success (Bextine & Thorvilson 2002). Beauveria bassiana was delivered to S. invicta colonies by workers that had fed on pellets. The objective of this study w as to develop an effective delivery method that would transfer viral infection to S. invicta colonies. Thus, SINV-1 (TX5) w as encapsulated in pellets and the delivery method assessed. While not tested directly, the potential of this strategy is to provide the means to infect the majority of ants with a high titer of virus, consequently inducing colony collapse. M ATERIALS AND M ETHODS Colony Collection and Viral Detection Solenopsis invicta colonies were collected during Ma y and Aug 2008 from Smith, Cherokee, Hunt, and Gregg Counties, Texas. A total of 58 colonies were collected from Smith County, 15 colonies from Cherokee County, 9 colonies from Hunt County, and 7 colonies from Gregg County; all colonies were of the polygyne phenotype. To rigorously evaluate variability of acceptance of these pellets by re ants, ant colonies were collected from widely disparate locations to increase the probability of including behavioral and genetic variations in the ant colonies tested. All colonies collected were tested for the presence of the Solenopsis invicta virus 1 (SINV-1) by reverse transcription polymerase c hain reaction (RTPCR) and gel electrophoresis. RNA was extracted from entire S. invicta colonies (workers, queens, and brood) using TRIzol reagent (Invitrogen, Carlsbad, CA) following manufacturers protocol. Samples were then tested for virus using a SuperScript One-Step Reverse Transcriptase PCR (RTPCR) kit (Invitrogen, Carlsbad, CA). RT-PCR was completed using a specic primer set (p62 and p63; Valles & Strong 2005) for a short segment (326 bp) of SINV-1 and performed in duplicate (Tufts et al. 2010). Puried, active virus was extracted from colonies which tested positive for SINV-1 using a modied protocol by Hunter, USDA, ARS (Tufts et al. 2010). Development of Sodium Alginate Pellets The virus extract was microencapsulated using a 1% sodium alginate suspension (Bextine & Thorvilson 2002). The solution was prepared by dissolving 2.5 g sodium alginate (Spectrum, Gardena, CA) and 2 g corn meal (Quaker Oats Co., Chicago, IL) in a solution of 10 mL 95% ethanol and 8.5 ml puried virus extract, the mix was then brought to a nal volume of 100 mL using autoclaved, nano-pure ltered water. The gelatinous solution was mixed vigorously for 10 min and then slowly dripped into an aqueous solution of 0.25 M calcium gluconate with a sterile 10 mL disposable pipette. After 5 min, the pellets were strained out of the gluconate solution by a sieve and allowed to dry at 22C on 2 sheets of wax paper for 24 h. Control pellets were produced in the same manner, substituting autoclaved, nano-pure

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Tufts et al.: Viral Pellets as Biological Control for S. invicta 239 ltered water for viral extract. Pellets were observed to shrink substantially from their original size over the following 24 h and were stored in airtight plastic vials at 22C, in the dark. Dried pellets had an average weight of 5.7 mg and an average diameter of 2 mm ( n = 20). Introduction of Pellets to Laboratory Colonies All colonies were tested by RT-PCR for the presence or absence of SINV, protocols as stated above. A sample of 20 worker ants from each colony was tested for SINV presence, for a total of 1780 ants. Ten of the established laboratory colonies which were negative for SINV infection were randomly chosen; 5 with brood present and 5 colonies without brood. All 10 treated colonies were offered 5 virus pellets. An additional 5 colonies, which also tested SINV negative, were chosen and offered 5 control pellets. All pellets (control and treated) were coated with 200 L of Vienna sausage liquid (Libbys, Chicago, IL) and placed approximately 16 cm away from the brood box (14 cm 10 cm 4 cm). A circle drawn around the pellets on the underside of the ant s observation tray (57.5 cm 41 cm 14.5 cm) delineated pellet movement. Throughout the experimental trial period (30 d) 2 samples of 10 ants each (a combination of foragers and workers) were collected every 5 d from each colony (control and treated), resulting in a total of 120 ants tested per colony. RNA was extracted and analyzed immediately for the presence or absence of virus using specic primer sets (p62 and p63; Valles & Strong 2005). Chisquare analysis of SINV positive ants between those with or without brood was performed with pooled data from the experimental groups. The experiment was replicated with pellets (control and treated) that were stored in dark, air tight containers at room temperature (22C) for 12 mo. Fifteen new colonies were collected, screened for SINV presence, and established as before. The experimental period for the aged pellets was 35 d. At the conclusion of the second replicated experiment, queens, current brood, and males from 2 infected colonies were sacriced and tested for the presence of virus. R ESULTS Of the 89 colonies collected only 26 were found to be positive for SINV, 13 from Smith County, 9 from Cherokee County, 2 from Hunt County, and 2 from Gregg County. Overall ant colonies within 2 counties in Texas (Smith and Cherokee, Co) showed a higher incidence of SINV-1 in Cherokee County with 9 of 15 colonies testing positive for SINV (60%), while Smith County had 13 of 58 colonies test positive (22.4%). Sample sizes for Hunt and Gregg, Co were not large enough to make statistical inferences. Ants quickly accepted the pellets and within 24 h of pellet introduction, some colonies had moved most of the pellets into the brood box. By d 15 all colonies including control colonies (100%) had moved all 5 pellets into their brood boxes. By d 5, post-feeding one set of forager/worker ants (20 individuals) tested positive for SINV. By d 10, post-feeding 2 additional colonies (40 individuals) tested positive, and by d 15 post-feeding, forager/worker groups in 4 of the 10 colonies (80 individuals) had tested positive for SINV (Fig. 1). Additionally, none of the control colonies tested positive for SINV infection, during or after the experiment. After 30 d the experiment was terminated, no additional colonies tested positive for SINV after d 15, resulting in a 40% infection rate of foragers/workers tested for the 10 experimental colonies. The presence or absence of Fig. 1. Gel electrophoresis of the RT-PCR from d 15 post-feeding. This gel illustrates 2 samples as duplicates in neighboring lanes, thus displaying 2 of the 4 colonies that were SINV-1 positive over the duration of the testing period (30 d). Each sample depicts 10 forager/worker ants that were offered 2 d old pellets. A 1Kb ladder (TrackIt 1Kb Plus DNA Ladder, Invitrogen, Cat. no. 10488-085) (lanes 1 and 34), ants given control pellets for 5 colonies (lanes 2-11), ants given viral pellets for 10 colonies (lanes 12-31), lanes 24-27 illustrate positive detection of virus for two colonies (~300bp), and negative controls for the RT-PCR (lanes 32-33). Multiple gels are shown.

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240 Florida Entomologist 94(2) June 2011 brood in a colony did not appear to have an effect on colony infection or detection of SINV. For the experiment with aged pellets, it was found that 30% of tested foragers/workers from the colonies tested positive for SINV 5 d postfeeding. No additional colonies tested positive for SINV over the duration of the trial (Fig. 2) and no control colonies were found to be SINV positive. Upon termination of the second experiment, queens, remaining brood, and males did not test positive for the presence of virus. Based on Chisquare analysis, a signicant difference was observed between the virus exposed and unexposed colonies ( 2 = 4.565, df = 1, P = 0.03). D ISCUSSION We have successfully demonstrated an inexpensive method to produce pellets containing ant-infecting virus for delivery to re ants. Pellets with attractive avor and virus were produced by a simple method which provides potential application for increasing the efcacy of currently used biological control agents of S. invicta The virus containing pellets provide substantial evidence that this method ma y have potential, after further renement, as an effective tool to introduce SINV and other viruses to re ants in the eld. The longevity of these viral pellets was shown to be at least 1 year when storing pellets in dark, airtight plastic containers, at room temp (22C). Delivery of virus to multiple colonies for both the freshly prepared and extendedly stored pellets (2 d and 12 mo, respectively) was successful. A crude virus preparation was used to produce these pellets, therefore a precise virus titer was not obtained, calculations based on nanodrop readings showed a 139.5 ng of protein/ L of inoculum was used. Even though a total of 600 ants were sampled in virus exposed colonies, detection of SINV-1 was sporadic when sampling workers that may or may not have yet been exposed to the virus through food sharing, i.e., trophallaxis. A plausible explanation for this anomaly is that SINV-1 was only being ingested and not transferred at 100% success from queen to offspring or worker to worker. However, trials were terminated after 30 d and infections may have persisted. Long term trials are needed but were beyond the current scope of this trial. External contamination is not a likely candidate for explaining infection because colonies that tested positive for SINV-1 remained positive throughout the experimental period. If external contamination were contributing to the detection of infection we would expect more colonies to have tested positive randomly or only during a single sampling time. Queens, males, and brood tested negative for the presence of virus; increasing the likelihood that SINV was not being transferred by contact between individuals but by ingestion alone. An individual may have become infected with the virus and died, but due to rapid mortality under laboratory conditions the virus may not have spread to nest mates. Conversely, the experiment clearly demonstrated that delivery of virus to foragers in a colony using this pellet formulation method was possible. Future studies will evaluate combinations of microencapsulated virus with the fungus, Beauveria bassiana previously shown to also reduce S. invicta colonies (Bextine & Thorvilson 2002) and various c hemical control agents (e.g. Amdro (Tetrahydro-5,5-dimethyl-2(1H)-pyrimidinone [3[4(triuoromethyl)phenyl]-1-[2-[4-(triuoromethyl)phenyl]ethyenyl]-2-propenylidene]hydrazone), and Over-and-Out (5-amino-1-(2,6dichloro-4-(triuoromethyl) phenyl)-4-((1,R,S)(triuromethyl) sulnyl)-1-H-pyrazole-3-carbonitrile)). Given that SINV-1 is taxonomically related to IAPV, adding additional immune stressors to S. invicta colonies may be able to induce a colony collapse effect, thus effectively decreasing populations of S. invicta Fig. 2. Gel electrophoresis of the RT-PCR from d 5 presented in duplicate, illustrating 3 different colonies with positive bands for viral infection. Each sample depicts 10 forager/worker ants that were offered 12-month-old pellets. A 1Kb ladder (TrackIt 1Kb Plus DNA Ladder, Invitrogen, Cat. no. 10488-085) (lanes 1 and 34), ants given control pellets for 5 colonies (lanes 2-11), ants given viral pellets for 10 colonies (lanes 12-31), lanes 22-23 and 2831 conrm positive detection of virus for 3 colonies (~300bp), and negative controls for the RT-PCR (lanes 32-33). Multiple gels are shown.

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Tufts et al.: Viral Pellets as Biological Control for S. invicta 241 A CKNOWLEDGMENTS We thank our anonymous reviews for constructive criticism and Christopher Powell for assistance in the laboratory. Funding was provided by a University of Texas at Tyler research grant. The mention or use of products within does not imply nor guarantee an endorsement by the USDA, ARS, to the exclusion of other similar, suitable products. R EFERENCES C ITED B AIRD R., W OOLFOLK S., AND W ATSON C. E. 2007. Survey of Bacterial and Fungal Associates of Black/Hybrid Imported Fire Ants from Mounds in Mississippi. Southeast Nat. 6(4): 615-632. B EXTINE B. R., AND T HORVILSON H. G. 2002. Field applications of bait-formulated Beauveria bassiana alginate pellets for biological control of the red imported re ant (Hymenoptera: Formicidae). Environ Entomol 31(4): 746-752. C OX -F OSTER D. L., et al. (21) 2007. A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318: 283-287. G RAHAM L. C., P ORTER S. D., P EREIRA R. M., D OROUGH H. D., AND K ELLEY A. T. 2003. Field releases of the decapitating y Pseudacteon curvatus (Diptera: Phoridae) for control of imported re ants (Hymenoptera: Formicidae) in Alabama, Florida, and Tennessee. Florida Entomol. 86(3): 334-339. H UNTER W. B., K ATSAR C. S., AND C HAPARRO J. X. 2006. Nucleotide sequence of 3 -end of Homalodisca coagulata V irus-1. A new leafhopper-infecting virus from the glassy-winged sharpshooter J. Insect Sci. 6.28 (Online: insectscience.org/6.28/). H UNNICUTT L. E., H UNTER W. B., C AVE R. D., P OWELL C. A., AND MOZORUK, J. J. 2006. Complete genome sequence and molecular characterization of Homalodisca coagulata virus-1, a novel virus discovered in the glassy-winged sharpshooter (Hemiptera: Cicadellidae). Virology 350: 67-78. JOHNSON, R. 2010. Honey Bee Colony Collapse Disorder. Congressional Research Service Report for Congress (www.crs.gov). OI, D. H., AND WILLIAMS, D. F. 2002. Impact of Thelohania solanapsae (Microsporidia: Thelohaniidae) on polygyne colonies of red imported re ants (Hymenoptera: Formicidae). J. Econ. Entomol. 87: 623630. OI, D. H., AND VALLES, S. M. 2008. Fire ant control with entomopathogens in the USA, pp. 237-257 In H. M. T. Hokkanen, A. E. Hajek, T. R. Glare, and M. OCallaghan [eds.], Use of Microbes for Control and Eradication of Invasive Arthropods. Springer Netherlands (doi: 10.1007/978-1-4020-8560-4). TABER, S. W. 2000. Fire Ants. Texas A&M University Press, College Station. TUFTS, D. M., HUNTER, W. B., AND BEXTINE, B. 2010. Discovery and effects of the Solenopsis invicta virus (SINV-TX5) on red imported re ant populations. J. Invertebr. Pathol. 104: 180-185. VALLES, S. M., et al. (7) 2004. A picorna-like virus from the red imported re ant, Solenopsis invicta : initial discovery, genome sequence, and characterization. J. Virol. 328: 151-157. VALLES, S. M., AND STRONG, C. A. 2005. Solenopsis invicta virus-1A (SINV-1A): Distinct species or genotype of SINV-1? J. Invertebr. Pathol. 88: 232-237. VALLES, S. M., et al. (20) 2007. Phenology, distribution, and host specicity in Solenopsis invicta virus-1. J. Invertebr. Pathol. 96: 18-27. VALLES, S. M., AND HASHIMOTO, Y. 2009. Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported re ant, Solenopsis invicta. Virology 388: 354-361. VANENGELSDORP, D., et al. (12) 2009. Colony collapse disorder: A descriptive study. PLoS One 4(8): e6481. Doi:10.1371/journal.pone.0006481.

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242 Florida Entomologist 94(2) June 2011 SUGARCANE PLANTING DATE IMPACT ON FALL AND SPRING SUGARCANE BORER (LEPIDOPTERA: CRAMBIDAE) INFESTATIONS J. M. B EUZELIN 1 A. M SZROS 1 W. A KBAR 1,2 AND T. E. R EAGAN 1 1 Department of Entomology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA 2 Current address: Monsanto Company, 700 Chesterfield Pkwy West GG3E, Chesterfield, MO 63017, USA A BSTRACT In a two-year eld study, sugarcane was planted on 4 dates ranging from the rst week of Aug to the third week of Nov, reproducing sugarcane phenologies associated with planting and harvesting operations in Louisiana. Sugarcane planted in early Aug offered an extended period of plant availability for sugarcane borer, Diatraea saccharalis (F.), infestations during the fall. Periodic sampling throughout the fall showed that early Aug plantings had higher ( P < 0.05) D. saccharalis -caused deadheart densities than later planted sugarcane. Destructive sampling conducted in early Oct showed that Aug plantings harbored greater deadheart densities ( P < 0.05 in fall 2007) and D. saccharalis infestations ( P < 0.05 in fall 2006 and 2007) than Sep plantings Data from this study suggest a potential for increased D. saccharalis overwintering populations in early plantings associated with greater infestations during the fall. However, differences in deadhearts and D. saccharalis infestations in deadhearts were not detected ( P > 0.05) during the spring. Three commercial sugarcane cultivars (L 99-226, L 97-128, HoCP 95-988) were studied. Differences in D. saccharalis injury or infestations as affected by cultivar were detected ( P < 0.05) only in early Oct 2007 when HoCP 95-988 harbored 2.3-fold greater infestations than L 99-226. Key Words: Diatraea saccharalis (F.), cultural practices, sugarcane IPM R ESUMEN En un estudio de campo de dos aos, se sembro caa de azcar en cuatro fechas desde la primera semana de agosto hasta la tercera semana de noviembre, que reproduce la fenologa de la caa de azcar asociada con las operaciones de siembra y cosecha en Louisiana. La caa de azcar sembrada en el principio de agosto ofreci un perodo extenso de disponibilidad de la planta para infestaciones por el barrenador de la caa, Diatraea saccharalis (F.), durante el otoo Muestras peridicas tomadas durante el otoo mostr que las siembras del principio de agosto tuvieron una mayor densidad (P < 0.05) de caas con corazones muertos causados por D. saccharalis que la caa de azcar sembrada mas tarde. El muestreo destructivo realizado en el principio de octubre mostr que las siembras de agosto albergaba ma yores densidades de corazones muertos (P < 0.05 en el otoo de 2007) e infestaciones de D. saccharalis (P < 0.05 en el otoo de 2006 y 2007) que las siembras de septiembre. Los datos de este estudio sugieren un posible aumento de poblaciones invernantes de D. saccharalis en siembras tempranas asociadas a una ma yor infestacin durante el otoo. Sin embargo, no se detectaron (P > 0.05) diferencias en los corazones muertos y las infestaciones de D. saccharalis en los corazones muertos durante la primavera. Se estudiaron tres variedades comerciales de caa de azcar (L 99-226, L 97-128, HoCP 95-988). Se detectaron (P < 0.05) diferencias en el dao causado por D. saccharalis o de infestaciones afectadas por el tipo de variedad solamente al principio de octubre del 2007, cuando HoCP 95-988 albergaba las infestaciones de 2.3 veces mayor que la L 99-226. The sugarcane borer, Diatraea saccharalis (F.), historically has been the most damaging arthropod in Louisiana sugarcane (hybrids of Saccharum L. spp .) (Hensley 1971; Reagan 2001). With the widespread use of susceptible high-yielding sugarcane cultivars, current D. saccharalis management is ac hieved by judiciously timed chemical control of economically damaging infestations, conservation of natural enemies, and cultural practices (Posey et al. 2006; Beuzelin et al. 2009, 2010). In Louisiana, sugarcane is grown in a 4to 6year rotation cycle, i.e., 3 to 5 crops are harvested from a single planting and are followed by a fallow period (Salassi & Breaux 2002). Sugarcane vegetative seed pieces are planted from Aug to Oct, with the traditional peak in Sep. However, as farms grow larger and more diversied, planting operations have become less exible due to simultaneous harvesting and planting activities (Garrison et al. 2000). In addition, late season production of sugarcane seed pieces has become more challenging due to early lodging of recently developed cultivars. Therefore, producers currently plant both earlier and later in the growing season

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Beuzelin et al.: Sugarcane Planting Dates and the Sugarcane Borer243 (Garrison et al. 2000; Viator et al. 2005b). Planting borer-free sugarcane seed pieces is a recommended D. saccharalis management tactic to reduce overwintering populations (LSU AgCenter 2010). Because of the onset of low temperatures beginning about mid-Nov, the growing and milling seasons are approximately 9 months and 3 to 4 months, respectively. Thus, harvest in Louisiana begins in Sep and is completed by early Jan. Sugarcane stalks are harvested close to the soil surface, and growers may leave post-harvest crop residue in the eld. Diatraea saccharalis larvae infesting crop residues at that time are exposed to cold temperatures and natural enemies which increase overwintering mortality (Kirst & Hensley 1974, Bessin & Reagan 1993). Sugarcane stubble in fallow elds should be plowed out as quickly as possible to reduce the number of overwintering larvae (LSU AgCenter 2010). For non-fallow elds, burning of crop residue occurs mostly in the early spring. With standard sugarcane management practices, early planting typically provides a better root establishment and higher yields (Viator et al. 2005a). Viator et al. (2005b) conducted a study to determine how Aug, Sep, and Oct planting dates impacted the yield of 5 sugarcane cultivars in Louisiana. Plant cane sugar yields for cultivar LCP 85-384 did not differ with planting dates, whereas for HoCP 85-845 and CP 70-321 sugar yields were higher for the Aug planting date. Charpentier & Mathes (1969) reported that elds planted in Aug show increased D. saccharalis infestations because they are highly suitable for moth oviposition. Fall sugarcane shoots (plant cane crop) and fall stubble (ratoon cane crop) are not considered to be D. saccharalis overwintering habitats but can serve as means of entry for lar vae into seed pieces and stubble portions underground where overwintering occurs (Kirst 1973). The earlier sugarcane is planted or harvested, the greater the period of time during the late summer and fall that shoots are available for D. saccharalis oviposition and larval establishment. Early planted and early harvested elds ma y, therefore, represent a substantial refuge for overwintering D. saccharalis and serve as a source of borers in the spring Two eld experiments were conducted between 2006 and 2008 to determine the effect of sugarcane eld phenology associated with planting and harvesting dates on D. saccharalis infestations from the fall to the spring M ATERIALS AND M ETHODS Planting Date Experiment 2006-2007 A eld experiment was conducted from 2006 to 2007 near Patoutville (N 29.872, W 91.744) in Iberia Parish, LA. A randomized split-plot complete block design with 10 blocks (1 replication per block) was used. Each block was 36.9 m long and 11.0 m wide (6 rows) with 4 main plots, each containing 2 subplots. The range of phenological conditions occurring throughout the Louisiana sugarcane industry was mimicked by assigning early Aug, early Sep, early Oct, and late Nov planting dates to main plots. Each main plot was 6.4 m long and 11.0 m wide (6 rows), separated by a 1.2-m gap. Subplots were planted either with cultivar L 97-128 ( D. saccharalis susceptible, White et al. 2008) or L 99-226 ( D. saccharalis moderately resistant, White et al. 2008). Each subplot w as 6.4 m long and 3 rows wide. Sugarcane was planted as whole stalks on Aug 4, Sep 2, Oct 5, and Nov 22 at a density of 6 stalks per 6.4m row. For each subplot, sugarcane density (shoot counts) and growth (height) were recorded from the center row during subsequent planting dates. On the third planting date (Oct), the number of D. sacc haralis -caused deadhearts was recorded from the center row of eac h subplot for the rst and second planting dates. Deadhearts are shoots with dead whorl leaves caused by herbivores damaging the apical meristem before above ground internodes are formed (Bessin & Reagan 1993). Insects such as the lesser cornstalk borer ( Elasmopalpus lignosellus (Zeller) Lepidoptera: Pyralidae) and wireworms (Coleoptera: Elateridae) also cause deadhearts in sugarcane. Therefore, only deadhearts exhibiting entrance holes and frass characteristic of D. saccharalis but no silken tubes (c haracteristic of E. lignosellus ), were recorded. Additionally, a 2.1-m long section of row w as randomly selected from 1 outer row of each subplot, and plants from this section were destructively sampled for D. saccharalis The number of injured shoots injured shoots turned into deadhearts, as well as the abundance and size of D. saccharalis immatures found within the injured shoots were recorded. The size of D. saccharalis larvae was visually determined, with small, intermediate, and large larvae corresponding approximately to rst-second, third, and fourth-fth instars, respectively. On the fourth planting date (Nov), the number of D. sacc haralis -caused deadhearts was recorded from the center row of eac h subplot from the rst, second, and third planting dates. The following spring (May 18 and Jun 7), numbers of shoots and deadhearts found in the center row were recorded. Deadhearts were collected and dissected for D. saccharalis immatures, whose number and size were recorded. Planting Date Experiment 2007-2008 A second eld experiment was conducted from 2007 to 2008 near Bunkie (N 30.950, W 92.163) in Avoyelles Parish, LA. A randomized split-plot complete block design with 4 blocks (1 replication per block) was used. Each block was 53.6 m long

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244 Florida Entomologist 94(2) June 2011 and 14.6 m wide (8 rows), and contained 4 main plots, 1 for each planting date. Main plots were 12.5 m long and 14.6 m wide (8 rows), separated by a 1.2-m gap. Subplots were planted with cultivar HoCP 95-988 ( D. saccharalis susceptible, White et al. 2008) or L 99-226. Each subplot was 12.5 m long and 7.3 m wide (four rows). Sugarcane was planted as whole stalks, at a density of 14 to 20 stalks per 12.5-m row, on Aug 6, Sep 5, Oct 10, and Nov 21. Sugarcane emergence and growth data collection was conducted on the 2 center rows of each subplot in the same manner as that of the 2006-2007 experiment. On the third planting date, the number of D. saccharalis caused deadhearts w as recorded from the 2 center rows of each subplot from the rst and the second planting dates. Additionally, sugarcane shoots for each subplot were examined from one randomly selected outer row. The number of injured shoots, injured shoots turned into deadhearts, and the abundance and size of D. saccharalis immatures found within the injured shoots were recorded. On the fourth planting date the number of D. sacc haralis -caused deadhearts was recorded from the 2 center rows of eac h subplot from the rst, second, and third planting dates. The following spring (May 12 and 28), numbers of shoots and deadhearts found in the 2 center rows were recorded. Deadhearts were collected and dissected for D. saccharalis immatures, with immature number and larval size recorded. Data Analyses Data from experiments initiated in 2006 and 2007 were analyzed separately. Analyses of variance (ANOVAs) were conducted with Proc GLIMMIX (SAS Institute 2008), and linear regressions were conducted by Proc REG (SAS Institute 2008). Data collected in early Oct from destructive sampling ( D. saccharalis -caused deadheart, D. saccharalis -injured shoot, and D. saccharalis immature counts), and data collected during the spring (shoot, D. saccharalis -caused deadheart, and D. saccharalis immature counts) were compared in two-w ay ANOVAs with planting date and cultivar as factors. Shoot count, plant size, and deadheart count data collected from periodic sampling of subplot center rows during the fall were compared by three-way repeated measures ANOVAs with planting date, cultivar, and observation date as factors. A variance component covariance structure was used to model the effects of repeated measures. In the experiment initiated in 2007, each of the 2 subplot center rows was considered a sampling unit. The Kenward-Roger adjustment for denominator degrees of freedom was used in all the ANOVA models to correct for inexact F distributions (Proc GLIMMIX, SAS Institute 2008). When ANOVA effects were detected ( P < 0.05), least square means were separated by the least signicant difference (LSD = 0.05). Least square means standard errors on a per hectare basis are reported. Linear regressions were conducted to deter mine whether a relationship between D. saccharalis and deadheart counts (recorded from destructive sampling in early Oct) w as detected. In addition, linear regressions between fall (late Nov) and spring deadheart counts (recorded from subplot center rows) were conducted to investigate the relationship between end and beginning of the year D. saccharalis infestations in newly planted sugarcane R ESULTS Sugarcane Availability Planting date, observation date, and planting date by observation date interaction effects were detected ( P < 0.05) for plant availability estimates (shoot density and plant height) from periodic sampling during the fall of 2006 and 2007 (T able 1). In 2006, differences in shoot densities between cultivars L 99-226 and L 97-128 were not detected ( F = 0.00; df = 1,54; P = 0.984). August plantings had 33,178 1,764 shoots/ha (LS mean SE) by early Sep In early Oct, Sep plantings had emerged with 47% lower shoot densities (Fig. 1) than the Aug plantings. In late Nov, the Oct plantings had the lowest shoot densities, 5.1fold and 2.9-fold less than Aug and Sep plantings, respectively. Plant height followed a pattern similar to that observed for shoot density (Fig. 1). In early Sep, Aug plantings measured 47.0 1.3 cm (LS mean SE). By late Nov, the Oct plantings had the smallest plants, 3.7-fold and 2.3-fold smaller than Aug and Sep plantings, respectively. In addition to a numerical trend ( F = 3.19; df = 1,27; P = 0.085) for L 99-226 plants being taller than L 97-128 plants, a signicant cultivar by planting date two-way interaction was detected ( F = 7.87; df = 2,27; P = 0.002). L 99-226 plants from Aug plantings were 9% taller than L 97-128 plants whereas cultivar differences were not detected in other plantings. Whereas shoots growing from the rst 3 plantings were available during the fall, shoots from the Nov plantings did not emerge until the following year (Fig. 1). Shoot density and plant height during the fall of 2007 showed patterns comparable to those observed in 2006, with early plantings having increased availability and the last planting not emerging until the following year (Fig. 1). In early Sep, the Aug plantings had 53,808 2,538 shoots/ ha that measured 50.7 1.9 cm. In late Nov, Aug plantings shoot density was 1.4-fold and 10.9-fold greater than that of Sep and Oct plantings, respectively. August plantings were 1.9-fold and 5.9-fold taller than those from Sep and Oct plantings, respectively. Shoot density and plant height

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Beuzelin et al.: Sugarcane Planting Dates and the Sugarcane Borer245 were also affected by cultivar ( F = 5.41; df = 1,18; P = 0.032 and F = 49.99; df = 1,9; P < 0.001, respectively), with L 99-226 showing greater density (13%) and height (23%) than HoCP 95-988. However, two-way and three-way interactions involving cultivar effects also were detected ( P < 0.05). Although L 99-226 generally had higher shoot densities than HoCP 95-988 (Fig. 1), the cultivar by collection date interaction ( F = 3.38; df = 2,84; P = 0.039) and the planting date by collection date by cultivar ( F = 12.34; df = 4,84; P < 0.001) interaction showed that differences in shoot density between L 99-226 and HoCP 95988 at each collection date changed to varying extents for each planting date (Fig. 1). For Aug plantings, L 99-226 had 50% higher shoot densities than HoCP 95-988 in early Sep; however, differences were not detected (LSD P > 0.05) during later sampling For Sep plantings, L 99-226 had 39 and 31% higher shoot densities than HoCP 95988 in early Oct and late Nov, respectively. For Oct plantings, differences in shoot densities between L 99-226 and HoCP 95-988 in late Nov were not detected (LSD P > 0.05). The cultivar by collection date (F = 4.66; df = 2,108; P = 0.011), cultivar by planting date (F = 9.45; df = 2,9; P= 0.006), and the three-way (F = 2.95; df = 4,108; P = 0.023) interactions showed that differences in plant height between L 99-226 and HoCP 95988 at each collection date changed to varying extents for each planting date (Fig. 1). For Aug plantings, L 99-226 was 35, 22, and 13% taller than HoCP 95-988 in early Sep, early Oct, and late Nov, respectively. For Sep plantings, L 99226 was 24 and 26% taller than HoCP 95-988 in mid-Oct and late Nov, respectively. For Oct plantings, L 99-226 was 51% taller than HoCP 95-988 in late Nov.Diatraea saccharalis Fall InfestationsPlanting date, collection date, as well as planting date by observation date two-way interaction effects were detected (P < 0.05) for D. saccharalis caused deadheart densities from periodic sampling during the fall of 2006 and 2007 (Table 1). Differences in deadheart densities as affected by sugarcane cultivar were not detected ( F = 0.26; df = 1,54; P = 0.614 in 2006 and F = 0.51; df = 1,9; P = 0.492 in 2007). In early Sep, deadhearts in Aug plantings were not observed in 2006 and 2007 (Fig. 2). In early Oct, Aug plantings had higher deadheart densities than Sep plantings (4,313 vs. 43 and 1,093 vs. 0 deadhearts/ha in 2006 and 2007, respectively). In late Nov 2006, Oct plantings had the lowest deadheart densities, 37.8-fold and 9.8-fold less than Aug and Sep plantings, respectively. September plantings had intermediate deadheart densities, 3.9-fold less than Aug plantings (Fig. 2). Diatraea saccharalis adult emergence holes, indicating life cycle completion, were observed in deadhearts from sugarcane planted in Aug (641 1,069 exit holes/ha [mean SD]). In late Nov 2007, deadhearts were not observed in Oct plantings whereas early Sep plantings had 13.0-fold less deadhearts than Aug plantings (Fig. 2). In early Oct 2006, after shoot examination and destructive sampling from border rows of Aug and Sep plantings, differences in deadheart densities were not detected (Table 2). Even in the absence of deadheart symptoms, some sugarcane shoots were injured with D. saccharalis feeding signs in leaf sheaths and boring into the stem. The density of these nondeadheart injured sugarcane shoots was greater (2.3-fold) in Aug vs. Sep plantings (Ta-TABLE 1. SELECTED STATISTICAL COMPARISONS FOR SHOOT DENSITIES, PLANT HEIGHT, AND DEADHEART DENSITIES FROM SUGARCANE PLANTED ON 4 DATES RANGING FROM EARLY AUG TO LATE NOV, 2006 AND 2007. Comparison Fall 2006 Fall 2007 F df P > FF df P > F Shoot density Planting date 746.462,54<0.001504.342,18<0.001 Observation date 993.332,108<0.001541.072,84<0.001 Planting date Observation date 105.034,108<0.001115.354,84<0.001 Plant height Planting date 1047.712,18<0.001853.932,6<0.001 Observation date 1141.932,108<0.001890.502,108<0.001 Planting date Observation date 74.334,108<0.001113.464,108<0.001 Deadheart density Planting date 54.232,54<0.00111.672,90.003 Observation date 20.811,54<0.00113.131,42<0.001 Planting date Observation date 4.202,540.0208.492,42<0.001

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246 Florida Entomologist 94(2)June 2011ble 2). In addition, there were differences in D. saccharalis infestations (Table 2), with Aug plantings harboring 4.7-fold more borers than Sep plantings. Differences between cultivars L 99-226 and L 97-128 for deadheart densities, non-deadheart injured shoot densities, and D. saccharalis infestations were not detected ( P > 0.05, Table 2). Among the D. saccharalis larvae that were collected in Aug and Sep plantings, 25 and 27% were small, 40 and 18% were intermediate, 35 and 55% were large, respectively. A linear regression ( F = 9.09; df = 1,38; P = 0.005; R2 = 0.193) showed that D. saccharalis infestations in early Oct (dependent variable) were Fig. 1. A) Sugarcane shoot density (LS means SE) and B) plant height (LS means SE) during the fall fro m planting date eld experiments in Patoutville, LA (2006) and Bunkie, LA (2007). *Cultivar L 97-128 for 2006 plantings and HoCP 95-988 for 2007 plantings.

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Beuzelin et al.: Sugarcane Planting Dates and the Sugarcane Borer247positively correlated with deadheart densities (slope: 0.694, 95% C.I. = 0.228, 1.161; intercept: 0.655, 95% C.I. = -0.331, 1.642). In early Oct 2007, shoot examination and destructive sampling from border rows showed that more D. saccharalis -caused deadhearts (24.0fold) occurred in Aug than in Sep plantings (Table 2). There was a numerical trend for greater deadheart differences between Aug and Sep plantings in cultivar HoCP 95-988 (P < 0.10 for the planting date by cultivar two-way interaction, Table 2) than in L 99-226. More D. saccharalis larvae were collected in Aug than in Sep plantings (19.0fold), and in HoCP 95-988 than in L 99-226 (2.3fold). The signicant ( P < 0.05) planting date by cultivar interaction showed that differences in D. saccharalis infestations between Aug and Sep plantings occurred to a greater extent in cultivar HoCP 95-988 than in L 99-226 (Table 2). Among the D. saccharalis larvae that were collected from Aug plantings, 3, 11, and 86% were small, intermediate, and large, respectively. All larvae recovered from Sep plantings were large. A linear regression ( F = 241.60; df = 1,14; P < 0.001; R2 = 0.945) showed that D. saccharalis infestations in early Oct (dependent variable) were positively correlated with deadheart densities (slope: 0.500, 95% C.I. = 0.431, 0.569; intercept: 0.158, 95% C.I. = -0.396, 0.712). Destructive sampling data collected in Oct 2006 did not differentiate D. saccharalis in deadhearts from D. saccharalis in nondeadheart injured shoots. However, data from 2007 showed that 68% of recovered borers were infesting deadhearts from the Aug planting date. Despite the presence of deadhearts, all D. saccharalis larvae collected from the Sep planting date were feeding in non-deadheart injured shoots.Diatraea saccharalis Spring InfestationsDifferences in sugarcane shoot densities during the spring changed with planting dates (Table 3, Fig. 3). During the spring of 2007 and 2008, sugarcane planted in Aug (2006 and 2007, respectively) had higher shoot densities than that planted in Sep (14 and 25%, respectively), Oct (51 and 76%, respectively), and Nov (87 and 97%, respectively). Sugarcane planted in Sep (2006 and 2007) had higher shoot densities than that planted in Oct (33 and 41%, respectively) and Nov (65 and 58%, respectively). However, the effect of planting dates during the spring of 2007 occurred to a different extent in L 99-226 vs. L 97-128 (Fig. 3), as shown by the signicant two-way planting date by cultivar interaction (Table 3). In addition, shoot densities in L 99-226 plots were 30% higher than those in HoCP 95-988 plots during the spring of 2008 (Fig. 3). Differences in deadheart densities and D. saccharalis infestations from deadhearts during the spring were not detected among planting dates (Table 3). Among D. saccharalis immatures infesting deadhearts during the spring of 2007, 25% were intermediate, 71% were large, and 4% were Fig. 2. Diatraea saccharalis-caused deadheart densities (LS means SE) during the fall in sugarcane fro m planting date eld experiments in Patoutville, LA (2006) and Bunkie, LA (2007). *Cultivar L 97-128 for 2006 plantings and HoCP 95-988 for 2007 plantings.

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248 Florida Entomologist 94(2)June 2011TABLE 2.DEADHEART DENSITIES, NON-DEADHEART INJURED SHOOT DENSITIES, AND D. SACCHARALIS INFESTATIONS (LS MEAN/HA SE) OBSERVED IN EARLY OCT FROM SUGARCANE PLANTED IN EARLY AUG AND EARLY SEP 2006 AND 2007. Fall 2006Fall 2007 Sugarcane Deadheart density Non-deadheart injured shoot density D. saccharalis density Deadheart density Non-deadheart injured shoot density D. saccharalis density Planting date Early Aug 1,196 3842,306 422 a2,220 541 a3,933 990 a819 3262,076 432 a Early Sep 1,068 384 982 422 b 470 541 b164 990 b 55 326 109 432 b F10.06 4.92 5.24 7.25 3.59 12.46 P > F0.8170.0330.0340.0360.1550.039 Cultivar L 99-2261,110 3311,708 4221,324 4811,475 786492 274656 362 b L 97-128/HoCP 95-98821,153 3311,580 422 1,366 4812,622 786 382 2741,530 362 a F30.01 0.05 0.01 2.57 0.32 8.73 P > F0.9110.8310.9430.1600.5950.026 Planting date Cultivar Early Aug L 99-2261,110 4682,477 5972,050 6802,622 1,112874 3541,093 480 b L 97-128/HoCP 95-98821,281 4682,135 597 2,391 6805,244 1,112765 3543,059 480 a Early Sep L 99-226 1,110 468 939 597 598 680328 1,112109 354 219 480 b L 97-128/HoCP 95-98821,025 4681,025 597 342 680 0 1,112 0 354 0 480 b F30.11 0.13 0.26 4.25 0.00 13.64 P > F 0.739 0.723 0.615 0.085 1.000 0.0101df = 1,18; 1,36; 1,18; 1,6; 1,3; and 1,3, respectively,2Cultivar L 97-128 for fall 2006 and HoCP 95-988 for fall 2007,3df = 1,18; 1,36; 1,18; 1,6; 1,6; and 1,6, respectively. LS means in columns followed by the same letter are not different (LSD, = 0.05).

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Beuzelin et al.: Sugarcane Planting Dates and the Sugarcane Borer249pupae. Pupae were recovered from deadhearts collected from Sep and Nov plantings. Among D. saccharalis larvae infesting deadhearts during the spring of 2008, 26% were intermediate and 74% were large. No pupae were recovered. Linear regressions conducted on data from experiments initiated in 2006 and 2007 did not detect a correlation (F = 0.30; df = 1,78; P = 0.583; R2 = 0.004 and F = 3.74; df = 1,62; P = 0.058; R2 = 0.057, respectively) between deadheart densities observed during the fall (late Nov) and the subsequent spring (May-June). DISCUSSIONIn this two-year study, sugarcane was planted on 4 dates from the rst week of Aug to the third week of Nov to reproduce sugarcane phenologies associated with planting and harvesting operations in Louisiana. Because several crops are harvested from a single planting, 25-30% of the Louisiana sugarcane production area is replanted each year with vegetative seed pieces produced from the harvest of 6.5% of the acreage (Legendre & Gravois 2001, 2006, 2010). This study showed that sugarcane elds planted (or harvested) in early Aug offer an extended period of plant availability for D. saccharalis infestations, with higher shoot densities and taller plants (increased biomass) than elds planted (or harvested) later in the summer or fall. Late Nov plantings did not produce vegetation until the following spring, suggesting that sugarcane elds planted (or harvested) after late Nov preclude the growth of aTABLE 3.STATISTICAL COMPARISONS FOR SHOOT DENSITIES, DEADHEART DENSITIES, AND D. SACCHARALIS INFESTATIONS IN DEADHEARTS FROM SUGARCANE PLANTED ON 4 DATES RANGING FROM EARLY AUG TO LATE NOV. Spring 2007Spring 2008 ComparisonF df P > FF df P > F Shoot density Planting date 38.433,27<0.00119.263,24<0.001 Cultivar 5.501,360.02513.581,240.001 Planting date Cultivar 15.623,36<0.0010.523,240.675 Deadheart density Planting date 0.803,720.4971.513,90.277 Cultivar 1.081,720.3030.491,440.486 Planting date Cultivar 0.553,720.6472.073,440.118 D. saccharalis density Planting date 1.163,360.3370.973,90.448 Cultivar 0.281,360.6010.001,441.000 Planting date Cultivar 1.543,360.2211.753,440.170 Fig. 3. Shoot densities, deadheart densities, and D. saccharalis infestations in deadhearts (LS means SE) dur ing the spring from sugarcane planted on 4 dates ranging from early Aug to late Nov, 2006 and 2007. Planting dates within a year followed by the same letter are not different (LSD, = 0.05). *Cultivar L 97-128 for 2006 plantings and HoCP 95-988 for 2007 plantings.

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250 Florida Entomologist 94(2)June 2011suitable host substrate for D. saccharalis oviposition. Sampling throughout the fall showed that early Aug plantings had higher D. saccharalis deadheart densities than later planted sugarcane. This suggests that sugarcane earlier availability and greater biomass associated with early plantings increased D. saccharalis infestations. Destructive sampling conducted in early Oct conrmed that greater deadheart densities were associated with higher D. saccharalis infestations. Although Charpentier & Mathes (1969) commented that Aug planting dates were associated with increases in D. saccharalis infestations in Louisiana, our study is the rst to quantify and compare fall infestations in newly planted sugarcane under current Louisiana production practices. Data from this study suggested a potential for increased D. saccharalis overwintering populations in early plantings associated with greater infestations during the fall. However, differences in deadhearts and D. saccharalis infestations in deadhearts were not detected during the spring. Four to 5 overlapping D. saccharalis generations occur annually in Louisiana (Hensley 1971). After being induced within the rst 2 larval stadia (Roe et al. 1984), D. saccharalis enters a form of diapause as a large larva, with a peak incidence (63 to 71% of eld populations) between Oct and Dec under Louisiana conditions (Katiyar & Long 1961). Although crop residues that are left in the eld after harvest may initially be infested with larvae, they decay rapidly and do not serve as habitat for overwintering D. saccharalis populations (Kirst & Hensley 1974). The main overwintering habitats are underground portions of vegetative seed pieces and stubble. Because D. saccharalis larvae can use fall shoots to gain access to their underground overwintering habitat (Kirst & Hensley 1974) and greater fall infestations were found in early plantings, differences in deadhearts and D. saccharalis infestations were expected during the spring. Deadheart incidence estimates the level of D. saccharalis infestations that occur during the spring in sugarcane (Bessin & Reagan 1993). Diatraea saccharalis larvae found in spring deadhearts from our study were a combination of intermediate and large larvae, indicating that both overwintering and rst generation borers were infesting the deadhearts. Although deadhearts provide appropriate estimates for D. saccharalis spring infestations, they were not adequate for determining infestations that had successfully overwintered in newly planted sugarcane. In addition, the small size of our experimental plots likely increased the redistribution rate of adults among plots in the late fall and spring, thus mitigating potential differences in overwintering larval infestations. Red imported re ants (Solenopsis invicta Buren), the primary D. saccharalis natural enemies in Louisiana sugarcane (Bessin & Reagan 1993; Beuzelin et al. 2009), were not articially suppressed and may also have increased variability in spring D. saccharalis infestations. Some overwintering mortality factors (i.e., temperature, ooding) likely impacted overwintering populations to the same extent regardless of D. saccharalis densities. However, density dependent mortality factors (i.e., predation, parasitism) may have decreased infestations to a greater extent in more heavily infested sugarcane. Because of methodological weaknesses and potential interactions among overwintering mortality factors, a better assessment of overwintering populations should have been conducted during the winter and spring. During the experiment initiated in 2006, destructive sampling of underground seed pieces was conducted in Jan from 2.1-m long sections of border row for each subplot. Only one overwintering D. saccharalis larva was recovered and sampling was extremely labor intensive. The use of eld cages collecting moths emerging from overwintering larvae may assist in better determining the role of sugarcane phenology during the fall on D. saccharalis overwintering populations (e.g., Kr et al. 1989). Although a practice of some insect pest management programs (Pedigo 2002), the manipulation of planting dates is more often associated with the agronomic management of crops. Because sugarcane stalks are the shortest in Aug, greater areas have to be harvested for seed piece production to achieve optimal planting rates. However, seed pieces are easier to harvest and plant in Aug before sugarcane stalks bend due to lodging (Viator et al. 2005a, 2005b). In addition, early planted sugarcane tends to produce higher yields (i.e., cane tonnage, sucrose concentration, sugar yield) associated with better root establishment (Viator et al. 2005a, 2005b; Hoy et al. 2006). Nevertheless, the effect of planting dates on yields is dependent on cultivar, with cultivar-specic optimal planting dates. Different cultivars may also show varying degrees of yield response to planting dates. In addition, planting date effects on yields vary with planting methods (Viator et al. 2005a; Hoy et al. 2006). In our study, sugarcane was planted as whole stalks. Louisiana growers also plant sugarcane as billets (stalk sections of 50-60 cm, Viator et al. 2005a). The yield response to planting dates of billetvs. whole stalk-planted sugarcane seems less consistent (Viator et al. 2005a; Hoy et al. 2006). Whereas early planted sugarcane may increase regional D. saccharalis populations during the spring, better root establishment and greater biomass may help compensate for borer injury during the spring, which might help protect yields. Early planting dates have also been reported to reduce losses associated with root injury from wireworms (Charpentier & Mathes 1969).

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Beuzelin et al.: Sugarcane Planting Dates and the Sugarcane Borer251L 99-226, L 97-128, and HoCP 95-988 are 3 commercial sugarcane cultivars, respectively, grown over 11, 17, and 5% of the Louisiana sugarcane production area (Legendre & Gravois 2010). These cultivars have shown varying levels of resistance to D. saccharalis (White et al. 2008) and differences in shoot population and growth during the fall and spring were observed in our study. However, differences in D. saccharalis injury or infestations as affected by cultivar were only detected in early Oct 2007 when HoCP 95-988 harbored greater (2.3-fold) infestations than L 99226. In a previous study, Bessin & Reagan (1993) observed greater deadheart densities in CP 6137 (D. saccharalis susceptible) than in CP 70330 (resistant) during the spring. Cultivar resistance to D. saccharalis has traditionally been determined based on measures of mature stalk injury (% bored internodes), adult production (number of moth exit holes in stalks), and tolerance to injury (% yield loss relative to % bored internodes) (Bessin et al. 1990; White et al. 2008). When comparing 10 sugarcane cultivars with varying levels of resistance, White & Dunckelman (1989) found limited differences in D. saccharalis deadheart injury. However, the percentages of deadhearts were typically consistent with resistance rankings based on independent assessment of stalk injury levels (% bored internodes). Although differences in D. saccharalis resistance levels may not be observed when deadhearts occur (i.e., early in sugarcane phenology before the formation of elongated internodes), the potential of cultivars with increased resistance to minimize fall and spring borer infestations deserves further research. Diatraea saccharalis infestations in newly planted sugarcane and stubble growth during the fall do not contribute directly to economic damage and have not been considered in management (Hensley 1971). Diatraea saccharalis late summer and fall populations are the source for overwintering borers, which will emerge in the spring the following year and cause economic damage. Our study showed that early planting and harvesting enhance late summer and fall D. saccharalis populations, thus having the potential for enhancing overwintering populations and subsequent economic damage. In areas where D. saccharalis is a severe problem, when susceptible cultivars are planted, or when insecticides cannot be applied, optimization of planting dates (e.g., Sep) may help minimize D. saccharalis population build-up. ACKNOWLEDGMENTSThis work was supported by USDA CSREES CropsAt-Risk IPM program grant 2008-51100-04415. We thank sugarcane growers Gerald Quebedeaux, Patoutville, Louisiana and Blake Newton, Bunkie, Louisiana for letting us use their farmland and for technical assistance. We thank D. C. Blouin (Louisiana State University) for assistance with data analyses, J. W. Hoy, N. A. Hummel, and B. E. Wilson (Louisiana State University) for review of earlier versions of the manuscript. This paper is approved for publication by the Director of the Louisiana Agricultural Experiment Station as manuscript number 2011-234-5534.REFERENCES CITEDBESSIN, R. T., AND REAGAN, T. E. 1993. Cultivar resistance and arthropod predation of sugarcane borer (Lepidoptera: Pyralidae) affects incidence of deadhearts in Louisiana Sugarcane. J. Econ. Entomol. 86: 929-932. BESSIN, R. T., REAGAN, T. E., AND MARTIN, F. A. 1990. A moth production index for evaluating sugarcane cultivars for resistance to the sugarcane borer (Lepidoptera: Pyralidae). J. Econ. Entomol. 83: 221-225. BEUZELIN, J. M., AKBAR, W., MSZROS, A., REAYJONES, F. P. F., AND REAGAN, T. E. 2010. Field assessment of novaluron for sugarcane borer (Lepidoptera: Crambidae) management in Louisiana sugarcane. Crop Prot. 29: 1168-1176. BEUZELIN, J. M., REAGAN, T. E., AKBAR, W., FLANAGAN, J. W., CORMIER, H. J., AND BLOUIN, D. C. 2009. Impact of Hurricane Rita storm surge on sugarcane borer (Lepidoptera: Crambidae) management in Louisiana. J. Econ. Entomol. 102: 1054-1061. CHARPENTIER, L. J., AND MATHES, R. 1969. Cultural practices in relation to stalk moth borer infestations in sugar cane, pp. 163-174 In J. R. Williams, J. R. Metcalf, R. W. Mungomery, and R. Mathes [eds.], Pests of Sugarcane. Elsevier publishing Co., New York, New York. GARRISON, D. D., DUFRENE, E. O., AND LEGENDRE, B. J. 2000. Effect of planting date on yields of sugarcane cultivars grown in Louisiana. J. American Soc. Sugar Cane Technol. 20: 115. HENSLEY, S. D. 1971. Management of sugarcane borer populations in Louisiana, a decade of change. 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252 Florida Entomologist 94(2)June 2011LEGENDRE, B. L., AND GRAVOIS, K. A. 2006. The 2005 Louisiana sugarcane variety survey. The 2005 Louisiana sugarcane variety survey, pp. 89-98 In Sugarcane Research Annual Progress Report 2005. Louisiana State University Agricultural Center, Baton Rouge, Louisiana. LEGENDRE, B. L., AND GRAVOIS, K. A. 2010. The 2009 Louisiana sugarcane variety survey. The 2009 Louisiana sugarcane variety survey, pp. 83-96 In Sugarcane Research Annual Progress Report 2009. Louisiana State University Agricultural Center, Baton Rouge, Louisiana. LSU AGCENTER, LOUISIANA STATE UNIVERSITY AGRICULTURAL CENTER. 2010. Louisiana recommendations for control of sugarcane insects, pp. 130-131 In Louisiana Insect Pest Management Guide 2010. Louisiana State University Agricultural Center, Pub. 1838, Baton Rouge, Louisiana. PEDIGO, L. P. 2002. Entomology and Pest Management (4th edition). Prentice Hall, Upper Saddle River, New Jersey. POSEY, F. R., WHITE, W. H., REAY-JONES, F. P. F., GRAVOIS, K., SALASSI, M. E., LEONARD, B. R., AND REAGAN, T. E. 2006. Sugarcane borer (Lepidoptera: Crambidae) management threshold assessment on four sugarcane cultivars. J. Econ. Entomol. 99: 966971. REAGAN, T. E. 2001. Integrated pest management in sugarcane. Louisiana Agric. 44(4): 16-18. ROE, R. M., HAMMOND, A. M., JR., DOUGLAS, E. E., ANDPHILOGENE, B. J. R. 1984. Photoperiodically induced delayed metamorphosis in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Pyralidae). Ann. Entomol. Soc. America 77: 312-318. SALASSI, M. E., AND BREAUX, J. 2002. Economically optimal crop cycle length for major sugarcane varieties in Louisiana. J. American Soc. Sugar Cane Technol. 22: 53-62. SAS INSTITUTE. 2008. Users Manual, version 9.2. SAS Institute, Cary, North Carolina. VIATOR, R. P., GARRISON, D. D., DUFRENE, E. O., JR., TEW, T. L, AND RICHARD E. P., JR. 2005a. Planting method and timing effects on sugarcane yield. Crop Management: online (doi:10.1094/CM-2005-062102-RS). VIATOR, R. P., RICHARD, E. P., JR., GARRISON, D. D., DUFRENE, E. O., JR., AND TEW, T. L. 2005b. Sugarcane cultivar yield response to planting date. J. American Soc. Sugar Cane Technol. 25: 78-87. WHITE, W. H., AND DUNCKELMAN, J. W. 1989. The response of sugarcane selections to sugarcane borer in the greenhouse and field. J. American Soc. Sugar Cane Technol. 9: 56-61. WHITE, W. H., VIATOR, R. P., DUFRENE, E. O., DALLEY, C. D., RICHARD, E. P., JR., AND TEW, T. L. 2008. Reevaluation of sugarcane borer (Lepidoptera: Crambidae) bioeconomics in Louisiana. Crop Prot. 27: 1256-1261.

