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Ortego et al.: New Aphid Species Recorded from South America


SIX ALIEN APHID SPECIES (HEMIPTERA: APHIDIDAE)
RECORDED FOR THE FIRST TIME FROM SOUTH AMERICA


JAIME ORTEGO1, NICOLAS PEREZ HIDALGO2, JUAN M. NIETO NAFRiA2 AND M. PILAR MIER DURANTE2
1INTA EEA Junin, CC. Nro. 78; 5570 San Martin, Mendoza, Argentina

2Departamento de Biologia Animal, Universidad de Le6n, 24071 Le6n, Espaia

ABSTRACT

Six aphid species: Saltusaphis scirpus Theobald, Myzocallis boerneri Stroyan, Macrosipho-
niella absinthii (Linnaeus), Macrocrosiphoniella abrotani (Walker), Macrosiphoniella
pseudoartemisiae Shinji and Macrosiphoniella tapuskae (Hottes & Frison) are recorded for
the first time in South America. They were all collected in Argentina. Comments for each
species and identification keys for Myzocallidina and Macrosiphoniella known in South
America are given.

Key Words: Hemiptera, Aphididae, Neotropical, alien species

RESUME
Se citan por vez primera para Sudam6rica seis species de pulgones: Saltusaphis scirpus
Theobald, Myzocallis boerneri Stroyan, Macrocrosiphoniella abrotani (Walker), Macrosi-
phoniella absinthii (Linnaeus), Macrosiphoniella pseudoartemisiae Shinji y Macrosipho-
niella tapuskae (Hottes & Frison). Todas ellas recogidas en la Argentina. Se presentan
comentarios de cada especie y claves de identificaci6n para las species de Myzocallidina y
de Macrosiphoniella conocidas en Sudam6rica.


The distribution of most aphid species (Hemip-
tera: Aphididae) is limited to holarctic territory.
Only those belonging to the subfamilies Greeni-
deinae, Lizeriinae, Neophyllaphidinae, Pterasterii-
nae, and Spicaphidinae (with approximately 150,
33, 12, 5, and 13 species, respectively) and most of
the subfamily Hormaphidinae (with approximately
180 species) are found in gondwanic territories.
The species in the remaining subfamilies are char-
acteristic of holarctic territories with some penetra-
tion in neighboring Oriental, Ethiopian, or Neotro-
pical regions. The number of species in the subfam-
ilies Calaphidinae, Eriosomatinae, Chaitophorinae,
Lachninae, Saltusaphidinae (with, respectively,
332, 319, 165, 124, and 68 species, according to Eas-
top 1998) and the large subfamily Aphidinae (with
around 2800 species) recorded in one or another
southern territory is limited or very low. Some of
these species may be considered as natives of these
territories due to the evolution into lineages of hol-
arctic origin (see Nieto Nafria et al. 2002 for several
South American macrosiphins). Many others, the
alien species, inhabit these territories due to direct
introduction from holarctic populations.
The introduction of these species could be the
result of (1) natural invasions during recent geo-
logical periods, as in the case of species colonizing
(especially high regions) northern Neotropical and
northern Oriental regions and the suboriental bor-
der of the Ethiopian region; and (2) human activi-
ties and displacements, which, in the case of South


America date to no more than to 500 years. In
most cases, however, this period of time is no more
than 100 or 150 years, when traffic to and from
other parts of the world increased and journeys
lasted much shorter periods of time (Remaudiere
et al. 1985; Dixon 1998; Blackman & Eastop 2000;
Rapoport 2000). The exotic component of aphid
fauna in continental Argentina is considerable, ac-
counting for just over 75% according to the latest
review (Ortego et al. 2004), and is still growing.

MATERIALS AND METHODS

In this paper we follow the classification of the
family Aphididae used by Remaudiere & Re-
maudiere (1997), completed by Quednau (1999),
with the nomenclatural adaptations by Nieto
Nafria et al. (1998a).

RESULTS AND DISCUSSION

After studying specimens collected during re-
cent years in the Andean Argentinian strip we re-
corded another six alien aphid species, also re-
corded for the first time in South America.

New Data for the subfamiliy Saltusaphidinae:

The subfamily Saltusaphidinae Baker, 1920 is
of holarctic origin, though there is currently one
exclusive species in Chile (Quednau 1990):







Florida Entomologist 89(2)


Thripsaphis (T) unciniae Quednau, 1990. A few
species have been recorded in other regions (Aus-
tralia, New Zealand, sub-Saharan Africa) as a re-
sult of human activities and displacements (Eas-
top 1986). Saltusaphidinae live on species of the
family Cyperaceae or more unusually, Juncaceae,
Poaceae, or other monocotyledonous species. They
are generally very globose or flattened aphids,
sometimes elongate and even bacilliform, with
scarcely or no discernible siphunculi and a clavi-
form cauda; they are an attractive yellow or green
(in different shades) color mixed with black; mi-
croscopic examination (or even stereoscopic mag-
nification) reveals numerous dorsal setae, mostly
claviform, spatulate, flabelliform, or fungiform
and often short or very short.