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Reinert et al.: Differential Grasshopper on Turfgrass and Landscape Plants253 THE DIFFERENTIAL GRASSHOPPER (ORTHOPTERA: ACRIDIDAE) ITS IMP ACT ON TURFGRASS AND LANDSCAPE PLANTS IN URBAN ENVIRONS J AMES A. R EINERT 1 W AYNE M ACKAY 2 M. C. E NGELKE 1 AND S TEVE W. G EORGE 1 1 Texas A&M AgriLIFE Research & Extension Urban Solutions Center, 17360 Coit Road, Dallas, TX, 75252-6599, U.S.A. E-mail: j-reinert@tamu.edu 2 Mid-Florida Research & Education Center, 2725 S. Binion Road, Apopka, FL, 32703-8504 U.S.A. A BSTRACT The differential grasshopper, Melanoplus differentialis (Thomas) (Orthoptera: Acrididae), frequently migrates from highw ay rights-of-way, pastures, and harvested elds to feed in urban/suburban landscapes and retail/wholesale nurseries across the southern and southwestern U.S.A., as these areas dry down during hot dry summers. Nine selected turfgrasses and 15 species of landscape plants were evaluated for their susceptibility or resistance to this grasshopper. Grasshoppers were collected from stands of Johnsongrass, Sorghum halepense which was used as a standard host for comparison in both experiments. Based on feeding damage number of grasshopper fecal pellets produced, and their dry weight, Zoysia matrella cv. Cavalier was the least preferred grass followed by Buchloe dactyloides cv. Prairie and Z. japonica cv. Meyer. Festuca arundinacea was signicantly the most preferred host and sustained the most feeding damage followed by Poa pratensis P. arachnifera cv. Reveille and 2 Cynodon spp. cultivars, Tifway and Common. Among the landscape plants, Hibiscus moscheutos cv. Flare, Petunia violacea cv. VIP, Phlox paniculata cv. John Fanick, Tecoma stans cv. Gold Star, and Campsis grandiora were the least damaged or most resistant. Plumbago auriculata cv. Hullabaloo, Glandularia hybrid cv. Blue Princess, Canna generalis, Johnsongrass, and Cortaderia selloana cv. Pumila sustained the most damage. Based on the number of fecal pellets produced and their weights Canna generalis and Glandularia hybrid cv. Blue Princess were the most preferred landscape plants tested. K ey Words: turfgrass, lawns, landscape plants, nursery plants, host plant resistance, Melanoplus differentialis R ESUMEN El chapuln diferencial, Melanoplus differentialis (Thomas) (Orthoptera: Acrididae), frecuentemente emigra desde los derec hos de va, pasturas y terrenos cosechados hacia jardines urbanos y viveros comerciales en busca de alimento, principalmente donde las reas comienzan a secarse en el verano del sur y sureste de Estados Unidos La susceptibilidad o resistencia a la alimentacin de chapulines fue evaluada en nueve pastos para csped y otras quince plantas ornamentales. Los chapulines se colectaron en Johnsongrass, Sorghum halepense el cual se uso como un hospedero estndar en ambos experimentos. Con base a los datos del dao al alimentarse numero y peso de las heces fecales producidas, Zoysia matrella cv. Cavalier es el menos preferido, seguido de Buchloe dactyloides cv. Prairie y Z. japonica cv. Meyer. El mas preferido signicativamente, con el mayor dao al alimentarse fue Festuca arundinacea seguido de Poa pratensis P. arachnifera cv. Reveille y dos pastos de Cynodon spp. cv. Tifway y Common. En el grupo de plantas ornamentales, Hibiscus mosc heutos cv. Flare, Petunia violacea cv. VIP, Phlox paniculata cv. John Fanick, Tecoma stans cv. Gold Star, y Campsis grandiora presentaron la mayor resistencia. Plumbago auriculata cv. Hullabaloo, Glandularia hybrid cv. Blue Princess, Canna generalis Johnsongrass y Cortaderia selloana cv. Pumila presentaron el mayor dao signicativamente. Con los parmetros de numero y peso de heces fecales Canna generalis y Glandularia hybrid cv. Blue Princess fueron las plantas mas preferidas. Translation provided by Carlos Campos, Texas A&M AgriLIFE Res. & Ext. Center, Dallas, TX The differential grasshopper, Melanoplus differentialis (Thomas) (Orthoptera: Acrididae), does not y long distances like the migratory grasshopper Melanoplus sanguinipes (Fabricius) (Shotwell 1930). However, as highway rights-ofway, pastures, and harvested elds dry down during hot dry summers, M. differentialis adults y from them to nearby urban/suburban landscapes and retail/wholesale nurseries to consume the foliage of turfgrasses and many landscape plants

PAGE 132

254 Florida Entomologist 94(2) June 2011 across the Southern U.S.A. Based on limited surveys during summers and autumns since 1998, we have recorded the differential grasshopper as the most frequently encountered species occurring in urban areas of Dallas, Texas. M. differentialis is also 1 of the most important grasshopper species causing economic injury to corn, wheat, alfalfa, and several other eld crops (Anonymous 1994; Isely 1944; Harvey & Thompson 1993). A single adult of this species feeding on a small potted or landscape plant can defoliate it practically overnight, and the invasion of many adults can devastate an entire landscape after just a few days and nights of feeding. Such sudden damage to nursery production can render the planting stock unsellable for the remainder of the season. The extremely hot and dry summers in the Southern and Southwestern U.S.A. create ideal conditions for extensive outbreaks across many states. Dense migrating populations do not occur every year, but when conditions are right, large and quite devastating populations do occur across the region. As pastures and eld crops are either harvested or desiccated from drought in late summer and early autumn, M. differentialis readily disperse into plant nurseries and the urban landscapes in searc h of food (Reinert et al. 2001). As a result, extensive damage is common on many landscape plant species, and effective grasshopper control strategies for the urban landscape, and especially plant nurseries, are often required to protect valuable plants that contribute signicantly to high property values (Merchant & Cooper 2010; Reinert et al. 2001; Royer & Edelson 2004). Several studies have been conducted to determine the feeding preferences of selected species of grasshoppers on various grasses and herbaceous plants; however, most of them have dealt with range or pasture grasses, weeds, and cultivated eld crops. Isely (1938) determined that the short-horned grasshoppers (Acrididae), including M. differentialis have mandible patterns possessing both graminivorous and forbivorous c haracteristics, which allows them to readily feed on both grasses and forbs. Specic host feeding studies have also been conducted with M. differentialis Isely (1944) fed nymphs of M. differentialis on 2 native grasses ( Andropogon saccharoides Swartz and Sprorobolus heterolepis A. Gray) and on Johnosongrass, Sorgham halepense (L) Pers; bermudagrass, Cynodon dactylon L. Pers; and corn, Zea mays L. He also fed them on 5 weeds: Helianthus annuus (L.) (Asteraceae); common sunower, Ambrosia aptera (DC) (Asteraceae); giant ragweed, Lactuca virose (L.) (Asteraceae); wild lettuce, Gaillardia pulc hella (Four.) (Asteraceae); and Parthenium hysterophorus (L.) (Asteracaeae) that were commonly present in stands of J ohnsongrass. Isely (1944) did not report on the preference of 1 grass or herb over another, but only that M. differentialis matured an average of 12 d faster in cages with forbs than in cages with only grasses In another set of studies with 12 species of plants in Maryland, M. differentialis showed a strong preference for common dandelion, Taraxacum ofcinale F. H. Wigg. (Asteraceae). Plantago rugellii Dcne. (Plantaginaceae); Dactylis glomerata L.; and Cyperus strigosus L. (Cyperaceae) also served as good hosts (Kaufmann 1968). Goldenrod, Solidago altissima L. (Asteraceae), was only nibbled by the grasshoppers (Kaufmann 1968). Kaufmann also showed that this grasshopper could develop and reproduce by feeding only on species of Poaceae; but development was slower and adults were smaller than when they fed on both grasses and forbs. M. differentialis also showed a preference for some corn hybrids over others in c hoice eld experiments (Brunson & Painter 1938; Harvey & Thompson 1993). Even though under eld conditions M. differentialis feeds heavily on alfalfa, Medicago sativa L. (Fabaceae), it was found to be an inadequate host for complete development (Barnes 1963). M. differentialis showed strongest preference for the common sunower Helianthus annuus L. (Asteraceae) compared to the following offered food plants: fava bean, Faba vulgaris Moench. (Fabaceae); kale, Brassica oleracea L. (Brassicaceae); and tomato, Solanum Iycopersicum L. (Solanaceae) (Howard 1995). However, in another test M. differentialis preferred giant ragweed, Ambrosia trid
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Kendra et al.: Ambrosia Beetle Diversity


DIVERSITY OF SCOLYTINAE (COLEOPTERA: CURCULIONIDAE)
ATTRACTED TO AVOCADO, LYCHEE, AND ESSENTIAL OIL LURES


PAUL E. KENDRA1*, JORGE S. SANCHEZ1, WAYNE S. MONTGOMERY1, KATHERINE E. OKINS2, JEROME NIOGRET1,
JORGE E. PENA3, NANCY D. EPSKY1 AND ROBERT R. HEATH'
'USDA-ARS, Subtropical Horticulture Research Station, Miami, FL 33158
2Florida Department of Agriculture and Consumer Services, DPI, CAPS, Gainesville, FL 32608

3University of Florida, Tropical Research and Education Center, Homestead, FL 33031

*Corresponding author; E-mail: paul.kendra@ars.usda.gov

ABSTRACT

The redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Sco-
lytinae), is an exotic wood-boring insect that vectors laurel wilt, a lethal vascular disease of
trees in the Lauraceae, including avocado (Persea americana) and native Persea species (red-
bay, swampbay). As part of research to identify host-based attractants for X. glabratus, we
discovered that a diverse array of non-target ambrosia beetles was attracted to the same
substrates asX. glabratus. During Sep-Dec 2009, several field tests were conducted in north
Florida (in woodlands with advanced stages of laurel wilt) with traps baited with commer-
cial lures of the essential oils, manuka and phoebe, and with freshly-cut wood bolts of avo-
cado (a known host) and lychee (Litchi chinensis, a non-host high in the sesquiterpene a-
copaene, a putative host attractant). In addition, manuka-baited traps were deployed in av-
ocado groves in south Florida to monitor for potential spread ofX. glabratus. The combined
trapping results indicated that none of these substrates was specific in attraction ofX. gla-
bratus. Numerous non-target ambrosia beetles were captured, including 17 species repre-
sentative of 4 tribes within the subfamily Scolytinae. This report provides photo-
documentation and data on the species diversity and relative abundance for a group of
poorly-studied beetles, the scolytine community in Florida Persea habitats.

Key Words: ambrosia beetles, Persea americana, Litchi chinensis, manuka oil, phoebe oil

RESUME

El escarabajo de la ambrosia del laurel rojo (redbay), Xyleborus glabratus Eichhoff (Coleop-
tera: Curculionidae: Scolytinae), es un insecto ex6tico barrenador de madera que transmite la
marchitez del laurel, una enfermedad vascular mortal de arboles de la familiar Lauraceae, los
cuales incluyen el aguacate (Persea americana) y las species nativas del g6nero Persea (re-
dbay, swampbay). Como parte de la investigaci6n para identificar las substancias quimicas
que atraen a X glabratus, fue descubierto que un arsenal divers de otros escarabajos de la
ambrosia que no eran de interns econ6mico fue atraido a las mismas substancias. Entre sep-
tiembre y diciembre del 2009, se realizaron varias pruebas en el norte de la Florida (en arbo-
ledas con etapas avanzadas de la marchitez del laurel) usando trampas con cebos comerciales
de los aceites esenciales manuka y de phoebe, y con madera de aguacate recien cortada (un
hu6sped conocido) y del lychee (Litchi chinensis, que no es hu6sped, pero es alto en el sesqui-
terpeno a-copaene, una substancia quimica atractiva). Ademas, trampas con manuka fueron
desplegadas en arboledas de aguacate en el sur de la Florida para supervisor la extension po-
tencial delX. glabratus. Los resultados de las captures combinados indicaron que ninguna de
estas substancias eran especificas en la atracci6n delX. glabratus. Numerosos escarabajos de
la ambrosia fueron capturados, incluyendo 17 species que representan cuatro tribus dentro
de la subfamilia Scolytinae. Este informed proporciona la foto-documentaci6n y datos en la di-
versidad de la especie y la abundancia relative para un grupo de escarabajos poco estudiado,
la comunidad del Scolytinae en los habitats de Persea de la Florida.

Translation provided by the authors.


The redbay ambrosia beetle, Xyleborus glabra- males are flightless and remain within the host
tus Eichhoff (Coleoptera: Curculionidae: Scolyti- tree, while diploid females (typically sibling-
nae), is an exotic wood-boring insect that vectors mated) disperse to colonize new hosts. Unlike
laurel wilt, a lethal vascular disease of trees in most ambrosia beetles, female X. glabratus are
the Lauraceae (Fraedrich et al. 2008). Haploid primary colonizers, capable of attacking healthy







Florida Entomologist 94(2)


unstressed trees. During gallery excavation, fe-
males introduce spores of a symbiotic fungus,
Raffaelea lauricola T.C. Harr., Fraedrich &
Aghayeva (Harrington et al. 2008), carried in my-
cangial pouches located at the base of the mandi-
bles (Fraedrich et al. 2008). The fungus provides
food for both larvae and adults, but it also invades
the host vascular system and results in systemic
wilt and ultimately tree death. Native to south-
eastern Asia, X. glabratus was first detected in
the U.S. in 2002 near Port Wentworth, Georgia
(Rabaglia et al. 2006). Since then, the vector-dis-
ease complex has spread along the coastal plain
into South Carolina and Florida, and has been re-
ported from a single county in Mississippi
(USDA-FS 2010). In northern Florida, high mor-
tality has occurred in native Persea species, in-
cluding redbay (P. borbonia (L.) Spreng.) and
swampbay (P. palustris (Raf.) Sarg.), and the
rapid southward spread of the pest complex cur-
rently threatens commercial groves of avocado (P.
americana Mill.), a confirmed susceptible host
(Mayfield et al. 2008). Florida's avocado produc-
tion, centered in Miami-Dade County, is worth
$13 million annually (USDA-NASS 2010), and re-
placement costs of all avocado trees (commercial
and backyard) in Miami-Dade, Broward, Palm
Beach, and Lee Counties have been estimated at
$429 million (Evans & Crane 2008).
Due to the serious economic threat posed byX.
glabratus, there is a critical need for effective at-
tractants to detect, monitor, and control the
spread of this invasive pest. Preliminary research
provided no evidence of an aggregation phero-
mone and no strong attraction to its fungal sym-
biont, to its frass, or to ethanol (a standard attrac-
tant for ambrosia beetles); suggesting that host
tree volatiles are the primary attractants for dis-
persing females (Hanula et al. 2008). Additional
studies identified manuka and phoebe oils (essen-
tial oil extracts from the tea tree, Leptospermum
scoparium Forst. & Forst., and the Brazilian wal-
nut, Phoebe porosa Mez., respectively) as effective
baits for field monitoring ofX. glabratus in South
Carolina (Hanula & Sullivan 2008). Based on
comparisons of volatile chemicals emitted from
chipped redbay wood, manuka oil, and phoebe oil,
Hanula & Sullivan (2008) hypothesized that 2
sesquiterpenes, a-copaene and calamenene, were
likely the primary host attractants.
While conducting research to evaluate attrac-
tion of female X. glabratus to wood volatiles and
essential oil lures, we discovered that a diverse
number of non-target ambrosia beetles (both en-
demics and exotics established in Florida) were
attracted to the same substrates as X. glabratus.
Several field tests were conducted in north-cen-
tral Florida (Alachua and Marion Counties) in
natural stands of redbay and swampbay with
known infestations of X. glabratus and visible
signs of laurel wilt disease. We used 4-funnel


Lindgren traps and/or sticky panels baited with
commercially available essential oil lures
(manuka and phoebe) or with freshly-cut wood
bolts of avocado (a confirmed host) and lychee
(Litchi chinensis Sonn., a presumed non-host).
Lychee is in the family Sapindacaeae; it lacks the
typical aromatic laurel volatiles, but it has a high
content of a-copaene (Niogret et al. unpublished).
During that same period, monitoring traps
(Lindgren traps baited with manuka lures) were
deployed in avocado groves in south Florida (Mi-
ami-Dade County). This report summarizes and
illustrates the ambrosia beetles captured over a
4-month period (Sep-Dec 2009) in Florida to (1)
provide a tool for action agencies and field scien-
tists to facilitate identification of non-target spe-
cies captured in X.glabratus monitoring traps, (2)
document the species diversity and relative abun-
dance for the scolytine community in Persea hab-
itats, and (3) identify potential secondary coloniz-
ers of Persea hosts subsequent to initial attack by
X. glabratus.

MATERIALS AND METHODS

Field test 1 was conducted in Citra, Marion
County, FL at the University of Florida Agricul-
tural Experiment Station (PSREU). The back
edge of the station bordered an upland wooded
area dominated by mature live oak (Quercus uir-
giniana Mill.) with an understory that included
redbay trees symptomatic for laurel wilt. Test 1
was run from 27 Aug-22 Oct 2009 and consisted of
6 treatments: a commercial manuka lure (Syn-
ergy Semiochemicals, Burnaby, BC), wood bolts
from 3 avocado cultivars representative of the 3
horticultural races ('Simmonds', West Indian
race; 'Brooks Late', Guatemalan race; and 'Seed-
less Mexican', Mexican race), bolts from lychee
(cv. 'Hanging Green'), and an unbaited control.
Wood bolts were collected from the USDA-ARS
germplasm collection at the Subtropical Horticul-
ture Research Station (SHRS), Miami, FL 1 d
prior to test deployment. The ends of the bolts
were coated with wax to prevent desiccation, and
then both ends re-cut when used as baits at the
start of the test. All baits were deployed in four-
funnel Lindgren traps (BioQuip, Rancho
Dominguez, CA) with 300 mL of an aqueous solu-
tion of 10% propylene glycol (Low-Tox antifreeze;
Prestone, Danbury, CT) added to the collection
cup. For the manuka treatment, a single lure was
hung from the trap lid by a wire twist tie. For the
wood substrates, 2 freshly-cut bolts (5 cm diam x
15 cm length) were suspended with wire from the
lid on opposite sides of the trap. Experimental de-
sign was a randomized complete block, with 5 rep-
licate blocks arranged in a linear array along the
fence at the back of the research station. Within a
block, traps were spaced 10 m apart, 1.5 m above
the ground, and spacing was 50 m between repli-


June 2011







Kendra et al.: Ambrosia Beetle Diversity


cate blocks. Traps were checked every 2 weeks for
a total of 8 weeks. At each sampling date, the re-
tention solutions (with insect captures) were col-
lected, a thin layer was sawed from the lower end
of each bolt (to "renew" release of wood volatiles),
the collection cups were refilled, and trap posi-
tions were rotated sequentially within each block
to minimize potential positional effects on beetle
capture.
Field tests 2 and 3 were conducted in Cross
Creek, Alachua County, FL at the Lochloosa Wild-
life Conservation Area (St. John's River Water
Management District). The study site consisted of
mesic flatwoods composed of an overstory of slash
pine (Pinus elliottii Englem) with a mixed under-
story that included numerous swampbay trees ex-
hibiting advanced stages of laurel wilt. Test 2 was
conducted from 7 Oct-2 Dec 2009 and evaluated
the same 6 treatments described above. Test 3
was conducted from 5 Nov-29 Dec (at a site adja-
cent to test 2) and contained a commercial phoebe
lure (Synergy Semiochemicals) in addition to the
6 treatments above. However, with tests 2 and 3
there were differences in trap type and trap lay-
out. The essential oil lures were still deployed in
four-funnel Lindgren traps, but the wood bolts
were paired and hung vertically with 2 white
sticky panels (23 cm x 28 cm, Sentry wing trap
bottoms; Great Lakes IPM, Vestaburg, MI) sta-
pled back-to-back at the bottom of the bolts.
Sticky panels were further secured with several
binder clips around the edges. Tests 2 and 3 fol-
lowed randomized complete block design, with 5
replicate blocks arranged in a rectangular grid.
Each block consisted of a row of traps hung ~2 m
high in non-host trees, with a minimum of 10 m
spacing between adjacent traps in a row, and with
50 m spacing between rows. Both tests were 8
weeks in duration and checked every 2 weeks. At
each check, the retention solutions and sticky
panels were collected, a thin layer was sawed
from the bottom of each bolt, the solutions/panels
were replaced, and the trap positions were ro-
tated sequentially within each row.
In addition to the field tests conducted in north
Florida, monitoring/survey traps were deployed
in several avocado groves in Miami-Dade County,
FL during the fall of 2009. All monitoring traps
consisted of four-funnel Lindgren traps baited
with manuka lures, which were hung ~2 m above
ground within the canopy of avocado trees. Traps
were checked at 2-week intervals and sites in-
cluded the SHRS avocado germplasm collection in
Miami and 3 commercial groves in Homestead.
All sample collections (from monitoring traps
and field tests) were sorted in the laboratory at
SHRS, and scolytine species were counted, photo-
graphed, and stored in 70% ethanol. Specimens
removed from sticky panels were soaked over-
night in histological clearing agent (Histo-clear
II; National Diagnostics, Atlanta, GA) prior to


storage in alcohol. Beetle identifications were
confirmed at FDACS-DPI (Gainesville, FL) by K.
E. Okins, and voucher specimens were deposited
at both DPI and SHRS.

RESULTS

The combined trapping results from field tests
and monitoring traps totaled 659 ambrosia bee-
tles, consisting of 17 species from 4 tribes within
the subfamily Scolytinae (Table 1). More than
90% of the captures were from the tribe Xyle-
borini, and only 1 specimen (Coccotrypes distinc-
tus (Motshulsky)) was representative of the tribe
Dryocoetini. With the exception of X. glabratus
(Fig. 1), most beetles were captured in fairly low
numbers, with many species represented by only
a single capture, despite significant trapping ef-
forts with a variety of host-based attractants. Xy-
leborus glabratus comprised the majority of cap-
tures in north Florida, as expected due to its inva-
sive pest status, but the percentages varied by
site (15% in Marion County with test 1; 75.4%
and 86.2% in Alachua County with tests 2 and 3,
respectively). Four other species of Xyleborus
were captured (Fig. 2), with X. ferrugineus (Fabr-
icius) (Fig. 2A) and X. affinis (Eichhoff) (Fig. 2B)
the 2 most abundant species after X. glabratus.
There were several other representatives within
the tribe Xyleborini (Fig. 3), with Ambrosiodmus
obliquus (LeConte) (Fig. 3A) a dominant species
at both the Alachua site and in the Miami-Dade
avocado groves.
The tribe Cryphalini was represented by Hy-
pothenemus dissimilis (Zimmerman) and several
other Hypothenemus species difficult to discern to
species level (Fig. 4). Hypothenemus beetles were
major components at the Marion site and in Mi-
ami-Dade County. Within the tribe Corthylini, 5
species were captured, of which 4 are presented in
Fig. 5. Two of those ambrosia beetles had distinc-
tive morphological features. Females of Corthylus
papulans Eichhoff(Fig. 5C) have greatly enlarged
terminal antennal segments which bear several
long, recurved setae (Fig 5E); males of C. papu-
lans lack the characteristic setae. In Pityoborus
comatus (Zimmerman) (Fig. 5D), females are
unique in that the mycangia are located on the
pronotum, and consist of a pair of large shallow
depressions covered with dense pubescence (Fig.
5F; Furniss et al. 1987).

DISCUSSION

The subfamily Scolytinae contains 2 function-
ally distinct groups of beetles bark beetles which
feed on phloem from the inner bark of host trees,
and ambrosia beetles which cultivate and feed on
symbiotic fungi within the xylem layers (Rabaglia
2002). Among the bark beetles, there are major
forest pests which have been well studied, includ-







Florida Entomologist 94(2)


June 2011


TABLE 1. AMBROSIA BEETLES (CURCULIONIDAE: SCOLYTINAE) CAPTURED IN MARION, ALACHUA, AND MIAMI-DADE
COUNTIES, FL FROM SEP-DEC 2009, ARRANGED ACCORDING TO LAWRENCE & NEWTON (1995).

Marion Co. Alachua Co. Miami-Dade Co

Test 1 Test 2b Test 3b Monitoring

Tribe Dryocoetini
Coccotrypes distinctus (Motschulsky) 1
Tribe Xyleborini
Ambrosiodmus lecontei Hopkins 1
Ambrosiodmus obliquus (LeConte) 6 14 22
Premnobius cavipennis Eichhoff 1
Theoborus ricini (Eggers) 1
Xyleborus affinis (Eichhoff) 2 11 4
Xyleborus californicus Wood 2 1
Xyleborus ferrugineus (Fabricius) 3 34 22 1
Xyleborus glabratus Eichhoff 3 193 287
Xyleborus volvulus (Fabricius) 7 3

Tribe Cryphalini
Hypothenemus dissimilis (Zimmerman) 1 1
Hypothenemus spp. 9 1 1 22

Tribe Corthylini
Subtribe Corthylina
Corthylus papulans Eichhoff 1
Monarthrum mali (Fitch) 1
Subtribe Pityophthorina
Pityoborus comatus (Zimmerman) 1
Pseudopityophthorus minutissimus (Zimmerman) 1
Pseudopityophthorus pruinosus (Eichhoff) 1

a8-wk field test in redbay; Lindgren traps baited with wood bolts (avocado, lychee) or manuka oil lures.
'8-wk field test in swampbay; Sticky traps baited with wood bolts (avocado, lychee); Lindgren traps baited with manuka/phoebe
oil lures.
'Monitoring in avocado groves; Lindgren traps baited with manuka oil lures.


ing the southern pine beetle, Dendroctonus fron-
talis Zimmerman (Chellman & Wilkinson 1980),
the western pine engraver, Ips pini (Say) (Kegley
et al. 1997), and several other Ips spp. found in
the southeastern U.S. (Conner & Wilkinson
1998). In contrast, the ambrosia beetles are gen-
erally not of economic importance and conse-
quently have received less attention. They are
minute beetles, spend the majority of their life
concealed within host trees, and typically attack
stressed or dying trees. Despite the large number
of described species (e.g., >500 currently recog-
nized Xyleborus spp. worldwide, Rabaglia et al.
2006), much is unknown regarding the basic biol-
ogy, ecology, host range, fungal symbionts, and
population dynamics of many endemic ambrosia
beetles. Far less is known about exotic invasive
species, which are not pests in their native lands
but may acquire pest status when introduced into
new environments, as is the case with X. glabra-
tus in the U.S.
Although our research was focused on identifi-
cation of attractants specifically for detection and
control ofX. glabratus, information was obtained


concurrently on the species diversity and relative
abundance for the ambrosia beetles found in na-
tive Persea habitats in north-central Florida. In
south Florida the trapping effort was less inten-
sive, but preliminary data was also obtained for
the species composition in avocado groves. This
summary report identifies the species of Scolyti-
nae most likely to be encountered while monitor-
ing for X. glabratus, and the photo-documenta-
tion provides fellow researchers (non-taxono-
mists) and action agency personnel with a conve-
nient tool for preliminary identification of non-
target captures.
Some of the non-target beetles identified
herein are species that may potentially function
as secondary vectors of the laurel wilt pathogen.
Once healthy trees are attacked by X. glabratus,
the stressed trees are susceptible to further at-
tack by secondary colonizers that can contribute
to the rapid mortality seen in laurel hosts. Obser-
vations made on dead swampbay trees at the
Lochloosa Conservation Area (Kendra et al. un-
published) indicated that multiple wood-boring
species attacked those Persea trees, as evidenced







Kendra et al.: Ambrosia Beetle Diversity


Fig. 1. The redbay ambrosia beetle Xyleborus glabratus Eichhoff vector of a lethal wilt fungus (Raffaellea lau-
ricola) causing high mortality of trees in the Lauraceae in the southeastern U.S. A. Female. B. Male. (Note: Males
ofX. glabratus are flightless; this specimen was obtained from host wood, not from a flight trap.)


Fig. 2. Four species of Xyleborus not of economic importance. A. X ferrugineus (Fabricius). B.X. affinis (Eich-
hoff). C. X. olvulus (Fabricius). D. X californicus Wood. (All specimens female.)


B 1 Imm


A _, _
", 1 lmn







Florida Entomologist 94(2)


I mm








A,
Bmm









Imm








CP

Fig. 3. Ambrosia beetles within the tribe Xyleborini.
A. Ambrosiodmus obliquus (LeConte). B. Theoborus ri-
cini (Eggers). C. Premnobius cavipennis Eichhoff. (All
specimens female.)


by bore holes of various diameters. These second-
ary colonizers may potentially pick up Raffaelea
from the host xylem and transport it to new trees,
accelerating the spread of laurel wilt. In north
Florida, X ferrugineus, X affinis, and A. obliquus
were the most abundant species in native Persea
habitats; in avocado A. obliquus and Hypothene-
mus spp. were dominant. In South Carolina, Ha-
nula et al. (2008) found that Xylosandrus cras-
siusculus (Motschulsky) was attracted to
wounded redbay trees. Further research should
evaluate these additional beetle species to (a) de-
termine if stressed (diseased) trees in the Lau-
raceae can serve as hosts, and if so, then (b) deter-
mine if Raffaelea spores can be recovered from
the mycangia of ambrosia beetles that developed
within Raffaelea-infected hosts. In other systems,
there is evidence that exchange or "cross contam-
ination" of symbiotic fungi may occur among am-
brosia beetle species that occupy a common breed-
ing site (Gebhardt et al. 2004). Alternatively, Raf-
faelea spores may potentially be transported pas-


sively by the setae and/or cuticular asperities
(protuberances) commonly found on the anterior
slope of the female pronotum, as has been demon-
strated for Hypothenemus hampei (Ferrari) and
spores ofFusarium solani (Martius) (Morales-Ra-
mos et al. 2000).
The commercial lures currently available for
X. glabratus are non-specific in attraction, so
high numbers of non-target captures are likely
to be encountered with the monitoring system
(manuka-baited Lindgren traps) employed by
the State of Florida. Manuka and phoebe oil
lures were originally developed for field moni-
toring of another (phylogenetically distant)
wood-boring beetle, the emerald ash borer Agri-
lus planipennis Fairmaire (Coleoptera: Bupres-
tidae) (Crook et al. 2008). Preliminary research
(Kendra et al. unpublished) indicated that these
essential oil lures were not only non-specific, but
may have limited field life for attraction of X.
glabratus. With the data set presented here, ap-
proximately 30% of the captures were non-tar-
get species of Scolytinae. Development of effec-
tive strategies for early detection and control
(i.e., attract-and-kill systems) ofX. glabratus is
contingent on identification of specific attracta-
nts. In the absence of species-specific phero-
mones or food-based attractants for X. glabratus
(Hanula et al. 2008), this will be a difficult chal-
lenge.

CONCLUSIONS

Multiple trapping studies targeting the redbay
ambrosia beetle, Xyleborus glabratus, effectively
generated a survey of the overall scolytine com-
munity resident in Florida Persea habitats. These
ambrosia beetle species that co-occur with X. gla-
bratus, are attracted to the same host-based vola-
tile chemicals; and they are the non-target spe-
cies likely to be encountered in traps set out to
monitor for X. glabratus. Those species that can
function as secondary colonizers of Persea hosts
should be evaluated as potential secondary vec-
tors for transmission of the laurel wilt pathogen,
Raffaelea lauricola.

ACKNOWLEDGMENTS

We gratefully acknowledge David Long, Mike Win-
terstein (USDA-ARS; Miami, FL), Rita Duncan (Univ.
Florida; Homestead, FL), Gurpreet Brar, and Stephen
McLean (Univ. Florida; Gainesville, FL) for technical
assistance; Patti Anderson (FDACS-DPI, Gainesville,
FL) for Persea identifications; Bud Mayfield (USDA-
Forest Service; Asheville, NC) for advice on field trap-
ping; Ray Schnell (USDA-ARS; Miami, FL) for advice
on avocado germplasm samples; David Jenkins
(USDA-ARS; Mayagiiez, PR) and 2 anonymous re-
viewers for suggestions with the manuscript; Pansy
Vazquez-Kendra and Elena Schnell for translation of
the abstract; and Connie Rightmire (St. John's River


June 2011







Kendra et al.: Ambrosia Beetle Diversity


Fig. 4. Ambrosia beetles within the tribe Cryphalini. A. Hypothenemus dissimilis (Zimmerman). B. Hypothene
mus sp. (Both specimens female.)


Fig. 5. Ambrosia beetles within the tribe Corthylini. A. Monarthrum mali (Fitch). B. Pseudopityophthorus pru-
inosus (Eichhoff). C. Corthylus papulans Eichhoff. D. Pityoborus comatus (Zimmerman). E. Detail of C. papulans
(anterior end; ventral view) showing enlarged terminal antennal segments bearing long recurved setae. F. Detail
of P. comatus (anterior end; dorsal view on left, lateral view on right) showing pronotal mycangia (oval pits covered
with dense setae), the storage site for symbiotic fungal spores. (All specimens female.)


Water Management District) for assistance in obtain-
ing a special use permit for the Lochloosa Wildlife
Conservation Area. This work was supported in part
by the USDA-ARS National Plant Disease Recovery


System and the Florida Avocado Administrative Com-
mittee. This report presents the results of research
only; mention of a proprietary product does not con-
stitute an endorsement by the USDA.


I mm 1mm


1 MM


I MM







Florida Entomologist 94(2)


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FruiNu-01-22-2010_revision.pdf>


June 2011







Ferreira & Scheffrahn: Light Attraction Behavior of a Drywood Termite 13:



LIGHT ATTRACTION AND SUBSEQUENT COLONIZATION BEHAVIORS OF
ALATES AND DEALATES OF THE WEST INDIAN DRYWOOD TERMITE
(ISOPTERA: KALOTERMITIDAE)

MARIA TERESA FERREIRA'2 AND RUDOLF H. SCHEFFRAHN1'
'Fort Lauderdale Research and Education Center, University of Florida,3205 College Avenue, Davie, FL 33314

2Azorean Biodiversity Group (CITA-A), Departamento de Ciencias Agrdrias, Universidade dos Acores,
Pico da Urze, Angra do Heroismo, Portugal

ABSTRACT

Laboratory studies were conducted during the 2007 and 2008 dispersal seasons of the West
Indian drywood termite Cryptotermes brevis (Walker), a serious urban pest of wooden struc-
tures. Attraction to light and subsequent colonization of this species were studied by observ-
ing the response of alates to lit and dark chambers. Several intensities of light were tested
to determine if light intensity had a role in the alates' attraction to light and subsequent col-
onization. A bioassay was conducted with semi-shaded wood blocks to quantify negative pho-
totaxis for the dealates. We found that the alates of C. brevis preferred flying into lit areas
for colonization, and that the number of colonizations was highest in the high light intensity
treatments. Negative phototaxis of the dealates was observed because these preferred to col-
onize in the dark habitat treatments. This information is important when deciding what con-
trol methods may be used to prevent C. brevis from colonizing wood structures. Traps with
a high intensity light to attract C. brevis alates and to prevent infestation may be a way to
monitor and control this urban pest.

Key Words: Cryptotermes brevis, phototaxis, colonization, alates, dealates

RESUMO
Estudos num laborat6rio foram realizados durante a 6poca de voo de dispersao de 2007 e
2008 para a t6rmita da madeira seca Cryptotermes brevis (Walker), uma praga urbana de es-
truturas de madeira s6ria. A atraccgo a luz e subsequent colonizacio desta esp6cie foi es-
tudada, observando a resposta dos alados a cameras de preferencia de luz. Varias
intensidades de luz foram testadas para determinar se a intensidade da luz tinha um papel
na atraccgo dos alados pela luz e subsequent colonizacio. Um ensaio usando blocos de ma-
deira semi-cobertos para quantificar o comportamento fototactico negative dos dealados foi
conduzido. N6s observamos que os alados de C. brevis preferem areas com maior iluminacao
para colonizarem, e que o numero de colonizacoes era maior no tratamento com maior inten-
sidade de luz. O comportamento fototactico negative dos dealados foi observado porque os
dealados preferem colonizar nos tratamentos de habitats escuros. Esta informanao 6 impor-
tante quando se tem de decidir que m6todos de control podem ser usados para prevenir a
t6rmita C. brevis de colonizar estruturas de madeira. Usar armadilhas com uma intensidade
luminosa elevada para atrair alados de C. brevis e prevenir uma infestacio poderd ser uma
forma de monitorizar e controlar esta praga.


Translation provided by the authors.


The West Indian drywood termite Cryptoter-
mes brevis (Walker) (WIDT) is a serious urban
pest that causes significant levels of damage to
wooden structures. This termite was first de-
scribed in Jamaica in 1853 and has a tropicopoli-
tan urban distribution except in Asia (Scheffrahn
et al. 2008). Like most Kalotermitidae the WIDT
nests in its food source, wood, where it spends
most of its life cycle. A colony of drywood termites
can vary in size from hundreds to a few thousand
termites (Nutting 1970), and several colonies can
be found inside a single piece of wood. Myles et al.
(2007), for example, found as many as 30 colonies
of WIDT in a single floor board.


Drywood termites are major pests, accounting
for about 20% of the budget spent on termite con-
trol in the United States (Su & Scheffrahn 1990).
One of the main methods for controlling these
pests has been the use of fumigation to eliminate
existing colonies. Fumigation, however, does not
prevent new infestations, and therefore it is ben-
eficial to combine fumigation with additional pre-
ventative control methods. Preventing this spe-
cies from founding a new colony during the dis-
persal flight season is an important technique for
the control of this pest. This study aims to develop
a better understanding of the behavior of the
WIDT during the dispersal flight season, the time







Florida Entomologist 94(2)


when WIDT is most accessible to physical, chem-
ical, or behavioral management efforts.
The life cycle of WIDT includes a dispersal
flight where the mature winged forms (alates)
leave their previous colony to form new colonies.
The dispersal flights are the only occasion when
this species is found outside of wood (Kofoid 1934)
because it never leaves the nest to forage for new
food sources (Korb & Katrantzis 2004). After fly-
ing, the alates shed their wings and associate as
pairs of female and male dealates. These pairs
will crawl on the substrata in search for a suitable
place to start a new colony (Snyder 1926; Wilkin-
son 1962; Nutting 1969; Minnick 1973).
According to Light (1934a) most termites
castes are negatively phototactic, but the alates,
attracted to light, seek to emerge into openings
and fly toward light. However, there are no data
correlating colonization sites with lighting condi-
tions. Positive phototaxis of C. brevis and conge-
nus alates is followed by negative phototaxis of
the dealates (Wilkinson 1962; Minnickl973), al-
though no data have been produced to confirm
this observation.
The present study quantified the phototactic
colony site selection of C. brevis alates and
dealates. The first hypothesis tested was that fa-
vorable colonization sites in lighted areas are
more likely to be selected by alates of C.brevis,
and that colonization will be higher at higher
light intensities. The second hypothesis tested
was that dealates search for darker areas on the
substrate to colonize.

MATERIALS AND METHODS

Termites

Experiments were conducted at the University
of Florida, Fort Lauderdale Research and Educa-
tion Center (FLREC), Davie, Florida, in a room
partially filled with wood infested by C. brevis
that originated from several sites around South
Florida. The wood was stored in a dark room at
ambient temperature (average 25.6'C) and ambi-
ent relative humidity (average 73.5%). The ter-
mite alates used in the experiments dispersed
naturally from the infested wood. The room re-
mained dark except for the time the experiments
were conducted, and the alates were free to fly
anywhere in the room. The first experiments took
place between Apr and Jul 2007, and the second
experiments between Apr and Jun 2008.

Light Intensity Experiments

Twenty four transparent plastic boxes (36 x 23
x 28 cm) served as light preference chambers. The
boxes were wrapped in aluminum foil to isolate
the light box from adjacent boxes. The boxes were
placed with the open side facing the infested wood


and a hole was cut in the side of each box in order
to fit (Fig. 1) an electrical cord for a set of white
Light Emitting Diodes (LED) (HolidayLEDSTM
model No TS-70) strung in a series of 5 single
light bulbs attached with tape and hung through
the hole. The LEDs were all connected to each
other in a continuous string of lights mounted in
a series and connected to a single power source.
Electrical tape was used to position the light
bulbs in place as well as prevent light from dis-
persing through the cut hole, so that the only
light source was inside each box. The LED sets
were randomly distributed among 12 lit boxes
and 12 dark boxes. The lit boxes had a light inten-
sity of approximately 40 lux as measured by a
light meter (Extech Instruments model No
403125) and the dark boxes had approximately
0.11 lux (due to some contaminating light from
nearby experiments occurring at the same time).
A cube of wood (5 x 5 x 5 cm) with 6 drilled holes
was placed in the center of each box (Fig. 1). Each
2.3 mm diam hole was 1.5 cm deep, and there was
a single hole per face of the block. Four thumb
push pins were placed on the underside of the
block to allow for enough space (3 mm) for the ter-
mite to access the hole on the underside.
The difference in colonization between differ-
ent light intensities was analyzed with the same
24 boxes previously described. The LED lights
were also used but this time there were 4 differ-
ent set ups for the different light intensities with
6 replicates per light intensity (measured by the
light meter): 6 of the boxes were dark with (-0.11
lux); 6 boxes had 1 LED light bulb with an inten-
sity of approximately 11 lux; 6 boxes had 5 LEDs
together (-40 lux); 6 boxes had 10 LEDs attached
with an intensity of approximately 480 lux. The
boxes with the different light intensities were
randomly distributed. A block of wood (15x2x9
cm) with 24 holes (12 on top and 12 on the bottom,
and each 1.5 cm deep and 2.3 mm diam) was
placed in the center of each box. After 3 months,
the blocks were collected and the number of colo-
nized holes per block was counted. A hole was con-
sidered to be colonized when a complete fecal seal
(covering of the hole with hardened fecal material
from the termites) was present.

Negative Phototaxis Experiment

The negative phototaxis of the dealates was
studied with a white PVC pipe (51 cm x 7 cm in-
side diam) wrapped with electrical tape and
closed on one end. A 102 x 2 x 5 cm board with 40
holes, each 2.3-mm diam and drilled 2.5 cm apart,
was placed inside the pipe. The outermost holes
were 1 cm from the edge of the board. Of the 40
holes, 20 were always exposed to light (regular
fluorescent indoor lighting) and 20 were exposed
to decreasing levels of light toward the closed end
of the PVC pipe (Fig. 2). The board was visually


June 2011







Ferreira & Scheffrahn: Light Attraction Behavior of a Drywood Termite


Fig. 1. Arena for light intensity experiments. Aluminum foil isolated the box and the LED lights were placed
above the block of wood.


divided into 10-cm sections (excluding 1 cm at
each end). Each section had 4 holes available for
colonization. A board with the same dimensions
and number of holes was used as a control and
was completely exposed to the same light inten-
sity. The 10-cm sections were lettered from A to J
with sections A, B, C, D, and E inside the PVC pipe
and sections F, G, H, I, and J outside (Table 1). This
experimental protocol was replicated 4 times. Light
measurements were made inside and outside the
PVC pipe (Table 1). After dispersal flight season
was over the number of colonized holes was counted
for each 10-cm section based on the previously de-
scribed colonization criteria.

Statistical Analysis

The data for the lit versus dark boxes were an-
alyzed by a non-parametric Wilcoxon Matched
Pairs test (SAS Institute 2003) to test whether
the numbers of colonizations in the dark and lit


areas were different. Colonization differences be-
tween the light intensities were tested with Stu-
dent's t-test (SAS Institute 2003).
A Chi-squared test for independence (SAS In-
stitute 2003) was used to test whether the distri-
bution of colonizations was dependent on the light
intensity. A Student's t-test for dependent sam-
ples was used to determine whether the differ-
ences between the numbers of colonizations in
each 10-cm section were significant, (SAS Insti-
tute 2003).

RESULTS

Light Intensity Experiments
Lit vs dark boxes. A total of 43 holes were col-
onized. There were significantly (t-test = 4.01E-
05, P < 0.0001) more holes colonized in the lighted
boxes (2.8 + 0.3, mean SEM) than in the dark ar-
eas (0.8 0.2 (mean SEM)).







Florida Entomologist 94(2)


Fig. 2. Arena for negative phototaxis experiment. PVC pipe wrapped in black tape covered half of the wood block.
Controls had no PVC pipe cover.


Light intensity. A total of 76 holes were colo-
nized in the light intensity experiment. A higher
number of colonizations was recorded in high
light intensity boxes. There were significant (t-
test, P < 0.05) differences in colonizations among
the various light intensities (Fig. 3).

Negative Phototaxis Experiment

A total of 175 holes were colonized during the
negative phototaxis experiment. There were no
significant differences in number of colonizations
between the 10-cm sections (P > 0.29) in the con-
trol boards. The distribution of number of coloni-
zations was independent of the section where the
colonizations occurred (Chi-squared = 5.9541, P =
0.75; n.s.) with no section of the control board pre-
ferred over any other section.
In the boards placed in the PVC pipes coloniza-
tion distribution was not independent of the sec-
tion where it occurred (Chi-squared = 33.3939, P =
0.001) with a significantly higher number of colo-
nizations occurring in the dark sections (Fig. 3).
Sections A, B, C, and D inside the PVC pipe had a
significantly higher number of colonizations than
sections G, H, I, and J outside the PVC pipe (P <
0.05). Section E (inside the PVC pipe) was not sig-
nificantly different (P > 0.16) from sections G-J
(outside the PVC pipe); and section F (outside the
PVC pipe) was not significantly different (P > 0.08)
from sections A-D (inside the PVC pipe) (Fig. 4).


DISCUSSION

The results of the light versus dark experi-
ment confirmed the hypothesis that colonization
of the alates occur more frequently in lit areas.
The termite C. brevis in South Florida flies
mainly between 1:00 and 2:00 AM (unpublished
data) when it is very dark. These results showed
that colonization sites located in areas that are lit
during the night may be more susceptible to infes-
tation by C. brevis during dispersal flights. The
presence of artificial lights may cause a change of
behavior for some species of animals (Longcore &
Rich 2004), but artificial lights may be beneficial
to structure infesting termites like C. brevis. The
attraction of these termites to artificial lights
puts structures that have a continuous nighttime
light source at higher risk of being infested than
structures near by that are not lit.
The light intensity experiment showed that in-
creasing light intensities increased the number of
termite colonizations (Fig. 3). Minnick (1973) re-
ported differences in the wavelength of light pre-
ferred by C. brevis, and Guerreiro et al. (2007) re-
ported differences in color preference for the
alates. Neither of them reported results based on
light intensity. The fact that we found that 11 lux
of light intensity was significantly different from
480 lux shows that the increase in light intensity
caused an increase in colonization of the wood
blocks by the termites. Cryptotermes brevis has


TABLE 1. DISTANCES OF 10-CM WOOD SECTIONS FROM THE CLOSED END OF 51-CM PVC PIPE AND THE LIGHT INTENSITY
AT THE CENTER OF EACH SECTION.