Saltusaphis scirpus Theobald, 1915

Studied Material: Maipui (Mendoza), yellow
water trap, 28-II-2004, 1 alate viviparous female
(J. Ortego leg.); Cyperus rotundus Linnaeus, 1753,
29-IV-2004, alate and apterous viviparous females
and nymphs (J. Ortego leg.). Viviparous females
greenish-black, relatively large (usually over 2
mm), somewhat depressed and oval-shaped, with
a concave front and eyes fairly separated from the
antennal insertion (very common characters in
the subfamily). Antennae almost reaching body
length, fore femur much more voluminous than
the others, siphunculi subcylindrical, low, pig-
mented and very rough. Dorsum of apterae with
small and abundant segmental sclerites (with pig-
mentation very similar to that of intersegmental
sclerites) and numerous short and flabelliform se-
tae, except the marginal ones on final segments,
which are long (in particular on the two blunt tu-
bercles of abdominal segment VIII) and claviform,
blunt or pointed. Alatae with marginal and spinal
(one or two) sclerites and small pleural plates on
each abdominal segment, antennal segment III
bearing 9 to 21 oval-transverse secondary senso-
ria, and wings with slightly bordered veins and
spots on edge of pterostigma and tip of the veins.
Saltusaphis scirpus has been recorded in most
of Europe (Bulgaria, Czech Republic, France, Ger-
many, Greece, Hungary, Italy, Poland, Portugal,
Rumania, Russia [with doubts], Slovakia, Spain,
Sweden, and the Ukraine) and sub-Saharan
Africa (Angola, Burundi, Kenya, Lesotho, Malawi,
Mozambique, South Africa, Sudan, and Zimba-
bwe), several Asian countries (from the Mediter-
ranean to India) and North America (U.S.A.), ei-
ther under its valid name, or as Hiberaphis iberica
Bdrner, 1949, Saltusaphis africana Eastop, 1953
and Bacillaphis afghanica Narzikulov & Umarov,
1970 (Nieto Nafria et al. 1998b). It is easily differ-


entiated from Thripsaphis unciniae, so far the
only species in the subfamily recorded from South
America, by its dorsal sclerotization and pigmen-
tation and shape of abdominal segment VIII (in
apterae and alatae) and by the pigmentation of
the wings. The presence of this species in Argen-
tina could be due to (1) eggs in the soil of ornamen-
tal plants living in water or nearby, or in soil ad-
hered to plant bulbs or tubercles, or (2) eggs or
specimens living on ornamental Cyperaceae, for
example Cyperus. The latter option is the most
likely. The invasion could have originated from
Europe, North America, or even South Africa.

New Data on the Subtribe Myzocallidina (Calaphidinae:
Panaphidini):

The subfamily Calaphidinae Oestlund, 1918 is
of holarctic origin, though some if its species now
inhabit other parts of the world. Most of the gen-
era and nearly all the species of Myzocallis Pas-
serini, 1860 live on Fagaceae. This genus includes
42 taxa of species level, divided into 10 subgenera
(Quednau 1999), two of which, M. (Agrioaphis)
castanicola Baker, 1917 (specifically, the nomino-
typical subspecies) and M. (M.) coryli (Goeze,
1778), have been recorded in Argentina, Brazil,
and Chile (Blackman & Eastop 1994; Quednau
1999). There are records (Fuentes-Contreras et
al. 1997; Perez Hidalgo et al. 1998; Bergmann et
al. 2002; Ortego et al. 2004) of another four spe-
cies of the subtribe in South America: Hoplocallis
picta (Ferrari, 1872) in Argentina and Chile,
Tuberculatus (T) querceus (Kaltenbach, 1843) in
Argentina, Tuberculatus (Nippocallis) kuricola
(Matsumura, 1917) in Brazil, and Tuberculatus
(Tuberculoides) annulatus (Hartig, 1841) in
Argentina, Brazil, and Chile.

Myzocallis (Myzocallis) boerneri Stroyan, 1957

Studied Material: Junin (Mendoza), Quercus
suber Linnaeus, 1753, 27-VIII-2004, alatae females
and nymphs (J. Ortego leg.). Myzocallis boerneri
was recorded (Nieto Nafria & Mier Durante 1998;
Blackman & Eastop 1994; Quednau 1999) on vari-
ous species of Quercus canariensiss, castaneaefolia,
cerris, ilex, infectoria, faginea, macedonica, persica,
rotundifolia, suber, and variabilis) in most of Eu-
rope, the Macaronesian Islands; Lebanon, Israel,
Iran; South Africa; New Zealand, and U.S.A. (Cali-
fornia). The species almost certainly entered Ar-
gentina via seedlings of European species of Quer-
cus from Europe. It is differentiated from the other
6 species recorded for South America by the follow-
ing key. Any doubts can be clarified by consulting
the key by Quednau (1999).


la. Abdomen with spinal tubercles ..