Inside of PVC pipe Outside of PVC pipe

Section A B C D E A B C D E

Distance from closed end of PVC pipe (cm) 10 20 30 40 50 60 70 80 90 100
Light Intensity (lux) 0.01 0.04 0.08 0.20 0.70 600 600 600 600 600


June 2011







Ferreira & Scheffrahn: Light Attraction Behavior of a Drywood Termite


Ej1



oil 11 4D Ws

Fig. 3. Average number of holes colonized by C.
brevis per light intensity (n = 43). Different letters rep-
resent significant differences at P < 0.05.


been observed to have as many as 2 founded colo-
nies in a total of 22 nuptial chambers (Scheffrahn
et al. 2001) which means that there is a 9% suc-
cess rate of colonization. This shows that invest-
ing in preventing C. brevis alates from founding
colonies is important because their success rate is
high enough to make prevention methods neces-
sary. The attraction to light by alates can be used
to create light traps as a form of preventing infes-
tations and re-infestations by C. brevis alates and
as a means of partial control of this species. Such
light traps inside structures that are already in-
fested may minimize the spread of the infestation,
and the results of this study showed that more in-
tense the light used the more alates will be at-
tracted; thereby making the use of light a possible
alternative to use of chemicals for prevention. Fur-
ther studies with different light wavelengths can
help improve light traps as an alternative method
to prevent the founding of colonies by C. brevis.
Previous experiments with C. havilandi (Sjdst-
edt) (Wilkinson 1962) and C. brevis (Minnick
1973) showed negative phototaxis in dealates of
these species. After landing, the dealates search
and colonize dark areas. Due to the nature of
wood structures, it could be argued that this be-
havior is not really negative phototaxis but that
because the cracks and holes are usually hidden
and in dark areas the dealates end up colonizing
there. If so, the behavior would be dependent not
on light intensity but on the locations of a good


Inside PVC pipe Outside PVC pipe


E -25
2
S0
b
>0-

A B C D E F G H I J
10 cm section.
Fig. 4. Average number of C. brevis colonized holes
per section (n = 175). Sections A, B, C, D, and E were in-
side the PVC pipe and sections F, G, H, I, and J were
outside the PVC pipe. Bars with the same letter were
not significantly different at P < 0.05.


places to colonize. However, the present study
showed that negative phototaxis of dealates did
occur. Independent distributions of colonizations
occurred in the controls, but independent coloni-
zations did not occur in the semi-shaded blocks;
this confirmed the negative phototaxis hypothe-
sis. The lighter segment of the wood block in-
serted in the PVC pipe had significantly less colo-
nizations than the darker segment. However, the
numbers of colonizations in section F (immedi-
ately outside the PVC pipe) were not significantly
different from the numbers in the darker areas
inside the PVC pipe.
Colonization of section F might have occurred
because the termites colonizing that area had
searched for colonizing sites in the dark area in-
side the PVC pipe where earlier colonizers had al-
ready taken all suitable sites, i.e., a site satura-
tion. Also they may have landed near the PVC
pipe cueing in on the darker area nearby and col-
onizing the sites near that dark area. However,
further studies on this are needed to understand
why the section closest to the PVC pipe on the
light side was significantly more colonized than
the section immediately inside the dark PVC
pipe; where it would be expected considering the
negative phototaxis behavior. One way to ap-
proach this might be to use a higher density of col-
onizing holes or fewer termites, so that the num-
ber of holes is not a limiting factor.
In termites, both the alates and dealates have
well developed compound eyes (Light 1934b) and
their behavior during flight season has shown
that they do respond to light while in flight (posi-
tive phototaxis) and to have negative phototaxis
after landing. Further studies on the behavior of
C. brevis during the flight season can help im-
prove different methods to prevent colonization
and subsequent infestation by this species.
This study has shown that alates fly to and col-
onize more in higher light intensity areas, while
the dealates have an opposite behavior colonizing
more in darker areas. This knowledge is useful to
improve light traps as a control method against
colony foundation from C. brevis.

ACKNOWLEDGMENTS

We thank the late Boudanath Maharajh for techni-
cal support, and Roxanne Connelly, Jonathan F. Day,
and Paulo A. V. Borges for reviewing an early version of
this manuscript. We also thank Dr. James L. Nation and
anonymous reviews whose invaluable critical comments
helped improve this manuscript. Financial support for
this research was provided in part by the University of
Florida and the Portugal Foundation for Science and
Technology (FCT-SFRH/BD/29840/2006).

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LIGHT, S. F. 1934a. The constitution and development of
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genia, biogeografia e ecologia das t6rmitas dos
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NUTTING, W. L. 1969. Flight and colony foundation, pp.
233-282 In K. Krishna and F. M. Weesner [eds.], Bi-
ology of Termites.Volume I. Academic Press, London
and New York, 598 pp.
NUTTING, W. L. 1970. Composition and size of some ter-
mite colonies in Arizona and Mexico. Ann. Entomol.
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SCHEFFRAHN, R. H., BUSEY, P., EDWARDS, J. K., KRE-
CEK, J., MAHARAJH, B., AND SU, N-Y. 2001. Chemical
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CHINI, P. 2008. Endemic origin and vast anthropo-
genic dispersal of the West Indian drywood termite.
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914p8530t781632n/fulltext.html. Accessed July 2008.
SNYDER, T. E. 1926. The biology of the termite castes.
Quart. Rev. Biol. 1: 522-552.
Su, N.-Y., AND SCHEFFRAHN, R. H. 1990. Economically
important termites in the United States and their
control. Sociobiology 17: 77-94.
WILKINSON, W. 1962. Dispersal of alates and establish-
ment of new colonies in Cryptotermes havilandi
(Sjostedt) (Isoptera, Kalotermitidae). Bull. Entomol.
Res. 53: 265-288.


June 2011







Pereira et al.: Male A. suspense Lipid and Protein Content


INFLUENCE OF METHOPRENE AND DIETARY PROTEIN ON MALE
ANASTREPHA SUSPENSE (DIPTERA: TEPHRITIDAE) LIPID AND
PROTEIN CONTENT

RUI PEREIRA1', JOHN SIVINSKI2, JEFFREY P. SHAPIRO AND PETER E. A. TEAL2
'Entomology and Nematology Department, University of Florida, Gainesville, Florida

2Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, Florida

ABSTRACT

Because both the application of a juvenile hormone analog, methoprene, and the addition of
protein to the adult diet increased the sexual success of male Caribbean fruit fly,Anastrepha
suspense (Loew) (Diptera: Tephritidae), it was hypothesized that both might also impact
male nutritional status. Total content of lipid and of protein inA. suspense males were mea-
sured to discover if there was an effect of these treatments alone or in combination on the
content of each of these substances. In the first 24 hours following adult emergence, 6 dif-
ferent treatments were applied (all possible combinations of methoprene in acetone solution
or acetone alone, and protein-diet enrichment). Adult weight was determined for all treat-
ments at 5, 10, 15, 20, 25, 30 and 35 d post-emergence. Dietary protein had a positive effect
on the weight and total lipid and protein contents during the first 35 d of adult male life.
There were minimal negative impacts from methoprene applications. Even though males
were more active sexually, there was no significant change in weight or protein content dur-
ing the study period. However, total lipid content decreased with age. The usefulness of me-
thoprene to enhance the sexual performance of mass-reared tephritids destined for sterile
release appears to outweigh any physiological costs/limitations that such treatment might
confer.

Key Words: adult age, adult weight, Caribbean fruit fly, hydrolyzed yeast, juvenile hormone,
sexual maturation

RESUME

Debido a que tanto la aplicaci6n de un andlogo de la hormona juvenile, metopreno, y la adi-
ci6n de proteinas a la dieta del adulto aumento el 6xito sexual del macho de la mosca de la
fruta de Caribe, Anastrepha suspense (Loew) (Diptera: Tephritidae), se planted la hip6tesis
de que ambos tambi6n podrian afectar el status nutricional masculine. Se midi6 el conte-
nido total de lipidos y de proteinas en los machos deA. suspense para descubrir si habia un
efecto de estos tratamientos solos o en combinaci6n sobre el contenido de cada una de estas
sustancias. En las primeras 24 horas despu6s de la emergencia de adults, se aplicaron 6 di-
ferentes tratamientos (todas las combinaciones posibles de metopreno en soluci6n de ace-
tona en soluci6n o solo acetona y con el enriquecimiento de proteinas en la dieta). Se
determine el peso adulto para todos los tratamientos a los 5, 10, 15, 20, 25, 30 y 35 dias des-
pu6s de la emergencia. Las proteinas diet6ticas tuvo un efecto positive sobre el peso y el total
de lipidos y proteinas durante los primeros 35 dias de vida de los machos adults. Hubo un
minimo de impacts negatives de las aplicaciones de metopreno. A pesar de que los machos
eran mas activos sexualmente, no hubo ningun cambio significativo en el peso o el contenido
de protein durante el period de studio. Sin embargo, el contenido de lipidos totales dis-
minuyeron con la edad. La utilidad de metopreno para mejorar el desempeio sexual de mos-
cas tefritidas criadas en masa destinadas para programs que liberan los machos est6riles
parece superar los costs fisiol6gicos y las limitaciones que dicho tratamiento puede conferir.


Topical application of the juvenile hormone an- the underlying causes) has yet to be investigated
alog, methoprene, on the dorsal surface of adult experimentally. When methoprene and protein
male Caribbean fruit flies, Anastrepha suspense are combined there is an additive increase in
(Loew) (Diptera: Tephritidae), increases male male sexual performance, and males are ~ 4 times
sexual success (Pereira et al. 2009), apparently more likely to mate than males not exposed to me-
because it increases the production of male sex thoprene nor given access to protein (Pereira et
pheromone. In addition it accelerates sexual mat- al. 2010).
uration by several days (Teal & Gomez-Simuta Presumably, increased pheromone production
2002). The addition of protein to the adult diet occurring at an earlier age, as well as accelerated
has a similar effect on sexual performance, but sexual activity, is energetically demanding and







Florida Entomologist 94(2)


may affect the balance of the metabolic com-
pounds (Teal et al. 2000). One hypothesis fre-
quently mentioned for the relatively long, some-
times more than 2 weeks, pre-reproductive period
found in many adult frugivorous Tephritidae is
that the time is used to acquire resources needed
for reproduction (Sivinski et al. 2000). Decreases
in resource foraging time due to accelerated sex-
ual maturation and increases in body nutrients
resource expenditure might be expected to result
in substantive changes in a fruit fly's nutritional
status that could affect longevity and long-term
sexual performance. We further supposed that
these expenses could be particularly difficult to
incur in the absence of a protein enriched adult
diet.
As a result, we hypothesized that the nutri-
tional effects of methoprene and diet, both alone
and in combination, might have an important ef-
fect on sexual performance of male flies reared for
sterile insect technique (SIT) programs. It is from
this perspective that we address the following
questions: (1) What influence does methoprene
treatment have on male A. suspense weight and
lipid/protein nutrient stores over a period of 35 d;
~ 33% of males survive to this age under labora-
tory conditions (Sivinski 1993); and (2) do protein
enriched and protein deprived diets affect male
weight and lipid/protein content, and is there an
interaction between diet and methoprene treat-
ment?
Considering the potential importance of adult
diet to SIT, relatively little nutritional work has
been done with A. suspense. The rate and the
temporal patterns of consumption of carbohy-
drates, proteins, and amino acids by adults
(Sharp & Chambers 1984; Landolt & Davis-Her-
nandez 1993), as well as the role of food availabil-
ity and quality on male pheromone production
have been studied to some extent (Epsky & Heath
1993; Teal et al. 2000; Teal & Gomez-Simuta
2002). A positive influence of sucrose on male
pheromone calling (Landolt & Sivinski 1992) and
survival (Teal et al. 2004) has been reported.
However, no work has been done specifically on
the nutritional impact of protein incorporation
into adult diets. This is the first study of male te-
phritid nutritional balance challenged by artifi-
cially elevated "juvenile hormone" titers to im-
prove sexual performance.

MATERIAL AND METHODS

Insects

The Caribbean fruit flies used in this study
were obtained from laboratory colony at the Cen-
ter for Medical, Agricultural and Veterinary Ento-
mology (CMAVE) USDA-ARS, at Gainesville, FL.
At the time of the study, the colony was 3 years
old and had been produced according to the con-


ventional mass rearing protocols (FDACS 1995).
Pupae were collected from the colony and sorted
by size in a pupal sorting machine (FAO/IAEA/
USDA 2003). Pupal size was homogenized to re-
duce male size and weight variability; large males
have been shown to have a sexual advantage over
smaller males (Burk & Webb 1983; Burk 1984;
Webb et al. 1984; Sivinski & Dodson 1992; Sivin-
ski 1993). Males used for this experiment were
from pupal size class of 10.9 0.71 mg (n = 30) in
weight. This is considered a mid-size pupal
weight for field collected A. suspense males in in-
fested guava fruits (Hendrichs 1986). Throughout
the experiment flies were maintained in a labora-
tory room with a photoperiod of 13L:11D (light
from 0700 to 2000 h), a light intensity of 550 50
lux, a temperature of 25 1C and a relative hu-
midity of 55 5%.

Diet and Hormonal Treatments

Following emergence, males were subjected to
1 of the following 6 diet and hormonal treat-
ments:

*M'P: topical methoprene in acetone;
access to sugar and hydrolyzed yeast
*M'P-: topical methoprene in acetone;
access to sugar
*M-P: topical acetone; access to sugar and
hydrolyzed yeast
*M P: topical acetone; access to sugar
*P+: no topical application; access to sugar
and hydrolyzed yeast
*P: no topical application; access to sugar.

Methoprene (5 pg in 1 pL acetone) was applied
topically within the first 24h after emergence.
Controls consisted of application of 1 pL acetone
only (M-) or no topical application (P' and P-). In
order to conduct the topical application, males
were immobilized in a net bag, and the solution
was applied through the mesh on the dorsal sur-
face of the thorax from a micro-pipette. No anaes-
thesia was used to immobilize the flies. Precau-
tions were taken to avoid cross contaminations
among experimental subjects. Male flies exposed
to the different treatments were maintained in
screen cages (30 cm by 30 cm by 30 cm), with a
maximum male density of 200 flies/cage. Flies
were allowed free access to food (according to
above treatments) and water. In protein-deprived
treatments (P-) flies were only provided with
sugar. Protein was provided to the flies in the
form of hydrolyzed yeast mixed with sugar (1:3
parts, respectively). This mixture is considered a
high quality diet for Anastrepha species (Jacome
et al. 1995; Aluja et al. 2001).
Experimental cages were maintained for up to
35 d. For weight and chemical analysis, male flies


June 2011







Pereira et al.: Male A. suspense Lipid and Protein Content


were sampled at the following ages: 5, 10, 15, 20,
25, 30, and 35 d of adult age. For each age and
treatment, 5 flies were randomly sampled and
stored at -84'C until used for analysis. In order to
obtain the base line information after emergence,
5 newly emerged (without access to any food or
water) and untreated flies were collected as well.
Because lipid content in Ceratitis capitata (Wied.)
has been found to vary according to the time of
the day, due to the different activities in which
males were engaged (Warburg & Yuval 1997), we
sampled males at the same time each day (16:30
h, immediately before the beginning of the calling
period). Flies were weighed individually prior to
homogenization for lipid and protein determina-
tion.

Quantification of Lipids and Proteins

Individual male flies were homogenized in a
solution of PBS buffer at pH 7.25 (8.77 g of 0.15 M
NaCl and 7.1 g of 50 mM Na2HPO4 in 1 L of wa-
ter). The homogenate was then brought up to 4.0
mL with PBS. Lipids were extracted from the ho-
mogenate by adding 40 mg of Na2SO4 to half of the
initial volume, and 3.75 mL of chloroform: meth-
anol (1:2) (Bligh & Dyer 1959) was used to sepa-
rate polar and non-polar constituents of the ho-
mogenate. An additional 1.25 mL of chloroform
was added to the homogenate and vortexed for 4
min at 4,000 rpm. The non-polar chloroform
phase was collected. The remaining solution was
re-extracted with chloroform (1.875 mL), vor-
texed and collected. Chloroform was evaporated
in a Speed Vac device (Thermo Savant, San Jose,
CA).
Lipid contents were determined by the vanillin
reagent method (Van Handel 1985; Warburg &
Yuval 1996), with triolein being used as a stan-
dard. Quantification of lipids was done by react-
ing 10 pL of sample with 190 pL of vanillin re-
agent. Lipid content was determined colorimetri-
cally at 530 nm in a spectrophotometer (Bio-tek
Instruments, Winooski, VT).
Protein determination was done according the
Pierce BCA protein assay (Pierce, Rockford, IL).
One mL of the polar fraction of the homogenate
was centrifuged for 1 min at 14,000 rpm. Half the
volume was mixed with 100 pL of sodium deoxy-
choate reagent (0.15 w/v) and 100 pL of 72% (w/v)
tricloroacetic acid (TCA) to precipitate the pro-
teins. After incubation at room temperature for 10
min and centrifugation for 10 min at 14,000 rpm
the supernatant was discarded. The precipitate
was dissolved and reacted with 50 pL of 5% (w/v)
sodium dodecyl sulfate (SDS) and 1 mL of Pierce
micro BCAT protein assay reagent (Pierce, 1999).
After incubation in a water bath at 37'C for 30
min, proteins in samples and standards were de-
termined colorimetrically at 562 nm in a spectro-
photometer (Bio-Tek Instruments, Winooski, VT).


Statistical Analyses

Data were analyzed by two-way analysis of
variance (ANOVA) to detect the interactions be-
tween age and treatment for the parameters
studied, independently (weight, lipid content, and
protein content). These analyses were followed by
an ANOVA to detect differences between means
in the treatments. Tukey's test was used to sepa-
rate means (Ott & Longnecker 2001). Statistical
analyses were performed with R software (ver-
sion 2.1.0, www.r-project.org).

RESULTS

Male weight

Average adult weight varied between 5.8 mg
and 11.6 mg (Fig. 1). There was no interaction be-
tween treatment and adult age (F35,192 = 1.16, P =
0.256), and no effect of age (F7,192 = 1.59, P = 0.140;
Table 1) on adult weight. There was, however, a
significant effect of treatment (F5 192 = 24.46, P <
0.05). Protein-fed males generally had signifi-
cantly higher fresh weights than sugar fed males
(Fig. 1, Table 2).

Lipid content

Significant effects of treatment (F5,19= 131.37,
P < 0.001), adult age (F,192 = 83.14, P < 0.001), and
the interaction of adult age and treatment (F35,92 =
6.34, P < 0.001) were found. Male lipid content
per treatment per age (Fig. 2) differed both
among ages and for different treatments (Table 1)
and among treatments for different ages
(Table 2). In protein-deprived males, lipid con-
tents dropped at 5 d after emergence, while pro-
tein-fed males maintained stable lipid levels dur-
ing the first 10 d of adult life (Fig. 2). Afterwards,
lipids dropped to lower levels. Methoprene treat-
ment did not affect lipid levels in either protein-
fed or protein-deprived male flies.

Protein Content

Significant effects of treatment (F,192 = 44.63, P
< 0.001), adult age (F, 192= 15.00, P < 0.05), and the
interaction of adult age and treatment (F3,92 =
4.77, P < 0.001) were found. Significant differ-
ences in protein content among the different ages
were found within each treatment except for
treatment M-P' (Fig. 3, Table 1), and among treat-
ments at all ages (Table 2). Protein-fed males
maintained higher protein levels than protein-de-
prived males (Fig. 3). In protein-fed males, pro-
tein content steadily increased through time,
while in protein-deprived males, protein levels
declined. Methoprene did not affect the level of
protein in either protein-fed or protein-deprived
males.







Florida Entomologist 94(2)


p M-P+
p P+


--M+P-
- M-P-
- P-


0 5 10 15 20 25 30 35
Adult age (days)

Fig 1. Mean (SD) adult weight (n = 5) of male Caribbean fruit flies at different adult ages among the 6 treat-
ments featuring methoprene (M) and protein (P) and their various combinations.


DISCUSSION

In male A. suspense there was a clear effect of a
protein-enriched diet on weight, total lipid, and to-
tal protein content over the first 35 d of adult life.
In contrast, there was no effect of methoprene or
acetone application on the studied parameters. Re-
gardless of diet type, weight and total protein con-
tent were relatively stable during adult life. In con-
trast, total lipid content steadily decreased with
age. This decline began later, however, in flies fed a
protein-enriched diet (10 d after emergence).


In all the treatments, consumption of protein
resulted in insects able to regulate their weight,
protein levels, and lipid content at higher level
than insects without a protein food source. This is
broadly consistent with what has been observed
in Tephritidae in general and other Anastrepha
spp. in particular (Aluja et al. 2001). In nature,
adult tephritids feed on a variety of carbohy-
drates and proteins derived from fruit juices, hon-
eydew, and bird feces (Hendrichs et al. 1991; War-
burg & Yuval 1997; Yuval & Hendrichs 2000).
Protein enhances reproductive performance in C.


TABLE 1. ANALYSIS OF VARIANCE (ANOVA) FOR MALE CARIBBEAN FRUIT FLY WEIGHT, TOTAL LIPIDS, AND TOTAL PRO-
TEINS AMONG DIFFERENT AGES IN 6 DIFFERENT TREATMENTS FEATURING METHOPRENE (M) AND PROTEIN (P)
AND THEIR VARIOUS COMBINATIONS (NS, NON SIGNIFICANT DIFFERENCES, P > 0.05; 0.01< P < 0.05; *** P
< 0.001).

Treatments Male weight Total lipids Total proteins

M+P+ F,32 = 1.1559 (ns) F7,32 = 7.5932 *** F7,32 = 3.2711*
M+P- F7,32 = 1.9367 (ns) F7,32 = 32.76 *** F7,32 = 8.6007 ***
M-P+ F7,32 = 1.8041 (ns) F7,32 = 11.124*** F7,3 = 2.0186 (ns)
M-P- F7,32 = 2.0277 (ns) F7,32 = 35.906 *** F7,32 = 7.7853 ***
P+ F7,32 = 1.0071 (ns) F7,32 = 12.504 *** F7,32 = 2.9685*
P- F7,32 = 0.1291 (ns) F7,32 = 20.805*** F7,32 = 4.8732 ***


June 2011







Pereira et al.: Male A. suspense Lipid and Protein Content


350


300


250


1 200


* 150 -
-I




100


50


0


-0M-P+ -e-M-P-
-i-P+ -- P-


0 5 10 15 20 25 30 35
Adult age (days)

Fig. 2. Mean (SD) total lipid content (n = 5) of male Caribbean fruit flies at different adult ages among the 6
treatments featuring methoprene (M) and protein (P) and their various combinations.


capitata (Warburg & Yuval 1997; Kaspi et al.
2000; Shelly & Kennelly 2002; Shelly et al. 2002;
Yuval et al. 2002), and protein-fed males start to
call earlier in life (Papadopoulos et al. 1998). Pro-
tein-fed males are more competitive in terms of
post copulatory sexual selection as well (Taylor &
Yuval 1999). In Bactrocera dorsalis (Hendel), in-
corporation of protein into adult diet significantly
increases survival and mating success (Shelly el
al. 2005). Among Anastrepha species, Aluja et al.
(2001) evaluated the effects of different adult nu-
trients, including protein and sugar, on male sex-


ual performance in adults of 4 species, (A. ludens
(Loew), A. obliqua (Macquart), A. serpentina
(Wied.), A. striata Schiner). Overall, protein-fed
males were more sexually successful than pro-
tein-deprived, except for A. ludens where no dif-
ferences were found. Neither did male diet influ-
ence A. ludens female reproductive potential fol-
lowing trophalaxis (Mangan 2003). However, in a
more recent study protein did improve A. ludens
sexual performance (Aluja et al. 2008).
Thus, perhaps not surprisingly, protein-en-
hanced diets typically, but not always, enhance


TABLE 2. ANALYSIS OF VARIANCE (ANOVA) FOR MALE CARIBBEAN FRUIT FLY WEIGHT, TOTAL LIPIDS, AND TOTAL PRO-
TEINS AMONG TREATMENTS FOR DIFFERENT AGES FEATURING JUVENILE HORMONE (JH) AND PROTEIN (P)
AND THEIR VARIOUS COMBINATIONS (* 0.01< P < 0.05; ** 0.001< P < 0.01; *** P < 0.001).

Adult age (days) Male weight Total lipids Total proteins

5 F5,24 = 4.546 ** F5,24 = 26.285 *** F5,24 = 3.5202*
10 F5,24 = 4.566 ** F5,24 = 28.881*** F5,24 = 26.629 ***
15 F5,24 = 5.521** F5,24 = 12.548 *** F5,24 = 10.277 ***
20 F5,24 = 2.624 F5,24 = 13.873 *** F5,24 = 8.9948 ***
25 F5,24 = 4.370 ** F5,24 = 50.275 *** F5,24 = 4.2223 **
30 F5,24 = 4.429 ** F5,24 = 21.339 *** F524 = 8.9493 ***
35 F5,24 = 5.120 ** F5,24 = 36.197 *** F524 = 9.5048 ***







Florida Entomologist 94(2)


900

800


700



500


E 400
0
3O
400

C 300

200

100

0


-c-M+P+ ---M+P-
-0M-P+ -e--M-P-
---p+ --- P-


0 5 10 15 20 25 30 35
Adult age (days)
Fig. 3. Mean (SD) total protein content (n = 5) of male Caribbean fruit flies at different adult ages among the
6 treatments featuring methoprene (M) and protein (P) and their various combinations.


sexual success. However, there are perhaps re-
vealing differences in physiology and foraging
tactics for food and mates among males of differ-
ent species with different diets and activities. For
instance, unlike A. suspense, protein-fed C. capi-
tata males have lower lipid content than those
that are protein-deprived (Kaspi et al. 2000). In
addition, while lekking males are heavier and
contain significantly more protein and sugar than
resting males, they do not contain more lipids
(Yuval et al. 1998). Lipids (fatty acids, phospho-
lipids, and sterols) have a nutritional role distinct
from carbohydrates and proteins. Yuval et al.
(1994) described lipids metaphorically as an ener-
getic trust fund, whereas carbohydrates are com-
parable to a readily accessible cash account. Per-
haps the difference between A. suspense and C.
capitata in their use of protein for lipogenesis re-
flects a difference in energy use patterns, with A.
suspense putting more reserves in "long-term" ac-
counts for future use. This in turn might reflect
more predictably encountered food sources for C.
capitata or a lower daily chance of mortality forA.
suspense that leads in turn to "planning" for the
future.
Many kinds of fatty acids and phospholipids
are synthesized by insects, but all insects require
sterols in their diet (Chapman 1998). Reduction


of total lipid content with age in A. suspense can
be the result of somatic activities, since lipids rep-
resent stored energy, even if some restoration of
lipid reserves occurs by lipogenesis (Warburg &
Yuval 1996). In male A. suspense, at least in the
first 10 days of adult life of protein-fed males,
there is a slight increase in lipid content. The
same phenomenon occurs in C. capitata (Warburg
& Yuval 1996) and A. serpentina (Jacome et al.
1995). Total lipid content declined following male
A. suspense sexual maturation. Sharp decreases
indicate that males started to utilize their meta-
bolic reserves, and this seems likely to correspond
to the energetic requirements of any number of
sexual and agonistic behaviors and processes
(e.g., Sivinski et al. 2000). One of these potential
expenditures that can be indirectly examined
with the present data is pheromone production.
Nestel et al. (1986) suggested that lipid re-
serves in male C. capitata may play an important
role in the regulation and production of sex pher-
omone. Nestel et al. (2005) found a decrease in
lipid body content after sexual maturation (as the
present data reveal for A. suspense), but later on
the content displayed a harmonic pattern where
total lipid content increased and decreased at a
periodicity of 10 days. In A. suspense, male pher-
omone production increases when methoprene is


June 2011







Pereira et al.: Male A. suspense Lipid and Protein Content


applied (Teal et al. 2000). However, we found that
application of methoprene did not affect lipid con-
tent of flies maintained on different diet treat-
ments.
The differences in total protein content be-
tween protein-fed and protein-deprived males
may be influenced by ingested protein in the gut.
However, the gradual increase in total protein
content over time in both protein-deprived (after
10 d as adult) and protein-fed males is both diffi-
cult to explain and inconsistent with artificial-
diet protein alone accounting for the difference.
Tephritids are known to feed on animal excre-
ment (Prokopy et al. 1993; Epsky et al. 1997), and
perhaps the consumption of bacteria from their
own feces or bacteria growing on dead flies or on
food sources, inadvertently provided them with a
protein source. Regardless of the origin of this ad-
ditional protein, the difference in protein contents
on the different diets suggests it was not suffi-
cient to completely satisfy nutritional require-
ments.
The findings of this study have implications for
SIT programs. Among the most important is that
while the addition of methoprene has male sexual
advantages it appears to have no immediate nu-
tritional detriments. Thus it is a relatively "cost-
free" means of improving the performance of
mass-reared and released flies. The incorporation
of dietary protein has a positive effect on adult
weight and lipid and protein content all of which
are plausibly related to performance as well
(Yuval et al. 1998). Due to these effects, the incor-
poration of protein in adult diet for SIT programs
is also recommended.

ACKNOWLEDGMENTS

We thank David Nestel (IPP-The Volcani Center,
Beit-Dagan, Israel), Nikos Papadopoulos (University of
Thessaly, Magnisia, Greece), and Steve Ferkovich
(CMAVE, USDA-ARS, Gainesville-FL, USA) for critical
reviews of an earlier version of this manuscript. This
project was funded in part by the International Atomic
Energy Agency (Research Contract 12863). Financial
support was provided to RP by the Centro de Ci6ncia e
Tecnologia da Madeira through the Ph.D. grant BD I/
2002-004.

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June 2011







Lee & Thomas: Taxonomic Status of Cucujus clavipes


CLARIFICATION OF THE TAXONOMIC STATUS OF CUCUJUS CLAVIPES
WITH DESCRIPTIONS OF THE LARVAE OF C. C. CLAVIPES AND
C. C. PUNICEUS (COLEOPTERA: CUCUJIDAE)

JONGEUN LEE' AND MICHAEL C. THOMAS2
'Department of Biological Science, College of Natural Sciences, Andong National University, Andong, 760749
E-mail: Korea jelee@andong.ac.kr

2Florida State Collection of Arthropods, Division of Plant Industry, Gainesville, Florida 326147100, USA
E-mail: michael.thomas@freshfromflorida.com

ABSTRACT

The larvae of Cucujus c. clavipes Fabricius and C. c. puniceus Mannerheim are fully de-
scribed and illustrated in detail for the first time. Based on larval and adult morphology the
present recognition of two subspecies is maintained.

Key Words: taxonomy, Cucujus, larva, North America

RESUME

Por primera vez se described e ilustran las larvas de Cucujus c. clavipes Fabricius y C. c. pu-
niceus Mannerheim. Basandose en la morfologia larval, se acepta el reconocimiento de las
dos subespecies.


Translation provided by the authors.


Cucujus clavipes Fabricius (1781) was de-
scribed from "America boreali." Cucujus pu-
niceus Mannerheim (1843) was described from
"insula Sitkha", now Baranof Island in south-
eastern Alaska and the site of the modern city
of Sitka. Both descriptions are of adults only,
are based on the adult stage and are brief and
relatively uninformative. Of C. clavipes, Fabri-
cius wrote: "ruber, thorace fuscato, femoribus
clavatis rufis" (red, thorax dark, femora clavate,
red); of C. puniceus, Mannerheim wrote: "elong-
atus, depressus, laete sanguineus, antennis ni-
grofuscis, pectore abdomineque rufoferrugineis,
thorax subrotundato, lateribus leviter denticu-
lato, supra obsolete bisulcato" (Elongate, de-
pressed, rich red, antennae nigro-fuscus, abdo-
men rufo-ferrugineous; thorax rounded, later-
ally weakly denticulate, above obsoletely bisul-
cate).
LeConte (1854, 1861, 1863) consistently treated
C. puniceus as a valid species. Casey (1884) re-
duced it to a variety of C. clavipes and said of it:
"The body is more elongated, and usually of a
brighter color. The first joint of the antennae is
usually of a dark testaceous, while in clavipes it is
black. The antennae are slightly longer, and the
neck slightly narrower in puniceus." Leng (1920)
treated C. puniceus as either a variety or subspe-
cies of C. clavipes [In the Leng Catalogue, a let-
tered taxon following a numbered species name
could be ". . variety, subspecies, race, etc." (Leng
1920: v)] and Hetschko (1930) followed Casey in
treating it as a variety of C. clavipes. Schaeffer


(1931) described Cucujus clavipes subnitens as a
variety from Arizona and Utah. Thomas (1993) in a
list of Nearctic Cucujidae treated C. puniceus as a
subspecies of C. clavipes and Schaeffer's taxon as a
variety as previously described.
In an effort to resolve the status of Cucujus
clavipes we examined adults and larvae from both
eastern and western North America.

Larvae

Larvae of Japanese Cucujus coccinatus
Lewis were described and illustrated by Ha-
yashi (1980, 1986) and the larva of C. mniszechi
Grouvelle was described by Lee and Sato
(2007).
Larvae of C. clavipes Fabricius were briefly and
partially illustrated (head and mandible) by B0v-
ing and Craighead (1931) and Klausnitzer (2001).
Peterson (1951) provided extensive illustrations of
C. clavipes but provided only a brief description. In
neither case was the origin of the specimen illus-
trated provided. Lawrence (1991) re-used Peter-
son's illustrations and added scanning electron mi-
crographs of mouthparts of a specimen from Cali-
fornia. The larva of both North American subspe-
cies of C. clavipes Fabricius are fully illustrated
and described for the first time in the present pa-
per. The larva ofC. clavipes is similar to C. mnisze-
chi (Lee and Sato 2007), but can be distinguished
by absence of a distinct epicranial stem and pres-
ence of a sharp prostheca. In C. mniszechi the epi-
cranial stem is present and the prostheca is blunt.







Florida Entomologist 94(2)


Larvae of C. clavipes are reported to be preda-
ceous (Smith and Sears 1982) or facultatively pre-
daceous (Lawrence 1991). Their extreme cold tol-
erance, which increases with increasing latitude,
has been extensively studied (Sformo et al. 2010,
and references therein).

MATERIALS AND METHODS

The larvae were preserved in 70% ethyl alco-
hol, cleared in 10% KOH solution for 1 hour,
rinsed in water, and dissected under a stereo-
scopic microscope (Leica MS5). Slide mounting
procedures were carried out according to LeSage
(1984), and the larval terminology follows
Lawrence (1991). Specimens were measured with
an ocular micrometer and the measurements
were transferred to graph paper. The illustrations
were then sketched in pencil, the sketches inked,
and assembled into plates, which were optically
scanned and cleaned up in a graphics editor. Spec-
imens examined are deposited in the Florida
State Collection of Arthropods (FSCA) and the
University of Alberta E. H. Strickland Entomo-
logical Museum (UASM).

Descriptions

Cucujus clauipes clauipes Fabricius, 1781 (Fig. 1 AJ)

Diagnosis: See this section under C. c. puniceus.

Material examined: 37 total from: INDIANA:
Morgan Co.: Martinsville (10); Tippecanoe Co. (1);
OHIO: Champaign Co. (1); Columbiana Co. (1);
WISCONSIN: Calumet Co.: Forest Junction (1);
Ingham Co.: Dansville State Game Area (1); Sha-
wano Co.: Shawano (16); Shawnee Co.: Tilleda (6)
(all deposited in the FSCA).
Description: Late instar (Fig. 1A). Body 22.0 -
26.0 mm long, elongate, subparallel, strongly dor-
soventrally flattened with strongly forked median
process at abdominal apex (Fig. 1A). Head and
abdominal segment 8 moderately sclerotized, yel-
lowishbrown to brown, tergite of abdominal seg-
ment 9 strongly sclerotized and brown.
Head (Fig. 1B): prognathous, strongly trans-
verse and dorsoventrally flattened. Lateral mar-
gin rounded. Median endocarina absent; epicra-
nial stem present but very short; frontal sutures
lyriform, strongly curved; bases contiguous. St-
emmata well-developed, 6 on each side of head
(Peterson (1951) reported 5 on each side; we count
6 but 1 is small and difficult to see). Frontoclypeal
suture absent. Fronotoclypeal region with 3 long
setae anterior to angles of frontal arms, 1 pair an-
terior to the apex of the frontal arms on each side
of the head, 1 pair medially between the frontal
arms, and 1 pair at the apex of the frontoclypeal
region near the clypeolabral suture. Clypeolabral


suture complete. Labrum (Fig. 1G) free, with 3
pairs of setae and anterior border fimbriate.
Epipharynx glabrous medially, with 5 anterior se-
tae on each side. Antennae 3segmented, ratio of
lengths of antennomeres 1, 2, and 3 about 1.0: 1.2:
1.0. Mandibles (Fig. 1H) heavily sclerotized, sym-
metrical, apices bidentate with a smaller subapi-
cal tooth; with 2 dorsolateral mandibular setae;
prostheca acuminate, spinelike, with a broad
base; mola with numerous setae medially and
penicillus posteriorly (The scanning electron mi-
crographs in Lawrence (1991: 464, figs. 34.528, c-
f) show a conspicuous patch of microtrichia on
both the dorsal and ventral surfaces of the man-
dible near the base; these are virtually invisible
in liquid and are not illustrated here). Maxilla
(Fig. 1E) with cardo triangular, divided by an in-
ternal ridge, basal portion trapezoidal, 1 moder-
ately elongate seta near latero-basal margin; sti-
pes elongate; mala falciform with 5 apical spines
and a medial brush composed of several thick se-
tae; maxillary palpus 3segmented, segment 1 ase-
tose, segment 2 with 2 setae, segment 3 with 4
minute apical setae. Labium (Fig. 1F) with con-
spicuous mentum and prementum; mentum
about as long as wide, with 2 pairs of setae and
prementum with 1 pair of setae and 1 pair of sen-
silla; ligula rounded anteriorly, 1 pair of setae and
microtrichia anteriorly; labial palpi 2-segmented
and widely separated at base.
Thorax: Meso and metathorax tergites, and
abdominal tergites and ventrites 18 each with 1
transverse ridge near anterior margin, ridge on
ventral surface of abdominal segment 1 lightly
sclerotized. Prothorax subquadrate, transverse,
0.5 times as long as wide, sides slightly curved,
dorsal surface smooth; prosternal surface smooth,
3 setae (1 elongate) at anterolateral angles and 2
short setae at posterolateral angles; prosternum
trapezoidal, sides oblique, posterior margin
straight, pair of medial setae present posterior to
posterior margin of presternum. Meso- and met-
athorax transverse, both 0.5 times as long as
wide, sides curved, dorsal surface of both tergites
smooth with 3 short setae at anterolateral angles
and 2 short setae at posterolateral angles; both
sterna without well-defined subdivisions, each
smooth with a pair of discal setae near anterior
margin; spiracular sclerite projecting strongly
from lateral margin, spiracles (Fig. 1C) annular
and angled posterolaterally. Legs (Fig. 1D) mod-
erately long, 5segmented; claw falciform, large.
Abdomen: Segments 17 transverse, tergite
surface smooth with 2 setae anterior to spiracles
and 2 setae posterior to spiracles; ventrite surface
with 3 setae, 2 anteriorly and 1 posteriorly. Seg-
ment 8 slightly enlarged, tergite (Fig. 11) with a
stout spicule at each posterolateral margin, pos-
terolateral angles with 4 long and 4 short setae, 3
pairs of short setae anteromedially; sternite (Fig.
1J) with 7 pairs of setae and with large stout pro-


June 2011






Lee & Thomas: Taxonomic Status of Cucujus clavipes


E

WE


0-0.5 M











H
A E F 0.5mm G















0.5 mm

Fig. 1. Larva of Cucujus c. clavipes. A, habitus, dorsal view; B, head, dorsal view; C, A7 spiracle, D, prothoracic
leg; E, left maxilla, dorsal view; F, labium, ventral view; G, labrum, dorsal view; H, left mandible, dorsal view; I, ab-
dominal segments 89, dorsal view; J, same, ventral view.







Florida Entomologist 94(2)


cess posteriorly with many minute setae apically.
Tergum 9 with a basally forked process, directed
dorsad; base of process with a pair of short, api-
cally forked processes, 1 short seta at apex of
forked process; anterior margin with laterally
curved processes projecting from tergum 8; ven-
trite 9 reduced and concealed from above.

Cucujus clauipes puniceus Mannerheim
(Fig. 2 AJ)

Diagnosis. The larva of this species is very sim-
ilar to that of Cucujus c. clavipes, but can be dis-
tinguished by the ratio of the 8th abdominal seg-
ment length vs length of the forked process (4:3 in
C. c. puniceus; 1:1 in C. c. Clavipes), and the ratio
of the 8th abdominal segment width vs the width
of forked process (measured at tips) (5:3 in C. c.
puniceus; 3:2 in C. c. clavipes).
Material examined: 7 total, from: CANADA:
ALBERTA: George Lake (2, UASM); USA: CALI-
FORNIA: El Dorado Co.: Blodgett Forest (1,
FSCA); Tulare Co.: Sequoia National Park,
Stoney Cr. Picnic Area (2, FSCA);UTAH: Cache
Co.: Logan Valley (2, FSCA)
Description: Late instar larva (Fig. 2A). Body
21.0-24.0 mm long, elongate, subparallel,
strongly dorsoventrally flattened with forked me-
dian process at abdominal apex (Fig. 2A). Head
and abdominal segment 8 moderately sclerotized,
brown, tergum 9 strongly sclerotized and dark
brown.
Head (Fig. 2B): prognathous, strongly trans-
verse and dorsoventrally flattened. Lateral mar-
gin rounded. Hind corners of epicranium slightly
produced posteriorly. Median endocarina and epi-
cranial stem very short; frontal sutures lyriform,
strongly curved; bases contiguous. Stemmata
well-developed, 6 present on each side of head. Fr-
ontoclypeal suture absent. Fronotoclypeal region
with 3 long setae anterior to angles of frontal
arms, 1 pair anterior to the apex of the frontal
arms on each side of the head, 1 pair medially be-
tween the frontal arms, and 1 pair at the apex of
the frontoclypeal region near the clypeolabral su-
ture. Clypeolabral suture complete. Labrum free
(Fig. 2G), with 5 pairs of setae. Epipharynx medi-
ally glabrous, 6 anterior setae on each side. An-
tennae 3segmented, ratio of lengths of antenno-
meres 1, 2, and 3 about 1.0: 1.4: 1.0. Mandibles
(Fig. 2H) heavily sclerotized, symmetrical, apices
bidentate with a smaller subapical tooth; with 2
dorsolateral mandibular setae present; prostheca
acuminate, spinelike, with a broad base; mola
with numerous setae medially and posteriorly.
Maxilla (Fig. 2E) with cardo, divided by an inter-
nal ridge, basal portion trapezoidal, with 1 mod-
erately elongate seta near basal margin; stipes
elongate; mala falciform, mala falciform with 5
apical spines and a medial brush composed of sev-
eral thick setae; maxillary palpus 3segmented,


segment 1 asetose, segment 2 with 3 setae, seg-
ment 3 with 1 seta and 4 minute apical setae. La-
bium (Fig. 2F) with conspicuous mentum and pre-
mentum; mentum about as long as wide, with 3
pairs of setae, prementum with 3 pairs of setae;
ligula transverse, with anterior microtrichia; la-
bial palpi 2 segmented.
Thorax: Meso and metathorax tergites, and
abdominal tergites and ventrites 18 each with 1
transverse ridge near anterior margin, ridge on
ventral surface of abdominal segment 1 smaller
lightly sclerotized. Prothorax subquadrate, trans-
verse, 0.5 times as long as wide, sides curved, dor-
sal surface smooth; prosternal surface smooth, 3
setae (1 elongate) at anterolateral angles and 2
short setae at posterolateral angles; prosternum
trapezoidal, sides oblique, posterior margin
straight, a pair of medial setae present posterior
to posterior margin of presternum. Meso- and
metathorax transverse, both 0.5 times as long as
wide, sides curved, surface of both tergites
smooth with 3 short seta at anterolateral angles
and 2 short setae at posterolateral angles; both
sterna without well- defined subdivisions, each
smooth with a pair of discal setae near anterior
margin; spiracular sclerite projecting strongly
from lateral margin, spiracles (Fig. 2C) annular
and angled posterolaterally. Legs (Fig. 2D) mod-
erately long, 5segmented; claw falciform, with 2
setae.
Abdomen: Segments 17 transverse, tergite
surface smooth with 2 setae anterior to spiracles
and 2 setae posterior to spiracles; ventrite surface
with 3 setae, 2 anteriorly and 1 posteriorly. Seg-
ment 8 enlarged, tergite (Fig. 21) with a stout sp-
icule at each posterolateral margin, posterolat-
eral angles with 8 short setae, 3 pairs of short se-
tae anteromedially, 2 pairs of short setae postero-
medially. Ventrite (Fig. 2J) with 9 pairs of setae
and large stout process posteriorly with numer-
ous minute setae apically. Tergite 9 with a basally
forked process, directed dorsad, as wide as long;
base of process with a pair of short, apically
forked processes, 1 short seta at apex of forked
process; anterior margin with lateral curved pro-
cesses projecting from tergite 8; sternite 9 re-
duced and concealed from above.

Adults

Given the differences discovered in the larvae
of the 2 subspecies, we examined adults to deter-
mine if there were corresponding adult differ-
ences. We examined 120 adult specimens of C. c.
clavipes in the FSCA from the following states
and provinces: CANADA: Ontario; USA: Colo-
rado, Illinois, Indiana, Iowa, Kansas, Maine,
Maryland, Massachusetts, Michigan, Mississippi,
Missouri, New York, New Jersey, North Carolina,
Ohio, Pennsylvania, Virginia, Wisconsin. We ex-
amined 46 adult specimens of C. c. puniceus in the


June 2011








Lee & Thomas: Taxonomic Status of Cucujus clavipes


E


1.0 mm B


0.5 mm


0.5 mm


0.5 mm


Fig. 2. Larva of Cucujus c. puniceus. A, habitus, dorsal view; B, head, dorsal view; C, A7 spiracle, D, prothoracic
leg; E, left maxilla, dorsal view; F, labium, ventral view; G, labrum, dorsal view; H, left mandible, dorsal view; I, ab-
dominal segments 89, dorsal view; J, same, ventral view.







Florida Entomologist 94(2)


FSCA from the following states and provinces:
CANADA: Alberta, British Columbia; USA:
Alaska, California, Idaho, Oregon.
As noted in previous literature, C. c. clavipes
has a black scape, while C. c. puniceus has a red
scape. However, specimens of C. c. puniceus from
Alaska have black scapes. We had formed the im-
pression that individuals from the western U.S.
were on average more elongate than those from
the eastern part of the country. Measurements of
series from both populations revealed consider-
able overlap in body proportions, with specimens
of the C. c. puniceus slightly more elongate, rang-
ing in size from 12.5mm to 16.6mm, while speci-
mens of C. c. clavipes ranged in size from 9.5mm
to 14.6mm.
Lee and Sato (2007) found taxonomically use-
ful genitalic differences among Asian species of
Cucujus. Male genitalia from specimens of C.
clavipes from all parts of its distribution were ex-
amined and found to be indistinguishable.

CONCLUSIONS

Despite the larval differences, the lack of con-
sistent and significant morphological differences
in the adults suggests that at this point given the
state of our knowledge, the present treatment of
these 2 populations as subspecies of the same spe-
cies is valid. Research into molecular differences
may prove useful in understanding the limits of
both taxa.

ACKNOWLEDGMENTS

We thank Chi Feng Lee and John Marris and 2 anon-
ymous reviewers for reviewing a previous draft of this
manuscript. George Ball generously lent larvae from
the University of Alberta collection. This is Entomology
Contribution No. 1180 of the Bureau of Entomology,
Nematology, and Plant Pathology, Florida Department
of Agriculture and Consumer Services. This research
was supported by a grant to the senior author from the
2008 Academic Exchange Program of Andong National
University.

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FABRICIUS, J. C. 1781. Species insectorum, exhibentes
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two new species and the larvae of Cucujus mniszechi
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leoptera. Bull. Broooklyn Entomol. Soc. 26: 174176.
SFORMO, T., WALTERS, K., JEANNET, K., WOWK, B.,
FAHY, G. M., BARNES, B. M., AND DUMAN, J. G. 2010.
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to -100 C in the Alaskan beetle Cucujus clavipes pu-
niceus (Coleoptera: Cucujidae) larvae. J. Exper. Biol.
213: 502-509
SMITH, D. B., AND SEARS, M. K. 1982. Mandibular struc-
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similar coleopterous larvae Cucujus clavipes (Cu-
cujidae), Dendroides canadensis (Pyrochroidae), and
Pytho depressus (Salpingidae). Can. Entomol. 114:
173175.
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i-viii and 1-93.


June 2011







Garcia & Norrbom.: Tephritoid Flies and Their Host Plants in Southern Brazil 151



TEPHRITOID FLIES (DIPTERA, TEPHRITOIDEA) AND THEIR PLANT HOSTS
FROM THE STATE OF SANTA CATARINA IN SOUTHERN BRAZIL

FLAVIO R. M. GARCIA1 AND ALLEN L. NORRBOM2
1Federal University of Pelotas, Institute of Biology, Department of Zoology and Genetics
Laboratory of Insect Ecology, 96010-900 P.O. Box: 354, Pelotas, RS, Brazil
E-mail: flavio.garcia@pq.cnpq.br

2Systematic Entomology Laboratory, c/o National Museum of Natural History, MRC-168,
P.O. Box 37012, Washington, DC 20013-7012, USA
E-mail: allen.norrbom@ars.usda.gov

ABSTRACT
A total of 12,540 ripe fruits belonging to 46 species in 25 plant families were sampled from
either the trees or the ground in 6 municipalities in the state of Santa Catarina, Brazil be-
tween 2002 and 2006 to determine which fruit fly species developed on various host plants.
Each fruit was weighed and placed into a plastic flask filled with sterilized sand 7 cm deep,
and the opening of the flask was covered with sheer fabric. The flasks were kept under con-
trolled conditions (25 3 C, 70 10% RH and 12h photophase). After 7 d, the pupae were
sifted from the sand and transferred to Petri dishes lined with filter paper. Twenty-one spe-
cies of Tephritoidea were recovered consisting of 13 species ofTephritidae, 6 of Lonchaeidae,
and 2 of Ulidiidae. We present new host records for some species of fruit flies.

Key Words: Tephritidae, Lonchaeidae, Ulidiidae, fruit pests, new host records

RESUME
Este trabajo dirigido a la evaluaci6n de las species de moscas de la fruta y sus plants hos-
pederas en el estado de Santa Catarina Brasil. Un total de 12.540 frutos maduros que per-
tenecen a 46 species y 25 families de arboles o del suelo en seis municipios del estado de
Santa Catarina, Brasil entire 2002 y 2006 fueran muestradas. Cada fruto fue pesado y se co-
loca en un frasco de plastico cubierto con Voil, con 7 cm de arena esterilizada. Los frascos fue-
ron mantenidos en condiciones controladas (25 3 C, UR 70 10% y 12h de photophase).
Despu6s de siete dias, la arena se tamiza y la pupas fueron transferidas a places de petri con
papel filtro como sustrato. Veintiun species de Tephritoidea fueron recuperados 13 espe-
cies de Tephritidae, seis species de Lonchaeidae, y dos de Ulidiidae. Se presentan los regis-
tros de para algunas species de fruta o moscas.


Translation provided by the authors.


Approximately 70 species of Tephritidae are
considered important pests of fruit production
worldwide. The majority of the species of eco-
nomic importance belong to 5 genera: Anas-
trepha, Bactrocera, Ceratitis, Dacus, and Rhago-
letis (Garcia 2009). The genus Neosilba of the
family Lonchaeidae (McAlpine & Steyskal 1982)
includes 16 described species (Strikis & Prado
2005), some of which cause severe damage to cer-
tain species of fruit crops in the American trop-
ics.
Field surveys of fruit flies (Tephritoidea) and
their host plants and parasitoids are essential for
understanding the bioecology of the economically
important genera and species in this superfamily
(Bateman 1972). The creation of the common
market, Mercosul, involving Brazil, Argentina,
Paraguay and Uruguay, has elevated the impor-
tance of such studies because knowledge of these
pest species, their hosts and natural enemies is
key to containing their destructive effects as


trade in fruits between these countries expands.
In Brazil, most of the pest tephritids belong to the
genus Anastrepha, but host plants are known for
only 44% of the species (Zucchi 2007).
Santa Catarina has the most host plant
records, 81, for species of Tephritidae among the
Brazilian states (Garcia 2011). However, only 46
plant species belonging to 18 families are re-
corded in the state as hosts for fruit flies in the ge-
nus Anastrepha (Nora et al. 2000).
This work reports new information from a sur-
vey of fruit fly species and their host plants in the
state of Santa Catarina, Brazil.

MATERIALS AND METHODS

Fruit Sampling

Between 2002 and 2006, a total of 12,540 ripe
fruits from 46 plant species belonging to 25 fami-







Florida Entomologist 94(2)


lies were sampled. Fruits were picked from the
plants, or freshly fallen fruits were gathered from
the ground below them. Sampling occurred in 6
municipalities of Santa Catarina, Brazil: Anchi-
eta (26' 53'S and 53' 33'W), Chapec6 (27' 06'S and
53' 16'W), Cunha Pora (26' 07'S and 53W 16'),
Palmitos (27' 06'S and 53' 16W'), Sao Carlos (27'
07' S and 53' 00' W), and Xanxer6 (26' 87' S and
52'W' 40). Each fruit was weighed and placed into
a plastic flask containing 7 cm of sterilized sand,
and the opening of the flask was covered with
sheer fabric. The flasks were kept under con-
trolled conditions (25 3C, 70 + 10% RH and 12h
photophase). After 7 d, the sand was sifted and
the pupae transferred to Petri dishes with filter
paper as substrate.

Identification of fruit flies and host plants

Characters of the females, primarily of the ac-
uleus, and body and wing markings, were consid-
ered in identifying species ofAnastrepha (Zucchi
2000) identified by Garcia and Zucchi. Ceratitis
capitata (Wiedemann) is the only species of Cer-
atitis in Brazil and was easily recognized by the
description by Zucchi (2000). Lonchaeidae were
identified by Dr. Pedro Strikis, and other Te-
phritidae and Notogramma cimiciforme Loew
(Ulidiidae) were identified by Norrbom. The host
plant species were identified by the botanists Dr.
Sergio Augusto de Loreto Bordignon, Dr. Rosiane
Berenice Denardin, and Licia Salengue. Some
voucher specimens of fruit flies and host plants
were deposited at the Zoobotanic Museum of the
University of Chapec6.

Data Analysis

The infestation indexes were calculated in 2
ways: (1) by dividing the total number of puparia
obtained by the number of fruits in the sample
(puparia/fruit); or (2) by dividing the total num-
ber of puparia by the total mass (kg) of fruits in
the sample (puparia/kg). The host plants ofAnas-
trepha obtained in this work were compared to
the lists of hosts assembled by Norrbom (2004)
and Zucchi (2007, 2008) with the aim of providing
new host records for Brazil.

RESULTS AND DISCUSSION

Twenty-one species of Tephritoidea were re-
covered: 13 species of Tephritidae, 6 of Lon-
chaeidae, and 2 of Ulidiidae (= Otitidae) (Table 1).
The species, Parastenopa guttata Aczel and P.
montana Aczel, are new records of fruit flies for
the state of Santa Catarina, and the total number
of known species of Tephritidae from the state is
now 81 (Garcia 2011). The development of flies
from the fruit of yerba mate, Ilex paraguariensis
A. St. Hil., is reported for the first time. Two spe-


cies of the genus Parastenopa, P. guttata and P.
montana, were reared. The only Parastenopa spe-
cies previously known to attack this plant were
reared from stems or from leaf galls of the Para-
guay tea psyllid, Gyropsylla spegazziniana Lizer
& Trelles (Hemiptera, Psyllidae) (Blanchard
1929; psyllid as Metaphalara spegazziniana), al-
though the North American P. limata (Coquillett)
breeds in the fruit of several Ilex species (Ben-
jamin 1934; Phillips 1946). Araticum,Annona ru-
gulosa (Schltdl.) H. Rainer (Annonaceae), Inga
sellowiana Benth. (Fabaceae), and the iguana
hackberry, C. iguanaea (Jacq.) Sarg. (Ulmaceae)
are recorded for the first time as host plants of
Anastrepha fraterculus (Wiedemann). Rio Grande
cherry, Eugenia involucrata DC., is recorded for
the first time as a host plant ofAnastrepha obli-
qua (Macquart); and sete-capas, Campomanesia
guazumifolia (Cambess.) O. Berg. (Myrtaceae), is
recorded as a host plant ofAnastrepha sororcula
Zucchi. Strawberry guava, P. cattleianum Sabine
(Myrtaceae), is recorded for the first time as host
plant ofbothA. obliqua andA. sororcula in Brazil.
Previously strawberry guava had been reported
as a host ofA. obliqua in Guatemala (Eskafi &
Cunningham, 1987).
The greatest infestations based on the number
of puparia per fruit were found in pumpkin, Cu-
curbita pepo L. (6.59), followed by pineapple
guava, Acca sellowiana (0. Berg) Burret (6.23),
and common guava, Psidium guajava L. (6.16).
Regarding the parameter puparia/kg, the great-
est infestations occurred in strawberry guava, P.
cattleianum (422), followed by pineapple guava,
P cattleianum (278), yerba mat6, I. paraguarien-
sis A. St. Hil. (260), and wild cherry, P avium (L.)
L. (232). Considering both parameters, pineapple
guava, P cattleianum, was the species most in-
fested by fruit flies.
The highest number of plant hosts was re-
corded for A. fraterculus (20 plant species from 8
families) (Table 1); predominantly fig, Ficus car-
ica L. (Moraceae) (75.0% of the total of samples
collected were infested); guavirova, Campomane-
sia xanthocarpa 0. Berg, (60.7%); guaviju, Myr-
cianthes pungens (0. Berg) D. Legrand (57.1%);
Surinam cherry, Eugenia uniflora L. (55.3%); wild
cherry, P. avium (L.) L. (Rosacae) (52.0%); pineap-
ple guava, P cattleianum (51.7%); common guava,
P cattleianum (51.4%); guava (48.0%), Cam-
pomanesia guazumifolia (45.4%) (Myrtaceae);
and carambola, Averrhoa carambola L. (Oxali-
daceae), (35.3%).
Nine new host plants ofA. fraterculus were re-
corded in Brazil: araticum, A. rugulosa (Annon-
aceae); Inga sellowiana (Fabaceae); common fig,
F carica (Moraceae); pineapple guava, P. cattle-
ianum (Myrtaceae); jaboticaba, Myrciaria cauli-
flora (Mart.) O. Berg (Myrtaceae); Campomanesia
guazumifolia (Myrtaceae); wild cherry, P avium
(Rosaceae); bergamot orange, Citrus reticulata


June 2011









TABLE 1. PLANTS SAMPLED WITH THEIR RESPECTIVE ORIGIN (0), FRUIT WEIGHT (FW), NUMBER OF FRUITS SAMPLED (N), NUMBER OF PUPAE (P), AVERAGE NUMBER OF PUPAE
PER FRUIT (P/N), AND AVERAGE NUMBER OF PUPAE PER KG (P/KG). N = NATIVE AND E = EXOTIC. NUMBER IN PARENTHESES FOLLOWING FLY SPECIES NAMES = NUMBER
OF SPECIMENS REARED.

FW # fruits #pupae
Plant Species O (kg) n P P/n SE P/kg SE Tephritidae Lonchaeidae & Ulidiidae


Annonaceae
Araticum, Annona rugulosa
Aquifoliaceae
Erva-mate, Ilex paraguariensis
Cactaceae
Pereskia aculeata
Cucurbitaceae
Ab6bora, Cucurbita pepo



Chuchu, Sechium edule


Melancia, Citrullus lanatus
Melao, Cucumis melo
Pepino, Cucumis sativus
Ebenaceae
Caqui, Diospyros kaki


Euphorbiaceae
Mandioca, Manihot esculenta
Fabaceae
Inga, Inga sellowiana


N 4.64 102 33 0.32 0.1 7.10 2,4 A. fraterculus (3)


N 1.00 2465 259 0.11 0.1 259.70 20.5 Parastenopa spp.(254)


N 0.37 50 45 0.90 0.2 121.62 10.4 A. barbiellinii (19)

E 139.77 68 448 6.59 2.1 3.21 1,8 A. grandis (310)



E 8.94 120 46 0.38 0.1 5.15 3.2


E 58.30
E 10.80
E 8.22


0.14 0.1
0.92 0.3
0.26 0.1


0.03 0.1 A. grandis (2)
1.11 0.7
1.34 0.8


E 9.47 126 367 2.91 1.1 38.74 12.0 A. fraterculus (11)
C. capitata (293)


N 0.52 210 2 0.01 0.0 3.83 1.3 A. montei (2)


N 1.75 246 49 0.20 0.2 27.97 6.2 A. fraterculus (5)
C. capitata (4)


Moraceae
Figo, Ficus carica E 1.22 52 22 0.42 0.2 18.10 8.3 A. fraterculus (16)


Myrtaceae
Araca, Psidium cattleianum


N 5.67 670 2393 3.57 1.3 421.99 25.1 A. fraterculus (1220)
C. capitata (10)


Neosilba zadolicha (5)
Neosilba padroi (7)
Neosilba sp. (6)


Neosilba zadolicha (18)


Dasiops sp. (8)
Euxesta sp.(12)
Neosilba padroi (40)
Euxesta sp. (22)
Lonchaea sp. (12)
Neosilba padroi (10)

Neosilba padroi (9)
Euxesta sp. (7)


Lonchaea sp. (12)
Neosilba sp. (19)








TABLE 1. (CONTINUED) PLANTS SAMPLED WITH THEIR RESPECTIVE ORIGIN (0), FRUIT WEIGHT (FW), NUMBER OF FRUITS SAMPLED (N), NUMBER OF PUPAE (P), AVERAGE NUM-
BER OF PUPAE PER FRUIT (P/N), AND AVERAGE NUMBER OF PUPAE PER KG (P/KG). N = NATIVE AND E = EXOTIC. NUMBER IN PARENTHESES FOLLOWING FLY SPECIES
NAMES = NUMBER OF SPECIMENS REARED.

FW # fruits #pupae
Plant Species O (kg) n P P/n SE P/kg SE Tephritidae Lonchaeidae & Ulidiidae


Cereja, Eugenia involucrata

Goiaba, Psidium guajava



Goiaba-do-campo,Acca sellowiana
Guaviju, Myrcianthes pungens
Guavirova, Campomanesia xanthocarpa
Jabuticaba, Myrciaria cauliflora
Pitanga, Eugenia uniflora
Sete-capotes, Campomanesia guazumifolia


Uvaia, Eugenia pyriformis

Oxalidaceae
Carambola,Averrhoa carambola

Passifloraceae
Maracuja, Passiflora edulis


Rosaceae
Ameixa, Prunus domestic
Cereja-do-mato, Prunus avium
Nespera, Eriobotrya japonica

Pera, Pyrus communis
Pessego, Prunus persica


Rutaceae
Bergamota, Citrus reticulata


N 2.85 516 155 0.30 0.1 54.47 13.0 A. fraterculus (79)
C. capitata (15)
N 12.47 236 1454 6.16 2.4 116.64 10.9 A. fraterculus (697)
A. obliqua (14)
A. sororcula (7)
C. capitata (13)
N 1.79 80 498 6.23 3.2 277.80 23.2 A. fraterculus (254)
N 0.25 52 21 0.40+ 0.1 84.31 13.3 A. fraterculus (12)
N 2.61 717 53 0.07 0.0 20.27 6.8 A. fraterculus (32)
N 0.16 25 3 0.12 0.1 18.75+ 7.7 A. fraterculus (3)
N 4.49 1699 406 0.24 0.1 90.37 15.6 A. fraterculus (223)
N 4.51 398 799 2.01 1.0 177.08 23.1 A. fraterculus (360)
A. obliqua (4)
A. sororcula (5)
N 1.60 334 148 0.44 0.2 92.48 17.9 A. fraterculus (51)
C. capitata (43)

E 3.31 65 25 0.38 0.1 7.56+ 3.12 A. fraterculus (9)
A. obliqua (2)

N 26.58 298 628 2.11 0.5 23.63 15.7 A. dissimilis (9)
A. pseudoparallela (363)
C. capitata (12)

E 5.24 148 267 1.80 1.2 50.94 10.8 A. fraterculus (148)
E 0.40 36 94 2.61 1.1 232.45 27.9 A. fraterculus (47)
E 12.79 1263 1285 1.02 0.7 100.44 30.1 A. fraterculus (218)
C. capitata (816)
E 9.85 96 52 0.54 0.2 5.28 2.6 A. fraterculus (33)
E 27.32 652 1151 1.77 0.9 42.13 18.1 A. fraterculus (372)
C. capitata (322)


E 8.67 138 44 0.32 0.1 5.07 3.3 A. fraterculus (12)


Neosilba padroi (6)

Neosilba padroi (29)








Neosilba padroi (12)
Neosilba padroi (5)


Neosilba padroi (3)


Neosilba padroi (12)


Lonchaea sp. (185)
Neosilba padroi (29)


Neosilba sp. (14)




Lonchaea sp. (14)
Neosilba zadolicha (43)
Neosilba sp. (41)


Neosilba padroi (12)



















TABLE 1. (CONTINUED) PLANTS SAMPLED WITH THEIR RESPECTIVE ORIGIN (0), FRUIT WEIGHT (FW), NUMBER OF FRUITS SAMPLED (N), NUMBER OF PUPAE (P), AVERAGE NUM-
BER OF PUPAE PER FRUIT (P/N), AND AVERAGE NUMBER OF PUPAE PER KG (P/KG). N = NATIVE AND E = EXOTIC. NUMBER IN PARENTHESES FOLLOWING FLY SPECIES
NAMES = NUMBER OF SPECIMENS REARED.

FW # fruits #pupae
Plant Species O (kg) n P P/n SE P/kg SE Tephritidae Lonchaeidae & Ulidiidae

Notogramma cimiciforme (9)
Laranja, Citrus sinensis E 17.20 176 105 0.60 0.2 6.10 4.0 Neosilba padroi (69)
Notogramma cimiciforme (29)
Sapindaceae
Camboata-vermelho, Cupania vernalis N 5.80 63 2 0.03 0.0 0.34 0.2 Neosilba padroi (2)
Sapotaceae
Aguaf, Chrysophyllum gonocarpum N 0.24 87 9 0.10 0.1 37.50 12.3 A. elegans (7)
Solanaceae
Jod, Solanum sisimbrifolium N 0.22 32 13 0.41 0.2 59.09 29.5 Neosilba padroi (12)
Tomate, Lycopersicum esculentum E 6.57 193 107 0.55 0.3 16.29 8.2 Neosilba padroi (52)
Ulmaceae
Esporao-de-galo, Celtis iguanaea N 911.66 807 608 0.75 0.3 0.67 0.2 A. fraterculus (3) Neosilba padroi(3)
R. pastranai (577)







Florida Entomologist 94(2)


Blanco (Rutaceae); and iguana hackberry, C.
iguanaea (Jacq.) Sarg. (Ulmaceae).
Pereskia aculeata Mill., also known as Ora-
pro-nobis or Barbados gooseberry, was found to
be a host plant for Anastrepha barbiellinii Lima;
and Campomanesia guazumifolia (Myrtaceae)
was recorded for the first time as a host plant for
both A. obliqua and A. sororcula.
Native plant species served as hosts of 12 fruit
fly species from 4 genera of Tephritidae, whereas
exotic plant species served as hosts of only 4 spe-
cies from 2 genera. Ceratitis capitata developed in
9 plant species from 5 families, with the following
order of predominance: khaki, Diospyros kaki
Thunb. (Ebenaceae) (93.1% of the fruits sampled
were infested); medlar, Eriobotrya japonica
(Thunb.) Lindl. (Rosaceae) (63.5%); uvaia, Euge-
nia pyriformis Cambess. (Myrtaceae) (29.2%);
and peach, P persica (L.) Batsch (28.1%). Some
fruit fly species occurred exclusively in 1 plant
species: Anastrepha barbiellinii in ora-pro-nobis,
Pereskia aculeata; Anastrepha grandis (Mac-
quart) only in pumpkin, C. pepo; Rhagoletotrypeta
pastranai Aczel only in esporao-de-galo, Celtis
iguaneae (Jacq.) Sarg.; Anastrepha dissimilis
Stone and A. pseudoparallela (Loew) only in Pas-
siflora edulis Sims;Anastrepha montei Lima only
in cassava, Manihot esculenta Crantz; and Paras-
tenopa guttata and P montana only in yerba
mate, I. paraguariensis St. Hil.
Lonchaeid flies were recorded from 22 host
plant species from 9 families of which 12 were na-
tive and 10 exotic. Araujo & Zucchi (2002) have
also described the indiscriminate infestation of
native and exotic fruits by Lonchaeidae. Neosilba
padroi, a species described recently by Strikis &
Lerena (2009), had the highest number of host
species (7 native, 15 exotic) belonging to 8 fami-
lies; the lance fly, Lonchaea sp., had 4 host species
(2 native and 2 exotic) in 4 families; Neosilba
zadolicha McAlpine & Steyskal had 3 host species
(1 native and 2 exotic) in 3 families; and Dasiops
sp. occurred only in C. pepo (exotic). Neosilba
zadolicha occurred in araticum, A. rugulosa (An-
nonaceae), araci, P cattleianum (Myrtaceae), and
peach, P persica (Rosaceae). Spondias spp. (Anac-
ardiaceae) (Santos et al. 2004) and medlar, Erio-
botrya japonica (Strikis & Prado 2009) also may
serve as hosts ofN. zadolicha.
The Ulidiidae occurred only in 5 exotic species
of Rutaceae and Cucurbitaceae; Euxesta sp. oc-
curred only in Cucurbitaceae and N. cimiciforme
Loew only in Rutacaeae. Euxesta sp. occurred on
3 plant species, with predominance in chayote,
Sechium edule (Jacq.) Sw., (48.9%). N. cimici-
forme occurred only in bergamot orange, C. retic-
ulata Blanco, and orange, Citrus sinensis (L.) Os-
beck. This species has a wide geographic range in
the New World and is a scavenger recorded from
a wide variety of plants (Steyskal 1963). Unlike
our results, Uch6a-Fernandes et al. (2003) and


Aguiar-Menezes et al. (2004) obtained specimens
ofN. cimiciforme in passion fruit (Passiflora sp.),
with occurrences also in tangerine, C. reticulata,
and orange, C. sinensis. Such differences may be
due to the interpopulation differences or seasonal
availability of host plants in different regions (Se-
livon 2000).
Pumpkin was infested by 4 species of flies be-
longing to 3 families. Guava, passion fruit, and
peach were infested by 5 species each, and these
fruits were found to support infestations only of
species of Tephritidae and Lonchaeidae.
Under the conditions in which this research
was conducted, we conclude that a wide diversity
of fruit-bearing plant species in the state of Santa
Catarina was attacked by 22 species of tephritoid
flies. The most predominant fly was A. fratercu-
lus, and P cattleianum was the host species most
frequently infested by these flies.

ACKNOWLEDGMENTS

We thank the National Council of Technological and
Scientific Development of Brazil (CNPq) for the Schol-
arship of Research Productivity; Biologist Pedro Strikis
from Unicamp for Lonchaeidae identifications; Prof. Dr.
Roberto Antonio Zucchi for some species of Tephritidae
confirmations, and Professors Dr. S6rgio Bordignon
from Unilasalle, and Dra. Rosiane Denardin and Lucia
Verona from Unochapec6, for plant identifications.

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Florida Entomologist 94(2)


SUSCEPTIBILITY OF GENERA AND CULTIVARS OF TURFGRASS TO
SOUTHERN CHINCH BUG BLISSUS INSULARIS (HEMIPTERA: BLISSIDAE)


JAMES A. REINERT, AMBIKA CHANDRA AND M. C. ENGELKE
Texas AgriLife Research & Extension Center, Texas A&M System, 17360 Coit Road, Dallas, TX 75252-6599 USA
E-mail: j-reinert@tamu.edu

ABSTRACT

The southern chinch bug (Blissus insularis Barber) is the most damaging insect pest of St.
Augustinegrass (Stenotaphrum secundatum Walt. Kuntze), across the southern U.S.A.
Susceptibility to the southern chinch bug and reproductive potential of the bugs on 24 cul-
tivars from 7 genera in 8 turfgrasses were evaluated under greenhouse conditions. Steno-
taphrum secundatum ('Raleigh', 'Texas Common', and 'Captiva') cultivars were the most
susceptible among all the turfgrass genera and each produced populations 97.5 bugs per
15-cm diameter plant within the 11-week test period from Jul to Sep 2008. Substantial
populations also developed on zoysiagrass (Zoysia spp.) ('Emerald', 'Empire', 'Palisades',
and'Zorro') cultivars and on '609' buffalograss (Buchloe dactyloides (Nutt.) Engelm.). Low
population development was recorded on cultivars of bermudagrass (Cynodon spp.), centi-
pedegrass (Eremochloa ophiuroides (Munro) Hack.), seashore paspalum (Paspalum vagi-
natum Swartz), bahiagrass (Paspalum notatum Flugge), and tall fescue (Festuca
arundinacea Schreb.).

Key Words: turfgrass pests, host plant resistance, host range, pest management, host plants

RESUME

La chinche surefia del past (Blissus insularis Barber) es el insecto mas danino para St. Au-
gustingrass (Stenotaphrum secundatum Walt. Kuntze), en el sur de U.S.A. La susceptibili-
dad de materials y el potential reproductive del insecto fueron evaluados en 24 cultivares
pertenecientes a siete g6neros de past para c6sped en condiciones de invernadero. Los cul-
tivares de S. secundatum ('Raleigh','Texas Common', y'Captiva') fueron los mas susceptibles
con poblaciones de 97.5 chinches por plant en maceta de 15 cm de diametro, en 11 sema-
nas de evaluacion dejulio a septiembre 2008. Considerables niveles de poblaciones tambi6n
fueron registrados en zoysiagrass (Zoysia spp.) (cultivares 'Emerald', 'Empire', 'Palisades', y
'Zorro'), asi como en el cultivar '609' de buffalograss (Buchloe dactyloides (Nutt.) Engelm.).
Bajos niveles de desarrollo se registraron en cultivares de bermudagrass (Cynodon spp.),
centipedegrass (Eremochloa ophiuroides (Munro) Hack.), seashore paspalum (Paspalum va-
ginatum Swartz), bahiagrass (Paspalum notatum Flugge), y tall fescue (Festuca arundina-
cea Schreb.).


The authors provided the translation by Carlos Campos


The southern chinch bug (SCB) (Blissus insu-
laris Barber) (Hemiptera: Blissidae) is the most
damaging insect pest of St. Augustinegrass
(Stenotaphrum secundatum Walt. Kuntze), across
the southern U.S.A., Bermuda, Mexico, and
throughout the Caribbean Archipelago (Henry &
Froeschner 1988; Sweet 2000). In the U.S.A. it is
found from South Carolina to Florida, westward
to Oklahoma and along the Gulf Coast to Texas
and in California, Hawaii, Puerto Rico, and Guam
(Reinert et al. 1995; Mortorell 1976; Vittum et al.
1999).
This pest begins to damage St. Augustine
lawns as early as Mar in parts of Southern Flor-
ida and Texas and first instars have been found
during all 12 months in Southern Florida (Rein-
ert, unpublished data). Damage begins as small
patches of dead grass early in the season, with en-


tire lawns killed as the summer progresses. Dur-
ing heavy infestations, large populations will
progress from one lawn to another as they move
from one city block to the next (Reinert & Kerr
1973). According to Painter (1928) B. leucopterous
L. damages grasses by "removal of the synergic
food-bearing solutions which flow to the roots by
way of the phloem; the stopping up of the sieve
tubes, and perhaps also the removal of water from
the xylem, together with the stoppage of the trac-
heids." It is believed that this same process takes
place when SCB feeds at the node and the crown
area of Stenotaphrum, which mimics the effects of
a toxin being injected into the plant. SCB infesta-
tions soon turn the grass yellow, brown, and it
eventually dies within a few days. Both nymphs
and adults feed in aggregates in localized areas
early in the season, with these areas coalescing


June 2011







Reinert et al.: Susceptibility of Turfgrasses to Southern Chinch Bug


into large dead areas or entire lawns as the sea-
son progresses (Reinert et al. 1995).
Stenotaphrum is cultivated extensively in sub-
tropical and tropical climates around the world
(Busey 2003; Sauer 1972). It is used widely across
the southern U.S.A. as a turfgrass in urban land-
scapes, including residential and commercial
lawns, parks, some sports complexes, and as a
pasture grass (Busey 2003; Sauer 1972). Steno-
taphrum has long been considered the primary
host of the SCB (Reinert & Kerr 1973; Reinert et
al. 1995; Vittum et al. 1999). The SCB has been
identified on 9 other grass hosts (Cherry & Na-
gata 1997; Slater 1976).
Resistant cultivars 'Floratam', 'Floralawn',
'FX-10', and 'Captiva' have been developed and
deployed to help manage this pest (Busey 1993;
Cherry & Nagata 1997; Dudeck et al. 1986; Horn,
et al. 1973; Reinert & Dudeck 1974). Recently,
populations of SCB have been identified that have
overcome the resistance in each of these cultivars
(Busey & Center 1987; Cherry & Nagata 1997;
Reinert 2008).
This study was established to characterize
the reproductive potential and development of
the SCB on 24 cultivars ofturfgrass from 7 gen-
era in 8 turfgrasses used across the Southern
U.S.A.

MATERIALS AND METHODS

This study was conducted under greenhouse
conditions during Jul-Sep 2008 at the Texas
AgriLife Research and Extension Center at Dal-
las, TX, U.S.A. A total of 24 cultivars (Table 1) in-
cluding St. Augustinegrass, (5) zoysiagrass (Zoy-
sia spp.) (5), bermudagrass (Cynodon spp.) (5),
buffalograss (Buchloe dactyloides (Nutt.) En-
gelm.) (2), centipedegrass (Eremochloa ophiuroi-
des (Munro) Hack.) (1), seashore paspalum
(Paspalum vaginatum Swartz) (2), bahiagrass
(Paspalum notatum Flugge) (2), and tall fescue
(Festuca arundinacea Schreb.) (2) were evaluated
for their susceptibility to SCB infestation and de-
velopment.
Plugs of grass grown either in the field or
greenhouse were divided and planted into 18-cell
trays and allowed to grow to cover the whole cell.
Cells measured 7.5 x 7.5 cm and 4 cm deep. Plants
were fertilized bi-monthly during establishment
with Miracle-Gro All Purpose fertilizer (24-8-16 +
B (200 ppm), Cu (700 ppm), Fe (1500 ppm), Mn
(500 ppm), Mo (5 ppm), Zn (600 ppm)) (Scotts,
14111 Scottslawn Road, Marysville, Ohio) at
~8.25 kg of N ha-1 month-1. Once sufficient growth
was achieved to provide near complete coverage of
the entire cell (ca. 14 weeks), plugs from 4 cells of
each cultivar were repotted into 15-cm diam plas-
tic pots and allowed to establish for 2 weeks. Each
pot was filled with soil within 2.5 cm of the top.
Potted plants were then fitted with a cylindrical


plastic cage (a modification of Starks & Burton
1977) to exclude extraneous insects and to confine
the SCB. Cages were made of Lexan 8010 Film
(0.2 mm thickness) (General Electric Plastics,
4600 AC Bergen op Zoom, The Netherlands) and
measured 32.5 cm tall and 2.5 cm in diam and
were vented on opposite sides with two, 8-cm di-
ameter ventilation holes to allow air circulation
within the cage. Ventilation holes and the top end
of the cage were covered with Voile 118" Decora-
tor Fabric in White # 235-004-81 (Hancock Fab-
rics, Plano, Texas, hancockfabrics.com) cut 15 mm
larger than the holes and secured with glue.
On 6-8 Jul 2008, 10 adults (5 male and 5 fe-
male) were introduced into each cage. Before bugs
were introduced, each pot was filled to the top
with fine topdressing sand. When each cage was
inserted over the plant in the pot the area be-
tween the cage and the wall of the pot was back-
filled with additional sand to form an escape-
proof barrier to the confined insects.
Pots were maintained in the greenhouse in a
randomized complete block design with 4 repli-
cates and held on full size aluminum sheet pans
that were 45 cm x 65 cm 18 gauge (WINCO Indus-
tries Co., Lodi, New Jersey). Potted plants were
provided sub-surface irrigation by filling the pans
with 1.5-2.0 cm of water as needed to avoid wilt-
ing of the test grasses. Watering was done every
3-4 d. After the pots were allowed to soak-up wa-
ter for about 2 h, the excess water was drained to
avoid causing deterioration of the root system.
Cages had to be opened about every 2 weeks so
the grass could be clipped, since there was not
enough room for the continued plant growth. The
clipping process was done over one of the alumi-
num sheet pans so that any SCB adults or
nymphs that were removed with the clippings or
that tried to escape could be collected with a hand
aspirator and returned to the grass when the cage
was put back in the pot.
SCB for this experiment were collected by vac-
uum sampling the bugs from a residential lawn of
S. secundatum in the Houston, Texas area. A
modification of the procedure for vacuuming (Na-
gata & Cherry 2007; and personal communica-
tion) was used. An Echo Shred'N' Vac model ES-
210 (Echo Inc., Lake Zurich, Illinois) leaf blower/
vacuum was modified by cutting-offthe distal 15-
cm end of the vacuum tube. This unit has an 87.5
cm long intake tube (11.25 cm diam) and pro-
duces 225.31 km/h (140 mph) of vacuum. A 20-cm
long piece of French drain pipe (10.3 cm outside
diameter) that fit loosely within the intake tube
was shimmed to fit the inside diam of the vacuum
tube by wrapping it with duct tape, close to each
end, so it would fit snugly inside to reattach the 2
pieces of the intake vacuum tube. When the 2
pieces of tube were re-joined, a 20-cm diameter
piece of polyester Tricot interlocking netting
(mesh size ca. 9.6 x 8 per cm, 24 x 20 per inch) cut








Florida Entomologist 94(2)


June 2011


TABLE 1. RATE OF REPRODUCTION AND DEVELOPMENT OF SOUTHERN CHINCH BUG ON CULTIVARS AND GENERA OF
TURFGRASS.

Nymphs

Genera of grasses Total nymphs',b Adults',b Totala'b
Cultivars 1st ab 5tha,b (N) (A) (N + A)


Stenotaphrum secundatum (St Augustinegrass)
Raleigh
TX Common
Captiva
FX-10
Floratam

Zoysia spp. (Zoysiagrass)
Palisades
Emerald
Zorro
Empire
Cavalier

Buchloe dactyloides (Buffalograss)
609
Prairie

Festuca arundinacea (Tall Fescue)
Rebel
Paladin

Cynodon spp. (Bermudagrass)
Tifton 10
Tifway
Texturf 10
TifSport
Common

Paspalum notatum (Bahiagrass)
Argintine
Pensacola

Paspalum vaginatum (Seashore Paspalum)
Seadwarf
AZ-1

Eremochloa ophiuroides (Centipedegrass)
Tifblaire


34.3 a*
25.3 a
30.7 a
1.0 bc
0.8 bc


5.8 b
1.3 bc
1.3 bc
0.8 bc
0.0 c


45.8 a*
30.0 b
18.5 b
0.0 c
0.3 c


3.7 bc 0.3 c
0.8 bc 0.0 c


2.0 bc 1.0 c
2.8 bc 0.0 c


1.3 bc
0.0 c
0.3 c
0.0 c
0.0 c


1.8 bc 0.0 c
0.0 c 0.0 c


0.3 c 0.0 c
0.0 c 0.0 c


0.0 c 0.3 c


163.0 a*
117.5 ab
93.3 b
1.3 d
1.1 d


16.5 c
8.8 cd
8.0 cd
4.3 cd
0.3 d


7.5 cd
1.0 cd


5.0 cd
3.3 cd


2.8 cd
1.3 cd
0.5 d
0.0 d
0.0 d


2.0 cd
0.0 d


1.5 cd
0.0 d


0.3 d


17.8 a*
4.3 b
4.3 bc
0.0 d
0.0 d


2.5 bcd
1.0 cd
0.0 d
1.3 bcd
0.8 d


180.8 a* A**
121.8 b A
97.6 b A
1.3 dB
1.1 dB


19.0 cA
9.8 cd AB
8.0 cd AB
5.6 cd AB
1.1 dB


2.5 bcd 10.0 cd ns
0.8 d 1.8 d


1.0 cd 6.0 cd ns
0.8 d 4.1 cd


0.3 d
0.0 d
0.0 d
0.0 d
0.0 d


3.1 cd ns
1.3 d
0.5 d
0.0 d
0.0 d


0.0 d 2.0 d ns
0.0 d 0.0 d


0.5 d 2.0 cd ns
0.0 d 0.0 d


0.3 d


0.6 d


"Mean number of 1st, 5th instars, total nymphs, adults, and total population on each turfgrass cultivar after an 11-week devel-
opment period.
bData in each column was transformed as V (n + 0.001) for analysis; untransformed means are reported.
*Means in a column followed by the same lower case letter are not significantly different by Fishers protected LSD (P = 0.05)
(Analysis among all turf groups).
**Means in the total column for each grass followed by the same upper case letter are not significantly different by Fishers pro-
tected LSD (P = 0.05) or by Student's t-test. (Analysis within a turf group only).


from the material of a BioQuip superior aerial
net (Cat. No. 7215NA, BioQuip Products, Ran-
cho Dominguez, CA), material was inserted at the
outer end of the French drain insert to form a 15-
cm deep collecting basin to catch insects that
were dislodged as the grass was vacuumed.
The potted plants were maintained in the
greenhouse until the week of 22 Sep 2008, when
the total number of bugs produced on each plant


was assayed. Plants in the experiment (1 repli-
cate at a time) were individually submerged in
18.9-liter plastic buckets of water (the plant was
weighted with a stone so it would stay sub-
merged) and all SCB nymphs and adults that
floated to the surface within 30 min were re-
moved, identified by instars, and counted. After a
plant had been submerged for 5 min and again at
15 min, the canopy of the plant was agitated by







Reinert et al.: Susceptibility of Turfgrasses to Southern Chinch Bug


hand to dislodge any bugs that had failed to let
loose and float to the surface. This procedure was
a modification of the flotation method that has
been used widely to accurately assay field popula-
tions of SCB for chemical efficacy tests (Reinert
1974, 1982).

Data Analysis and Statistics

Data for the number of SCB for each growth
stage for each cultivar were analyzed by Analysis
of Variance (ANOVA) (PROC GLM) for a random-
ized complete block design with 4 replications to
test for differences in the number of progeny that
had developed on each cultivar. Data for cultivars
were also grouped by species of grass (Zoysia and
Cynodon each contained 2 species, but were ana-
lyzed by genus only) and analyzed to determine
suitability among the 8 types of turfgrass tested.
The transformations / (n + 0.001) was used on
each data set to achieve normality and homogene-
ity of variance before analysis (Kuehl 2000) but
untransformed means are presented. Means were
compared at the 5% level of significance with
Fisher's least-significant difference (LSD) multi-
ple range test. For the total population column,
means were also compared within each grass gen-
era by Student's t-test (SAS Institute 2009).

RESULTS AND DISCUSSION

This method of caging the SCB on potted grass
plants in a no-choice experiment worked well to
assay the reproductive and developmental poten-
tial on each cultivar. Caging was necessary to con-
fine the bugs on the grasses, but the main prob-
lem with this type and size of cage was that cages
were not large enough to accommodate the
growth potential of several of the grasses, and
they had to be opened during the experiment to
clip and remove leaf material from many of the
cultivars. The modified Echo@ blower/vacuum
also worked well for collecting large numbers of
SCB specimens for this type of study.

Stenotaphrum (St. Augustinegrass)

Stenotaphrum, as expected, served as the best
host among the 8 turfgrass groups. The highest
population was produced on 'Raleigh' with all 5
instars and adults present in substantial num-
bers for a mean of 163.0 nymphs, 17.8 adults, and
a total population of 180.8 bugs per plant after
the 11-week test period (Table 1).'Texas Common'
was the second best host (117.5 nymphs, 121.8 to-
tal), followed by 'Captiva' with 93.3 nymphs and
97.5 total bugs produced on it. Analysis conducted
across all 8 groups of turfgrass showed that Ra-
leigh produced significantly more SCB than ei-
ther Texas Common or Captiva, but all 3 cultivars
serve as good reproductive hosts and all 3 mean


populations far exceeded the accepted damage
threshold level of 20 to 30 bugs per 0.1 m2 (Reinert
1972; Buss & Unruh 2006). The highest individ-
ual SCB population on any of the replicate plants
of Raleigh, Texas Common, and Captiva was 311,
252, and 155, respectively. Neither 'FX-10' nor
'Floratam' served as an acceptable host with this
population of SCB and they yielded only an aver-
age of 1.3 and 1.1 total bugs, respectively. More-
over, all of the bugs on these 2 cultivars had devel-
oped on only 1 replicate plant. Additionally, they
were all first instars, except for 1 third instar on
1 of the FX-10 replicate plants and 1 fifth instar
on 1 Floratam replicate plant. Analysis conducted
only on the 5 cultivars of Stenotaphrum showed
that population levels on FX-10 and Floratam
were not significantly different, and they were
significantly lower than those on the 3 susceptible
cultivars (Raleigh, Texas Common, and Captiva).
In a related study in a lab no-choice experi-
ment with the same population of SCB, adult sur-
vival was high on Raleigh, Texas Common, and
Captiva (72-78%), but survival on Floratam and
FX-10 was only 48 and 58%, respectively, after 7 d
of confinement (Reinert unpublished data). How-
ever, when Floratam and FX-10 were first re-
leased (Horn et al. 1973; Busey 1993), both culti-
vars consistently provided >80% antibiosis within
7 d for populations of SCB adults that were col-
lected from lawns in Florida (Reinert & Dudeck
1974; Reinert 1978). More recently, however,
Cherry & Nagata (1997) showed that oviposition
of eggs was high and survival on Floratam,
Seville, Bitterblue, and FX-10 cultivars was 88.6
to 75.6% for populations of SCB collected from
Florida lawns.

Zoysia (Zoysiagrass)

Among the 5 Zoysia cultivars, 'Palisades'
served as the best host for SCB with the develop-
ing population consisting of all 5 instars and
adults. A mean of 16.5 nymphs and 19.0 total
bugs had developed on this cultivar during the
11-week test period. When the Zoysia cultivars
were analyzed either among the total cultivars or
separately for the genus, the same statistical sep-
arations were recorded (Table 1). Palisades pro-
duced a significantly higher number of SCB than
'Cavalier' (mean total of only 1 SCB), but the pop-
ulation on Palisades was not significantly higher
than either 'Emerald', 'Zorro', or 'Empire'.
Although Zoysia is not normally considered a
primary host of the SCB (Reinert et al. 1995), this
study shows certain cultivars, Palisades along
with Emerald, Zorro, and Empire can serve as ac-
ceptable reproductive hosts with mean develop-
ment of >5.5 total bugs during this study. This
would be an equivalent of 31 bugs per 0.1 m2,
which is within the considered threshold of dam-
age on Stenotaphrum. Three of the replicate







Florida Entomologist 94(2)


plants of Palisades had total populations >23
SCB. Also, 1 replicate plant each of Zorro, Emer-
ald, and Empire had total populations of 27, 26,
and 14 SCB, respectively. Population develop-
ment on Cavalier in the present study with B. in-
sularis was the lowest among the Zoysia cultivars
tested with an average of 1.1 bugs per replicate
plant.
Studies with a related Blissus species, the
western chinch bug (B. occiduus Barber), showed
that Zoysia and particularly the cultivars 'Ze-
nith', 'Meyer', and 'Crowne', serve as acceptable
hosts for that species as well (Eickhoffet al. 2006,
2007). Populations of B. occiduus preferred both
Buchloe and Zoysia. Cavalier along with Emerald
and Zorro produced the lowest number ofB. occid-
uus in their greenhouse study and these cultivars
were listed as moderately resistant (Eickhoff et
al. 2007). Cavalier expresses good resistance to
both species of Blissus.

Buchloe (Buffalograss)

For the 2 cultivars of Buchloe, only'609" served
as a good host for SCB with 7.5 nymphs and a to-
tal of 10.0 bugs per plant (Table 1). This would be
equivalent to 56.5 bugs 0.1 m2 which is within the
threshold of damage on Stenotaphrum. Although
there was a large difference between the mean
number of SCB that developed on the 2 cultivars,
there was no significant difference due to a large
amount of variance among the replicates.

Other Turfgrass Genera

Surprisingly, both cultivars of Festuca,
'Rebel', and 'Paladin', did support low develop-
ment of SCB with total mean numbers of 6 and
4.1 bugs per replicate plant, respectively (Table
1). The development on the 5 Cynodon cultivars
was very low. Poor development of SCB has also
been reported on Cynodon (no cultivar identi-
fied) by Cherry & Nagata (1997). Slater (1976) in
his study of host relationships of Blissinae de-
scribed Cynodon as a breeding host for the SCB.
However, Kelsheimer & Kerr (1957) reported Cy-
nodon to be rarely attacked by the SCB. One of
the authors has received numerous reports of
chinch bug feeding on and damage to Cynodon in
Florida, Texas, and in island nations throughout
the Caribbean, but most likely these populations
and their damage were caused by another Blis-
sus species. Cynodon has been reported as a host
of B. leucopterus leucopterus Say (Lynch et al.
1987).
The 2 cultivars of P. notatum, 2 cultivars of P.
vaginatum, and 1 cultivar of Eremochloa did not
support much SCB development (<2.0 bugs per
plant) in this study. Also, only 1 adult developed
on 1 of the Eremochloa cv. 'Tifblaire' replicate
plants. Kelsheimer & Kerr (1957) reported Er-


emochloa to be an occasional host for the SCB.
Additionally, Kerr (1966) reported that SCB will
attack other lawn grasses (P. notatum, Cynodon,
Eremochloa, and Zoysia) but mostly it is a prob-
lem on Stenotaphrum. Other common hosts in-
clude crabgrass (Digitaria spp.), torpedograss
(Panicum repens L.), and pangolagrass (Digi-
taria eriantha Steud) (Slater & Baranowski
1990).
This study confirms the high suitability of
Stenotaphrum as a developmental host for the
SCB. We also show that 4 cultivars of Zoysia and
the cultivar 609 Buchloe serve as good breeding
hosts and may have potential for damage by SCB.

ACKNOWLEDGMENTS

This study was supported in part by grants from the
Texas Turfgrass Research, Extension, and Education
Endowment. Appreciation is extended to J. E. McCoy for
his technical assistance.

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Florida Entomologist 94(2)


June 2011


TOMARUS SUBTROPICUS (COLEOPTERA: SCARABAEIDAE)
LARVAL FEEDING HABITS


OLGA S. KOSTROMYTSKA1 AND EILEEN A. BUSS2
1Entomology Department, Rutgers, The State University of New Jersey, 93 Lipman Drive,
New Brunswick, NJ 08901

2Entomology and Nematology Department, University of Florida, P.O. Box 110620, Gainesville, FL 32611

ABSTRACT

The importance of soil organic matter for Tomarus subtropicus Blatchley larval development
and survival, the amount of damage larvae could cause on turfgrasses, and potential larval
host range were investigated in greenhouse experiments. First instars were reared individ-
ually in seedling trays containing sand or peat, with or without St. Augustinegrass. Sur-
vival, developmental stage, and final weight were recorded 1 month after introduction. First
instars died in the pots with peat but no grass, so it appears that grass roots were critical for
larval growth and development. Soil organic matter did not significantly affect grub weight
gain and development, but more root loss occurred with grass grown in sand. In host range
tests (2005 and 2006), first and third instars were reared on 6 species of warm season
grasses and ryegrass. Grub weight gain, development, survival and grass root reduction
were determined 2 months after introduction. Larval survival ranged from 62-93% if grubs
were reared on warm season grasses to only 40% if reared on ryegrass. Grubs reared on
warm season grasses gained weight and successfully developed into third instars, indicating
that all of the tested warm season turfgrasses were suitable for larval T subtropicus growth
and development. Grub feeding caused significant root reduction of all grasses in our study,
which ranged from 36 to 87% and differed among grass species. As result, quality ratings
and clipping yields decreased for most of the turfgrasses after 5 weeks of infestation, but ba-
hiagrass and seashore paspalum were less affected by T subtropicus root feeding, compared
to the other grass species.

Key Words: sugarcane grub, host range, bermudagrass, sugarcane, St. Augustinegrass,
ryegrass, root reduction, soil organic matter, grub weight gain and development

RESUME

La importancia de material organica en el suelo para el desarrollo y sobrevivencia de larvas
de Tomarus subtropicus Blatchley, la cantidad de dano que las larvas puedan causar en el
c6sped y el rango potential de hospederos por las larvas fueron investigados en experiments
realizados en invernaderos. Se criaron los primeros instares individualmente en bandejas
usadas para plantillas con arena o turba y con o sin el c6sped San Augustin. El sobreviven-
cia, el estadio de desarrollo y el peso final fueron anotados 1 mes despu6s de la introducci6n.
Los primeros instares murieron en las macetas con turbo y sin grama, esto parece indicar
que las raices de grama son basicas para el crecimiento y desarrollo de las larvas. La material
organica del suelo no afecto el aumento en el peso y el desarrollo de las larvas, pero hubo una
mayor p6rdida de las raices de la grama sembrada en arena. En pruebas del rango de los hos-
pederos (2005 y 2006), se criaron los primeros y terceros instares sobre 6 species de grama
de la estaci6n calida y sobre centeno. Se determinaron el aumento en el peso de las larvas,
el desarrollo, la sobrevivencia reducci6n en las raices de la grama 2 meses despues de la in-
troducci6n. El sobrevivencia de las larvas fue entire 62-93% en las larvas criadas sobre
grama de la estaci6n calida y solo 40% en larvas criadas sobre centeno. Las larvas criadas
sobre grama de la estaci6n calida aumentaron en peso y se desarrollaron exitosamente en
instares de tercer estadio, que indica que todas las classes de grama de la estaci6n calida pro-
badas fueron apropiadas para el crecimiento y desarrollo de larvas de T subtropicus. En
nuestro studio, la alimentaci6n de las larvas caus6 una reducci6n significativa de las raices
de 36 a 87% y varian entire las species de grama. Como un result, el indice de la calidad y
el rendimiento de las cortadas de grama diminuy6 para la mayoria de las classes de grama
despu6s de 5 semanas de infestaci6n, pero el c6sped Bahia y el paspalum costero fueron me-
nos afectados por la alimentaci6n de T subtropicus sobre las raices, comparados con las otras
species de grama.


Tomarus subtropicus Blatchley is a destructive Coasts, but is also distributed along coastal Ala-
turfgrass pest along Florida's Gulf and Atlantic bama, Georgia, South Carolina, and North Caro-







Kostromytska & Buss: Tomarus subtropicus Host Range


lina (Cartwright 1959). As with other grub spe-
cies, T. subtropicus grubs directly damage turf by
their root-feeding and tunneling behaviors
(Ritcher 1966; Tashiro 1987; Braman & Pendley
1993). This pest is univoltine, with eggs present
from late Jun to early Aug, first instars from Jul
to Aug, second instars from Aug to Sep, and
mostly third instars from Oct to Feb in Florida
(Kostromytska & Buss 2008). Tomarus subtropi-
cus attacks the roots of sugarcane (Saccharum
spp.) (Gordon & Anderson 1981), St. August-
inegrass (Stentaphrum secundatum (Walt.
Kuntze)) (Kostromytska & Buss 2008), and ber-
mudagrass (Cynodon dactylon (L.)) (Summers
1974; Reinert 1979; Prewitt & Summers 1981),
resulting in crop yield loss and large patches of
dead turfgrass. Its potential to feed on and injure
other warm season turfgrasses has not been as-
sessed. Tomarus subtropicus grubs also feed on
plant roots in ornamental plant beds, and cause
plant dieback (E. Buss, personal observation).
White grub feeding habits vary depending on
the species. Some species (e.g., Cotinis nitida L.)
obtain nutrients from soils high in organic matter
(Brandhorst-Hubbard et al. 2001), while others
need live plant roots (e.g., Popilliajaponica New-
man, Rhizotrogus majalis (Razumowsky), Cyclo-
cephala spp.,Phyllophaga spp.). Some scarab spe-
cies consume soil organic matter as first instars,
then switch to live roots in later instars (Litsinger
et al. 2002). The larval feeding habits of T sub-
tropicus have not been clearly described. Tomarus
subtropicus females oviposit and their offspring
develop in soil with a high organic matter content
(e.g., muck soil in sugarcane fields) (Cherry & Co-
ale 1994), but they also survive and develop in
sandy soils with low organic matter in residential
environments (Kostromytska & Buss 2008).
Because the urban landscape is a complex sys-
tem with many plant species, understanding the
feeding preference of key pests helps to explain
pest distribution, abundance, and damage related
to feeding, and can affect the management strat-
egies used against them. We conducted no-choice
tests to assess the effect of soil organic matter on
T subtropicus survival and development, and to
determine which other warm season turfgrasses
could be hosts for T subtropicus grubs.

MATERIALS AND METHODS

Effect of 2 Soil Types on First Instars

A no-choice test with a nutrient-poor soil
(sand) and an organic soil (Black Velvet peat
(Black Gold Compost Co., Oxford, Florida)) was
conducted from Jul to Aug 2005 to evaluate T
subtropicus larval growth and survival. The roots
of 'Palmetto' St. Augustinegrass plugs were
washed, and grass plugs were replanted into the
seedling tray cells (8 x 8 x 8 cm) with either sand


or peat (48 cells for each growing media). Another
24 cells were filled with peat, but no grass. Grass
was maintained in the greenhouse for 2 weeks be-
fore the experiment. Cells were arranged in a ran-
domized complete block design.
Adult T subtropicus were collected from in-
fested St. Augustinegrass lawns in Punta Gorda
(Charlotte County) and Fort Myers (Lee County),
Florida, and held in the laboratory to obtain eggs
and young larvae (Kostromytska 2007). Grubs (2-
6 d old; mean weight: 0.028 0.002 g) were ran-
domly assigned to the following treatments: peat
only, peat and grass, or sand and grass (24 repli-
cates or cells for each). Cells of grass planted in
sand or peat (24 cells of each) were uninfested
controls. Individual first instars were placed in a
depression (2.5 cm deep, 0.7 cm in diameter)
made in the center of each cell and covered with
soil. Each cell was provided 30 mL of water daily.
After 1 month, cells were visually inspected, and
surviving grubs were weighed. Grass roots were
washed with a #10 sieve, oven-dried in paper bags
for 48 h at 55C, and root dry weights were re-
corded. Data were analyzed by an ANCOVA (SAS
Institute 2004) with soil type as a factor, post-
treatment grub weight as a dependent variable
and initial grub weight as a covariate. A two-way
ANOVA was also conducted with soil type and
grub presence as factors and dry root weight as a
dependent variable. Tukey's HSD test was con-
ducted for mean separation.

Survival and Growth of Third Instar T subtropicus
Reared on Different Turfgrass Species

Six warm season and 1 cool season turfgrasses
were evaluated as possible hosts for T subtropi-
cus grubs. Tested grasses included Palmetto St.
Augustinegrass, 'Tifway' bermudagrass (C. dacty-
lon x transvaalensis Burtt-Davy), 'Empire' zoysia-
grass (Z. japonica Steud.), common centipede-
grass (Erimochloa ophiuroides (Munro) Hack),
'Pensacola' bahiagrass (Paspalum notatum
Flugge), 'Sea Dwarf' seashore paspalum
(Paspalum vaginatum Swartz), and 'Gulf' annual
ryegrass (Lolium multiflorum Lam.). Thirty plugs
(15 cm diameter) of each warm season grass spe-
cies were obtained from the University of Florida
Plant Science Unit in Citra (Marion County),
Florida, in Aug 2005. Soil was washed off the
roots and the grass plugs were planted with Fa-
Fard mix #2 (Conrad Fafard, Inc., Agawam, Mas-
sachusetts) in plastic pots (15 cm diameter). An-
nual ryegrass was seeded at a rate of 0.05 kg/m2.
Grass was watered daily and fertilized monthly
with 24.4 kg of N per ha during 2 months of estab-
lishment. Pots were arranged in a randomized
complete block design in the greenhouse.
Initial grub weights were obtained, then 1 re-
cently molted (<7 d old) third instar was put in a
shallow depression on the soil of each of 15 pots







Florida Entomologist 94(2)


for each turfgrass species. Grubs that failed to dig
into the soil within 10 min were replaced. Larval
survival, weight, and weight gain were deter-
mined after 8 weeks. Daylight was supplemented
with lights to provide a photoperiod of 16:8 h
(L:D) and the average ambient greenhouse tem-
perature was ~23.4C. Pots were watered with
150 mL of tap water every other day. Each turf-
grass species was maintained at its recommended
height (Turgeon 2002): 1.3 cm for seashore
paspalum, 2.5 cm for bermudagrass, 5.1 cm for
centipedegrass and zoysiagrass, and 7.6 cm for
annual ryegrass, St. Augustinegrass and bahia-
grass. To assess the amount of feeding damage,
grass clippings were collected weekly and fresh
weights were taken within 2 h. Clippings were
then oven-dried for 48 h at 55C, and weighed. Af-
ter 8 weeks of infestation, grass roots were cut
within 2 mm of the plant crown, washed with a
#20 sieve, oven-dried for 48 h (55C), and dry root
weights were recorded.

Tomarus subtropicus Neonate Survival, Growth and
Development on 6 Warm Season Grasses

The warm season turfgrass species noted in
the previous section were tested as potential
hosts for T subtropicus larvae in a greenhouse ex-
periment in 2006. Grass plugs were planted into
10-cm plastic pots with native soil (94% sand, 4%
clay, 2% silt and 1.6% organic matter), and al-
lowed 2 months to establish. First instars (1-3 d
old) were weighed immediately before being indi-
vidually placed in a hole (8 mm diameter, 5 cm
deep) in the soil of each pot, and were covered
with soil. Grass was watered daily as needed and
fertilized weekly (0.6 kg of N per ha, Miracle
Gro Scotts Miracle-Gro Products Inc., Marys-
ville, OH). Four replicates per grass species were
arranged and each replicate included 6 infested
and 6 uninfested control pots. Grubs were re-
moved from the pots after 8 weeks, and larval sur-
vival, weight, and head capsule width were deter-
mined. Grass clippings were collected and pro-
cessed weekly, as previously described. Grass
color and density were visually assessed on a
scale from 1 (yellow, sparse) to 9 (dark green, very
dense) and total grass quality was calculated by
averaging the two scores. After the grubs were re-
moved, grass roots were cut to within 2 mm of the
plant crown, washed with a #20 sieve, and placed
in paper bags. The remaining plant parts were
collectively placed into paper bags, and oven-
dried for 48 h (55C). Dry root weight and dry to-
tal plant yield were recorded.

Statistical Analysis

The correlation between initial and final grub
weights was tested before analysis. If the 2 vari-
ables were significantly correlated, the ANCOVA


GLM procedure (SAS Institute 2004) was used to
analyze the effect of turf species on grub final
weight with a correction for initial weight for all
experiments. Percent of root reduction was calcu-
lated as averaged root weights of controls minus
root weights of infested plants and divided by av-
eraged weights in controls. Analysis of variance
(GLM procedure, SAS Institute 2004) was used to
determine the effect of turf species on the percent-
age of grub survival and development, and effect
of grub presence on dry root weight. Proportion
data were arcsine square root transformed before
analysis. Clipping weights and grass quality rat-
ings were analyzed by a repeated measure analy-
sis (SAS Institute 2004) with time as a repeated
within-group factor and grub presence as a be-
tween-group factor. Means were separated by
Tukey's HSD.

RESULTS AND DISCUSSION

Effect of 2 Soil Types on First Instars

Tomarus subtropicus grubs fed on live St. Au-
gustinegrass roots, regardless of soil type, in this
test. Two grubs (8%) that were reared on peat
without grass survived, but they remained first
instars, and all other grubs in this treatment
died. All grubs provided with St. Augustinegrass
roots, regardless of soil type, were second instars
when the test was evaluated. Grub survival to the
second instar when reared on peat with grass was
83%, and 75% when reared on sand with grass.
Tomarus subtropicus body weights were statisti-
cally similar when grubs were reared on grass
grown in peat (0.87 0.07 g) or grass grown in
sand (0.74 0.08 g).
Grub feeding significantly reduced St. August-
inegrass dry root weight compared to uninfested
cells, regardless of soil type (F = 156.66; df= 1, 85;
P < 0.0001). Uninfested pots had statistically sim-
ilar dry St. Augustinegrass root weights (1.24
0.06 g in peat; 1.17 0.06 g in sand). The final dry
root weight of infested grass grown in sand (0.31
0.05 g) was significantly lower than in infested
grass grown in peat (0.59 0.05 g) (F = 4.59; df =
1, 85; P = 0.03), indicating that more root her-
bivory may have occurred in the pots with sand.
Peat, like muck soil, is an organic soil, consist-
ing of poorly decomposed animal and plant rem-
nants (Brown 2009) with organic matter content
ranging from 40 to 80% (Andriesse 1988; Litaor et
al. 2005; Kechavarzi et al. 2010) which can pro-
vide nutrients (e.g., carbon, nitrogen, sulfur, and
phosphorous) to plants and soil fauna (Andriesse
1988; Killham 1994). Although the nutrient con-
tent of the soils or turfgrass were not measured in
our test (fertilization and irrigation were consis-
tent across all treatments), it is possible that the
peat provided additional nutrients to the grass,
which could lead to either more efficient grub


June 2011







Kostromytska & Buss: Tomarus subtropicus Host Range


feeding or increased compensatory plant growth
(Radcliffe 1970; Brown and Gauge 1990; Steinger
& Miiller-Scharer 1992; Sparling et al. 2006). In
addition, grubs may, while feeding on grass roots,
acquire additional nutrients when ingesting soil
with greater organic matter content (Seastedt
1985; Brown & Gange 1990), and thus cause less
root damage. The tendency for insects to consume
more of a nutritionally inferior food to overcome
the lack of needed nutrients has been docu-
mented for many taxa (King 1977; Yang & Joern
1994; Obermaier & Zwolfer 1999; Berner et al.
2005).

Survival and Growth of Third Instar T subtropicus
Reared on Different Turfgrass Species

Third instar initial weights (1.97 0.08 g) were
statistically similar among treatments (F = 0.71;
df = 6, 76; P = 0.64). However, initial and final
grub weights were significantly correlated (Pear-
son's r = 0.37, P = 0.001) and were included as co-
variates in the analysis. Final grub weight dif-
fered statistically among grasses (Table 1). Third
instar weights when reared on ryegrass (1.96 +
0.2 g) and bermudagrass (2.1 0.2 g) were signif-
icantly lower than grub weights on any of the
other grasses tested (F = 8.51; df = 6, 76; P <
0.0001). However, 66.7% of the grubs survived on


bermudagrass and only 40% survived on
ryegrass. Analysis of grass dry root weight with
and without grubs indicated that there was a sig-
nificant root reduction in all pots with warm sea-
son grasses, but not in the pots with ryegrass
(Fig. 1). These data suggest that ryegrass was a
poor larval host for T subtropicus. Annual
ryegrass was the only C3 grass included in the
test. C3 grasses typically are more beneficial nu-
tritionally to herbivores than C4 grasses (Barbe-
henn & Bernays 1992), however physical proper-
ties could be a key factor for herbivore host selec-
tion (Scheirs et al. 2001). Suitability of annual
ryegrass as a host for T subtropicus larvae may
be affected by the physical structure of the grass
root system. Numerous ryegrass fine roots cre-
ated a dense mesh through the entire pot, which
may have reduced grub movement. At evaluation,
live grubs in ryegrass pots were in soil chambers
that were located near pot walls and not more
than 5 cm deep. In contrast, the other grasses had
thick main roots from which roots branched closer
to the pot walls and bottom, and grubs were often
found in the center of the pots at different depths.
Grass leaf growth changed differently among
grass species over time (summarized statistics
are in Table 2). For St. Augustinegrass, the
main effect of grub presence and the interaction
of grub feeding with time were significant. On


TABLE 1. LARVAL T. SUBTROPICUS SURVIVAL AND GROWTH ON DIFFERENT GRASS SPECIES AND ASSOCIATED ROOT RE-
DUCTION IN 2005 AND 2006.

% Grub Initial grub weight Final grub weight Proportional Root reduction
Grass species survival (g SEM)' (g SEMY weight gain3 (% SEMY

2005
Bahiagrass 93.3 6.7 1.8 0.2a 3.05 0.2b 1.8 0.1a 27.4 8.8bc
Bermudagrass 66.7 6.7 1.7 0.2a 2.10 0.2a 1.1 0.3ab 64.9 3.2a
Centipedegrass 66.7 24.0 1.9 0.3a 3.43 0.2b 2.1 0.3a 60.5 4.9a
Ryegrass 40.0 0.0 2.2 0.7a 1.96 0.2a 1.0 0.1b 17.2 4.9c
Seashore paspalum 86.7 6.7 2.1 0.2a 3.57 0.2b 1.8 + 0.1a 49.1 + 9.lab
St. Augustinegrass 66.7 6.7 2.2 0.2a 3.18 0.1b 1.4 0.1a 52.2 7.7ab
Zoysiagrass 86.7 6.7 2.0 0.1a 3.04 0.lb 1.8 0.2a 55.3 6.4ab

2006
Bahiagrass 79.3 7.9 0.028 0.002a 2.62 0.2abc 103.7 11.2abc 48.1 4.6bc
Bermudagrass 66.8 6.7 0.034 0.003a 2.20 0.2c 69.6 8.3c 87.5 4.6a
Centipedegrass 91.5 4.9 0.030 0.002a 2.43 0.2bc 87.6 0.7bc 36.3 4.2c
Seashore paspalum 62.5 10.5 0.031 0.003a 3.35 0.2a 126.2 15.6a 60.8 4.6ab
St. Augustinegrass 87.3 4.3 0.033 0.002a 2.62 0.2abc 92.2 7.9abc 65.0 6.8ab
Zoysiagrass 70.8 7.9 0.030 0.002a 3.13 0.lab 114.3 8.7ab 80.9 2.8a

Initial grub weight was not significantly different among treatments at a = 0.05 (2005: F = 1.64; df= 6, 69; P = 0.15 and 2006:
F = 1.84; df= 5, 143; P = 0.11).
'Means within columns with different letters are statistically different at a = 0.05 (2005: F= 8.51; df= 6, 76; P < 0.0001 and 2006:
F = 5.04; df= 5, 109; P = 0.0004).
'Proportions were calculated by dividing the final weights by initial weights. Means within columns with different letters are
statistically different at a = 0.05 (2005: F = 3.89; df= 6, 76; P = 0.002 and 2006: F = 3.95; df = 5, 109; P = 0.0026).
'Means within columns with different letters are statistically different at a = 0.05 (2005: F = 6.32; df = 6, 105; P < 0.0001 and
2006: F = 16.52; df= 5, 111; P < 0.0001).







Florida Entomologist 94(2)


4.5
4 EP aIft .l mbp
b



SI I
I tt I i


SI

Fig. 1. Reduction of root mass caused by third instar
T subtropicus feeding, 2005. Means marked with differ-
ent letter are different at a = 0.05 (F = 39.51; df= 1, 130;
P < 0.0001)


average, clippings collected from the infested
pots weighed less than clippings from unin-
fested control pots, and this difference in-
creased over time. For bahiagrass, bermuda-
grass, and centipedegrass the main effect of
grub presence was significant, whereas the in-
teraction between grub presence and time was
not significant. Clipping yield of these grasses
changed significantly over time in all pots, but
uninfested pots on average yielded more clip-
pings. For zoysiagrass, the main effect of grub
presence was not significant although the main
effects of time and the interaction of time and
grub presence were significant. Thus, decrease
in clipping weight over time was more pro-
nounced in the pots with grubs. Only the main
effect of time was significant for ryegrass and


seashore paspalum, so grass growth changed
over time, but variation of clipping yield was
not related to grub presence.

Tomarus subtropicus Neonate Survival, Growth, and
Development on 6 Warm Season Grasses

On average, 76.3% of grubs survived across
treatments and 70.8% of all (94% of survivors)
grubs reached the third instar (Table 1). Percent-
age survival, percent of grubs that reached the
third instar, and head capsule width did not differ
among the grasses (F = 1.64; df = 5, 23; P = 0.20;
F = 2.17; df = 5, 23; P = 0.10; and F = 1.73; df = 5,
109; P= 0.13).
Mean initial first instar weight (0.031 0.01 g)
did not correlate with mean grub final weight
(2.75 0.86 g) (r = 0.11, P = 0.22), and was not in-
cluded in the analysis as a covariate. Final grub
weight and proportional weight gain differed
among grasses. Similar to the result obtained in
the 2005-experiment, grubs feeding on bermuda-
grass gained less weight (weight gain about 70
times initial weight) when compared to grubs
reared on seashore paspalum (weight gain about
126 times initial weight) and zoysiagrass (weight
gain about 114 times) (Table 1).
Similar to the 2005-experiment, bermuda-
grass appeared to be a poorer host for T subtropi-
cus compared to the other warm season grasses.
However, T subtropicus larvae are reported to
damage bermudagrass (Reinert 1979), so despite
the slower grub growth, this grass may still be an
acceptable host. Reduced grub growth may be in-
fluenced by a smaller root mass in bermudagrass
(grubs consumed on average 87.5% of root mass).


TABLE 2. STATISTICS SHOWING EFFECTS OF TIME, GRUB FEEDING, AND THEIR INTERACTION ON CLIPPING YIELD DURING
AN 8-WEEK PERIOD IN 2005 AND 2006.

Grub feeding Time Time*Grub

Grass species F df P F df P F df P

2005
Bahiagrass 8.50 1, 27 <0.01 2.48 7, 21 0.05 1.05 7, 21 0.43
Bermudagrass 11.22 1, 23 <0.01 44.49 7, 17 <0.01 1.96 7, 17 0.12
Centipedegrass 12.80 1, 23 <0.01 3.20 7, 17 0.02 1.6 7, 17 0.20
Seashore paspalum 3.07 1, 19 0.10 20.12 7, 13 <0.01 2.57 7, 13 0.07
St. Augustinegrass 0.09 1, 27 0.76 2.92 7, 21 0.03 0.67 7, 21 0.74
Zoysiagrass 9.47 1, 23 <0.01 1.99 7, 17 0.11 2.69 7, 17 0.05

2006
Bahiagrass 1.56 1, 41 0.22 5.43 7, 35 <0.01 1.99 7, 35 0.08
Bermudagrass 8.10 1, 38 <0.01 5.38 7, 32 <0.01 2.47 7, 32 0.04
Centipedegrass 4.66 1, 44 0.04 6.09 7, 38 <0.01 0.36 7, 38 0.92
Seashore paspalum 0.30 1, 39 0.59 6.88 7, 33 <0.01 0.68 7, 33 0.69
St. Augustinegrass 1.49 1, 44 0.01 5.38 7, 32 <0.01 2.47 7, 32 0.04
Zoysiagrass 4.61 1, 46 0.04 9.48 7, 34 <0.01 1.29 7, 34 0.28


June 2011







Kostromytska & Buss: Tomarus subtropicus Host Range


Grub movement was also limited, so grubs could
not migrate in search of food after the previous
source had been exhausted.
Grub feeding caused significant root reduction
of all grasses in our study (F = 70.61; df = 6, 287;
P < 0.0001) (Fig. 2). The percent of root reduction
ranged from 36 to 87% and differed among
grasses (F = 16.52; df= 5, 111;P < 0.01) (Table 1).
However, the total plant yield was reduced only
for bahiagrass and bermudagrass (F = 22.81; df=
11, 287; P = 0.001) (Fig. 3). Root loss does not nec-
essarily reduce aboveground plant growth, and in
some cases, minor root damage can lead to in-
creased or compensatory foliage growth
(Humphries 1958; Seastedt et al. 1988; Brown &
Gange 1990; Bardgett et al. 1999; Blossey &
Hunt-Joshi 2003).
Measurements of grass yield over time demon-
strated that tested grasses responded differently
to T subtropicus herbivory (statistics are summa-
rized in Table 2). Centipede and zoysiagrass
tended to yield fewer leaf clippings if infested
with grubs, and clipping weights varied over time
(the main effects of infestation and time on clip-
ping yield were significant), but effect of grub
feeding was not significantly stronger with time
(interaction was not significant). The interaction
of the 2 factors was significant for bermudagrass
and St. Augustinegrass, so grub feeding de-
creased clipping yield beginning week 5. Grub
feeding did not affect clipping yield in pots of ba-
hiagrass and seashore paspalum.
Grub feeding reduced the quality ratings for
St. Augustinegrass, bermudagrass, zoysiagrass
and centipedegrass, but not for bahiagrass and
seashore paspalum (statistics are summarized in
Table 3). Differences in grass quality were appar-
ent 4 weeks (St. Augustinegrass, bermudagrass),
6 weeks (zoysiagrass), and 8 weeks (centipede-
grass) after grubs were introduced. Grass crowns



Pounwithoutlgrb b
b
6 b

i b




I I


P.y'i A.rAWi
Fig. 2. Reduction of root mass caused by larval T.
subtropicus feeding, 2006. Means marked with different
letters are different at a = 0.05 (F = 70.61; df = 1, 287; P
<0.0001)


"'4




a1




V,. . A. p..WB

Fig. 3. Effect of grub feeding on total plant yield by
warm season grasses, 2006. Means marked with differ-
ent letter are different at a = 0.05 (F = 5.51; df = 1, 253;
P = 0.02)



could be pulled easily from the pots with grubs,
but grass remained green in all pots.
Our study demonstrated that T subtropicus
can successfully survive and develop on bahia-
grass, bermudagrass, centipedegrass, zoysiagrass
and seashore paspalum, and confirmed that St.
Augustinegrass was an adequate host (Reinert
1979), regardless of soil organic content. Quality
ratings and clipping yields decreased for most of
the turfgrasses after 5 weeks of infestation, but
bahiagrass and seashore paspalum were less af-
fected by T subtropicus root feeding, compared to
the other grass species. It was previously re-
ported that 3 grubs per 0.1 m2 could severely dam-
age bermudagrass (Reinert 1979), but the grasses
in our study could tolerate approximately 5 and
12 third instar grubs per 0.1 m2 in 2005 and 2006,
respectively, despite >50% root reduction.
Environmental conditions (temperature, pho-
toperiod, herbivore aboveground grazing or mow-
ing, fertilization, and irrigation practices) can sig-
nificantly affect plant tolerance to root herbivory
in addition to plants characteristics and insect
density (Ladd & Buriff 1979; Seastedt et al. 1988;
Brown & Gange 1990; Potter et al. 1992; Crutch-
field et al. 1995; Crutchfield & Potter 1995; Bra-
man & Raymer 2006). For instance, 4 Japanese
beetle (Popillia japonica Newman) grubs per 15-
cm pot (~20 grubs per 0.1 m2) significantly re-
duced Poa pratensis L. clipping yield in a green-
house study (Ladd & Buriff 1979), but clipping
yield from other field and greenhouse tests was
unaffected by 60-90% root reduction from 40-60
grubs per 0.1 m2 and 24-30 grubs per 0.1 m2, re-
spectively (Potter et al. 1992; Crutchfield & Potter
1995).
During our experiment, grass was regularly ir-
rigated, and although the roots were dramatically
reduced and crowns could be easily removed from
infested pots, the foliage remained green. Most
third instar-feeding in the field occurs during the







Florida Entomologist 94(2)


June 2011


TABLE 3. STATISTICS SHOWING EFFECT OF TIME, GRUB FEEDING, AND THEIR INTERACTION ON TURFGRASS QUALITY IN
2006.

Grub feeding Time Time*Grub

Grass species F df P F df P F df P

Bahiagrass 0.6 1, 44 0.44 3.90 7, 40 0.15 0.95 7, 40 0.43
Bermudagrass 9.08 1, 44 <0.01 17.07 7, 40 <0.01 5.54 7, 40 <0.01
Centipedegrass 5.71 1, 44 0.02 13.51 7, 40 <0.01 8.66 7, 40 <0.01
Seashore paspalum 0.04 1, 44 0.83 4.61 7, 40 0.01 0.37 7, 40 0.70
St. Augustinegrass 9.08 1,44 <0.01 17.07 7,40 <0.01 5.54 7,40 <0.01
Zoysiagrass 32.23 1, 44 <0.01 14.08 7, 40 <0.01 9.82 7, 40 <0.01


fall (Kostromytska 2007), which coincides with
reduced rainfall and slower warm season turf-
grass growth. Thus, drought and/or other envi-
ronmental stresses during this time, in addition
to root damage by grubs, may more quickly over-
whelm the grass.

ACKNOWLEDGMENTS

We are grateful for the collection sites, assistance,
and cooperation provided by E. McDowell (Tony's Pest
Control), P. Quartuccio (All-Service Pest Management),
Pest Solutions Plus, N. Palmer (Master Gardener),
Greig Henry, and Trish Wood.

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Florida Entomologist 94(2)


June 2011


VAGILITY AS A LIABILITY: RISK ASSESSMENT OF THE LEAF-BLOTCHING
BUG EUCEROCORIS SUSPECTS (HEMIPTERA: MIRIDAE),
A PROSPECTIVE BIOLOGICAL CONTROL AGENT OF THE
AUSTRALIAN TREE MELALEUCA QUINQUENERVIA


GARY R. BUCKINGHAM1*, SUSAN A. WINERITER', JASON D. STANLEY2, PAUL D. PRATT3 AND TED D. CENTER3
1USDA-ARS Invasive Plants Research Laboratory, P.O. Box 147100, Gainesville, FL 32614-7100

2Division of Plant Industry, Florida Department of Agriculture and Consumer Services, P.O. Box 147100,
Gainesville, FL 32614-7100

3USDA-ARS Invasive Plant Research Laboratory, 3225 College Ave., Fort Lauderdale, FL 33314

*Retired

ABSTRACT

Melaleuca quinquenervia (Cav.) S.T. Blake (Myrtales: Myrtaceae) forms dense monocul-
tures that displace native vegetation in wetlands of southern Florida, USA. Faunal studies
in the tree's native Australian range revealed several prospective biological control agents,
including the leaf-blotching bug, Eucerocoris suspects Distant (Hemiptera: Miridae). This
herbivore was imported into quarantine to assess risk to Florida native and ornamental spe-
cies after preliminary Australian studies had indicated that it might be useful. Ornamental
Melaleuca spp. suffered heavy feeding in no-choice adult feeding trials, with moderate feed-
ing on some native Myrtaceae. Native species sustained light to heavy feeding in multi-
choice adult feeding trials and in a no-choice nymphal feeding trial. Feeding increased on na-
tive species in a large enclosure after M. quinquenervia was cut, allowed to dry, and then re-
moved. Nymphs completed development only on M. quinquenervia and ornamental
bottlebrushes, Melaleuca spp. However, inability to fully develop on non-target species is of
limited importance as a criterion for release of insects with highly mobile immature stages
as compared to less vagile species. Local movement from the host to other plant species could
result in unacceptable non-target damage despite seemingly adequate developmental spec-
ificity. This insect would clearly harm native and ornamental Myrtaceae and should there-
fore not be released.

Key Words: Biological control, Hemiptera, Eucerocoris suspects, host range, Melaleuca
quinquenervia, Miridae, Myrtaceae, risk assessment, weed control

RESUME

Melaleuca quinquenervia (Cav.) S.T. Blake (Myrtales: Myrtaceae) forma monoculturas den-
sas que desplazen la vegetaci6n native en las tierras humedas en el sur de la Florida, EEUU.
Estudios faunisticas realizados en el rango native Australiano del arbol revelan various agen-
tes de control biol6gico prospectivos, incluyendo un chinche que mancha las hojas, Euceroco-
ris suspects Distant (Hemiptera: Miridae). Este herbivoro fue importado al laboratorio de
cuarentena para evaluar su riesgo hacia las species nativas de la Florida y oramentales
despu6s de que studios preliminares en Australia indicaron que esta especie puede ser util.
Especies ornamentales de Melaleuca sufrieron niveles fuertes de alimentaci6n en pruebas
sin opci6n de los adults, con alimentaci6n moderada en plants nativas de la familiar Myr-
taceae. Las species nativas sostuvieron alimentaci6n leve y fuerte en pruebas de opciones
multiples de alimentos para los adults y en pruebas sin opci6n de alimentos para las ninfas.
La alimentaci6n aument6 sobre las species nativas en un cercado grande despu6s que la M.
quinquenervia fue cortada, puesta a secar y quitada. Las ninfas completaron su desarrollo
solamente sobre M. quinquenervia y species de Melaleuca ornamentales. Sin embargo, la
incapacidad para desarrollar completamente sobre species que no son el enfoque es de im-
portancia limitada como un criterio para la liberaci6n de insects con estadios de immaduros
altamente movies comparado con species menos movies. El movimento local de un hospe-
dero a otras species de plants puede resultar en daio no acceptable en plants que son el
enfoque a pesar de que la especificidad del desarrollo parece adecuada. Este insecto clara-
mente daiaria las Myrtaceae nativas y ornamentales y por ello no debe ser liberado.







Buckingham et al.: Risk Assessment for Eucerocoris suspects


Melaleuca quinqueneruia (Cav.) S. T. Blake is a
large tree of Australian origin and one of numerous
invasive plants threatening the Florida Everglades.
This introduced tree forms expansive monocultures
and spreads rapidly from prolific seed production.
Its presence and rapid spread hinders restoration of
many south Florida ecosystems including sawgrass
prairies, hardwood hammocks, and even pine up-
lands (Bodle 1998; Turner et al. 1998). Invaded hab-
itats are transformed into nearly pure stands of M.
quinquenervia trees, thereby altering the function
and structure of these systems.
A biological control program began in 1986 to
curtail the M. quinqueneruia invasion by inhibit-
ing its reproduction. Eucerocoris suspects Dis-
tant (Hemiptera: Miridae) seemed an excellent
candidate based upon the injury it caused to
young shoots (Fig. 1) in Australia (Burrows &
Balciunas 1999). It was introduced into quaran-
tine during 1996 to complete host range evalua-
tions by focusing on native and cultivated Myrta-
ceae. Herein, we report results of host range stud-
ies that led us to reject this species.


Burrows & Balciunas (1999) described the bi-
ology and life history of E. suspects as follows.
Females insert eggs into the young shoots.
Nymphal development progresses through 5 in-
stars and requires about 17 d but is influenced by
plant quality. A single female produces up to 163
progeny and adults live up to 72 d. Adults and
nymphs feed on the sap of young leaves and
shoots causing distinctive brown blotches on the
foliage. Their dispersive capacity is unknown but
both nymphs and adults are very active, making
them difficult to contain, and are readily able to
disperse onto nearby vegetation.

MATERIALS AND METHODS

Laboratory Cultures

Dr. Charles Turner and staff of the USDA-ARS
Australian Biological Control of Weeds Labora-
tory collected E. suspects adults near Brisbane,
Australia, during Jul and Aug 1996. A shipment
arrived in quarantine at Gainesville, Florida on


S


[1


r"


Fig. 1. A female Eucerocoris suspects and feeding scars on Melaleuca quinqueneruia.







Florida Entomologist 94(2)


12 Jul with 17 of 38 adults alive (5 males and 12
females). A second shipment arrived on 31 Aug,
which contained 4 of 75 adults alive (not sexed).
Adults were placed on seed-grown M. quinquen-
ervia saplings of varying sizes, usually less than 2
m tall. The saplings were sleeved with fine-mesh
netting (100 holes/cm2) and held in air-conditioned
quarantine greenhouses. Plants were fertilized
with an encapsulated fertilizer, watered regularly,
and occasionally sprayed with a soap-vegetable oil
mixture to control insect pests. They were not
sprayed after the bugs were added. During rear-
ing, adults and/or nymphs, which preferred new
growth, were removed and placed on new saplings,
depending upon the amount of damage and the
amount of remaining leaf material. They were
reared continuously from Jul 1996 to Jun 1997,
during which time 5 host range studies, desig-
nated I-V, were conducted. Each study consisted of
1 or more separate trials designated A-L.
No-choice Adult Feeding and Oviposition Trials (Study I)
Potted test plants (Table 1) 1-2 m tall bearing
new growth on the stem tips (hereafter referred to


as shoots) were individually caged in quarantine
greenhouses in sleeves of nylon netting. Groups of
5 pairs of adults were randomly assigned to the
cages. Most plant species were set up within 2 d of
the commencement of the study but 3 species
were set up 11-13 d later. Each group of plants (n
= 3 groups) was assigned a separate control plant
of M. quinquenervia to comprise trials A, B, and C
(Table 1, Test ID). Adults and nymphs were re-
moved at 7-11-d intervals and placed separately
on new plants. Each exposed plant was held to as-
sess further nymphal emergence. Adults were
transferred to a new plant of the same species as
many as 4 times (Table 1). Adult survival was re-
corded at each plant change. Nymphs were re-
moved, counted, and placed together on a new
plant of the same species. The number of shoots
bearing damaged leaves and the number of leaves
with feeding blotches were recorded for most spe-
cies. Damage results were categorized by a three-
tiered intensity scale: "+" = not damaged; "++" =
moderately damaged; and "+++" = heavily dam-
aged. The presence of eggs was noted at the end of
the test. Water-filled vials with bouquets of ex-


TABLE 1. RESULTS OF THE NO-CHOICE ADULT FEEDING AND OVIPOSITION TRIALS WITH POTTED MYRTACEAE AND LYTH-
RACEAE EXPOSED TO EUCEROCORIS SUSPECTS (STUDY I).

Trial No. Plants Time to 100% Feeding Nymphs
Test speciesD IDb', Tested Mortality (d) Intensityd Produced (no.)

Melaleuca citrina (Curtis) Dum. Cours A 4 27 +++ 91
Melaleuca citrina (broad-leaved) B 4 >42 +++ 184
Melaleuca viminalis (Sol. ex Gaertn.) Byrnes A 4 27 +++ 181
Melaleuca viminalis 'Little John' C 4 32 +++ 51
Calyptranthes pallens Griseb.* C 1 9 + 0
Calyptranthes pallens* B 1 10 + 0
Calyptranthes zuzygium (L.) Sw.* C 3 32 ++ 0
Eugenia axillaris (Sw.) Willd.* C 1 9 ++ 0
Eugenia confusa DC.* B 1 10 + 0
Eugenia foetida Pers.* C 1 9 ++ 0
Eugenia foetida* B 1 10 ++ 0
Eugenia uniflora L. B 2 >10 +++ 0
Lagerstroemia indica L. (Lythraceae) A 1 >25 + 0
Leptospermum scoparium J. R. Forst. & G. Forst. B 2 20 + 0
Melaleuca quinquenervia (Cav.) Blake C 4 >42 +++ 18'
Melaleuca quinquenervia B 4 >25 +++ 401
Melaleuca quinquenervia A 4 >25 +++ 2579
Myrcianthes fragrans (Sw.) McVaugh* B 1 10 ++ 0
Psidium friedrichsthalianum (0. Berg.) Nied. C 2 17 +++ 0
Psidium cattleianum Sabine C 3 32 +++ 0
Syzygium paniculatum Gaertn. A 1 10 ++ 0

"Florida natives are indicated with *
bPlants with the same letter had the same Melaleuca quinquenervia control plant.
'Five pairs of adults on a potted plant covered with mesh sleeve, adults moved weekly to a new plant, old plant was held for
nymphal emergence, total plants tested with those adults.
dSubjective feeding estimate, compared with feeding on M. quinquenervia; at first plant change M. quinquenervia reps. had 129-
168 leaves with >10 feeding spots.
'Total nymphs produced on all plants exposed to that cohort of adults.
Progeny of one female, four of the 5 females were trapped and died in the release vial.
The test ended when all adults were dead on the 3 companion plants. Two females were still alive on M. quinquenervia.


June 2011







Buckingham et al.: Risk Assessment for Eucerocoris suspects


cised shoots were tethered to Melaleuca citrina
(Curtis) Dum.Cours and Melaleuca viminalis
(Sol. ex Gaertn.) Byrnes at the third and fourth
plant changes to provide supplemental food as
the original plant material was insufficient fol-
lowing extensive feeding.

No-choice Nymphal Feeding Trials (Study II)

Techniques were similar to those for the previ-
ous adult test except that 10 first instars (1.3 to
1.7 mm in length) were placed on the plants in-
stead of adults. Supplemental bouquets of excised
shoots in water-filled vials were tethered to test
plants that had too few suitable flushing shoots to
support the insects after the first week. Four na-
tive Eugenia spp. were included with M. quin-
queneruia in trial D, and 2 non-target species of
Melaleuca along with M. quinqueneruia in trial E
(Table 2). Insect survival, the number of adults
produced, and the number of leaves attacked
were recorded weekly for 28 d. Adults that devel-
oped during the test were retained on the plants
with the remaining nymphs.

Multi-choice Adult Feeding and Oviposition Trials with-
out M. quinqueneruia (Study III)

In trial F, 2 bouquets of shoots in water-filled
vials of each of 10 test plant species (Table 3) were
placed into a glass-topped wooden cage (44.5 x
44.5 x 44.5 cm, 1 x w x h) in a greenhouse with
daily mean temperature 22-24C (range 19-33C),
and 78-81% RH (range 45-97%). Natural lighting
was supplemented with fluorescent lights to
maintain a 16L:8D photoperiod. One bouquet of


each species was randomized to one of 10 posi-
tions in each half of the cage. Ten females and 2
males were released in the cage. Feeding was sub-
jectively estimated on a scale of 0 to 5 from light
to heavy after 2 d when the bouquets were re-
placed and again after 3 d when trial F ended.
The plants were examined for eggs at the end of
the trial.
In trial G, 1 potted plant of each of 4 species
was placed in a cloth screen cage (0.6 x 0.6 x 1.2
m, 1 x w x h) in a greenhouse. The test species in-
cluded 3 Myrtaceae, pineapple guava (Feijoa sell-
owiana (0. Berg) O. Berg), bay rum tree (Pimenta
racemosa (Mill.) J. W. Moore), and java plum
(Syzygium cumini (L.) Skeels), and 1 Rutaceae,
lemon (Citrus limon (L.) Burm.f. (pro. Sp.) (med-
ica x aurantifolia)). Three pairs of adults were re-
leased in the cage. Two plants with extensive
feeding were removed during the second d and
the other 2 plants were left until d 11. The plants
were checked for eggs and nymphs when the test
ended.

Large Enclosure Multi-choice Adult Feeding Trials with
and without M. quinquenervia (Study IV)

Potted plants, 1-1.5 m tall, were exposed to
adults in a large walk-in screen enclosure (1.8 x
1.5 x 1.8 m, 1 x w x h) in a greenhouse. Plants were
randomized to 1 of 9 positions in 3 rows of 3 each,
in trials H and I. A M. quinquenervia plant was
placed next to the test plant in the center of the
enclosure at the start of each trial. The 15 test
plant species are listed in Table 4. All were Myrt-
aceae except Morella cerifera (L.) Small (Myri-
cales: Myricaceae), which was considered at risk


TABLE 2. NO-CHOICE NYMPHAL FEEDING AND DEVELOPMENT TRIALS ON NATIVE EUGENIA SPP. AND EXOTIC MELA-
LEUCA SPP. (STUDY II).

Survival (%)' after: Leaves attacked (cumulative no.)'

Test Plant 7 d 28 d No. Adultsb 7 d 28 d

Trial D
Eugenia axillaris 0 0 0 20 -
Eugenia confusa 0 0 0 6
Eugenia foetida 0 0 0 28 -
Eugenia rhombea (Berg) Krug & Urb. 30 0 1 34 76
Melaleuca quinquenervia 60 30 5 50+ 344+

Trial E
Melaleuca citrina 100 0 7 50 284+
Melaleuca citrina (broad-leaved) 10 0 0 20 20
Melaleuca viminalis 80 40 4 50+ 347+
Melaleuca viminalis 'Little John' 100 70 8 50 459
Melaleuca quinquenervia 80 50 6 50+ 446+

"Ten small nymphs per plant, new adults were left on the plant with the remaining nymphs.
bSome adults died before 28 d when trapped in the folds of the mesh sleeve.
'Plants with a "+" were difficult to count accurately so the count is a minimum.







Florida Entomologist 94(2)


TABLE 3. MULTI-CHOICE ADULT FEEDING AND OVIPOSITION TRIAL ON MYRTACEAE WITHOUT MELALEUCA QUINQUENERVIA
(STUDY III).

Feeding intensity'

Day 2b Day 5

Test species (Trial F) Bouquet 1 Bouquet 2 Bouquet 1 Bouquet 2 Eggs

Calyptranthes pallens 1 4 4 5 0
Calyptranthes zuzygium 0 0 0 0 0
Eugenia axillaris 2 1 5 4 6
Eugenia confusa 1 1 3 1 0
Eugenia foetida 0 0 1 1 2
Eugenia uniflora 2 3 2 2 0
Leptospermum scoparium 0 0 0 0 0
Myrcianthes fragrans 1 1 3 1 0
Psidium friedrichsthalianum 2 3 3 5 0
Psidium cattleianum 3 1 5 4 1

"Ten Y Y and 2 6 6 released in the cage. Feeding estimate: 1= Light, scattered feeding, no large blotches; 2 = Light-medium;
3 = Medium, noticeable feeding, on multiple leaves; 4 = Medium-heavy; 5 = Heavy, some leaves blackened, some abscinded.
bBouquets were randomized in each half of cage and were replaced in d 2.
Test terminated on d 5 when eggs were counted.


because of limited use by other Melaleuca herbi-
vores (Wheeler 2005; Pratt et al. 2009). Two spec-
imens of Eugenia DC. were placed together at the
same position in trial H because they had fewer
shoots than the others. Ten pairs of adults were
released on the M. quinquenervia plant. Dam-
aged leaves were counted daily except for trial H,
which was not assessed until the third d. The few
damaged leaves on the test plants were removed
daily to avoid duplicate counting. Damaged
leaves were not removed from M. quinquenervia
because this would have resulted in total defolia-
tion of the tree. The resultant cumulative counts
were therefore underestimates for M. quinquen-
eruia inasmuch as most leaves would have been
subjected to repeated feeding. The M. quinquen-
eruia was cut on the fifth day and the pieces were
tied to the trunk to allow the leaves to dry and the
bugs to disperse. The dried M. quinqueneruia was
removed on the seventh day and the trial termi-
nated on the tenth day. The plants in trial I were
examined for eggs when the test ended.
Melaleuca viminalis and the 2 forms of M. cit-
rina were randomized to 9 positions, 3 each, in
trial J. Due to a limitation of standard M. vimina-
lis plants, 1 M. viminalis cultivated variety 'Little
John' was also incorporated into the trial. We
placed 2 pots together at one position for M. cit-
rina and one for M. viminalis because they had
fewer shoots than the other plants. Eighteen
males were released on the ceiling in the center of
the cage. Three survivors were removed on the
fifth day, but 2 more were found on the seventh
day when damaged leaves were counted. Males
were used to avoid oviposition in order to preserve
the plants for other uses.


No-choice Starvation Trial with Nymphs and Adults
(Study V)

Three potted sugarcane plants, Saccharum of-
ficinarum L. (Cyperales: Poaceae), and 2 potted
lemon plants were individually caged in nylon
sleeves in the greenhouse (trial K). Three fe-
males, 2 males, and 6 medium-sized nymphs were
released in each cage where they remained until
they died. The number of leaves with feeding
spots was counted after all bugs were dead. An
additional trial (trial L) was conducted to deter-
mine if discoloration observed on the plants in
trial K was due to feeding damage. A leaf of a sug-
arcane plant and a lemon plant was covered with
a small net sleeve. One pair of adults and 2
nymphs were released in the small sleeve. Each
plant was enclosed in a larger sleeve. All plants in
trials K and L were examined for eggs when the
test ended.

RESULTS

No-choice Adult Feeding and Oviposition Trials (Study I)

Feeding was moderate to heavy on nearly all
plants tested (Table 1). The 2 non-target Mela-
leuca spp., the 2 Psidium spp., and Eugenia uni-
flora L. were most heavily attacked. Native Myrt-
aceae were noticeably damaged but, with the ex-
ception of Calyptranthes zuzygium (L.) Sw. on
which they survived for 32 d, the adult bugs died
within 10 d. Nymphs developed and eggs were
found only on non-target Melaleuca spp. and M.
quinquenervia. Lagerstroemia indica L. suffered
only light damage, although some nymphs lived


June 2011







Buckingham et al.: Risk Assessment for Eucerocoris suspects


TABLE 4. WALK-IN ENCLOSURE MULTI-CHOICE ADULT FEEDING TRIALS ON MYRTACEAE WITH AND WITHOUT MELA-
LEUCA QUINQUENERVIA (STUDY IV).

Leaves with feeding blotches, cumulative tally on day

Test Species 3 5 7 10 Eggs

Trial H. Ten adult pairs, potted plants at 9 positions, M. quinquenervia next to central plant
Calyptranthes pallens 1 1 5 11
Calyptranthes zuzygium 0 0 0 0 -
Eugenia axillaris 1 3 11 16 -
Eugenia confusa 0 1 14 22 -
Eugenia foetida 8 8 25 36 -
Eugenia rhombea 0 0 4 4 -
Melaleuca quinquenervia 164 293 Dryinga Removed -
Myrcianthes fragrans 2 2 30 36 -
Morella cerifera (L.) Small (Myricaceae) 0 1 1 1 -
Psidium longipes (Berg) McVaugh 0 0 22 22 -

Trial I. Set up same as Trial H but eggs were assessed at the end of evaluation
Melaleuca citrina
Melaleuca citrina (broad-leaved) 2 3 13 21 no
Melaleuca viminalis 3 28 104 254 yes
Eucalyptus camaldulensis Dehnh. 0 0 1 1 no
Eucalyptus camaldulensis 4c 11i 12' 23' yes
Leptospermum scoparium 0 0 0 0 no
Melaleuca quinquenervia 258 328 Drying Removed -
Psidium friedrichsthalianum 0 0 7 0 no
Psidium cattleianum 0 0 0 0 no

Trial J. Eighteen 5 5, potted plants at 9 positions, 3 of each species
Melaleuca citrina -- 23d
Melaleuca citrina (broad-leaved) -38
Melaleuca viminalis -127

aMelaleuca quinquenervia was cut on d 5 and left in cage to dry slowly.
bMelaleuca quinquenervia was removed from the cage on d 7.
'Number of stems fed upon. Feeding was on stems not on leaves.
Total of the 3 positions, 122 leaves attacked on C. viminalis were all on 1 plant.


at least 25 d on it. Damage was usually distrib-
uted throughout the plant with over 50% of the
shoots attacked on 7 of 10 species. In general, sur-
vival on most test plants was quite long (Table 1).

No-choice Nymphal Feeding Trials (Study II)

Small nymphs were dead by d 7 on 3 native
Eugenia spp. in trial D, but 30% survived on E.
rhombea Ridl. (Table 2). One became an adult,
but it and the remaining nymphs were dead by d
28. Only the newest leaves on E. rhombea were
attacked but they were heavily damaged and ab-
scinded. Similar feeding and damage was ob-
served on the other Eugenia spp., with few leaves
attacked due to low nymphal survival but heavy
damage levels (Table 2). Older leaves were also
attacked on E. axillaris (Sw.) Willd., but with lit-
tle damage. On M. quinquenervia, 60% of nymphs
survived to d 7 and 50% became adults. Two died


after being entrapped in the folds of the cloth
sleeve, so survivorship would probably have been
somewhat greater.
Survival and feeding on non-target Mela-
leuca spp. in trial E were similar to those on M.
quinquenervia except on the broad-leaved M.
citrina. All nymphs were alive on d 7 on M. cit-
rina and M. viminalis "Little John" and 80%
were alive on M. viminalis and on M. quinquen-
ervia. All were dead on M. citrina by d 28, but
40% and 70% were alive on the 2 M. viminalis
plants and 50% on M. quinquenervia. Most sur-
viving nymphs developed to adults on both non-
target Melaleuca spp. and M. quinquenervia,
and some adults died before 28 d. Feeding on 2
non-target Melaleuca spp. was comparable to
that experienced by M. quinquenervia (Table 2).
Feeding on the test plants was usually distrib-
uted throughout the canopy, with most foliage
damaged at shoot tips.







Florida Entomologist 94(2)


Multi-choice Adult Feeding and Oviposition Trials with-
out M. quinquenervia (Study III)

All species in trial F except C. zuzygium and
Leptospermum scoparium J. R Forst. & G. Forst.
sustained damage, but Eugenia foetida Pers. was
not damaged until after d 2 (Table 3). Most spe-
cies had light to medium damage by d 2. Four spe-
cies showed medium to heavy feeding on one bou-
quet on d 5 (Calyptranthes pallens Griseb., E. ax-
illaris, Psidium friedrichsthalianum (0. Berg.)
Nied., and P. cattleianum Sabine). Eggs were
found on 3 species: the natives E. axillaris and E.
confusa and the cultivated P cattleianum.
Two of the 4 species in trial G were attacked on
d 1. Five of 7 leaves on P. racemosa and 4 of 5
leaves on S. cumini had more than 5 feeding
blotches. The trial ended on d 11, with no feeding
on lemon but with 14 leaves damaged on A. sell-
owiana, half of which had more than 5 feeding
blotches. No live insects were recovered at the
end of trial G. Nymphs only emerged from eggs in
the stems of A. sellowiana. None emerged from
stems ofP. racemosa or S. cumini.

Large Enclosure Multi-choice Adult Feeding Trials With
and Without M. quinquenervia (Study IV)

With M. quinquenervia present in trials H and
I, there was little feeding on test plants by d 5 ex-
cept on one of 2 plants of M. citrina and 1 plant of
M. uiminalis (Table 4). However, the feeding on
both non-target plants was much less than that
on M. quinquenervia. Feeding increased on most
plants after M. quinquenervia was cut, but was
still minor except on the 2 plants already men-
tioned. Feeding on M. viminalis on d 10 was sim-
ilar to that on M. quinquenervia on d 3. Eggs were
found only on M. citrina, M. viminalis and M.
quinquenervia in trial I. There was little feeding
on the broad-leaved M. citrina in this trial, al-
though it was heavily attacked by adults in no-
choice trials. Feeding on plants of other non-tar-
get species was not particularly damaging, but
was noticeable.
Little feeding occurred on either variety of M.
citrina in trial J. Almost all feeding on M. vimina-
lis, 96%, and all on M. citrina occurred at one of
the 3 positions within the cage. Also, the attacked
leaves were on relatively few shoot tips, not
widely distributed over the plant (M. citrina 3 of
25 tips had feeding, M. citrina (broad-leaved), 5 of
10, and M. viminalis, 9 of 100).

No-choice Starvation Trial with Nymphs and Adults
(Study V)

All insects died on sugarcane plants (n = 3) by
the eighth d without feeding in trial K. On lemon
plants (n = 2) all insects were dead by the sixth d,
but 7 and 8 leaves were damaged on 2 of the


plants. This damage was slight, perhaps due to
test probing, and all damaged leaves had less
than 5 feeding blotches. Brown streaks were ob-
served along the veins on some sugarcane leaves,
but when additional insects were confined on new
leaves (trial L) to determine if this streaking was
symptomatic of feeding damage, none resulted.
No eggs were found on any of the test plants.

DISCUSSION

The host range of the Melaleuca leaf-blotching
bug, E. suspects, may be considered acceptable
based on the fact that the insect was able to com-
plete development only on M. quinquenervia,
other exotic Melaleuca spp. and once on E. rhom-
bea. However, the vagile nature of this insect
would enable it to feed on a wide variety of plants
that are not developmental hosts. Feeding dam-
age proved especially troublesome because of the
frequency that this insect probed or test fed on
non-target plants. Thus, even though E. suspects
is stenophagous, it presents a risk to rare en-
demic plant species such as E. rhombea that are
sympatric with M. quinquenervia in southern
Florida. The exact nature of this risk cannot be
known without further study but the precaution-
ary principal, which dictates conservative ac-
tions, would disqualify release of this insect. We
did not test unusually high numbers of insects
per cage, for instance, but unacceptably high lev-
els of collateral damage were observed on non-
target species in large cage trials. In contrast, and
contrary to cage tests, Burrows & Balciunas
(1999) observed that adults released on test
plants in a shade-house fed and reproduced only
on M. quinquenervia. Our concerns, however,
were confirmed through field observations by per-
sonnel at the Brisbane laboratory: damage was
observed on mixed Myrtaceae in a garden plot, all
stages ofE. suspects were found on bottlebrush,
Melaleuca spp., and damage to guava was severe
(Purcell et al. 2000). Bottlebrushes are relatively
common ornamentals in Florida and some other
states. These were originally placed in the genus
Callistemon but have since been synonymized
with Melaleuca (Craven 2006).
These results matched those of Burrows &
Balciunas (1999) quite closely in terms of common
genera tested in cage tests. They reported notice-
able feeding on Melaleuca (=Callistemon), Psid-
ium, and Syzygium, as did we. Nymphal survival
to adult was 47% on M. viminalis in their tests as
compared to 50% herein.
Damage from an equal amount of feeding on
Calyptranthes, Eucalyptus, Eugenia, and Psid-
ium was greater than that on Melaleuca. The
damaged young leaves and young stems dried
and abscinded on those genera, as they often did
on Melaleuca, but there were fewer leaves on non-
target plants than on the longer, foliose shoots of


June 2011







Buckingham et al.: Risk Assessment for Eucerocoris suspects


the target weed. Thus, an equal number of at-
tacked leaves among test plants resulted in a dis-
proportionate level of damage on non-target hosts
as compared to Melaleuca. However, this level of
non-target damage was limited to a few test spe-
cies as many more leaves were usually attacked
on Melaleuca.
The present data also have relevance to exper-
imental protocols for host range testing. Evalua-
tion of an herbivore's host range is an effort to
maximize predictive precision within the bounds
of practicality. Multiple replicated experiments
provide insight to variation in an herbivore's host
preferences, but limited financial resources ne-
cessitate abandoning continued testing (replica-
tion) at early stages of evaluation for herbivores
that demonstrate broad host ranges. Although de-
velopment of E. suspects appears to be confined
to Melaleuca, non-host feeding by the highly mo-
bile nymphs and adults is too broad and too dam-
aging for this bug to be used for biological control.
Therefore, many tests were terminated with few
replicates when it became apparent that this in-
sect was not a suitable candidate for release. Con-
tinuation of testing for the sake of additional rep-
licates would have wasted time and resources, so
testing was curtailed in favor of more suitable
candidates.

ACKNOWLEDGMENTS

We thank Mayana Roberg Anderson for assistance
with plant maintenance. We further acknowledge D. W.
Burrows, J. K. Balciunas, M. F. Purcell, K. E. Galway, J.
A. Goolsby, J. R. Makinson, D. Mira, and the late C. E.
Turner for significant contributions towards the study
of Eucerocoris suspects. Mention of trade names or
commercial products in this publication is solely for the
purpose of providing specific information and does not


imply recommendation or endorsement by the U.S. De-
partment of Agriculture. This research was supported,
in part, by grants from the South Florida Water Man-
agement District and the Florida Department of Envi-
ronmental Protection Bureau of Invasive Plant
Management.

REFERENCES CITED

BODLE, M. 1998. Dial M for melaleuca. Wildland Weeds.
1: 9-10.
BURROWS, D. W., AND BALCIUNAS, J. K. 1999. Host-
range and distribution of Eucerocoris suspects
(Hemiptera: Miridae), a potential biological control
agent for the paperbark tree Melaleuca quinquen-
eruia (Myrtaceae). Environ. Entomol. 28: 290-299.
CRAVEN, L. A. 2006. New combinations in Melaleuca for
Australian species of Callistemon Myrtaceae). No-
von 16: 468-475.
CRAVEN, L. A. 2009. Melaleuca (Myrtaceae) from Aus-
tralia. Novon 19: 444-453.
PRATT, P. D., RAYAMAJHI, M. B., CENTER, T. D., TIPPING,
P. W. AND WHEELER, G. S. 2009. The ecological host
range of an intentionally introduced herbivore: a
comparison of predicted versus actual host use. Biol.
Control 49: 146-153.
PURCELL, M. F., GALWAY, K. E., GOOLSBY, J. A., MAKIN-
SON, J. R., AND MIRA, D. 2000. Field plot experi-
ments, a method of assessing the host range of bio-
logical control agents for Melaleuca quinquenervia
in its native range, pp. 684-685 In N. R. Spencer [ed.]
Proc. X Inter. Symp. Biol. Control Weeds. Montana
State University, Bozeman Montana, USA. 1030 pp.
TURNER, C. E., CENTER, T. D., BURROWS, D. W. AND
BUCKINGHAM, G. R. 1998. Ecology and management
of Melaleuca quinquenervia, an invader of wetlands
in Florida, U.S.A. Wetlands Ecol. Manage. 5: 165-
178.
WHEELER, G. S. 2005. Maintenance of a narrow host
range by Oxyops vitiosa; a biological control agent of
Melaleuca quinquenervia. Biochem. Syst. Ecol. 33:
365-383.







Florida Entomologist 94(2)


June 2011


COMPARISON OF SYNTHETIC FOOD-BASED LURES AND LIQUID
PROTEIN BAITS FOR CAPTURE OF ANASTREPHA SUSPENSE
(DIPTERA: TEPHRITIDAE) ADULTS

NANCY D. EPSKY, PAUL E. KENDRA, JORGE PENA' AND ROBERT R. HEATH
Subtropical Horticultural Research Station, United States Department of Agriculture,
Agricultural Research Service, 13601 Old Cutler Road, Miami, FL 33158, U.S.A.

1University of Florida, Tropical Research and Education Center, 18905 SW 280th Street,
Homestead, FL 33031, U.S.A.

ABSTRACT

Field tests were conducted in south Florida to compare capture of the Caribbean fruit fly,
Anastrepha suspense (Loew), in Multilure traps baited with either of the liquid protein baits
torula yeast/borax or Nulure/borax, or with food-based synthetic lures including two-compo-
nent Biolure (ammonium acetate, putrescine) and three-component Biolure (ammonium ac-
etate, putrescine, trimethylamine). The highest relative proportion of females captured was
in traps baited with the two-component Biolure (44-61%), intermediate capture was in traps
baited with the three-component Biolure (14-24%) or torula yeast/borax (8-25%), and the
lowest capture tended to be in traps baited with Nulure/borax (0-19%). Similar results were
obtained for capture of males. Tests of the unipak two-component Biolure, which has a re-
duced ammonium acetate release rate and is a single package with both ammonium acetate
and putrescine sections, captured similar numbers of both females and males as Biolure for-
mulated in 2 individual packages. Traps baited with unipak Biolure combined with the ad-
dition of a trimethylamine lure captured fewer females than the unipak alone, but this was
greater than capture in traps baited with torula yeast/borax. Our studies confirmed that the
best lure for A. suspense is ammonium acetate and putrescine. However, C. capitata-tar-
geted traps baited with three-component Biolure should be as effective for A. suspense de-
tection and monitoring as traps baited with torula yeast/borax. The unipak two-component
Biolure will provide the improved handling that has been requested by users.

Key Words: Caribbean fruit fly, Biolure, ammonium acetate, unipak, torula yeast

RESUME

En pruebas de campo realizadas en el sur de Florida, se compararon resultados de capture
de la mosca de la fruta del CaribeAnastrepha suspense (Loew), capturadas en trampas Mul-
tilure que habian sido cebadas con cebos de protein liquid de levdura torula/borax o con
Nulure/borax, o con atrayentes alimenticios sinteticos los cuales incluian Biolure con dos
components (acetato de ammonia, putrescina) y Biolure con 3 components (acetato de am-
monia, putrescina, trimethylamina). El mas alto porcentajge de capture de hembras ocurrio
en trampas cebadas con Biolure de dos components (44-61%), un nivel de capture interme-
dio ocurrio en trampas cebadas con Biolure de 3 components (14-24%) o con los cebos de le-
vadura torula /borax (8-25%), mientras que la capture mas baja fue en aquellas trampas
cebadas con Nulure/borax (0-19%). Se obtuvieron resultados similares en la capture de ma-
chos. Pruebas unipak del Biolure de dos components, el cual da una tasa de liberation re-
ducida de acetato de ammonio y el cual es un atrayente individual con secciones de acetato
de ammonio y putrescina, no mostro diferencias en capture de machos o hembras entire el
Biolure de dos components formulado como atrayentes individuals o como unipak. La cap-
tura intermedia de las hembras fue obtenida en trampas cebadas con unipak Biolure com-
binado con un atrayente individual de trimethylamina, pero esta capture fue mayor que la
obtenida cuando se usaron trampas cebadas con levadura torula /borax. Nuestros studios
confirmaron que el mejor atrayente paraA suspense es el acetato de ammonio y putrescina.
Sin embargo, trampas destinadas a atrapar C. capitata y cebadas con acetato de amonio, pu-
trescina y trimethylamina deben ser tan efectivas como aquellas trampas cebadas con leva-
dura torula /borax para la deteccion y monitoreo deA. suspense. El unipak con Biolure de dos
components es igualmente efectivo y dara una mejor facilidad para manipulacion, la cual
ha sido pedida por los usuarios.


Translation provided by the authors.







Epsky et al.: Standard Baits for Anastrepha suspense


Tephritid fruit flies are among the most impor-
tant pests of fruits and vegetables in the world,
and use of traps and lures are important compo-
nents of fruit fly pest management programs.
Standard trapping protocols have been developed
for fruit fly detection and monitoring and, de-
pending on target species, different female-tar-
geted baits may be used (IAEA 2003). Selection of
trap and lure depends on the purpose of trapping,
availability of materials and cost. Although use of
a single trap type that would capture the highest
number of females and males of multiple species
would have many advantages, species-specific
traps that use lures specific to the target fruit fly
and environmental conditions are the best sys-
tems available currently (Diaz-Fleischer et al.
2009). Among the numerous liquid protein baits,
agencies primarily use torula yeast/borax solu-
tions for detecting and monitoring Anastrepha
spp. and NuLure/borax solutions for the Mediter-
ranean fruit fly, Ceratits capitata Weidemann
(Epsky et al. 1993; Heath et al. 1993, 1994).
Food-based synthetic attractants have been
developed based on volatile chemicals released
from liquid protein baits. A two-component Bi-
olure attractant comprised of ammonium acetate
and putrescine is used in traps that target Anas-
trepha spp. (Heath et al. 1995; Epsky et al. 1995,
Thomas et al. 2001). Addition of trimethylamine
is used in traps that target C. capitata (Heath et
al. 1997), and McPhail-type traps baited with the
Biolure three-component attractant are equal to
or better than liquid protein-baited traps for cap-
ture of C. capitata females (Epsky et al. 1999).
Capture ofAnastrepha spp. flies by traps baited
with either the two-component or three-compo-
nent attractant, however, is more variable (IAEA
2007). Initial studies found no difference in cap-
ture of the Mexican fruit fly, Anastrepha ludens
(Loew), in traps baited with Biolure ammonium
acetate and putrescine with or without the third
Biolure component, trimethylamine (Heath et al.
1997). However, Holler et al. (2006) found that ad-
dition of trimethylamine reduced capture of the
Caribbean fruit fly, Anastrepha suspense (Loew).
Regulatory agencies deploy McPhail-type
traps baited with the three-component Biolure
(ammonium acetate, putrescine, and trimethy-
lamine; Suterra LLC, Bend, OR), which is com-
mercially available, primarily to monitor for new
infestations of C. capitata in areas currently fly-
free, and questions remain on the effectiveness of
this trapping system for detecting and/or moni-
toringA. suspense. Due to problems with the de-
ployment of the synthetic attractants as individ-
ual lures, several versions of the components com-
bined in a single lure have been developed and
tested (Jang et al. 2007; Navarro-Llopis et al.
2008) including two-component and three-compo-
nent unipak versions of Biolure (Holler et al.
2009). We report results of field tests that were


conducted in south Florida to compare capture of
A. suspense in traps baited with either of the liq-
uid protein baits or with the Biolure two- and
three-component food-based synthetic attractant
(both individual lures and unipak lure) to deter-
mine relative effectiveness of these standard
trapping systems.

MATERIALS AND METHODS

Traps and Lures

MultiLure traps (Better World Manufacturing
Inc., Fresno, CA, USA) were used in all experi-
ments. Liquid protein baits included aqueous so-
lutions of torula yeast/borax (three 5-g pellets,
2.25:2.75 yeast:borax, in 300 mL water) (ERA In-
ternational, Freeport, NY) and NuLure (Miller
Chemical & Fertilizer Co., Hanover, PA) as a 300
mL aqueous solution of 9% NuLure (vol:vol) and
3% borax (wt:vol; sodium tetraborate decahy-
drate). Synthetic attractants included individual
component Biolure formulations of ammonium
acetate, putrescine, and trimethylamine, and the
unipak two-component Biolure formulation of
ammonium acetate and putrescine (Suterra LLC,
Bend, OR). The membrane-release area of the
ammonium acetate lure in the two-component
unipak has been reduced from ~35 mm diam. on
individual component lure to ~23 mm diam. on
unipak lure. This lowers the release rate of am-
monium acetate, which improves capture of the
Mexican fruit fly, Anastrepha ludens (Loew) but
has no effect on capture ofA. suspense (Thomas et
al. 2008). Traps baited with synthetic lures con-
tained 300 mL 10% polypropylene glycol (vol:vol;
LowTox, Prestone, Danbury, CT, USA) aqueous
solution to retain captured flies.

Field Tests

Field tests were conducted at the Tropical Re-
search and Education Center (TREC), University
of Florida, Homestead, FL. Experiment 1 com-
pared capture of flies in traps baited with (1) Nu-
Lure/borax, (2) torula yeast/borax, (3) two-compo-
nent Biolure (individual ammonium acetate and
putrescine lures), and (4) three-component Bi-
olure (individual ammonium acetate, putrescine,
and trimethylamine lures). Experiment 2 com-
pared capture of flies in traps baited with (1)
torula yeast/borax, (2) two-component Biolure, (3)
unipak two-component Biolure, and (4) unipak
two-component Biolure plus trimethylamine indi-
vidual Biolure. Tests were conducted in two hosts,
Surinam cherry, Eugenia uniflora L., and guava,
Psidium guajava L., for experiment 1; and in one
host, guava, for experiment 2. For the tests in
Surinam cherry, all 4 treatments were placed
around the periphery of a large tree in fruit.
There were 3 blocks (replicates) of traps, with 2 m







Florida Entomologist 94(2)


between traps within a block and 10 m between
blocks. For the tests in guava, there was 1 trap
per tree with the traps placed in 3 rows (blocks/
replicates) of trees. There were at least 10 m be-
tween rows and 10 m between traps within a row.
For both experiments, traps were sampled every 7
d, and numbers of male and female flies were re-
corded. Traps were sampled for 4 wk per sam-
pling period, for a total of 4 sampling periods in
experiment 1 (sample periods 1-2 in Surinam
cherry, sample periods 3-4 in guava), and 1 sam-
pling period in experiment 2. The protein bait so-
lutions were replaced every 7 d, but the synthetic
lures were not replaced during a sampling period.
A complete randomized design was used for trap
placement at the start of each sampling period.
Traps were rotated sequentially to the next posi-
tion within a block at time of sampling, so that all
treatments were in all positions within each sam-
pling period. Trees were in fruit throughout both
experiments, but the tests in Surinam cherry
were conducted toward the end of the fruiting
season (18 Jun to 13 Aug, 2008) and the experi-
ments in guava were conducted in the beginning
of the fruiting season (experiment 1 Jun 25 to
Aug 20, 2008; experiment 2 Aug 27 to Sep 17,
2009). Fruiting in guava trees began earlier in
2008 than in 2009 because the trees were
trimmed in spring 2009.

Statistical Analysis

Effect of treatment and either sample period
(experiment 1) or sample week (experiment 2)
were analyzed by two-way ANOVA in a factorial
model with interaction (Proc GLM, SAS Institute
2000) followed by LSD mean separation (P = 0.05)
for significant factors. When necessary, data were
transformed prior to analysis to satisfy conditions
of equal variance (Box et al. 1978). Numbers of to-
tal flies per trap per day and percentage females
for flies captured in traps baited with the Biolure
two-component attractant were analyzed as as-
sessments of population level. Summary statis-
tics are presented as average standard devia-
tion.

RESULTS

Experiment 1

Number of total A. suspense captured per trap
per day in traps baited with the two-component
Biolure was affected by sampling period (P = 9.08,
df = 3, 44; P < 0.0001; log (x + 1) transformed
data). Numbers of A. suspense in traps in Suri-
nam cherry decreased from 4.0 4.4 flies per trap
per day in sampling period 1 to 2.0 2.3 flies per
trap per day in sampling period 2. Numbers in
traps in guava increased from 0.4 0.2 in sam-
pling period 3 to 5.8 5.0 in sample period 4. Per-


centage of females captured in these traps was
also affected by sampling period (P = 3.27, df = 3,
48; P = 0.0321; square-root (x + 0.5) transformed
data). All captures were female biased, and per-
centage of females captured during sampling pe-
riods 1, 2, 3 and 4 were 86.6 + 10.6, 80.8 + 14.5,
60.8 32.0, and 76.1 + 16.7, respectively
Numbers of female and male flies per trap per
block were converted to relative trapping effi-
ciency to facilitate comparisons among the range
of population levels tested during the different
sampling periods (Epsky et al. 1999). There was a
significant interaction between sampling period
and treatment for female (F = 3.17; df= 9, 32;P =
0.0075) but not for male (F = 0.97; df = 9, 32; P =
0.4825) relative trapping efficiencies. Therefore,
one-way analyses were used to test effect of treat-
ment within each sampling period for females
and over all sampling periods for males. Traps
baited with the two-component Biolure had the
highest relative trapping efficiency for females for
all sampling periods (Table 1). The next highest
relative trapping efficiencies were for traps baited
with the three-component Biolure and with
torula yeast/borax solution. Relative trapping ef-
ficiency in traps baited with NuLure/borax was
significantly less than capture in traps with the
three-component attractant for the sampling pe-
riods in which the population was the lowest
(sample periods 2 and 3), Treatment also had an
effect on capture of males (F = 24.24; df = 3, 44; P
< 0.0001). The highest relative trapping efficiency
of males was 64.5 20.2% in traps baited with the
two-component Biolure, and this was higher than
traps baited with the three-component Biolure,
NuLure/borax or torula yeast/borax (12.4 + 10.9,
12.2 12.7, and 10.9 + 10.5, respectively).

Experiment 2

Number of total A. suspense captured per trap
per day in traps baited with the two-component
Biolure was affected by sample week (P = 23.83,
df= 3, 8;P = 0.0003; log (x + 1) transformed data).
Numbers increased from 6.5 0.6 in week 1 to
29.0 6.2 in week 4. Percentage of females cap-
tured in these traps was not affected by sampling
period (P = 1.62, df = 3, 8; P = 0.2591; square-root
(x + 0.5) transformed data). Overall, captures
were female biased, but the percentage of females
captured decreased slightly from 74.7 8.9 in
week 1 to 65.9 5.1 in week 4.
As in experiment 1, numbers of female and
male flies per trap per block were converted to rel-
ative trapping efficiency for subsequent analysis.
There was no interaction between treatment and
sample week, so data from all sample weeks were
pooled and effect of treatment was analyzed with
one-way ANOVA. Both individual and unipak
two-component Biolure formulations captured
more females than traps baited with torula yeast/


June 2011







Epsky et al.: Standard Baits for Anastrepha suspense


TABLE 1. RELATIVE TRAPPING EFFICIENCY (%) FOR CAPTURE OF FEMALE ANASTREPHA SUSPENSE IN FIELD TESTS CON-
DUCTED IN HOMESTEAD, FL. MULTILURE TRAPS WERE USED FOR ALL BAITS AND EACH SAMPLE PERIOD WAS
4 WEEKS. SURINAM CHERRY WAS AT THE END OF THE FRUITING SEASON IN SAMPLE PERIOD 2 AND GUAVA WAS
AT THE BEGINNING OF THE FRUITING SEASON IN SAMPLE PERIOD 3.

Surinam cherry** Guava

Bait* Sample period 1 Sample period 2 Sample period 3 Sample period 4

Two-component BioLure 44.1 4.5 a 60.2 10.2 a 60.9 12.8 a 60.9 20.5 a
Three-component BioLure 19.4 8.5 b 24.2 13.9 b 14.3 9.4 b 23.7 15.8 b
Torula yeast/borax 17.3 7.2 b 10.1 4.3 bc 24.8 12.6 b 7.5 2.2 b
NuLure/borax 19.2 12.8 b 5.5 2.9 c 0 c 8.0 4.4 b
F 4.22 21.56 27.17 11.06
df 3,8 3,8 3,8 3,8
P 0.0459 0.0003 0.0002 0.0032

*Ammonium acetate and putrescine alone (two-component Biolure) or with trimethylamine (three-component Biolure) in traps
with 300 mL 10% propylene glycol solution, 3 torula yeast/borax pellets in 300 mL water, 9% NuLure and 3% borax in 300 mL water.
**Means within a column followed by the same letter are not significantly different (LSD mean separation test on square root
(x + 0.5)-transformed data, non-transformed mean + standard deviation presented).


borax (Fig. 1 solid bars; F = 7.73; df = 3, 44; P =
0.0003), with intermediate capture with unipak
two-component Biolure plus trimethylamine.
There was no difference in number of males cap-
tured in traps baited with two-component Biolure
or unipak two-component Biolure (Fig. 1 shaded
bars; F = 3.37; df = 3, 44; P = 0.0268), and both
treatments captured more males than traps
baited with either with unipak Biolure plus trim-
ethylamine or torula yeast/borax.




45
a A a A females O males
A I
30 b B
w B
r 15 c B

0 n i n


Standard Unipak Unipak
AP AP AP+T


TY/borax


Treatments
Fig. 1. Relative trapping efficiency (%) for capture of
female (solid bars) and male (shaded bars) Anastrepha
suspense in field tests conducted in Homestead, FL.
Multilure traps were used for all baits and traps were
sampled for 4 weeks. Treatments included two-compo-
nent Biolure as individual ammonium acetate and pu-
trescine lures (Standard AP), unipak two-component
Biolure (Unipak AP), unipak two-component Biolure
plus trimethylamine individual Biolure (Unipak AP +
T) in traps with 300 mL 10% propylene glycol solution,
and 3 torula yeast/borax pellets (TY/borax) in 300 mL
water. Bars headed by the same lowercase (females) or
uppercase (males) letter are not significantly different
(LSD mean separation test on square root (x + 0.5)-
transformed data, non-transformed means presented).


DISCUSSION

Glass McPhail traps baited with torula yeast/
borax solution have been the standard trapping
system for Anastrepha spp. since early studies
found that torula yeast performed better than a
number of other yeast formulations (Lopez et al.
1971), and most studies have evaluated liquid
protein baits in these traps. Torula yeast/borax
captured more A. suspense than NuLure/borax in
tests with glass McPhail traps (Epsky et al.
1993). Plastic McPhail traps baited with two-com-
ponent Biolure captured more flies than glass
McPhail traps baited with either torula yeast/bo-
rax or two-component Biolure in tests in Florida
in Surinam cherry, loquat (Eriobotrya japonica
[Thunb.] Lindl.), and guava (Thomas et al. 2001;
Hall et al. 2005). Tests in grapefruit, Citrus para-
disi Macfady, in Florida found that captures in
Multilure traps baited with either two-compo-
nent Biolure or torula yeast/borax were higher
than in traps baited with NuLure/borax (Thomas
et al. 2008). Tests in sapodilla, Manilkara zapota
Van Royen, and mamey sapote, Pouteria sapota
(Jacq.), in Puerto Rico, however, found more A.
suspense were captured in Multilure traps baited
with torula yeast/borax than with two-component
Biolure (Pingel et al. 2006). In the only previous
test of three-component Biolure, Holler et al.
(2006) found that the highest capture was in plas-
tic McPhail traps baited with two-component Bi-
olure, and that there was no difference between
capture in plastic McPhail traps baited with
three-component Biolure or glass McPhail traps
baited with torula yeast/borax. Results from that
study, in which traps were placed in scattered
wild guava trees, were the same as the results
from our study.
Unipak versions of two-component and three-
component Biolure have been shown to be equal







Florida Entomologist 94(2)


to the same components formulated in single lure
formulations for capture of sterile C. capitata re-
leased in Florida and for capture ofA. suspense in
traps placed in backyard plantings of host fruit
trees in Sarasota/Bradenton and in Ft. Pierce
(Holler et al. 2009). We recorded similar results
for A. suspense in our tests in south Florida. Ad-
ditionally, we found that the unipak two-compo-
nent in combination with trimethylamine may be
better than torula yeast/borax for A. suspense
monitoring. This may be due to the lower release
rate of ammonia from the unipak two-component
formulation. The greater amount of ammonia
from the individual lure formulation used in ex-
periment 1, when combined with the amines from
the trimethylamine lure, may have been repel-
lant to A. suspense.
Our studies confirmed that the best lure for A
suspense is two-component Biolure ammonium
acetate and putrescine, however, C. capitata-tar-
geted traps baited with three-component Biolure
ammonium acetate, putrescine and trimethy-
lamine should be as effective as traps baited with
torula yeast/borax for A. suspense detection and
monitoring. The unipak two-component Biolure is
equally effective and will provide the improved
handling that has been requested by users.

ACKNOWLEDGMENTS

The authors thank M. Gill (USDA-ARS, Miami, FL)
for coordinating the field tests, and D. Long, J. Sanchez,
W. Montgomery, C. Allen, I. Filpo, and J. Tefel (USDA-
ARS, Miami, FL) for technical assistance; Joan Fisher
(Suterra LLC, Bend, OR) for supplying unipak two-com-
ponent Biolures; Jerome Niogret (USDA-ARS, Miami,
FL), David Jenkins (USDA-ARS, Mayaguez, PR) and
Donald Thomas (USDA-ARS, Weslaco, TX) for review-
ing an earlier version of this manuscript. This article re-
ports the results of research only. Mention of a
proprietary product does not constitute an endorsement
or recommendation by the USDA.

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Florida Entomologist 94(2)


June 2011


FOOD-BASED LURE PERFORMANCE IN THREE LOCATIONS IN PUERTO
RICO: ATTRACTIVENESS TO ANASTREPHA SUSPENSE AND A. OBLIQUA
(DIPTERA: TEPHRITIDAE)

DAVID A. JENKINS1, NANCY D. EPSKY2, PAUL E. KENDRA2, ROBERT R. HEATH AND RICARDO GOENAGA'
'USDA-ARS, Tropical Agriculture Research Station, 2200 Ave. P.A. Campos, Mayaguez, PR 00680-5470

2USDA-ARS-Subtropical Horticulture Research Station, 13601 Old Cutler Road, Miami, FL 33158

ABSTRACT
Lures based on odors released by hydrolyzed protein were assessed for their attractiveness
toAnastrepha obliqua andA. suspense at 3 locations in Puerto Rico in Aug through Oct 2009.
Lures compared included ammonium acetate combined with putrescine, hydrolyzed corn
protein (Nulure) with borax, freeze-dried Nulure, freeze-dried Nulure in combination with
ammonium acetate, freeze-dried Nulure in combination with ammonium acetate and pu-
trescine, and the Unipak lure, a single lure containing ammonium acetate and putrescine.
Where the distribution of trapped flies departed significantly from what would be expected
given an equal attraction of the baits, Nulure and freeze-dried Nulure always attracted
fewer flies than the other baits tested, regardless of species, sex, or location. Although all of
the baits or bait combinations containing ammonium acetate attracted more flies than the
Nulure or freeze-dried Nulure baits, there was a distinct trend of ammonium acetate and pu-
trescine and the Unipak lures to attract more flies after the 4th week of the study and for the
freeze-dried Nulure with ammonium acetate or in combination with ammonium acetate and
putrescine to attract more flies in the 1st 4 weeks of the study. This trial is unique in that it
was conducted in orchards of carambola, Aerrrhoa carambola (Oxalidaceae), a poor host for
both fly species. Our results are compared with other studies on lures ofA. obliqua andA.
suspense and the implications for monitoring/detecting pest Tephritidae are discussed.

Key Words: ammonium acetate, putrescine, Nulure, McPhail trap

RESUME
Trampas que trabajan a base de olores liberados por protein hidrolizada se evaluaron como
atrayentes de las moscas Anastrepha obliqua y A. suspense en tres localidades en Puerto
Rico durante agosto a octubre de 2009. Las trampas utilizadas en el studio incluyeron ace-
tato de amonio en combinaci6n con putrescina, protein hidrolizada de maiz (NuLure) con
b6rax, NuLure liofilizado en combinaci6n con acetato de amonio y putrescina, y la trampa
Unipak la cual contiene acetato de amonio y putrescina en una sola mezcla. Las trampas Nu-
Lure y NuLure liofilizada atrajeron menos moscas que el resto de las trampas irrespectiva-
mente de la especie, sexo, o localidad. Aunque todas las trampas o combinaciones de estas
con acetato de amonio atrajeron mas moscas que las trampas NuLure o NuLure liofilizada,
hubo una clara tendencia de las trampa de acetato de amonio y putrescina y la trampa Uni-
pak a atraer mas moscas despu6s de la cuarta semana a partir de comenzado el studio y de
las trampas NuLure liofilizadas con acetato de amonio o en combinaci6n con acetato de amo-
nio y putrescina a atraer mas moscas en las primeras cuatro semanas del studio. Este es-
tudio es unico en que se llev6 a cabo en huertos de carambola Averrrhoa carambola
(Oxalidacea), un cultivo que es un pobre hospedero de ambas species de moscas. Nuestros
resultados se comparan con otros studios con trampas deA. obliqua yA. suspense y las im-
plicaciones para el monitoreo y detecci6n de plagas Tephritidae son discutidos.

Translation provided by the authors.


Although less than 10% of the 199 described The island of Puerto Rico contains populations
species of Anastrepha are considered economi- of 2 economically important species; the Carib-
cally important (White & Elson-Harris 1992; bean fruit fly,A. suspense (Loew) and the West In-
Aluja 1994; Norrbom 2004), the occurrence of any dian fruit fly, A. obliqua (Macquart) (Jenkins &
of these economically important species in a re- Goenaga 2008). Although there are populations of
gion has a negative impact on growers. Growers A. suspense in Florida, there are no populations of
may be restricted from exporting their produce to A. obliqua there, making it risky to transport
certain markets, or may have to subject their fruit some Puerto Rican produce to Florida. Establish-
to expensive post-harvest sterilization measures ment of A. obliqua in Florida could jeopardize
(Simpson 1993). mango and other subtropical fruit crops.







Jenkins et al.: Food-based Anastrepha spp. Lures in Puerto Rico


Regulatory agencies spend considerable effort
and expense monitoring large areas for these and
other potentially invasive Anastrepha spp. (Anon-
ymous 2010). The need for effective monitoring/
detection devices has resulted in a long history of
studies on attractants for Anastrepha spp. (Heath
et al. 1993). Females of all frugivorous species of
Tephritidae that have been studied, including
Anastrepha spp., are anautogenous, i.e., they
need to consume protein as adults for ovary devel-
opment (Drew & Yuval 2000). Exploiting this
need for protein, a variety of potential lures based
on odors released from hydrolyzed proteins have
shown some degree of attractiveness, including
ammonia (released from ammonium acetate, am-
monium bicarbonate and urine) (Bateman & Mor-
ton 1981; Burditt et al. 1983), and hydrolyzed
torula yeast (Lopez et al. 1971; Burditt 1982). For
many years hydrolyzed torula yeast in a liquid
suspension, along with borax to reduce cadaver
decay, was used in 1 piece glass McPhail traps to
monitor and detect populations of Anastrepha
spp. (Anonymous 1989; Heath et al. 1993). A se-
ries of modifications to the trap and the lures
have improved the utility and effectiveness of the
trap (Epsky et al. 1993; Heath et al. 1995; Tho-
mas et al. 2001). Heath et al. (1995) identified
some common volatiles from baits and decompos-
ing fruit that were attractive to A. suspense.
These included ammonia, acetic acid (both re-
leased from ammonium acetate) and putrescine.
Although not attractive when deployed alone
(Heath et al. 2004), putrescine has been shown to
be a potent synergist to ammonium acetate for
capture ofbothA. ludens andA. supensa (Kendra
et al. 2008). Thomas et al. (2001) pointed out that
the design of the 1-piece glass McPhail trap was
difficult to service, especially with the new lures,
and prone to damage. A 2- piece plastic version of
the McPhail trap has since been widely adopted
by regulatory agencies. However, despite many
studies, no single bait has been identified to sat-
isfy the needs of regulatory agencies. Ideally, a
bait would be easy to apply, long-lasting, attrac-
tive to target species (often multiple target spe-
cies; regulatory agencies in Florida are currently
monitoring for A. obliqua and the Mediterranean
fruit fly, Ceratitis capitata Wied., among many
others) combined with low non-target attractive-
ness.
APHIS-PPQ in Puerto Rico currently deploys a
battery of traps and lures to detect and monitor
pest Tephritidae; trimethylamine and ammonium
acetate plus putrescine are used in Multilure
traps (2-piece plastic McPhail traps) to detect C.
capitata; methyl eugenol is used in Jackson traps
(tent-shaped sticky cards) to detect Oriental fruit
flies, Bactrocera dorsalis (Hendel) and carambola
fruit flies, B. carambolae Drew & Hancock, and
Cuelure is used in Jackson traps to detect melon
fruit fly, B. cucurbitae (Coquillett), and Queen-


island fruit flies, B. tryoni (Froggatt) (Anonymous
2010). In addition, torula yeast is still used at
some trap sites (Saez, personal communication).
Current lures for detecting/monitoring pest
Anastrepha spp. include Nulure (Miller Chemical
& Fertilizer, Hanover, PA.), a hydrolyzed corn pro-
tein lure (Gilbert et al. 1984), a freeze-dried prepa-
ration of Nulure (Heath et al. unpublished), am-
monium acetate combined with putrescine (Bi-
olure, Suterra LLC, Bend, Oregon), and the Uni-
pak (Suterra LLC), a single bait dispenser
containing ammonium acetate and putrescine
(Holler et al. 2009). Our objective in this study was
to compare these lures, as well as certain combina-
tions (freeze-dried Nulure combined with ammo-
nium acetate, or combined with both ammonium
acetate and putrescine) for relative attractiveness
to populations ofA. suspense and A. obliqua in Pu-
erto Rico. We chose to conduct these trials in car-
ambola, Averrhoa carambola (Oxalidaceae), be-
cause orchards of this fruit were available to the
researchers in 3 different regions of Puerto Rico.
Additionally, this is a poor host of both A. obliqua
andA. suspense; collections of thousands ofcaram-
bola fruit yielded no pupae ofA. supensa and rela-
tively few pupae of A. obliqua, principally when
preferred hosts, such as mango, were not available
(Jenkins & Goenaga 2008). Most lure trials are
conducted in orchards of preferred hosts; by con-
ducting these trials in a poor host environment we
evaluated efficacy of lures for detection of pest
Anastrepha at low population levels.

MATERIALS AND METHODS

Study Sites

Field trials were conducted in Sep and Oct of
2009, a time we had determined to be peak season
for both fly species (Jenkins, unpublished). All
trap blocks were set in experimental orchards of
carambola located at the USDA-ARS Tropical Ag-
riculture Research Experimental Station in Isa-
bela, PR, and at the University of Puerto Rico Ag-
ricultural Experiment Stations in Corozal and
Juana Diaz, PR. All orchards were planted in
1999 and were composed of 10 rows, each row con-
taining 22 trees. Trees were planted in a quin-
cunx system 3.7 m apart with 5.5 m between
rows. All of the 6 internal rows had 9 varieties of
tree planted randomly throughout the row. The
varieties, grafted onto Goldenstar rootstock, were
Arkin, B-10, B-16, B-17, Kajang, Kari, Lara, Sri-
Kembangan, and Thai Knight. The 2 rows of trees
on either side of these 6 internal rows were com-
posed entirely of Arkin grafted onto Goldenstar
rootstock. The first 2 trees and the last 2 trees of
each row were also Arkin grafted onto Goldenstar
rootstock. We have never recovered A. suspense
from thousands of carambola fruit and relatively
low numbers of A. obliqua have been recovered







Florida Entomologist 94(2)


from carambola fruit (Jenkins & Goenaga 2008);
nonetheless past experience has demonstrated
that both species can be trapped in relative abun-
dance from orchards of this fruit with no demon-
strable effects of fruit variety on trap catch (Jen-
kins, unpublished).

Traps and Lures

All baits were tested using plastic 2-piece Mul-
tilureTM traps (Better World Manufacturing, Inc.,
Fresno, CA). Commercial lures (Suterra, LLC,
Bend, OR) consisted of ammonium acetate and
putrescine (Biolure MFF), and the newly formu-
lated Unipak.
A total of 6 lures or lure combinations were
tested in each block as follows:

1. Ammonium acetate + putrescine (=AAPt)
2. Nulure with borax
3. Freeze-dried Nulure 7 (=FDN7)
4. Freeze-dried Nulure 7 + ammonium
acetate (=FDN7 + AA)
5. Freeze-dried Nulure 7 + ammonium
acetate + putrescine (=FDN7 + AAPt)
6. Unipak

For all treatments except the 9% Nulure with
borax, the trap fluid consisted of 200 mL of a 10%
solution of propylene glycol (Qualichem Technolo-
gies, GA) and water. The trap fluid for the 9% Nu-
lure lure (18 mL) consisted of 3% Borax (6 g)
mixed with 182 mL of water. The Nulure and
freeze-dried Nulure (9 g) were dissolved in the re-
spective trap fluid. Nulure and freeze-dried Nu-
lure baits were changed every 2 weeks (3 times
during the study). The ammonium acetate and
putrescine lures were changed every 4 weeks
(once during the study).

Trap Block Design

Traps were deployed in 3 rows of each orchard.
Rows with traps were at least 2 rows from the or-
chard border and separated by at least 1 trap-less
row from another row with traps. All 6 treatments
were represented in each of the 3 rows. Trees with
traps were separated from other trees with traps
by at least 1 tree. Traps were rotated to subse-
quent positions within rows each time they were
checked. Traps at all sites were checked for fruit
flies on Monday and Friday of each week between
24 Aug 2009 and 16 Oct 2009. All fruit flies were
returned to the laboratory for identification and
stored in 95% EtOH.

Statistical Analyses

The large number of independent variables we
were comparing combined with the low number of


replicates we were forced to use made the use of
an ANOVA unsuitable for our purposes. Chi-
square analyses were used to compare the ob-
served number of flies of each species and sex
trapped in each treatment to the expected num-
ber of flies under the assumption that flies would
be equally distributed among the treatments if
there was no difference in attraction. These anal-
yses were conducted for each sex of each species
for each week of the study (a total of 8 weeks) and
for the total number of flies trapped throughout
the study for each site. Chi-square probabilities
exceeding 0.05 were labeled as insignificant.
When the total number of flies trapped during a
given week or at a given site was less than 30, i.e.,
an expected distribution among the treatments
would be less than 5 (30 flies divided by 6 treat-
ments = 5), the result was regarded as too weak to
make inferences, even if the analyses indicated
significant departure from the null hypothesis (=
there was no difference in the distribution of flies
among the treatments).

RESULTS

Too few A. suspense were caught in traps at
the Isabela site (9 females and 1 male, total) to
merit analysis. A total of 294A. obliqua flies were
caught in Isabela, 167 of which were females
(58.0%) and 127 of which were males (42.0%).
Throughout the 8 weeks of the trial, the percent-
age of females trapped averaged 57.9% 4.7
(SEM). Of the 294 A. obliqua trapped at the Isa-
bela site during the experiment, 23% were in
traps baited with freeze-dried Nulure and ammo-
nium acetate and putrescine, 19% were in traps
baited with freeze-dried Nulure and ammonium
acetate, 19% in traps baited with UniPak lures,
17% were in traps baited with ammonium acetate
plus putrescine, 14% were in traps baited with
freeze-dried Nulure, and 7% in traps baited with
Nulure. Chi-square analyses indicated that the
distribution ofA. obliqua (combined sexes) among
the treatments departed significantly from the
null hypothesis, although this was not true for ev-
ery week of the study (Table 1).
A total of 231 Anastrepha spp. individuals
were trapped at the Corozal site. One hundred
and fifty nine (69%) of these were identified as A.
obliqua, of which 109 (69%) were females and 50
(31%) were males. Of the 72 A. suspense identi-
fied from traps at Corozal, 54 (75%) were female
and 18 (25%) were male. Throughout the 8 weeks
of the trial, the percentage of female A. obliqua
trapped averaged 70.2% 3.0 (SEM) and the per-
centage of female A. suspense trapped averaged
75.3% 3.8 (SEM).
Of the 159 A. obliqua trapped at the Corozal
site during the experiment, 30% were in traps
baited with ammonium acetate and putrescine,
28% were in traps baited with freeze-dried Nu-


June 2011
















TABLE 1. NUMBER OF FLIES CAPTURED BY WEEK AND BY BAIT AT THE ISABELA SITE. CHI-SQUARE ANALYSES WERE PERFORMED ON COMBINED SEXES WITHIN A SPECIES.

Number of flies

AAPt Nulure FDN7 FDN7 + AA FDN7 + AAt UniPak Total flies

Week Sex Male Female Male Female Male Female Male Female Male Female Male Female Male Female X2value X2prob

1 A. obliqua 0 1 3 5 1 3 7 3 2 2 0 2 13 16 12.6 0.0280
A. suspense 0 0 0 1 0 0 0 0 0 0 0 0 0 1 NA NA
2 A. obliqua 3 3 3 1 3 8 4 14 11 16 4 3 28 45 31.8 <0.0001
A. suspense 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NA NA
3 A. obliqua 4 1 2 0 4 1 3 3 9 8 6 3 28 16 18.7 0.0020
A. suspense 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NA NA
4 A. obliqua 2 5 2 2 4 3 6 1 2 0 3 4 19 15 4.1 0.5320
A. suspense 0 0 0 0 0 0 0 1 0 0 0 0 0 1 NA NA
5 A. obliqua 0 1 0 0 0 1 1 3 2 1 0 0 3 6 9.0 0.1090
A. suspense 0 1 0 0 0 0 0 1 0 2 0 0 0 4 NA NA
6 A. obliqua 3 1 1 0 2 3 3 1 2 6 5 10 16 21 19.3 0.0020
A. suspense 0 0 0 0 0 0 1 1 0 0 0 0 1 1 NA NA
7 A. obliqua 1 4 2 0 0 1 2 4 1 3 0 9 6 21 9.2 0.1000
A. suspense 0 0 0 0 0 0 0 1 0 1 0 0 0 2 NA NA
8 A. obliqua 9 13 0 2 2 4 0 1 2 2 1 5 14 27 28.3 <0.0001
A. suspense 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NA NA

Total A. obliqua 22 29 13 10 16 24 26 30 31 38 19 36 127 167 25.4 <0.0001
A. suspense 0 1 0 1 0 0 1 4 0 3 0 0 1 9 NA NA







Florida Entomologist 94(2)


lure and ammonium acetate and putrescine, 13%
were in traps baited with UniPak lures, 12% were
in traps baited with freeze-dried Nulure and am-
monium acetate, 11% were in traps baited with
freeze-dried Nulure, and 6% were in traps baited
with Nulure.
Of the 72 A. suspense trapped at the Corozal
site during the experiment, 33% were in traps
baited with ammonium acetate and putrescine,
26% were in traps baited with UniPak lures, 25%
were in traps baited with freeze-dried Nulure and
ammonium acetate and putrescine, 8% were in
traps baited with freeze-dried Nulure and ammo-
nium acetate, 6% were in traps baited with
freeze-dried Nulure, and 1% were in traps baited
with Nulure. As at the Isabela site, ammonium
acetate and putrescine, freeze-dried Nulure com-
bined with ammonium acetate or combined with
ammonium acetate and putrescine, and the Uni-
pak trapped more flies than the Nulure or the
freeze-dried Nulure (Table 2). This was true for
bothA. obliqua andA. suspense.
A total of 157 Anastrepha spp. individuals
were trapped at the Juana Diaz site. Ninety four
(59.9%) of these were identified as A. suspense, of
which 78 (83.0%) were female and 16 (17%) were
male. A total of 63 (40.1%) A. obliqua were
trapped at the Juana Diaz site, of which 49
(77.8%) were female and 14 (22.2%) were male.
Throughout the 8 weeks of the trial, the percent-
age of female A. obliqua trapped averaged 80.0%
+ 4.2 (SEM) and the percentage of femaleA. sus-
pensa trapped averaged 75.3% + 7.3 (SEM).
Of the 63A. obliqua trapped at the Juana Diaz
site during the experiment, 24% were in traps
baited with freeze-dried Nulure combined with
ammonium acetate and putrescine, 21% were in
traps baited with freeze-dried Nulure, 17% were
in traps baited with UniPak lures, 16% were in
traps baited with freeze-dried Nulure and ammo-
nium acetate and putrescine, 13% were in traps
baited with ammonium acetate and putrescine,
and 10% were in traps baited with Nulure.
Of the 94 A. suspense trapped at the Juana
Diaz site during the experiment, 27% were in
traps baited with freeze-dried Nulure and ammo-
nium acetate, 26% were in traps baited with am-
monium acetate and putrescine, 21% were in
traps baited with freeze-dried Nulure and ammo-
nium acetate and putrescine, 16% were in traps
baited with UniPak lures, 6% were in traps baited
with Nulure, and 4% were in traps baited with
freeze-dried Nulure. Generally, too few flies were
captured of either species to make a confident
analysis except when captures were summed for
the duration of the experiment (Table 3). No sig-
nificant departures from the null hypothesis were
detected in the distribution of A. obliqua flies
among the different baits.
At all 3 sites there was a consistent temporal
pattern in capture; freeze-dried Nulure combined


with ammonium acetate or combined with ammo-
nium acetate and putrescine caught more flies in
the first 4 weeks, whereas ammonium acetate
plus putrescine or the Unipak lures caught more
flies after the fourth week (Tables 1-3). The only
exception occurred at the Juana Diaz site when
the ammonium acetate and putrescine combina-
tion caught moreA. suspense than expected in the
first week (Table 3).

DISCUSSION

For all locations, species and sexes, where a
Chi-square analysis detected significant depar-
ture from equal distribution among the different
baits (and at least 30 flies were captured) Nulure
or freeze-dried Nulure consistently attracted the
fewest flies. This would suggest that the higher
attractiveness of the Unipak, ammonium acetate
and putrescine lures and the freeze-dried Nulure
in combination with either ammonium acetate or
ammonium acetate and putrescine is attributable
to the common factor of these lures, namely, the
presence of ammonium acetate in all of these
lures. However, bait attractiveness was not con-
stant over time, with a general trend of freeze-
dried Nulure in combination with ammonium ac-
etate or in combination with ammonium acetate
and putrescine attracting more flies in the first 4
weeks of the study and ammonium acetate and
putrescine or Unipak lures attracting more flies
in the fourth week and later. This appears to be
consistent with the anecdotal reporting that
freshly opened ammonium acetate lures are less
attractive than those that have been out a week
or more (Thomas et al. 2008). This is potentially
due to the dosage of ammonia released; Thomas
et al. (2008) demonstrated that higher doses of
the ammonia significantly reduced capture of A.
suspense and A. ludens compared to lower doses.
Also, Kendra et al. (2005) demonstrated that in-
creased doses of ammonia decreased the capture
of female A. suspense with undeveloped ovaries.
However, fresh ammonium acetate and pu-
trescine lures were placed in the field on the 5th
week of this study, approximately when they be-
gan to catch more flies. Also, the freeze-dried Nu-
lure combined with ammonium acetate or ammo-
nium acetate and putrescine caught more flies
early in the study, suggesting that the freshly
opened ammonium acetate packages are not too
strong, or that combined with the freeze-dried
Nulure, the ammonium acetate packages are at-
tractive at higher doses.
Many studies have been conducted on the at-
tractiveness of certain baits, but comparing these
studies in a meaningful manner is difficult and
subject to speculation. This is because these stud-
ies are often conducted in different regions, trap
different species of flies, different strains of flies
(wild versus lab-reared), test different combina-


June 2011
















TABLE 2. NUMBER OF FLIES CAPTURED BY WEEK AND BY BAIT AT THE COROZAL SITE. CHI-SQUARE ANALYSES WERE PERFORMED ON COMBINED SEXES WITHIN A SPECIES.

Number of flies

AAPt Nulure FDN7 FDN7 + AA FDN7 + AAt UniPak Total flies

Week Sex Male Female Male Female Male Female Male Female Male Female Male Female Male Female X2value X2prob

1 A.obliqua 0 1 0 1 2 0 1 6 0 6 0 0 3 14 15.1 0.0100
A. suspense 0 0 0 0 0 0 0 0 1 1 0 2 1 3 8.0 0.1560
2 A. obliqua 0 1 2 0 0 0 0 3 0 4 1 0 3 8 5.9 0.3150
A. suspense 0 0 0 0 0 0 1 2 0 0 0 0 1 2 15.0 0.0100
3 A. obliqua 3 4 2 1 0 6 3 5 7 16 0 0 15 32 51.5 <0.0001
A. suspense 0 1 0 0 1 0 0 1 0 5 0 1 1 8 10.3 0.0660
4 A.obliqua 6 9 0 2 0 0 0 0 3 3 0 3 9 17 37.2 <.0001
A. suspense 1 4 0 0 0 1 0 0 2 2 1 2 4 9 27.3 <0.0001
5 A. obliqua 0 3 0 0 1 2 0 1 3 1 0 2 4 9 5.0 0.4160
A. suspense 0 4 0 0 0 1 0 1 1 1 0 3 1 10 5.9 0.3150
6 A.obliqua 2 1 0 0 0 2 0 0 0 0 0 4 2 7 17.0 0.0050
A. suspense 2 2 0 0 0 1 0 0 0 2 0 0 2 5 11.0 0.0510
7 A.obliqua 3 3 0 1 0 0 0 0 0 0 1 5 4 9 42.7 <.0001
A. suspense 3 3 1 0 0 0 0 0 0 2 2 5 6 10 17.8 0.0030
8 A. obliqua 4 8 0 0 2 3 0 0 1 0 3 2 10 13 27.9 <.0001
A. suspense 1 3 0 0 0 0 0 1 0 1 1 2 2 7 9.0 0.1090

Total A. obliqua 18 30 4 5 5 13 4 15 14 30 5 16 50 109 48.3 <0.0001
A. suspense 7 17 1 0 1 3 1 5 4 14 4 15 18 54 37.5 <0.0001

















TABLE 3. NUMBER OF FLIES CAPTURED BY WEEK AND BY BAIT AT THE JUANA DIAZ SITE. CHI-SQUARE ANALYSES WERE PERFORMED ON COMBINED SEXES WITHIN A SPECIES.

Number of flies

AAPt Nulure FDN7 FDN7 + AA FDN7 + AAt UniPak Total flies

Week Sex Male Female Male Female Male Female Male Female Male Female Male Female Male Female X2value X2prob

1 A. obliqua 0 3 0 1 0 3 1 2 1 3 0 0 2 12 4.9 0.4332
A. suspense 1 9 2 3 0 0 0 4 1 6 1 1 5 23 13.6 0.0190
2 A. obliqua 0 0 0 0 1 0 0 0 0 2 0 0 1 2 7.0 0.2206
A. suspense 1 3 0 0 0 0 1 7 0 7 0 2 2 19 17.0 0.0050
3 A. obliqua 0 1 0 1 2 2 1 3 1 2 1 2 5 11 3.5 0.6234
A. suspense 0 4 0 1 0 2 0 8 0 2 2 1 2 18 9.4 0.0940
4 A. obliqua 0 1 1 0 0 0 1 1 0 5 1 0 3 7 9.2 0.1013
A. suspense 0 0 0 0 1 1 1 3 0 1 0 3 2 8 8.0 0.1560
5 A. obliqua 0 0 0 0 0 1 1 0 0 0 0 5 1 6 16.4 0.0057
A. suspense 1 1 0 0 0 0 0 0 0 0 1 2 2 3 10.6 0.0600
6 A. obliqua 0 1 0 0 0 0 0 0 0 1 0 0 0 2 4.0 0.5494
A. suspense 0 1 0 0 0 0 0 0 0 2 0 1 0 4 5.0 0.4160
7 A. obliqua 0 0 1 2 0 0 0 0 0 0 0 2 1 4 10.6 0.0599
A. suspense 0 1 0 0 0 0 0 0 0 0 1 0 1 1 4.0 0.5490
8 A. obliqua 0 2 0 0 1 3 0 0 0 0 0 0 1 5 14.0 0.0156
A. suspense 1 1 0 0 0 0 1 0 0 1 0 0 2 2 5.0 0.4160

Total A. obliqua 0 8 2 4 4 9 4 6 2 13 2 9 14 49 5.1 0.4038
A. suspense 4 20 2 4 1 3 3 22 1 19 5 10 16 78 25.9 <0.0001







Jenkins et al.: Food-based Anastrepha spp. Lures in Puerto Rico


tions of lures, are conducted in orchards of differ-
ent crop species, and at different times of the year,
all of which can impact the outcome. Nonetheless,
it is useful to summarize these studies because
baits will be used in a variety of conditions/loca-
tions/seasons to monitor/detect a variety of pest
Tephritidae.
Regionally, the experiments most similar to
the present study are those of Pingel et al. (2006)
comparing the attractiveness of ammonium ace-
tate plus putrescine with torula yeast plus borax
in commercial orchards of 3 crop species in south-
ern Puerto Rico, coinciding climactically and geo-
graphically with our Juana Diaz site. Conducted
in Apr to May of 2002, their study found that the
torula yeast outperformed the ammonium acetate
and putrescine combination in orchards of ma-
mey sapote and sapodilla, but the ammonium ac-
etate and putrescine lure attracted more flies
than the torula yeast in carambola orchards. The
difference between the effectiveness of the 2 lures
in the different orchards is striking and they
point out the preponderance ofA. obliqua in the
carambola orchard (94% trapped flies in the car-
ambola orchard were A. obliqua) whereas the
other orchards had higher relative populations of
A. suspense. Anastrepha suspense was more
abundant in the carambola orchard at the Juana
Diaz site during our study.
In a Colombian mango orchard A. obliqua was
caught in traps baited with Nulure and borax
more frequently than in traps baited with ammo-
nium acetate with putrescine, torula yeast, or
ammonium bicarbonate with putrescine (Epsky
et al. 2003). However, in another study in a Mex-
ican Pouteria sapota (Sapotaceae) orchard, traps
baited with ammonium acetate with putrescine
caught more A. obliqua than traps with the other
baits, and in a Mexican mango orchard Nulure
and ammonium acetate with putrescine both
caught more A. obliqua than traps baited with
other lures (Epsky et al. 2003). Furthermore,
traps baited with torula yeast in Costa Rica and
Honduras caught more A. obliqua than traps
baited with the other lures. Anastrepha suspense
does not occur in any of the locations of the cited
trial and so no comparison can be made with the
A. suspense results of our study. A similar study
indicated that ammonium acetate was the best
lure for detection of A. suspense in Florida but
that Nulure or torula yeast were the best lures for
A. obliqua in the Dominican Republic, based on
traps in mango orchards (Thomas et al. 2008).
In a study conducted in cages in Mexico, Diaz-
Fleischer et al. (2009) found ammonium acetate
and putrescine were more attractive toA. obliqua
than Nulure, but the ammonium acetate and pu-
trescine was not as attractive to A. ludens. Anas-
trepha ludens is an economic pest which regula-
tory agencies in the United States would like to
be able to detect. They also found that attractive-


ness varied according to whether the flies tested
were wild or reared in the laboratory for several
generations.
There is strong evidence that the attractive-
ness of a particular lure to a given fly is based on
that fly's physiological state, usually the stage of
ovary development and a possible explanation for
our results may be that populations varied phys-
iologically over the duration of the experiment.
Electroantennagram studies on A. suspense indi-
cated that immature females (females with little
ovary development) were more responsive to am-
monia and to ammonium bicarbonate lures, while
females with mature ovaries were more respon-
sive to putrescine and to carbon dioxide (Kendra
et al. 2005; Kendra et al. 2009). Diaz-Fleischer et
al. (2009) found that diet of the target fly did in-
fluence subsequent capture in traps, with more
protein-starved individuals being captured by
protein baits.
Nulure, followed by freeze-dried Nulure, con-
sistently attracted the fewest flies in our study,
regardless of species, sex, or location. This con-
trasts with the results obtained by Thomas et al.
(2008) in mango orchards in Dominican Republic,
where A. obliqua was most attracted to Nulure
and torula yeast baits. It is conceivable that Nu-
lure would attract more flies in different seasons.
Liquid lures, including Nulure and torula yeast,
have been shown to be more attractive in the dry
season than in the wet season (Heath et al. 1997)
and the Thomas et al. study was conducted at the
beginning of the wet season.
Diaz-Fleischer et al. (2009) recently concluded
that "there is no magic fruit fly trap," based on the
complex interactions of fly species, physiological
state and bait "preference," aggravated by low
trap efficiency. One particular short-coming was
the number of flies that entered a trap and suc-
cessfully escaped from it. It is certainly true that
these interactions are complex and that no single
bait or trap will suffice for all target species in all
regions. It has long been known that different te-
phritid species respond differently to hydrolyzed
protein from different sources; A. ludens was
more attracted to hydrolyzed cottonseed oil than
to hydrolyzed corn protein (Lopez and Becerril
1967). Anastrepha striata, A. serpentina, A. obli-
qua and A. balloui preferred baits of hydrolyzed
soy protein to torula yeast hydrolysate (Jiron and
Soto-Manitiu 1989). Despite all of the improve-
ments to the traps themselves and the lures, esti-
mates of percent capture have not changed over
more than 20 years; Calkins et al. (1984) and
Diaz-Fleischer et al. (2009) came up with esti-
mates of about 10% of the available population.
Kendra et al. (2010) were able to recapture up to
35% of released A. suspense, though. The results
of this study confirm what has been suspected;
that trap baits will have to be tailored based on
regional and seasonal use.







Florida Entomologist 94(2)


ACKNOWLEDGMENTS

We thank Elkin Vargas and Rosemarie Boyle
(USDA-ARS, Mayaguez, Puerto Rico) for technical as-
sistance; Amy Roda (USDA-APHIS, Miami, Florida)
and Paul Robbins (USDA-ARS, Fort Pierce, Florida) for
helpful suggestions with the manuscript. This report
presents the results of research only; mention of a pro-
prietary product does not constitute an endorsement by
the USDA.


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June 2011







Ovruski et al.: Host Preference By Diachasmimorpha longicaudata


HOST PREFERENCE BY DIACHASMIMORPHA LONGICAUDATA
(HYMNEOPTERA: BRACONIDAE) REARED ON LARVAE OF ANASTREPHA
FRATERCULUS AND CERATITIS CAPITATA (DIPTERA: TEPHRITIDAE)


SERGIO M. OVRUSKI, LAURA P. BEZDJIAN, GUIDO A. VAN NIEUWENHOVE, PATRICIA ALBORNOZ-MEDINA
AND PABLO SCHLISERMAN
Laboratorio de Investigaciones Ecoetol6gicas de Moscas de la Fruta y sus Enemigos Naturales (LIEMEN). Planta
Piloto de Procesos Industriales Microbiol6gicos y Biotecnologia (PROIMI) CCT Tucuman CONICET. Avda.
Belgrano y Pje. Caseros, (T4001MVB) San Miguel de Tucuman, Tucuman, Argentina


ABSTRACT

The preferences of Diachasmimorpha longicaudata (Ashmead) for larvae of Anastrepha
fraterculus (Wiedemann) and Ceratitis capitata (Wiedemann) were evaluated under labora-
tory conditions in no-choice and dual-choice tests, based on percent parasitism, proportion of
emerged parasitoids, proportion of female offspring, and number of parasitoid female visits
to and ovipositor probes on the artificial oviposition device as different measures of host pref-
erence. In no-choice tests D. longicaudata females did not demonstrate a significant prefer-
ence between C. capitata and A. fraterculus larvae. Nevertheless, D. longicaudata females
showed a strong preference forA. fraterculus larvae in dual-choice test. Although female bi-
ased parasitoid progeny resulted in all assays, significantly more D. longicaudata female off-
spring emerged from A. fraterculus pupae than from C. capitata pupae. Thus, this study
confirmed that both C. capitata andA. fraterculus are appropriate host for rearing D. longi-
caudata, but also provided evidence that female parasitoid progeny yield can be substan-
tially improved by usingA. fraterculus larvae as the host instead of C. capitata larvae.

Key Words: fruit flies, parasitoids, host preference, biological control, Argentina

RESUME

Se evalu6 la preferencia de Diachasmimorpha longicaudata (Ashmead) por larvas deAnas-
trepha fraterculus (Wiedemann) y Ceratitis capitata (Wiedemann) bajo condiciones de labo-
ratorio en situaciones de elecci6n y no-elecci6n. Las variables consideradas para el analisis
fueron el porcentaje de parasitismo, la proporci6n de parasitoides emergidos, la proporci6n
de descendientes hembras, el numero de hembras que visitaron la unidad artificial de ovi-
posici6n y el numero de hembras que realizaron pruebas con el ovipositor en la unidad. Los
resultados de los ensayos de no-elecci6n mostraron que las hembras de D. longicaudata no
tienen una significativa preferencia por las larvas de una u otra especie de tefritido. No obs-
tante, en el ensayo de elecci6n, las hembras del parasitoide manifestaron una significativa
preferencia por las larvas deA. fraterculus. En todos los ensayos realizados, la proporci6n de
descendientes hembras de D. longicaudata obtenida fue superior a la de los machos, aunque
significativamente mas hembras del parasitoide se obtuvieron de puparios deA. fraterculus.
El present studio confirm que tanto las larvas de C. capitata como las deA. fraterculus
son adecuadas para criar D. longicaudata en laboratorio, aunque tambi6n seiala que el em-
pleo de larvas de A. fraterculus mejoran sustancialmente la producci6n de descendientes
hembras del parasitoide.


Translation provided by the authors.


The South American fruit fly, Anastrepha
fraterculus (Wiedemann), and the Mediterranean
fruit fly, Ceratitis capitata (Wiedemann) are 2 of
the major pests currently affecting fruit crops in
Argentina (Guillen & Sanchez 2007). Early bio-
logical control attempts to suppress both te-
phritid pest species resulted in the use of exotic
parasitoids (Ovruski et al. 2000). Diachasmimor-
pha longicaudata (Asmead) is 1 of 5 exotic para-
sitoids introduced into Argentina from Costa Rica
and Mexico (Ovruski et al. 2003). It was originally
collected in the Malaysia-Philippine region and is


a solitary, koinobiont, larval-prepupal endopara-
sitoid of several tephritid species (Montoya et al.
2000). At present, D. longicaudata is considered 1
of the most significant biological control agents
for augmentative releases against economically
important fruit fly species in several Latin Amer-
ican countries (Montoya et al. 2007; Paranhos et
al. 2008; L6pez et al. 2009).
Although small scale releases of D. longicau-
data were made in the Citrus-growing areas of
northern Argentina during the 1960s (Ovruski et
al. 2000), the permanent establishment of this







Florida Entomologist 94(2)


opiine parasitoid on A. fraterculus has been veri-
fied as a direct result of early classical biological
control programs (Oroio & Ovruski 2007).
Currently, the suitability for successfully rear-
ing D. longicaudata on larvae of either C. capitata
or A. fraterculus is being studied in the PROIMI
insectary in San Miguel de Tucuman-Argentina,
as part of an augmentative release program
against both tephritid fruit fly species. Therefore,
the study here presented was conducted to evalu-
ate the effects of both C. capitata and A. fratercu-
lus on parasitism, parasitoid emergence, and sex-
ual ratio of offspring in D. longicaudata under
laboratory conditions. Furthermore, both the
number of visiting and oviposition events was
documented to assess the parasitoid female pref-
erence for 1 or the other host tephritid species.

MATERIALS AND METHODS

The study was performed at the Biological
Control Division of Planta Piloto de Procesos In-
dustriales Microbiol6gicos y Biotecnologia
(PROIMI) located in San Miguel de Tucuman, Ar-
gentina. The colony of D. longicaudata was origi-
nally established in 1999 with individuals im-
ported from Mexico (Ovruski et al. 2003), where
this colony had been reared in the laboratory on
Anastrepha ludens (Loew) larvae (Montoya et al.
2000). First, D. longicaudata was successfully
reared at the PROIMI laboratory on late-third in-
stars ofC. capitata. Then, in 2005 a second colony
of D. longicaudata was established on late-third
instars ofA. fraterculus. Parasitoid colonies were
held in cubical Plexiglas cages (30 cm) covered by
organdy screen on both lateral sides, at a capacity
of 300 pairs per cage at 25 + 1C; 75 + 5% RH, and
12:12 (L:D) h photoperiod. The parasitoid rearing
cage was provided with water and honey every
other day. The general C. capitata andA. fratercu-
lus rearing procedures were carried out as de-
scribed by Ovruski et al. (2003) and by Vera et al.
(2007), respectively. Both A. fraterculus and C.
capitata puparia were selected from different
samples and weighed for host quality evaluation.
Each species of fruit fly was exposed to 10
mated D. longicaudata females in cubical Plexi-
glas cages (30 cm) under both dual-choice and no-
choice assays. In the choice assay, an oviposition
unit (an organdy screen-covered petri dish, 8 cm
diameter, 0.8 cm deep) containing 300 laboratory-
reared third-instars of A. fraterculus (11 d old)
was placed on the floor of the test cage along with
another oviposition unit containing 300 labora-
tory-reared third-instars of C. capitata (6 d old).
Larvae of both fruit fly species were placed in the
units with artificial diet (brewer yeast + wheat
germ + sugar + water). Oviposition units were po-
sitioned in the central part of the test cage; each
unit was placed 1 cm from the side wall and sep-
arated by 10 cm from the other unit. In the no-


choice assays, an identical oviposition unit con-
taining 300 third-instars ofA. fraterculus (or 300
third-instars of C. capitata) was placed on the
floor of the central part of the test cage away from
the walls. All female parasitoids used in experi-
ments were 7-8 d old and deprived of any host lar-
vae before testing. The females used in no-choice
tests came either from parasitized puparia of A.
fraterculus or from parasitized puparia of C. cap-
itata. In the choice assay, 5 females stemming
from parasitized puparia of A. fraterculus and 5
females stemming from parasitized puparia of C.
capitata were used jointly. This combination of
parasitoids from different origins was used so as
to ameliorate a possible conditioned response by
the previous experience with the host on which it
was reared (Godfray 1994). Two control tests (no
parasitoids) were made to determine both natural
A. fraterculus and C. capitata mortality and
emergence rates. Each test, including control
treatments, was replicated 22 times. Each repli-
cate lasted 24 h. All assays were conducted in the
laboratory under the environmental conditions
described previously.
Behavioral observations can be used to provide
evidence of host preference for solitary parasitoids
(Mansfield & Mills 2004). For this reason, upon re-
lease of parasitoids into each test cage, the number
of female visits to and ovipositor probes in the ovi-
position units was recorded. Odor concentrations
of host fruit (Messing & Jang 1992) or oviposition-
deterring pheromone of tephritid fly (Prokopy &
Webster 1978) were not considered in the assays
because oviposition units with artificial diet were
used. The female parasitoids were observed once
every 15 min during the first 3 h and each observa-
tion lasted 30 s (Duan & Messing 2000a). A visit
was recorded each time a female arrived on the ovi-
position unit after release. An ovipositor probe was
confirmed each time a female parasitoid inserted
its ovipositor through the top organdy screen of the
oviposition dish. After the 3-h observations, all ovi-
position units remained exposed to female parasi-
toids for 21 h to finish a 24-h period (Duan & Mess-
ing 2000a). Then, all oviposition dishes were re-
moved from the cages, and fly larvae were directly
transferred into plastic cups (7 cm diameter, 6.7
cm deep) containing a 2 cm-vermiculite layer on
the bottom as pupation medium. Later, each cup
was tightly covered with a piece of organdy cloth
on the top. Thus, fly pupae were held within plastic
cups with moist, sterilized vermiculite until eclo-
sion. After that, the number and sex of the
emerged parasitoids, the number of emerged flies,
and the number of uneclosed puparia were
checked. Uneclosed puparia were dissected 2
weeks after emergence of the last adult parasitoid
in each cup to check for the presence or absence of
recognizable immature parasitoid stages (larvae,
prepupae, or pupae) and/or fully developed pher-
ate-adult parasitoids.


June 2011







Ovruski et al.: Host Preference By Diachasmimorpha longicaudata 197


Both the parasitism percentage and the num-
ber and sex ratio of emerged parasitoid progeny
were used as 3 suitable variables to measure host
preference, in addition to the behavioral observa- ,
tions (Mansfield & Mills 2004). Parasitism per- t -
centage was calculated by dividing the total num- s +1 +1 5
ber of emerged and unemerged parasitoids into v
the total number of larvae exposed in the oviposi-
tion unit. The proportion of emerged parasitoids
was calculated as the total number of emerged
offspring divided by the total number of recovered
pupae. The proportion of emerged flies was com-
puted as the total number of retrieved adult flies C t m
divided by the total number of recovered pupae. Z
The proportion of dead pupae was determined as m "
the total number of pupae that did not yield flies +1 +1
or parasitoids divided into the sum of closed and C t ft
uneclosed puparia. 2
Data on parasitism, parasitoid and fly emer-
gences, sexual ratio of parasitoid offspring (as
proportion of females), pupal mortality, and the
number of female visits to and ovipositor probes .
on the artificial oviposition device were analyzed
by a 2-sample unpaired t-test (P = 0.05) in no- H E 1 1 C
choice assays, and by a paired t-test (P = 0.05) in 0 0
the choice assay. Moreover, the numbers of P-
emerged adults and dead pupae recorded from
each fruit fly species per assay were statistically
compared with control treatments by means of
one-way analyses of variance (P < 0.05). Means <
were separated with a Tukey honest significant > 3 i
difference test (HSD) (P = 0.05). The proportion C. .
data were transformed to arcsine square root be- ? +i +I "
fore analysis. All untransformed means (+ SEM) z v
were presented in the text. Pupal weight differ- S "
ence between A. fraterculus and C. capitata was
analyzed by a Mann-Whitney Rank Sum test (P =
0.05).

RESULTS 8 Z

From the dual-choice test, significantly higher i C I m
parasitism and emerged adult parasitoid percent- +1 C d
ages were recorded from A. fraterculus than from ci v
C. capitata (Table 1). When these 2 fruit fly spe-
cies were analyzed in the no-choice tests, there
was no significant difference for either of these 2
measures of host preference (Table 1). Sex ratios
were female biased when D. longicaudata was
reared from either host fruit fly species. However, 1i C z
the proportion of female offspring was always sig- V = "
nificantly higher when the parasitoid was reared v
on A. fraterculus than on C. capitata (Table 1).
The proportion of emerged A. fraterculus and C.
capitata adults was significantly different between C
dual-choice, no-choice, and no-exposure control I
tests (F(2 = 260.0, P < 0.0001 forA fraterculus; F(
,, = 311.8, P < 0.0001 for C. capitata, Table 2). A sig- .g
nificantly higher proportion ofA. fraterculus adults 'G
were recovered from the no-choice test than from P
dual-choice test (Table 2). In contrast, significantly
Hr4







Florida Entomologist 94(2)


TABLE 2. MEAN (SEM) PROPORTION OF EMERGED ADULTS AND DEAD PUPAE FROM CERATITIS CAPITATA AND ANAS-
TREPHA FRATERCULUS RECORDED IN CHOICE, NO-CHOICE, AND CONTROL TESTS.

% emerged % emerged % dead % dead
Tests A. fraterculus adults C. capitata adults A. fraterculus pupae C. capitata pupae

Dual-choice 17.3 1.8 a 63.2 1.7 a 34.2 1.4 a 25.5 1.1 a
No-choice 24.6 2.6 b 25.1 1.9 b 32.3 2.0 a 37.4 t 2.5 b
Control 82.4 1.3 c 85.3 1.6 c 17.6 1.3 b 14.7 1.6 c

Values in the same column with the same latter are not significantly different (Tukey s test, P < 0.05).


2.5-times greater proportion of C. capitata adults
emerged from the dual-choice test than from no-
choice test (Table 2). The significantly lowest pro-
portion of dead fly pupae was recorded from no-ex-
posure control tests (F 2, 6 = 28.6, P < 0.0001 for A
fraterculus; F(2,63 = 38.9, P < 0.0001 for C. capitata,
Table 2). Significantly greater proportion of dead C.
capitata pupae was recorded from no-choice tests
than from dual-choice tests (Table 2).
Under both dual- and no-choice conditions, the
mean numbers ofD. longicaudata female visits to
the oviposition units containing A. fraterculus
larvae were significantly similar to those of para-
sitoid visits to the oviposition units containing C.
capitata larvae (paired-t = 1.89, df = 21.0, P =
0.0732 for dual-choice test; unpaired-t = 0.47, df=
42.0, P = 0.6435 for no-choice test; Fig. 1 A). Sim-
ilarly, in the no-choice assays, there were no sig-
nificant differences in the mean numbers of para-
sitoid females probing the oviposition artificial
devices (unpaired-t = 0.58, df = 42.0, P = 0.5631;
Fig. 1 B). In contrast, in the dual-choice test, a
significantly greater number of D. longicaudata
females were observed probing the oviposition
unit containing A. fraterculus larvae that the de-
vice containing C. capitata larvae (paired-t = 5.54,
df= 21.0, P < 0.0001; Fig. 1 B).

DISCUSSION

While D. longicaudata attacked both C. capitata
and A fraterculus larvae at similar rates when only
1 of the species was present, they preferred A
fraterculus when provided a choice. This divergence
may be suggestive of the relative host size differ-
ences. For example, A. fraterculus larvae used as
host in this study were twice as large as C. capitata
larvae (T = 60100.0, P < 0.0001, n = 200). Previous
studies conducted by Messing et al. (1993), Cancino
et al. (2002) and L6pez et al. (2009), found that D.
longicaudata females prefer large hosts. Eben et al.
(2000) also pointed to the progeny sex ratio as a
measure of host larva preference in D. longicau-
data. These authors found that D. longicaudata
reared from a larger species, A ludens (Loew), in
mango (Mangifera indica L.) had a much higher
proportion of female progeny than those parasitoids
that had developed in a smaller species, A. obliqua
(Macquart), infesting the same fruit.


A 100oo
90.
804 T

70
60O
; mova fonrairti e
2so C f rcarCapit
S40 cho
30
S120
10
o 70
Choice test No-choice lest



data female oviposit


d intas po40 f e btoc Ccapctat
540
30.

9 o10

Choice est N-echice tesl


Fig. 1 (A and B). Mean (t SEM) (A) number ofD. lon-

the oviposition units containing artificial diet plus
third-instars ofA. fraterculus or C. capitata recorded in
no-choice and dual-choice tests. Bars in each graph fol-
lowed by the same letter indicate no significant differ-
ences [unpaired t-test (P = 0.05) in the no-choice tests,
and paired t-test (P = 0.05) in the dual-choice test]



Behavioral observations provided further evi-
dence for a preference for A. fraterculus over C.
capitata larvae. In dual-choice tests D. longicau-
data females are more likely to exhibit oviposition
behaviors on devices containing A. fraterculus.
However, Silva et al. (2007) found that D. longi-
caudata females did not discriminate between the
volatiles produced by C. capitata or A. fraterculus
larvae. In contrast to the present study, the larvae
exposed by Silva et al. (2007) were feeding inside
infested guava fruits (Psidium guajava L.). It has
been repeatedly demonstrated that D. longicau-
data females respond to fruit volatiles, especially
from rotting fruits (Greany et al. 1977; Leyva et


June 2011







Ovruski et al.: Host Preference By Diachasmimorpha longicaudata


al. 1991; Messing & Jang 1992; Purcell et al.
1994; Eben et al. 2000; Carrasco et al. 2005).
Chemical cues derived from fermentation of the
artificial rearing medium can be exploited for
host searching byD. longicaudata (Duan & Mess-
ing 2000b). However, it is possible that differ-
ences in host larval substrates might have influ-
enced D. longicaudata's host detection ability.
Duan & Messing (2000b) found that C. capi-
tata larvae outside of the substrate on which they
fed generated vibration and chemical cues that
stimulated oviposition in Diachasmimorpha try-
oni Cameron, another generalist opiine fruit fly
larval parasitoid (Wharton 1989). In the case ofD.
longicaudata, chemical cues produced by C. capi-
tata larvae had little influence on probing behav-
ior (Duan & Messing 2000b). However, it is possi-
ble that D. longicaudata females may respond
more positively to chemical cues ofA. fraterculus
larvae than to those from C. capitata larvae. In
addition to larval frass, other parts of the host
larva such as hemolymph, alimentary canal, fat
bodies, labial glands, and mandibular glands may
be the source of 1 or more kairomones that stim-
ulate oviposition movements in larval parasitoid
species (Arthur 1981). Therefore, additional re-
search should be performed to further define
specificity ofD. longicaudata female responses to
chemical cues from both A. fraterculus and C. cap-
itata larvae. Based on this requirement, we plan
to conduct a second series of future experiments
with D. longicaudata and 2 neotropical opiine
fruit fly larval parasitoids.
Although dual-choice test results obtained in
the present study provide reliable information on
host rank order preferences for D. longicaudata,
the ecological considerations on preference can-
not be conjectured from this data. Therefore, we
are currently verifying the host preference by D.
longicaudata in field-cage tests using different
host fruit species which are commonly infested by
C. capitata and/or A. fraterculus larvae in the
field.
Finally, this study confirmed previous data in-
dicating that both C. capitata (Ovruski et al.
2003; Viscarret et al. 2006) and A. fraterculus
(Ovruski et al. 2007) are suitable hosts for labora-
tory rearing of D. longicaudata in Argentina. It
also provided evidence that female parasitoid
progeny yield can be highly improved by using A.
fraterculus larvae as host instead of C. capitata
larvae.

ACKNOWLEDGMENTS

I express my gratefulness to Arnaldo Mangeaud
(UNCo, C6rdoba, Argentina) for the statistical help and
to Carolina Chiappini, Natalia Salinas, and Josefina
Buonocore (PROIMI, Tucuman, Argentina) for technical
assistance. Special thanks to Jorge Cancino-Diaz and
Pablo Montoya (Mexican MOSCAMED Program,
Metapa de Dominguez, Chiapas, M6xico), and Pablo


Liedo (ECOSUR, Tapachula, Chiapas, M6xico) for al-
lowing me to introduce D. longicaudata specimens to
Argentina from M6xico, and to 2 anonymous referees for
helping us produce a better paper. I thank Teresa Vera
and Eduardo Willink (EEAOC, Tucuman, Argentina) for
providing me with the first A. fraterculus reared speci-
mens. This study was supported by Consejo Nacional de
Investigaciones Cientificas y T6cnicas de Argentina
(CONICET) (grant PIP/2005 No. 5129) and by Agencia
Nacional de Promoci6n Cientifica y Tecnol6gica de Ar-
gentina through Fondo Nacional de Ciencia y Tec-
nologia (FONCyT) (grant PICT/2006 No. 2402).

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lands.


June 2011







Lehnert et al.: Color Quantification


A NEW METHOD FOR QUANTIFYING COLOR OF INSECTS

MATTHEW S. LEHNERT'2, MURAT O. BALABAN3 AND THOMAS C. EMMEL2
'Department of Entomology and Nematology, University of Florida, Bldg. 970 Natural Area Drive,
P.O. Box 110620, Gainesville, FL 32611-0620

2McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida,
S.W. 34th Street and Hull Road, P.O. Box 112710, Gainesville, FL 32611-2710

3Fishery Industrial Technology Center, University of Alaska Fairbanks, 118 Trident Way, Kodiak, AK 99615

ABSTRACT

We describe a method to quantify color in complex patterns on insects, using a combination
of standardized illumination and image analysis techniques. Two color comparisons were in-
vestigated: (1) the percentage of blue in the submarginal band of the hindwing in yellow and
dark morph females ofPapilio glaucus L., and (2) the percentage of orange hues in the wings
of 2 putative subspecies of Eastern Tiger Swallowtail, P. g. glaucus L. and P. g. maynardi
Gauthier. Live specimens were photographed in a light-box with standardized lighting and
a color standard. Digital images were processed in LensEye software to determine the per-
centage of selected colors. No significant differences were found in the percentage of blue be-
tween yellow and dark morph females, but the percentage of orange hues between P. g.
glaucus and P. g. maynardi differed significantly. Color quantification can be a useful tool in
studies that require color analysis.

Key Words: color analysis, color quantification, butterfly comparison, digital image, Papilio
glaucus

RESUME

Se describe un m6todo para cuantificar el color en los patrons complejos de los insects, uti-
lizando una combinaci6n de iluminaci6n estandarizada y de la t6cnica de andlisis de imagen.
Se investigaron dos comparaciones de color: (1) el porcentaje de azul en la banda submargi-
nal de las alas posteriores en las hembras de forma amarilla y de forma oscura de Papilio
glaucus L. y (2) el porcentaje de tonos de color anaranjado en las alas de dos subespecies pu-
tativos de Papilio glaucus, P. g. glaucus L. y P. g. maynardi Gauthier. Se tomaron fotos de es-
pecimenes vivos en una caja de luz con iluminaci6n estandarizada y un estandar de color.
Las imagenes digitales fueron procesadas usando el program LensEye para determinar
el porcentaje de los colors seleccionados. No se encontraron diferencias significativas en el
porcentaje de color azul en las hembras de forma amarilla y de forma oscura, pero el porcen-
taje de tonos anaranjados entire P. g. glaucus y P. g. maynardi diferian significativamente.
Cuantificaci6n del color puede ser una herramienta util en los studios que requieren de un
andlisis de color.


Color and color patterns have been used to
study a wide range of ecological and evolutionary
topics, including sexual selection (Punzalan et al.
2008), aposematism (Brower 1958), industrial
melanism (Kettlewell 1961), and mimicry (Jig-
gins et al. 2001; Saito 2002). Color is used in the
classification of organisms to verify species and
population properties, and subspecies (Brower
1959). The color of butterfly life stages and wings
is used to understand evolutionary-developmen-
tal patterns and phenotypic plasticity (Star-
necker & Hazel 1999; Nice & Fordyce 2006; Otaki
2008). However, most of these studies are hin-
dered in their ability to quantify color.
When reporting quantified colors, RGB (red,
green, blue) and L*, a*, and b* values (L* = light-
ness, scale: 0-100; a* = green to red, scale: -120-


120; and b* values = blue to yellow, scale: -120-
120) are typically used. RGB are digitally repre-
sented by 256 values each, meaning a total of
more than 16 million possible color combinations
(Balaban 2008), but the colors produced by these
values are typically non-uniform and do not cor-
relate well to human vision (Pedreschi et al.
2006). However, L*, a*, and b* values are com-
bined together to represent a color that can be
used in a comparative context to other similar col-
ors (Pedreschi et al. 2006), and do account for the
way humans perceive color.
Existing methods for quantifying color include
simple visual estimates, with or without the use
of a book of color standards for reference such as
Munsell's (1976), spectrophotometry (Stevens et
al. 2007), color software with RGB applications







Florida Entomologist 94(2)


(Villafuerte & Negro 1998), and colorimetery (Ya-
giz et al. 2009). Human vision is color biased
(Wyszecki & Stiles 1982); factors such as lighting
condition, illumination, and color are context-de-
pendent (Endler 1990; Zuk & Decruyenaere
1994), and make color difficult to quantify. Speci-
mens need to be nearly homogenous in color and
have an almost flat surface to be accurately rep-
resented with colorimetry (Balaban 2008;Yagiz et
al. 2009), and common image software such as
Adobe Photoshop has limitations when stan-
dardizing or calibrating a digital image and when
quantifying the color patterns of complex images
with large color variation.
Our objective was to introduce the use of image
analysis with the LensEye software as a tool to
quantify the color of insects. LensEye software
was developed specifically for color quantification
purposes, which makes it more user-friendly than
other general color analysis programs such as
Adobe Photoshop. LensEye has been used in
food and agricultural sciences (Balaban 2008; Ya-
giz et al. 2009), but its application to entomologi-
cal studies is novel. To illustrate this process, the
wing colors of male and female Eastern Tiger
Swallowtail butterflies, Papilio glaucus L., were
analyzed in 2 comparisons: (1) the percentage of
blue on the hindwing between yellow and dark
morph females of P glaucus, and (2) the percent-
age of orange hues between males of the 2 subspe-
cies P. g. glaucus L. and P g. maynardi Gauthier.
In the first comparison, we predicted that the per-
centage of blue on the hindwing would be similar
in yellow and dark morph females, because to our
knowledge no previous reports have suggested a
larger amount of blue in either morph. In the sec-
ond comparison, we expected that males of P. g.
maynardi would have a significantly larger per-
centage of the wings represented by high a* and
b* values when compared with P g. glaucus, as a
combination of these values (reds and yellows)
likely produces the orange hues that are diagnos-
tic for this subspecies. To our knowledge, this is
the first report of color quantification of tiger
swallowtail butterflies.

MATERIALS AND METHODS

Study Species and Specimen Preparation

The Eastern Tiger Swallowtail, Papilio glau-
cus L. (Lepidoptera: Papilionidae), is a large
multi-colored butterfly found throughout the
eastern half of the USA (Scriber 1996). Females
are polymorphic and are either yellow with black
stripes or melanic (Clarke & Sheppard 1962;
Scriber 1996; Scriber et al. 1996); both forms have
blue scales along the submarginal region of the
dorsal side of the hindwings. Currently, 2 puta-
tive subspecies are recognized, P. g. glaucus and
P g. maynardi; the latter has a unique orange


background color rather than the yellow found on
the glaucus subspecies (Maynard 1891; Scriber
1986). Papilio g. maynardi is primarily found in
Florida, but occasionally is found in other south-
eastern states (Maynard 1891; Brower 1959;
Scriber 1986; Lindroth 1991). Ten yellow females
and 10 dark morph females of P. glaucus were
captured from Cedar Key and Lake Placid, Flor-
ida to compare the percentage of blue in the hind-
wings between these morphs. To compare the per-
centage of orange hues between the 2 subspecies,
10 males were collected from La Fayette, Georgia,
and 10 males from Lake Placid, Florida, to repre-
sent the P g. glaucus and P. g. maynardi subspe-
cies, respectively. All specimens were captured
during Apr-Jun, 2008, representing what is likely
the spring brood of P glaucus in these regions.
All butterflies were captured with a butterfly
net and placed into glassine envelopes for trans-
port. The live adults of P. glaucus were cooled in a
walk-in refrigerator at 4C, removed from the
glassine envelopes, and their wings spread at 4C
on white Styrofoam to expose the dorsal side of
the wings, positioned as if prepared for a profes-
sional insect collection. Spreading was facilitated
with insect pins placed near the costal and Al
veins of the forewing and the anal vein and distal
portion of M3 vein of the hindwing proximal to
the tail. No pins were inserted into the body. Once
a butterfly was spread, it was removed from the
walk-in refrigerator and walked to the equipment
for color analysis.

Protocol for Color Analysis

Each butterfly was placed individually in a
light-box with D65 standardized lighting (Lu-
zuriaga et al. 1997), and a Labsphere (North
Sutton, NH) yellow color standard was placed
next to the butterfly. Inside the light-box, a Ni-
kon D200 digital camera was fastened to a stand
approximately 0.3 m tall so that the camera
faced down, and was fixed at a specific height
and connected to a computer by a USB cable
(camera specifications listed in Table 1). The
light-box door was closed and a photograph was
taken of the butterfly. Once in the light-box, it
took less than 30 sec to process an individual
butterfly. The computer used Camera Control-
Pro software (Nikon, Tokyo, Japan) to control
the act of taking a photograph with the camera;
therefore, a photograph could be taken from the
computer while the camera was enclosed within
the light-box, and the picture would upload onto
the computer. Two types of software were used
for color analysis: Adobe Photoshop 6.0 (Adobe
Systems Inc, San Jose, California) used for im-
age adjustments, modifications, and edits, and
LensEye (Engineering and CyberSolutions,
Gainesville, Florida), used for color quantifica-
tion and analysis).


June 2011







Lehnert et al.: Color Quantification


TABLE 1. CAMERA SPECIFICATION USED FOR COLOR ANALYSIS OF LEPIDOPTERAN WINGS.


Image Quality
Image Size
Lens
Focal Length
Sensitivity
Optimize Image
High ISO NR
Exposure Mode
Metering Mode
Shutter Speed
Exposure Comp.: (in Camera)
Focus Mode
Exposure Comp.: (by Capture NX)
Sharpening
Tone Comp.
Color Mode
Saturation
Hue Adjustment
White Balance


The digital photographs (JPEG) (Fig. la) were
cleaned in Adobe Photoshop 6.0 to isolate the
images necessary for color analysis. The "eraser"
tool was used to remove insect pins, feces, and ad-
ditional artifacts created during photographing.
The image of the Labsphere color standard was
cleaned by selecting the "elliptical marquee" tool
that was used to highlight a yellow circular area
within the color standard, which was moved with
the "move" tool to the left of the butterfly, and the
remainder of the color standard was erased. This
process created 2 final images: a butterfly and a
yellow circle. The image resolution was adjusted
to 700 pixels wide by selecting "Image" in the
main toolbar, then "resize" and "image size", and
saved as a 24 bit BMP image (Fig. Ib). Females of
P glaucus were cleaned with the use of the
"eraser" tool until only one hindwing remained.
Males of P g. glaucus and P g. maynardi were
cleaned so the entire butterfly (minus antennae)
remained.
Cleaned images were opened and analyzed in
LensEye software. In Lenseye, the objects of
interest were separated from the background by
designating the background color to consist of any
pixel with RGB colors between 220 and 255, and
the "16 colors per axis (4096 color blocks)" option
was selected. This color information was dis-
played as the "% of total object area." Objects
smaller than a user-selected threshold of 100 pix-
els were ignored, ensuring only the butterfly and
color standard would be analyzed. In the color cal-
ibration option, the L*, a*, and b* values of the
color standard were entered (L*, a*, and b* value
of 90.17, -3.27, and 74.30, respectively), and the
image was calibrated by selecting the "Process
Image" tab. The software then calculated the av-
erage L*, a*, and b* values of the color standard


Compressed RAW (12-bit)
Large (3872 x 2592)
VR 18-200 mm F/3.5-5.6 G
35 mm
ISO 100
Custom
Off
Manual
Multi-Pattern
1/3 sec F/11
0EV
AF-S
0EV
Auto
Auto
Model
Normal
0
Direct Sunlight


from the uncalibrated image, and adjusted the
color of each pixel in the image so that the aver-
age color of the standard in the image would
equal that of the given reference values; this pro-
cess calibrated all objects in the image (Fig. Ic). A
spreadsheet was produced listing the percentage
of each color (color ID#) and the average and stan-
dard deviation of the L*, a*, and b* values based
on each pixel in the object. Each color ID # has a
unique L*, a*, and b* value (Table 2), and the in-


TABLE 2. SELECTED COLOR ANALYSIS RESULTS FROM
LENSEYE SOFTWARE OF LEPIDOPTERAN
WINGS.

Color ID#1 Color Standard Butterfly2

3472 0 1.534
3488 0 2.076
3744 0 8.74
3745 0 1.805
3762 0 0.271
Lab L* 90.17 71.61
StdDev L* 0.37 2.84
Lab a* -3.27 14.18
StdDev a* 0.71 1.72
Lab b* 74.3 78.42
StdDev b* 3.12 6.93
NBS name brilliant yellow strong orange yellow

'Each Color ID# represents a specific color (available in the
software) with a unique L*, a*, and b* value.
The numbers represent the percentage of each color (Color
ID#) in the image. Percentages do not equal 100, because this is
only a selected portion of the entire spreadsheet from the anal-
ysis. The numbers that correspond to the Lab L*, Lab a*, and
Lab b* represent the average L*, a*, and b* value of the image.
The NBS name represents the name of the color using the av-
erage L* a* and b* values.







Florida Entomologist 94(2)


Fig. 1. Example of sequential images produced dur-
ing color analysis. The raw image of the butterfly and
color standard (a) is saved as a JPEG and opened in
Adobe Photoshop where it is cleaned and saved as a
bitmap image 700 pixels wide (b). The cleaned image is
opened in Lenseye and calibrated and the colors quan-
tified (c).


formation for each color was provided in the "color
block information" in the software.
Both comparisons required the use of the "color
contours" option in LensEye software. For the first
comparison, the most abundant colors of blue (color
ID #) were selected from the spreadsheet and the
L*, a*, and b* values of these colors were searched
for in the "color block information" option. To ana-
lyze the calibrated image, it had to be reopened and
reprocessed in LensEye. The "show contours" op-
tion was selected revealing a table with options for
selecting thresholds, where the blue L*, a*, and b*
values were entered. On the image, the L*, a*, and
b* contour settings were manipulated by interac-
tively adjusting them and evaluating the quantity
of blue pixels that were highlighted in the image to
find the range of blue color values that encompassed
the entire blue area on the butterfly. After 2 images
of both yellow and dark morph females were manip-
ulated, the following settings were deemed best
suited for the task: L* contour greater than 20, a*
contour less than 19, b* contour less than 25. These
threshold values were entered for each of the 20 im-
ages and the software selected all the pixels that
met the above criteria (all blue areas were high-
lighted in red, Fig. 2A). The percentages of blue col-
ors of the total wing area were recorded for each im-
age by selecting the "report contour" option.
For the second comparison, we used a male of
P g. maynardi from Lake Placid, Florida, to de-
termine color composition to represent the may-
nardi subspecies. The image of this male was cal-
ibrated to receive the spreadsheet with the color
ID # information, and the color moderate-orange-
yellow (L*, a*, and b* values equal to 70, 9, and
60, respectively) was chosen to represent the
threshold to distinguish P g. maynardi from P g.
glaucus. This color was chosen because it was the
lightest orange hue represented by the specimen
in the image, and we also wanted to include
darker hues of orange in our analysis, as these
colors also may be present on the wings of P g.
maynardi. Calibrated images of the males were
reopened in LensEye and reprocessed. The
"show contours" option was selected and the L*,
a*, and b* contour values were entered into the
threshold space. All values greater than the cho-
sen threshold values were highlighted, because
these values (higher a* and b* values) would rep-
resent darker orange colors in the butterfly wings
than the moderate-orange-yellow color (Fig. 2B).
The "report contour" option was chosen to record
the percentage of wing area highlighted.

Statistical Analysis

We used a Welch's t test (two-tailed; P = 0.05)
to evaluate differences in the percentage of blue
between yellow and dark morph females, and the
percentage of orange on the wings of males of the
2 subspecies.


June 2011






Lehnert et al.: Color Quantification


F-1


7 7

L 1. f


L15


Fig. 2. Example of images used to determine the percentage of blue on hindwings of females of P. glaucus (A),
and to study color differences between subspecies of P. glaucus (B), with designated L*, a*, and b* color values. In
image A, the dark morph female (a) has more blue extending proximally from the submarginal band compared with
the yellow morph female (b). The regions of blue interpreted by Lenseye@ using specified values are highlighted in
red by the software. The percentage of blue in image (a) and (b) is 21.4% and 12.8%, respectively. In image B, the
same threshold for colors with a higher L*, a*, and b* value than moderate-orange-yellow were used for all males
of P. glaucus. Papilio g. glaucus (a) has less orange than P. g. maynardi (b), as indicated by the red. Image (a) and
(b) have 5.0% and 20.6% of the wings at or above the designated threshold. Both sets of images (A and B) display
the calibrated image on the left and the analyzed image on the right.







Florida Entomologist 94(2)


RESULTS

The dark morph and yellow females did not
differ significantly in the percentage of blue on
the hindwing (mean SE) (16.98 percent 3.10
and 14.2 percent + 1.4, respectively) (t = 1.5858; df
= 18;P = 0.1411). However, the pattern of blue dif-
fered between the morphs (Fig. 2A). All yellow fe-
males had blue scales restricted to the submar-
ginal area of the hindwing, resulting in less than
20% of blue color on the hindwing, which was sim-
ilar to some dark females, but other dark females
had blue that continued proximally and became
more random and scattered, resulting in a larger
variation of blue color in these morphs. Four of
the dark morph females had over 20% of blue
scales on the hindwing, synonymous with the
scattered blue scale phenotype, but the large vari-
ation in this morph led to an average quantity of
blue not significantly different from that of the
yellow morph.
Males of Papilio glaucus maynardi from Lake
Placid, Florida, had significantly more orange
than the butterflies from La Fayette, Georgia
(9.97 percent 2.18 and 0.52 percent + 0.90, re-
spectively) (t = 4.007; df = 18; P = 0.0021), 80% of
the analyzed P glaucus from La Fayette had 0%
of the wings at or above the designated L*, a*,
and b* threshold used to represent moderate-or-
ange-yellow. Although P. g. maynardi from Lake
Placid, Florida, was visually distinct from the
northern subspecies, the range of orange hues on
the wings would have been difficult to quantify
without a computer vision system and image
analysis software. Lenseye highlighted only the
areas of the wings we were interested in analyz-
ing. Even small patches of blue in the hindwing
were highlighted, verifying the software's sensi-
tivity to interpreting specified colors in an intri-
cate color pattern.

DISCUSSION

The application of image analysis software and
our methods open a new avenue for quantifying
color that could influence understanding of color
components in ecological and evolutionary sys-
tems. For instance, color associated with the ef-
fects of temperature or host plant (phenotypic
plasticity) (Price 2006), range distributions of hy-
brid zones (Blum 2002; Gay et al. 2008), floral
color changes in response to insect pollination
(Paige & Whitham 1985), and seasonal polyphen-
isms (Hazel 2002) can be quantified. This study
also provides a means to analyze color of live spec-
imens, which could have important implications
to studies of endangered species. In this study, the
butterflies seemed unaffected by the method, and
were capable of flight, copulation, and oviposition
after the study, verified by additional studies
(M.S.L., unpublished data). Our methods also


provide a protocol to quantify museum speci-
mens, for instance, in studying how color dynam-
ics of populations have shifted over time.
Our method allows the use of thresholds to
study colors of interest and to determine their per-
centage compared with the rest of the image. For
example, the blue scales scattered over the hind-
wing of a dark morph female were quantified, even
though these small blue spots were on a black
background. Additionally, similar, but different,
colors (yellow-orange) were quantified to distin-
guish 2 entities. Papilio glaucus maynardi is rela-
tively unstudied, and there are conflicting reports
concerning its distribution (Forbes 1960; Harris
1972; Howe 1975; Mather & Mather 1985; Scriber
1986; Lindroth et al. 1988). Our method could pro-
vide a means to determine its distribution. Other
aspects of its evolutionary history could be ad-
dressed, such as determining if the subspecies rep-
resent a color dine or a rapid shift in color, suggest-
ing similar dynamics of a narrow hybrid zone
where one phenotype rapidly shifts to the other.
The primary limitation of our method, and
other color quantification methods, is that stan-
dardized lighting is necessary; therefore, these
methods would not be reliable in all situations,
such as comparing the color of butterfly wings
from photographs taken outdoors under different
lighting conditions. We addressed this issue by
using a light-box with standardized lighting.
Other source and processing errors may have oc-
curred, such as instrumental inaccuracies of the
light-box, camera, and software; however, to min-
imize these errors we used the same camera and
light specifications for each individual. In addi-
tion, there may be a source error in that popula-
tions of P. glaucus may experience a seasonal
polyphenism, which could alter our interpreta-
tions of the data sets. We addressed this issue by
collecting the individuals from the various loca-
tions during a similar time period.

ACKNOWLEDGMENTS

We thank Jonathan Doyle and Matthew Standridge
(both at McGuire Center for Lepidoptera and Biodiver-
sity, University of Florida, Gainesville) for technical as-
sistance in cleaning up photographs for analysis and for
testing the protocol, and Alberto De Azeredo (Food Sci-
ence and Human Nutrition Department, University of
Florida, Gainesville) for camera, software, and photo-
graph assistance. Jonathan Doyle assisted in collecting
the P. glaucus. We thank Peter Adler (Department of
Entomology, Soils, and Plant Sciences, Clemson Univer-
sity, Clemson), and Richard Lehnert, and 3 anonymous
reviewers for editorial comments on the manuscript.

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Florida Entomologist 94(2)


June 2011


THE LARGE DECAPITATING FLY PSEUDACTEON LITORALIS
(DIPTERA: PHORIDAE): SUCCESSFULLY ESTABLISHED ON FIRE ANT
POPULATIONS IN ALABAMA

SANFORD D. PORTER', L. C. "FUDD" GRAHAM2, SETH J. JOHNSON3, LARRY G. THEAD4 AND JUAN A. BRIANO5
'Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, 1600 SW 23rd Drive,
Gainesville, FL 32608
2Department of Entomology and Plant Pathology, Auburn University, 301 Funchess Hall, Auburn, AL 36849-5413
3Department of Entomology, 400 Life Sciences Building, Louisiana State University Agricultural Center,
Baton Rouge, LA 70803
4Biological Control of Pests Research Unit, USDA-ARS, P.O. Box 67, Stoneville, MS 38776
Current Address: 10303 Wildcat Road, Collinsville, MS 39325
5South American Biological Control Laboratory, USDA-ARS, Bolivar 1559 (1686) Hurlingham,
Buenos Aires, Argentina

ABSTRACT

The large fire ant decapitating fly, Pseudacteon litoralis Borgmeier, from northeastern Ar-
gentina was successfully released as a self-sustaining biocontrol agent of imported fire ants
in south central Alabama in 2005. Five years later, this fly is firmly established at the orig-
inal release site and has expanded outward at least 18 km. Nevertheless, populations re-
main very low considering P. litoralis is one of the most abundant fire ant decapitating flies
in large areas of its range in South America. The reasons for low densities and why we were
only able to establish this fly at 1 of 9 release sites in 4 states (2003-2006) are unknown, but
problems with host-matching, release procedures, weather conditions, and competition with
previously released decapitating flies are discussed as possible factors.

Key Words: Solenopsis invicta, biological control, low population density

RESUME

La mosca grande del norte y centro-este de Argentina, Pseudacteon litoralis Borgmeier, de-
capitadora de la hormiga de fuego (hormiga brava), fue liberada exitosamente como agent
de control biol6gico de la hormiga de fuego importada en el sur-centro de Alabama en 2005.
Cinco aios despu6s, esta mosca se encuentra firmemente establecida en ese sitio y se ha ex-
pandido al menos 18 km; sin embargo, las poblaciones permanecen muy bajas considerando
que P. litoralis es una de las moscas decapitadoras de hormiga de fuego mas abundante en
su area de Am6rica del Sur. Se desconocen las razones de las bajas densidades y el por qu6
del establecimiento de esta mosca en s61o uno de los nueve sitios de liberaci6n en cuatro es-
tados (2003-2006), pero se discuten como posibles factors los problems de correspondencia
de hospederos, procedimientos de liberaci6n, condiciones climaticas y competencia con mos-
cas decapitadoras liberadas previamente.


Translation provided by the authors.


The decapitating fly Pseudacteon litoralis
Borgmeier (Fig. 1) is a parasitoid of the red im-
ported fire ant, Solenopsis invicta Buren, the
black imported fire ant, Solenopsis richteri Forel,
and 3 other species of saevissima complex fire
ants in southern Brazil, Paraguay, and northern
Argentina (Patrock et al. 2009). Pseudacteon lito-
ralis is the largest of the common Pseudacteon
species that attack fire ants and specializes in
parasitizing the largest sizes of fire ant workers
(Morrison et al. 1997). It is active throughout the
daylight hours, but prefers dawn and especially
dusk (Pesquero et al. 1996). As with several other
Pseudacteon phorids (e.g., P. tricuspis and P. no-


cens), sex is probably determined environmen-
tally, primarily by the size of the host, rather than
genetically like most other insects (Morrison et al.
1999). Males of P litoralis are not attracted to fire
ant mounds like P. tricuspis and P obtusus (Por-
ter & Pesquero 2001; Calcaterra et al. 2005). In
the lab, mating appeared to occur on and around
black objects in the top of the large attack boxes
(SDP, unpubl. obs.). This fly is one of the most
abundant fire ant decapitating flies throughout
much of its range in South America both numeri-
cally and spatially (Calcaterra et al. 2005; Pa-
trock et al. 2009, personal observations, SDP).
Like other species in the genus, P. litoralis is







Porter et al.: Fire Ant Decapitating Fly Established in Alabama


L


Fig. 1. Female Pseudacteon litoralis fly preparing to
oviposit in the thorax of a fire ant worker.


highly host-specific (Porter & Gilbert 2004;
Weissflog et al. 2008) probably because these flies
use fire ant alarm pheromones to find their hosts
(Vander Meer & Porter 2002) and also because of
their highly specialized life history of decapitat-
ing fire ant workers and then pupating inside
their empty head capsules (Porter et al. 1995).
The characteristics discussed above made P.
litoralis an attractive target for release as a self-
sustaining or classical fire ant biological control
agent. The objectives of this paper are to docu-
ment the release and establishment of P litoralis
in south central Alabama and to describe the fate
of 8 additional field releases conducted in Florida,
Mississippi, and Louisiana from the spring of
2003 to the summer of 2006.

MATERIALS AND METHODS
The original source population for the P. lito-
ralis flies discussed in this paper was from sev-
eral sites just off Route 11 about 6 kilometers


south of San Justo, Santa Fe, Argentina
(30.550S, 60.607oW). About 1,800 fire ant
workers parasitized with P. litoralis were
brought back to Gainesville, FL in Apr 2001.
The fire ants at the collection sites were S. in-
victa, although probably not the same biotype
as that found in the United States (Ross &
Trager 1991; Caldera et al. 2008). By the sum-
mer of 2001 the newly established P. litoralis
laboratory colony had dropped to about 1000 in-
dividuals (about 20-30 pupae per day, assuming
a 40-d life cycle) and remained at this level
through the end of 2001, after which numbers
began to gradually increase. In the winter of
2002, 100 or so males were added to the San
Justo colony from a collection site on the Para-
guay River near Herradura, Formosa, Argen-
tina (26.514S, 58.284oW). The S. invicta ants
at this site were probably more similar to the
U.S. biotype, but still not quite the same. By the
time releases had begun in the spring of 2003
the colony was producing about 500 pupae per
day. Maximum production was about 1,000 pu-
pae per day in Jan 2006.
Releases were conducted at sites where fire
ants were abundant (Table 1). We selected sites
with a large percentage of monogyne colonies be-
cause monogyne or single-queen fire ant colonies
have a higher percentage of the larger workers
preferred by P. litoralis females (Morrison et al.
1997). Most sites were near water sources and
had patches of tall grass or shrubbery that was
assumed to help protect fly pupae from being
killed in the sun. All of the sites were pastures ex-
cept the Florida Ironwood Golf Course (Table 1)
which was a mixture of fairways, lake edges, and
service roads along drainage canals. The Alabama
release site (Table 1) was drenched by Hurricane
Dennis just before the final groups of parasitized
ants were released in Jul 2005.


TABLE 1. FIELD RELEASE DATA FOR THE FIRE ANT DECAPITATING FLY PSEUDACTEON LITORALIS.

Site County, State Start Date Duration (days) Number Released Fate

Mickle Farm Alachua, FL May 2003 -3 -150 Failed
Morrill Farm Alachua, FL 15 May 2003 12 2,400 Failed
Whitehurst Farm- A' Marion, FL 15 Sep 2003 21 4,500b Failed'
Knox Sited'f Clay, MS 4 Aug 2004 20 6,400b Failed
Whitehurst Farm- B' Levy, FL 25 Apr 2005 27 5,200b Failed
Ironwood Golf Course' Alachua, FL 10 May 2005 32 4,800" Failed'
Biddle Farm Wilcox, AL 21 Jun 2005 18 4,600" Established
Idelwilde Res. Station E. Feliciana, LA 15 May 2006 20 5,200" Failed
Morrill Farm' Alachua, FL 18 May 2006 44 17,200 Failed

Adult flies released over disturbed mounds.
bEstimated parasitized fire ant workers.
'First-generation adult flies recovered at release site.
Fire ants at this site were primarily hybrids (black x red); ants at all the other sites were the red imported fire ant, S. invicta.
'Adult flies emerged from pupae in shaded emergence box in field.
Sites where P. curvatus flies were established prior to the release ofP. litoralis, P. tricuspis was previously established at all sites
except the Mississippi site.







Florida Entomologist 94(2)


Competing P tricuspis flies were present at all
of the P. litoralis release sites except the Missis-
sippi site where P tricuspis had been unable to es-
tablish on the hybrid fire ants (Table 1). At the
time P litoralis was released, P. curvatus flies
were not present at the Mickle and Morrill re-
lease sites in Florida, the Louisiana site, or the
Alabama site (until 2007).
The P litoralis flies were released at the first 2
sites (Table 1) as adult flies over disturbed fire ant
mounds as was the procedure for P tricuspis (Por-
ter et al. 2004). However, only a few of the females
were observed to hover over and attempt to ovi-
posit in the disturbed workers. The next 6 re-
leases (Table 1) were conducted by releasing
workers parasitized in the laboratory back into
their mother colonies as described for P curvatus
(Vazquez et al. 2006). The hope was that emerg-
ing females would naturally mate with nearby
males and then be attracted to attack fire ant
workers. At the final site (Table 1), pupae on
moist plaster trays were placed inside a large
emergence box (61 by 41 by 51 cm; height, width,
depth) in the field. This was done several days be-
fore the pupae were due to emerge. The box was
shaded to prevent overheating and placed on a
stand coated with Fluon to limit access for ants
and other arthropods. Upon emergence, the flies
flew to the light and exited through window
screen that protected the pupae from access of
larger organisms. Average emergence rates of
adult flies from pupae in this box was 84%, a
value comparable to that achieved with good rear-
ing procedures in the laboratory.
Initial surveys to determine whether the flies
had established were usually conducted in the
late afternoon or early evening by disturbing sev-
eral mounds at or near the release site and aspi-
rating all flies that were attracted to the mounds
(Porter et al. 2004; Vazquez et al. 2006). Begin-
ning in 2006, most surveying in Florida was ac-
complished with sticky traps (baited with live
ants) supplemented by aspiration (Puckett et al.
2007; Porter 2010). Sticky traps baited with ei-
ther live ants or freeze killed ants were also tried
in Alabama in 2008. We did not conduct prere-
lease surveys to detect the presence of P litoralis
at our release sites because none of the 20 or so
South American Pseudacteon species that attack
red imported fire ants have ever been found in
North America (unless they were intentionally re-
leased) despite extensive collections and observa-
tions over many years (Porter et al. 2004; Patrock
et al. 2009; Porter 2010; Plowes et al. 2011).

RESULTS

The decapitating fly P litoralis only became es-
tablished at the release site in Alabama (Table 1).
This site was a series of small weedy pastures en-
circled by trees and shrubbery (~7 ha). Releases


were conducted in overgrown areas near the tree
lines of the pastures. The first P litoralis fly was
recovered at this site on 20 Jun 2006. This collec-
tion occurred a year after the release even though
sampling had been conducted several times previ-
ously in both 2005 and 2006. The next flies were
detected a year later on 23 Jul (2 flies) and 31 Jul
2007 (7 flies). In 2008 (Jun and Jul) 3 years after
the release, P. litoralis flies were collected with
aspirators at 5 sites: the release site (1 fly), 6 km
south (1), 11 km south (2), 6 km west (1), and 18
km west (1). In the summer 2008 (Jun and Jul),
sticky traps were placed every half mile along
road right-of-ways for 10 miles in each of the 4
cardinal directions (80 total traps) for the sole
purpose of monitoring P. litoralis expansion. This
was repeated 3 times. Many P curvatus and P tri-
cuspis flies were found on the traps, but no P lito-
ralis flies. In Jun 2009, single flies were collected
2, 6, and 14 km north of the release site. In Jul
and Aug 2010, a total of 7 flies were collected on 3
different occasions at the release site. Throughout
this period, abundance of P litoralis was always
low; P litoralis was not collected at most of the
sites surveyed, and they were generally found in
only a small fraction of disturbed mounds in-
spected. However, 113 flies were aspirated at the
release site in the early morning on 16 Sep 2010,
an abundance that is equivalent to high densities
of this species in South America. To date, all P.
litoralis in Alabama have been collected with as-
pirators.
First generation, field-reared P. litoralis fe-
males were found about 6 weeks after 2 of the 6
Florida releases (Table 1). Unfortunately, re-
peated monitoring (2003-2010) failed to detect
any additional flies, including in the fall of 2010
when 4 sites near each of the 3 major release ar-
eas were checked twice for P. litoralis flies (Sep
and Oct, 74 total mounds). The Louisiana site was
first sampled 4 months after the release (Sep
2006). This release site was rechecked twice in
2009 (Apr and Sep) and twice in 2010 (Apr and
Sep) without finding P. litoralis. Five other sites
were sampled near the release site (1.6-5.2 km
away) in 2009 (Apr and Sep) and again in 2010
(Apr and Sep). Ten mounds were inspected at
each of the Louisiana sample sites, but no P lito-
ralis flies were collected even though both P. cur-
vatus and P tricuspis flies were collected. Flies
also were not detected at the Mississippi site
which was checked 11 times after the release
(Sept-Nov, 2004) and once in Jul 2005, almost a
year after the release. Three locations near the
Mississippi site were checked in Sep 2010, but
only a few dozen P curvatus flies were found.

DISCUSSION

The large decapitating fly, P. litoralis, is firmly
established on red imported fire ants in south


June 2011







Porter et al.: Fire Ant Decapitating Fly Established in Alabama


central Alabama. Populations of this species are
generally low, but they have survived through 5
winters and they have expanded at least 18 km
from the release site. This makes P litoralis the
third decapitating fly species released and suc-
cessfully established on imported fire ant popula-
tions in the United States. The first 2 Pseudacteon
species, P tricuspis, and P curvatus were released
at numerous sites across the Southeast and cur-
rently cover about 65% and 90% of the imported
fire ant range in the United States, respectively,
(Callcott et al. 2011). A fourth Pseudacteon spe-
cies, P obtusus, has been established in Texas and
Florida (Gilbert et al. 2008; SDP) and a fifth very
small species, P cultellatus, is currently being re-
leased in Florida (SDP). In addition to the flies
mentioned above, several other parasitic arthro-
pods (Williams et al. 2003), 2 species of
mermithid nematodes (Poinar et al. 2007), 2 spe-
cies of microsporidian pathogens, and at least 3
kinds of viruses, are being investigated as poten-
tial fire ant biocontrol agents (Oi & Valles 2009).
The expansion rate of P litoralis from the re-
lease site in Alabama has proven difficult to mon-
itor because low densities make this fly difficult to
detect at sample sites. Despite low densities, the
rate of expansion for P. litoralis in Alabama is
similar to expansion rates reported for P. tricuspis
in Texas and Louisiana, but probably less than
the very abundant P curvatus in Florida and Mis-
sissippi (Henne et al. 2007; Porter 2010). The low
densities of P litoralis at sites in Alabama is curi-
ous because P litoralis is consistently one of the
most abundant decapitating flies across most of
its range in South America both numerically and
spatially (Calcaterra et al. 2005; Patrock et al.
2009). The large number of flies recently collected
(Sep 2010) from the release site is encouraging,
but it is unknown whether this represents a new
trend or is just a temporal quirk.
The apparent failure to establish P litoralis at
the other 8 sites was disappointing. We made re-
leases at sites with a variety of habitats and cli-
mates in hopes that variety would increase the
probability of success. The Mississippi site was
chosen in hopes that the flies might do better on
the S. invicta x S. richteri hybrid fire ants found at
that site.
It is possible that populations have been estab-
lished at some sites listed in Table 1, but densities
are still too low to be easily detected, as has oc-
curred on several occasions with P curvatus (Gra-
ham et al. 2003; Vazquez et al. 2006). Neverthe-
less, this possibility seems unlikely at the Flor-
ida, Louisiana, and probably Mississippi sites
considering the frequency and duration of the
sampling efforts in those areas.
Repeated failures to establish P litoralis in the
field is reminiscent of failures to establish P. cur-
vatus collected from black fire ants in South
America on red fire ants in the United States


(Graham et al. 2003; Callcott et al. 2011). Perhaps
a biotype of P. litoralis better adapted to the bio-
type of red imported fire ants found in the United
States would have been more successful. How-
ever, we tried twice to establish additional labora-
tory colonies of P litoralis from flies collected
along the Parana River near Herradura, For-
mosa, Argentina (Apr 2003, 314 flies; Dec 2005,
1400 flies). Unfortunately, both attempts failed as
did other attempts to culture P. litoralis flies col-
lected in Sao Paulo State, Brazil (1997) and the
Corrientes area of Argentina (2004-2006). Ex-
actly why we were able to culture the flies col-
lected from San Justo, but not the P litoralis flies
collected elsewhere is unknown, although it may
be related to problems with mating since the
adult females seemed to be attracted normally to
the fire ant workers we provided to them in the
laboratory attack boxes.
While poor host matching may have been a
problem, other factors may also have been impor-
tant in the failure of P litoralis to establish at
some of release sites, especially since they did es-
tablish in Alabama and thus should have been
able to be established elsewhere on S. invicta fire
ants. Competition with previously released spe-
cies is one likely explanation. Our colleagues in
Texas provide strong evidence that the presence
of P curvatus at their release sites greatly dimin-
ished the success rate of establishing P. obtusus
(Plowes et al. 2011). Similarly in Florida, compe-
tition between P curvatus, P tricuspis, and the re-
cently released P obtusus appears to be greatly
reducing P. tricuspis populations (SDP and Lu,
unpublished). However, competition with P. cur-
vatus was not a problem with the first 2 releases
in Florida or with the releases in Alabama and
Louisiana because P litoralis was released at
these sites before P curvatus was present.
Poor weather conditions may have been an-
other factor at some of the failed sites. Examina-
tion of release records for P tricuspis (Callcott et
al. 2011) indicates that summer releases were
about half as successful as releases in the spring
or fall. Five of the 9 P litoralis releases, including
the successful one in Alabama (Table 1), were at
least partly carried out during hot summer
months (although rain and clouds from Hurricane
Dennis likely reduced negative impacts of sum-
mer heat for the Alabama release). Another possi-
ble problem is that U.S. fire ant populations may
not have enough major workers to sustain large
numbers of P litoralis, but intercontinental com-
parisons of worker polymorphism have not been
done to see if this is a real concern. Certainly, U.S.
fire ant colonies do have many workers in the size
range which P litoralis prefers to parasitize (Por-
ter & Tschinkel 1985; Morrison et al. 1997; Mor-
rison et al. 1999). Poor release technique is an-
other explanation. This would certainly seem to
be true for the first 2 releases, because the adult







Florida Entomologist 94(2)


flies did not show much interest in the disturbed
fire ant mounds and very few flies were used at
the first site. The large release box used in the
last release was an effort to try something differ-
ent than what had previously been done. The lack
of any first-generation field-reared flies at this re-
lease site was disappointing considering the num-
ber of flies released and the extended period of the
release.
In the fall of 2006, we made the decision to fo-
cus on other biocontrol agents with higher proba-
bilities of success. Nevertheless, P litoralis is
firmly established in Alabama and will presum-
ably expand into other states. While P litoralis
was locally abundant on one occasion in 2010, it
failed at most of the release sites and remained
rare in Alabama over most of the last 5 years, a
curious situation considering P litoralis is one of
the most abundant species of fire ant decapitating
flies throughout most of its range in South Amer-
ica (Calcaterra et al. 2005; Patrock et al. 2009).

ACKNOWLEDGMENTS

Vicky Bertagnolli, Kelly Ridley, Mel Leap, and Jen-
nifer Reese assisted with field releases and collections
in Alabama. Lloyd Davis, Darrell Hall, David Milne,
and Roberto Pereira assisted with field releases in Flor-
ida. Don Henne assisted with releases in Louisiana.
Evita Gourley, Mary Vowell and Dan Harsh assisted
with releases in Mississippi. Luis Calcaterra is thanked
for assistance with logistics in Argentina and field work
near Herradura.

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Florida Entomologist 94(2)


June 2011


HOST SPECIFICITY OF ANTHONOMUS TENEBROSUS (COLEOPTERA:
CURCULIONIDAE), A POTENTIAL BIOLOGICAL CONTROL AGENT OF
TROPICAL SODA APPLE (SOLANACEAE) IN FLORIDA

J. MEDAL', N. BUSTAMANTE', E. BREDOW2, H. PEDROSA2, W. OVERHOLT', R. DiAZ1 AND J. CUDA1

1University of Florida, Department of Entomology and Nematology, FL. 32611

2Universidade Federal do Parana, Curitiba, Brazil

ABSTRACT

Multiple-choice and no-choice tests were conducted at the Florida Department of Agricul-
ture quarantine facility to determine the host specificity of the South American flower bud
weevil, Anthonomus tenebrosus Boheman, intended for biological control of the exotic weed
tropical soda apple (TSA), Solanum uiarum Dunal in Florida, USA. Ninety-one plant species
in 21 families were included in multiple-choice feeding and oviposition experiments, includ-
ing the target weed and the 6 major cultivated Solanaceae: bell pepper (Capsicum annuum
L.), chili pepper (C. frutescens L.), tomato (Lycopersicon esculentum Mill.), tobacco (Nicoti-
ana tabacum L.), eggplant (Solanum melongena L.), and potato (Solanum tuberosum L.).
Plant bouquets with flower-buds of 8 to 10 randomly selected plant species, always including
TSA (S. viarum) were exposed to 10-20A. tenebrosus adults for 1 to 2 weeks. Oviposition and
feeding were observed twice a week. No-choice host-specificity tests were also conducted
withA. tenebrosus adults using potted flowering plants. Ten adults were exposed to 29 plant
species individually tested for 1 to 2 weeks. Plant species in each test were replicated 3 or 4
times. All tests showed that A. tenebrosus fed and laid eggs only on the target weed. No eggs
were deposited on any of the other of the 91 plant species tested. Host-specificity tests indi-
cated that a host range expansion ofA. tenebrosus to include any of the crops, and native
Solanaceae, and non-solanaceous plants tested is highly unlikely. A petition for field release
in the USA was submitted to the Technical Advisory Group for Biological Control Agents of
Weeds (TAG) in Oct 2007.

Key Words: host-specificity tests, weed biological control, Solanum viarum, Solanaceae

RESUME

Pruebas de ovoposici6n y alimentaci6n (con y sin elecci6n), se realizaron para evaluar la es-
pecificidad del picudo del bot6n floral, de origen suramericano, Anthonomus tenebrosus Bo-
heman, como agent potential para control biol6gico de bola de gato, Solanum viarum Dunal
en los Estados Unidos. Las pruebas se efectuaron en la cuarentena del Departamento de
Agriculture de la Florida. Noventa y una species de plants, en 21 families, fueron inclui-
das en las pruebas de especificidad de multiples elecci6n, incluyendo la maleza objetivo y las
seis plants cultivadas pertenecientes a la familiar Solanaceae mas importantes: chile dulce
(Capsicum annuum L.), chile (Capsicum frutescens L.), tomate (Lycopersicon esculentum
Mill.), tabaco (Nicotiana tabacum L.), berenjena (Solanum melongena L.), y papa (Solanum
tuberosum L.). En cada prueba se utilizaron racimos florales de ocho a diez plants escogidas
al azar incluyendo siempre la plant objetivo las cuales fueron expuestas a 10-20 adults de
A. tenebrosus por una a dos semanas. Registros de alimentaci6n y ovoposici6n fueron reali-
zados dos veces por semana. Pruebas de alimentaci6n/ovoposici6n sin elecci6n fueron tam-
bi6n realizadas usando plants en floraci6n. Diez adults fueron expuestos a 29 species de
plants en forma individual por una a dos semanas. Cada prueba tuvo tres o cuatro repeti-
clones. Las pruebas mostraron queA. tenebrosus se aliment6 y coloc6 posturas solo en bola
de gato. Ninguna postura fu6 depositada en las otras 90 species de plants evaluadas. Las
pruebas indicaron que la posibilidad deA. tenebrosus de llegar a ser una plaga de las Sola-
naceae cultivadas es muy remota. La solicitud al comit6 TAG para liberar el picudo en los Es-
tados Unidos fue presentada en octubre 2007.


Tropical soda apple (TSA), Solanum viarum Florida (Coile 1993; Mullahey & Colvin 1993); the
Dunal (Solanaceae), is an invasive weed native to introduction pathway is unknown. In 1993, a sur-
southeastern Brazil, northeastern Argentina, vey of beef cattle operations in south Florida esti-
Paraguay, and Uruguay that has invaded Florida mated 157,145 ha of infested pasture land, twice
grasslands and natural ecosystems. In 1988, TSA the infestation present in 1992 (Mullahey et al.
was first reported in the USA in Glades County, 1994). The infested area increased to more than







Medal et al.: Host Specificity ofAnthonomus tenebrosus


303,000 ha in 1995-96 (Mullahey et al. 1998).
Currently, more than 404,000 ha are believed to
be infested in Florida (Medal et al. 2010b). Due, at
least in part, to favorable environmental condi-
tions, the lack of natural enemies (herbivores and
pathogens), and seed dispersal by wildlife and
cattle feeding on the fruits. TSA has been spread-
ing rapidly and has been observed in the majority
of the counties in Florida and also in Alabama,
Georgia, Louisiana, Mississippi, North Carolina,
Pennsylvania, South Carolina, Tennessee, Texas,
and Puerto Rico (Bryson & Byrd Jr. 1996; Dowler
1996; Mullahey et al. 1993, 1998; Medal et al.
2003, 2010a). Although TSA has been reported in
Pennsylvania and Tennessee, it is highly probable
that does not overwinter in these states. Patter-
son (1996) studied the effects of temperatures and
photoperiods on TSA in controlled environmental
chambers and speculated that the range of TSA
could expand northward into the midwestern US.
S. viarum was placed on the Florida and Federal
Noxious Weed Lists in 1995.
TSA typically invades improved pastures,
where it reduces livestock carrying capacity. Foli-
age and stems are unpalatable to cattle; dense
stands of the prickly shrub prevent access of cat-
tle to shaded areas, which results in summer heat
stress (Mullahey et al. 1998). TSA control costs
for Florida ranchers were estimated at $6.5 to 16
million annually (Thomas 2007), and economic
losses from cattle heat stress alone were esti-
mated at $2 million (Mullahey et al. 1998). TSA is
a reservoir for at least 6 crop viruses (potato leaf-
roll virus, potato virus Y, tomato mosaic virus, to-
mato mottle virus, tobacco etch virus, and cucum-
ber mosaic virus) and the early blight of potato
and tomato fungus, Alternaria solani Sorauer
(McGovern et al. 1994a, 1994b; McGovern et al.
1996). In addition, major insect pests utilize TSA
as an alternate host; including Colorado potato
beetle, Leptinotarsa decemlineata (Say); tomato
hornworm Manduca quinquemaculata
(Haworth); tobacco hornworm, M. sexta (L.); to-
bacco budworm, Helicoverpa virescens (Fabri-
cius); tomato pinworm, Keiferia lycopersicella
(Walsingham); green peach aphid, Myzuz persicae
(Sulzer); silverleaf whitefly biotype B of Bemisia
tabaci (Gennadius); soybean looper, Pseudoplusia
includes (Walker); and the southern green stink
bug, Nezara viridula (L.) (Habeck et al. 1996;
Medal et al. 1999; Sudbrink et al. 2000). TSA also
reduces biodiversity in natural areas, ditch
banks, and roadsides by displacing native vegeta-
tion (Langeland & Burks 1998). TSA interferes
with restoration efforts in Florida by invading ar-
eas that are reclaimed following phosphate min-
ing operations (Albin 1994).
TSA Management practices in Florida pas-
tures primarily involve herbicide applications
and mowing (Sturgis & Colvin 1996; Mislevy et
al. 1996, 1997; Akanda et al. 1997). Herbicides or


mowing provide temporary weed suppression at
an estimated cost of $61 and $47 per ha, respec-
tively (Thomas 2007). However, application of
these control methods is not always feasible in
rough terrain or inaccessible areas.
In June 1994, the first exploration for TSA nat-
ural enemies in South America was conducted by
University of Florida and Brazilian researchers
(Medal et al. 1996). Sixteen species of insects
were found attacking the weed during this 2-
week survey. Host specificity tests were initiated
in 1997 by J. Medal (University of Florida) in col-
laboration with the Universidade Estadual
Paulista, Jaboticabal campus, Brazil, and the
USDA Biological Control Laboratory in Hurling-
ham, Buenos Aires province, Argentina, and in
Stoneville, MS. The South American leaf-feeder
Gratiana boliviana (Chrysomelidae) was ap-
proved for field release in Florida in summer
2003. In total, at least 230,000 beetles have been
released in 39 Florida counties since the summer
2003. The beetles established at almost all the re-
lease sites in central/south Florida and they are
having extensive defoliations and reducing the
weed fruit production on TSA plants (Medal &
Cuda 2010; Medal et al. 2010a; Overholt et al.
2009, 2010).
A second potential TSA biocontrol agent is the
flower-bud weevil Anthonomus tenebrosus Bohe-
man (Coleoptera: Curculionidae). This insect was
collected on TSA in Rio Grande do Sul, Brazil
(29.66465S, 50.80171oW) by the late Daniel Gan-
dolfo and Julio Medal in April 2000. The identity
of A. tenebrosus was confirmed by Drs. Wayne
Clark (Auburn University, AL) and Germano Ro-
sado Neto (Universidade Federal do Parand in
Curitiba, Brazil). Voucher specimens of A. tene-
brosus are deposited at Auburn University, Ala-
bama, at the Universidade Federal do Parand -
Curitiba campus, Brazil, and at the Florida State
Collection of Arthropods, Division of Plant Indus-
try in Gainesville, Florida. This species does not
have a common name in South America. The only
known A. tenebrosus host plants in South Amer-
ica are S. viarum and S. acculeatisimum.
The biology of A. tenebrosus was studied by
Davis (2007) at the quarantine facility in Gaines-
ville, Florida. Eggs are inserted individually into
TSA flower-buds, and hatch in 3-5 days. Larvae
are cream-colored with a yellowish brown head
capsule. They feed on the contents of the flower-
bud, and this feeding prevents the flower-bud
from opening. There is typically 1 larva, but occa-
sionally 2 larvae in a single flower-bud. As larval
feeding progresses, the flower-bud senesces and
drop from the plant. Three larval stadia are com-
pleted in 7-13 days. The pupal stage is completed
in 3-7 days inside the fallen flower bud. Pupae re-
semble the adult in form; they are cream-colored
but darken shortly before eclosion. Emerging
adults chew their way out of the flower-bud. De-







Florida Entomologist 94(2)


velopment from egg to adult stage lasts 11-69
days. Longer developmental times are apparently
not associated with seasonal differences as they
occurred throughout the year. Adults can live up
to 210 days under laboratory conditions. Adult
size appears to be related to food abundance dur-
ing development rather than beetle sex. Copula-
tion has been observed a few hours after adult
emergence and throughout the oviposition period.
At least 7-8 generations per year can occur under
laboratory conditions (temperature 24 + 3C, rel-
ative humidity 50-70%) conditions.

MATERIALS AND METHODS

Host Specificity Tests

Laboratory host specificity tests withA. tenebro-
sus adults were conducted from May 2000 to Janu-
ary 2003 at the Florida Department of Agriculture
and Consumer Services-Division of Plant Industry
quarantine facility in Gainesville, Florida. Open
field host-specificity tests were conducted at the
Universidade Federal do Parand Agricultural Ex-
periment Station in Parand state, Brazil from Oct
2005 to Mar 2007. For Florida tests, A. tenebrosus
adults were collected from TSA plants in Rio
Grande do Sul, Brazil and introduced onto caged
plants of TSA plants growing in 1-gallon pots to es-
tablish a laboratory colony in quarantine.
In this article we report the results of various
host-specificity tests with the flower-bud weevil
A. tenebrosus, to assess its possible use as biolog-
ical control agent of the non-native weed tropical
soda apple.

Multiple-Choice Feeding and Oviposition Tests

Ninety-one plant species in 21 families were
included in the feeding oviposition preference
tests at the Gainesville quarantine (Table 1).
Tested plants included 53 species in the family of
the target weed (Solanaceae), 26 of which were
from the genus Solanum and 27 from 14 other
genera that include plants of agricultural or eco-
logical importance. Ten species represented 5
families (Boraginaceae, Convolvulaceae, Ehreti-
aceae, Nolanaceae, Polemoniaceae) very close re-
lated to Solanaceae within the order Polemoni-
ales (Heywood 1993) were also included. Twenty-
eight plant species representing 15 families, most
of them with economic and/or environment value
in North America, were also tested. The target
weed (S. viarum), and other 9 plant species in
Solanaceae were tested at least 3 times (Table 1).
These included the natives Solanum donianum
Walpers, listed as a threatened plant in Florida
(Coile 1998), and S. americanum Mill, 2 non na-
tive-weeds (S. tampicense Dunal, S. torvum Sw.),
and the 5 major cultivated Solanaceae (bell pep-
per, Capsicum annuum L., tomato, Lycopersicon


esculentum Mill., tobacco, Nicotiana tabacum L.,
eggplant, Solanum melongena L., and potato,
Solanum tuberosum L.). Bouquets of leaves and
flower-buds of 8 to 10 plant species, always in-
cluding TSA were simultaneously exposed to 10-
20 A. tenebrosus adults (approximately 50%
males and 50% females) in clear plastic round
containers (26 cm diameter by 9 cm height, with
four 4-7 cm diameter vents drilled along the sides
of the container to allow for air circulation). At the
beginning of each test, the insects were placed at
the bottom center of each container to allow them
to choose any tested plants. Plant species in each
test were replicated 3-4 times (1 replication of
tested plants in each separate container). Bou-
quets were exposed toA. tenebrosus adults for 1 to
2 weeks. Observations of oviposition and feeding
were made twice a week, and consumed bouquets
were replaced as needed. Flower-buds were
checked for oviposition and eggs were counted
weekly. On the last day of each experiment,
flower-buds were scored for feeding damage, and
eggs laid on them were counted. Leaf and flower
bud area consumed was visually estimated using
a scale from 0 to 5 (0 = no feeding, 1 = probing or
<5% of area consumed, 2= light feeding or 5-20%
of the area, 3 = moderate feeding or 21-40%, 4 =
heavy feeding or 41-60%, and 5 = intense feeding
or >60% of the area consumed).

No-Choice Adult Feeding and Oviposition Tests

No-choice host specificity tests were also con-
ducted with A. tenebrosus adults at the Gaines-
ville-quarantine facility using potted plants (20-
60 cm height) in cages. Cages were made of clear-
plastic cylinders (15 cm diam, 50-60 cm height),
with a mesh screen at the top and covering 6 cir-
cular holes (6 cm diam) located in pairs at the bot-
tom, middle, and upper part of the cylinder to al-
low for air circulation. A. tenebrosus adults were
exposed to 29 plant species in 3 families, includ-
ing the native S. donianum, and all major culti-
vated Solanaceae (Table 2). Five to 7 plant species
with flower-buds were individually tested each
time due to limited cage numbers. Plants were ex-
posed to 10 A. tenebrosus adults (5 males, 5 fe-
males) for 1 to 2 weeks; each test plant was repli-
cated 3 or 4 times. Adults were F2 or F3 progeny
from adults originally collected in southern Brazil
and reared in quarantine on TSA. Adults had ei-
ther recently closed from pupae or were still
young less than 1 week old. Plants were replaced
as needed. At the end of the testing periods, feed-
ing and oviposition were recorded.

First Field Experiment in Brazil

A multiple-choice, open field experiment was
conducted at the Universidade Federal do Parana,
Agriculture Experimental Farm 'Canguiri'. A.


June 2011









TABLE 1. ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE MULTIPLE-CHOICE TESTS.

Common Names No. No. Feeding Eggs Laid per
Plant Family Species *indicates native species of Plants of Insects Score' Female

Category 1. Genetic types of the target weed species found in North America
SOLANACEAE
Tribe Solaneae
Genus Solanum
Subgenus Leptostemonum
Section Acantophora


Solanum uiarum Dunal


Tropical soda apple


Category 2. Species in the same genus as the target weed, divided by subgenera (if applicable).


Tribe Solaneae
Genus Solanum
Subgenus Leptostemonum
Section Acantophora
Solanum capsicoides All.
Section Lasiocarpum
Solanum quitoense Lam.
Solanum pseudolulo Heise
Solanum sessiliflorum Dunal
Section Micracantha
Solanum jamaicense Mill.
Solanum tampicense Dunal
Section Melongena
Subsection Lathyrocarpum
Solanum carolinense L.
Solanum dimidiatum Raf.
Solanum elaeagnifolium Cav.
Subsection Melongena
Solanum melongena L.
Cv.'Black Beauty'
Cv. 'Classic'
Cv. 'Market'
Cv. 'Asian Long Purple'
Section Persicariae


Red soda apple

Naranjilla
Falso lulo
Cocona nightshade

Jamaican nightshade
Wetland nightshade


Horse nettle*
Western horsenettle*
Silverleaf nightshade'

Eggplant


*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Solanaceous taxonomic categories were
taken from the Radboud University ofNijmegen, Netherland website (www.bagard.sci.kun.nl). Most of the plant common names are from: http:/www.plants.usda.gov.








TABLE 1. (CONTINUED) ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE MULTIPLE-CHOICE TESTS.

Common Names No. No. Feeding Eggs Laid per
Plant Family Species *indicates native species of Plants of Insects Score' Female


Subgenus Leptostemonum
Solanum bahamense
Section Torva
Solanum torvum Sw.
Solanum verbascifolium L.
Subgenus Solanum
Solanum americanum Mill.
Solanum diphyllum L.
Solanum erianthum Don.
Solanum jasminoides Paxt.
Solanum mauritianum Scop.
Solanum nigrescesns Mart. & Gal
Solanum nigrum L.
Solanum pumillum Dunal
Solanum seaforthianum Scop.
Solanum tuberosum


Bahama nightshade

Turkey berry
Mullein nightshade*

American nightshade*
2-leaf nightshade*
Potato tree*
White potato vine
Earleaf nightshade
Divine nightshade*
Black nightshade*
Rock outcrop Solanum'
Brazilian nightshade
L. Potato


Category 3. Species in other genera in the same family as the target weed, divided by subfamily (if applicable).
Genus Acnistus


Acnistus australe (Griseb.) Griseb.
Genus lochroma
lochroma sp.
Genus Physalis
Physalis angulata L.
Physalis arenicola Kearney
Physalis crassifolia Benth
Physalis gigantea L.
Physalis ixocarpa Brot.
Physalis pubescens L
Physalis walteri Nutt.
Tribe Daturae
Genus Brugmansia
Brugmansia sanguinea (Ruiz & Pav.) Don
Genus Datura


Acnistus

Iochroma

Cutleaf groundcherry
Cypresshead*
Yellow groundcherry
Strawberry groundcherry
Tomatillo
Husk tomato*
Walter's groundcherry


Angel's trumpet


*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Solanaceous taxonomic categories were
taken from the Radboud University ofNijmegen, Netherland website (www.bagard.sci.kun.nl). Most of the plant common names are from: http:/www.plants.usda.gov.








TABLE 1. (CONTINUED) ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE MULTIPLE-CHOICE TESTS.

Common Names No. No. Feeding Eggs Laid per
Plant Family Species *indicates native species of Plants of Insects Score' Female


Datura discolor Bernh
Datura metel L.
Datura meteloides D.
Datura stramonium L.
Tribe Lycieae
Genus Lycium
Lycium carolinianum Walter
Lycium fremontii Gray.
Genus Lycopersicon
Lycopersicon esculentum Mill.
Tribe: Nicandreae
Genus: Nicandra
Nicandra physaloides (L.) Gaertn.
Tribe Nicotianae
Genus Nicotiana
Nicotiana tabacum L.
Nicotiana rustica L.
Nicotiana sylvestris Speg. & Comes
Genus Nierembergia
Nierembergia scoparia Sendtri
Tribe Salpiglossidae
Genus Salpiglossis
Salpiglossis sinuata Ruiz & Pav
Genus Schizanthus
Schizanthus spp.
Tribe Solandeae
Genus Solandra
Solandra glandiflora Swartz


Desert thorn-apple
Devil's trumpet
Devil's weed*
Jimson weed*


Carolina desert-thorn
Fremont desert thorn

Tomato


Apple of Peru


Tobacco
Aztec tobacco
Woodland tobacco

Broom cupflower


Painted tongue

Butterfly flower


Showy chalicevine


Category 4. Threatened and endangered species in the same family as the target weed divided by subgenus, genus, and subfamily.
Section Torva


Mullein nightshade


Category 5. Species in other families in the same order that have some phylogenetic, morphological, or biochemical similarities to the target weed.
BORAGINACEAE

*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Solanaceous taxonomic categories were
taken from the Radboud University of Nijmegen, Netherland website (www.bagard.sci.kun.nl). Most of the plant common names are from: http:/www.plants.usda.gov.


Solanum donianum Walpers








TABLE 1. (CONTINUED) ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE MULTIPLE-CHOICE TESTS.

Common Names No. No. Feeding Eggs Laid per
Plant Family Species *indicates native species of Plants of Insects Score' Female


Heliotrope sp.
Myosotis alpestris Schmidt
Convolvulus purpurea L.
Ipomoea batata (L.) Lam.
Evolvulus muttallianus
EHRETIACEAE
Cordia sebestena L.
NOLANACEAE
Nolana paradoxa Lindl.
POLEMONIACEAE
Cobaea scandens Cav.
Gilia tricolor Benth
Phlox panuculata L.


Heliotrope
Forget-Me-Not*
Morning-glory*
Sweet-potato
Shaggy dwarf morning-glory*


Largeleaf geigertree*

Chilean bellflower


Catedral bells
Bird's-eye gilia
Fall phlox*


Category 6. Species in other orders that have some morphological or biochemical similarities to the target weed or that share the same habitat.


ANACARDIACEAE
Mangifera indica L.
Pistacia vera L.
APIACEAE
Daucus carota L.
ASTERACEAE
Helianthus annuus L.
Lactuca sativa L.
CAMPANULACEAE
Campanula persicifolia L
CRUCIFERAE
Brassica oleracea L. var. botrytis
CUCURBITACEAE
Citrullus lanatus (Thumb)
Cucurbita sativus L.
ERICACEAE
Vaccinium ashei Rende
FABACEAE
Glycine max (L.) Merrill


Mango
Cultivated pistachio

Carrot

Common sunflower
Lettuce

Peachleaf bellflower

Broccoli

Watermelon
Cucumber*

Rabbiteye blueberry*

Soybean


*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Solanaceous taxonomic categories were
taken from the Radboud University ofNijmegen, Netherland website (www.bagard.sci.kun.nl). Most of the plant common names are from: http:/www.plants.usda.gov.








TABLE 1. (CONTINUED) ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE MULTIPLE-CHOICE TESTS.

Common Names No. No. Feeding Eggs Laid per
Plant Family Species *indicates native species of Plants of Insects Score' Female


Phaseolus vulgaris L.
LOBELIACEAE
Lobelia cardinalis L.
LOGANIACEAE
Buddleia davidii Franch
POACEAE
Oryza sativa L.
Saccharum officinarum L.
Zea mays L.
ROSACEAE
Fragariax ananassa Duchesne
Malus pumilla Mill.
Rosa sp.
Rubus betulifolius Small
RUTACEAE
Citrus sinensis (L.) Osbeck
Citrus limon (L.) Burm.
Citrus paradise Mcfady
Murraya paniculata (L.) Jacq.
SCROPHULARIACEAE
Antirrhinum majus L.
Nemensia strumosa Benth


Kidney bean

Cardinalflower

Butterfly bush

Rice
Sugarcane
Corn

Garden strawberry
Paradise apple*
Miniature rose
Blackberry*

Sweet orange
Lemon
Grapefruit
Orange Jasmine

Garden snapdragon
Capejewels


Category 7. Any plant on which close relatives of the biological control agent (within the same genus) have been found or recorded to feed/ or reproduce.


MALVACEAE
Gossypium hirsutum L.
SOLANACEAE
Genus Capsicum
Capsicum annuum L.
Capsicum frutescens L.
Genus Solanum
Solanum sisymbriifolium Lam.


Cotton


Bell pepper
Chili pepper


Sticky nightshade


*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area). Solanaceous taxonomic categories were
taken from the Radboud University ofNijmegen, Netherland website (www.bagard.sci.kun.nl). Most of the plant common names are from: http:/www.plants.usda.gov.







Florida Entomologist 94(2)


June 2011


TABLE 2. ANTHONOMUS TENEBROSUS ADULT FEEDING AND OVIPOSITION ON SELECTED PLANTS IN QUARANTINE NO-
CHOICE TESTS.


Plant family Species

SOLANACEAE
Capsicum annuum
Capsicum frutescens
Lycopersicon esculentum
Nicotiana tabacum
Nierembergia scoparia
Physalis crassifolia
Solanum americanum
Solanum capsicoides
Solanum carolinense
Solanum citrullifolium
Solanum dimidiatum
Solanum diphillum
Solanum donianum
Solanum elaeagnifolium
Solanum heterodoxum
Solanum jamaicense
Solanum jasminoides
Solanum melongena
cv. Black Beauty
cv. Classic
cv. Market
cv. Asian Long Purple
Solanum nigrescens
Solanum pumilum
Solanum ptycanthum
Solanum retroflexum
Solanum scabrum
Solanum tampicense
Solanum toruum
Solanum tuberosum
Solanum viarum
MALVACEAE
Gossypium hirsutum L.
CONVOLVULACEAE
Ipomoea batata (L.) Lam.


Common names (*indicates
native Species)


Bell pepper
Chili pepper
Tomato
Tobacco
Broom cupflower
Yellow groundcherry
American nightshade*
Red soda apple
Horse nettle*
Watermelon nightshade*
Western horsenettle*
2-leaf nightshade
Mullein nightshade
Silverleaf nightshade*
Melonleaf nightshade*
Jamaican nightshade
White potato vine
Eggplant




Divine nightshade*
Rock-outcrop Solanum*
Wonder berry"
Sunberry
Garden huckleberry
Wetland nightshade
Turkeyberry
Potato
Tropical soda apple


Cotton


Sweet-potato


No. No.
of Plants of Insects


Feeding
Score*


9 90 0

q9 0 0


*0=No feeding, 1= Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4
tense (>60% area). Most of the plant common names are from: http:/www.plants.usda.gov.


tenebrosus adults were collected in Rio Grande do
Sul state in Dec 2005. These insects were reared in
screened cages (0.6 x 0.6 x 0.9 m) at the Neotropi-
cal Biological Control Laboratory in Curitiba on
TSA plants growing in 1-2 gallon pots to provide
progeny weevils for the experiment. One hundred
A. tenebrosus adults recently emerged from pupae
were released in the field (30 x 20m2) with 5 plant
species (TSA, eggplant cv.'Black Beauty', bell-pep-
per, potato, and tomato). Seven plants of each spe-
cies tested (35 plants/plot, 1m between plants,
35m2/plot, 4 plots, 10m between plots) were ran-
domly assigned in each of the experimental plots
following a Complete Block Randomized Experi-


: Heavy (41-60%), 5 = In-


mental Design with 4 replications. Test plants (n =
140) were transplanted in Oct 2005, and insects
were released when plants were flowering during
the last week of Dec 2005 on the ground approxi-
mately 1m from any plant. All plants were thor-
oughly examined weekly from 22 Dec 2005 to 31
Mar 2006, and number of adults, feeding, and
number of egg on the plants were recorded.

Second Field Experiment in Brazil

Another multiple-choice, open field experi-
ment exposing A. tenebrosus adults to flowering
eggplant cv.' Black Beauty', tomato, potato, and


Eggs/female