. . . . . . . .. . 2


lb. Abdomen without spinal tubercles ................ ................... ......................... 3


June 2006







Ortego et al.: New Aphid Species Recorded from South America


2a. Abdominal segment III with one pair of large spinal tubercles joined at base.
On Eurasian species of Quercus. When alive greyish-brown to brown with
abundant powdered to filamentous wax ................ ................ .Tuberculatus (T) querceus
2b. Abdominal segments I and II with one pair of pale or slightly pigmented spinal tubercles
and abdominal segment III with another bigger pigmented pair; all of them clearly
separated at the base. On Quercus spp. When alive, pale green to yellowish-green
or with shades of pink and even crimson coloring ............... Tuberculatus (Tuberculoides) annulatus
3a. Setae of the antennal segment III at least 4.0 times the joint width of article.
Siphunculi rising from marginal sclerites. On Eurasian species of Castanea
and Quercus. When alive, pale green to red and with whitish wax. Alar veins
(particularly the anterior ones) widely bordered ................... Tuberculatus (Nippocallis) kuricola
3b. Antennal setae much shorter (at most 1.0 times the joint width of article) .............................. 4
4a. Dorsum of the head and prothorax evenly dark and abdomen with a band of dark
spinal plates (each with a small central depigmented area), marginal plates
not close together and less pigmented than spinal ones. On Eurasian species
of Quercus. When alive, yellow (cream to lemon) and black ........................... Hoplocallis picta
4b. Abdomen without dark spinal band and head completely pale or with a dark spinal band .................. 5
5a. Dorsum completely pale or with very faint marginal spots on prothorax and/or several
abdominal segments. On Corylus. When alive, pale yellow or whitish-yellow ........ Myzocallis (M.) coryli
5b. Head and prothorax with a dark spinal band and abdomen with pairs of spinal
and marginal sclerites (spread out and well-pigmented in the nominotypical
subspecies). On species of Castanea and Quercus. When alive, lemon to
creamy yellow with black spots of varying size and intensity ......... Myzocallis (Agrioaphis) castanicola


5c. Prothorax pale or with a pair of marginal spots at most and abdomen with pairs
of marginal and spinal sclerites, less pigmented than the former. On Eurasian
species of Quercus. When alive pale yellow to yellowish-green or even very
pale greenish-yellow, with a cream-colored or dull brown thorax ............


New Data on the Tribe Macrosiphini (Aphidinae):
Genus Macrosiphoniella:

The genus Macrosiphoniella Del Guercio, 1911
is one of the big genera in the very extensive tribe
Macrosiphini, with approximately 140 species,
100 of which are classified in the nominotypical
genus and some are little known. It belongs to the
group of genera with long reticulate siphunculi,
like Macrosiphum Passerini, 1860 and Uroleucon
Mordvilko, 1914. Species of Macrosiphoniella
have monoecious and basically holocyclic cycles
(some are confirmed as being anholocyclic) and al-
most all of them live on species of Asteraceae. The
distribution of each species varies greatly within
the holarctic distribution of the genus as a whole.
Some, such as M. absinthii (Linnaeus, 1758), are
very widespread (possibly due to human activi-
ties spreading), while others have only been re-
corded in a few localities, for example, M. aetnen-
sis Barbagallo, 1979 (Nieto Nafria et al. 2004). To
date, four species from this genus had been re-
corded for South America (Smith & Cermeli 1979;
Remaudiere et al. 1991; Costa et al. 1993; Nieto
Nafria et al. 1994): Macrosiphoniella (M.) artemi-
siae (Boyer de Fonscolombe, 1841) and in particu-
lar its nominotypical subspecies (known from
Argentina only), M. (M.) sanborni (Gillette 1908)


Myzocallis (M.) boerneri


(recorded in Argentina, Brazil, Bolivia, Chile,
Colombia and Venezuela), M. (M.) tanacetaria
(Kaltenbach, 1843), and specifically its subspe-
cies bonariensis E. E. Blanchard 1922, (described
in Buenos Aires and former synonym of italica
Hille Ris Lambers, 1966, and recorded from Ar-
gentina and Chile), and M. (M.) yomogifoliae
(Shinji, 1924) (recorded in Brazil). Another four
species: M. (M.) abrotani (Walker, 1852), M. (M.)
absinthii (Linnaeus, 1758), M. (M.) pseudoartemi-
siae Shinji, 1933, and M. (M.) tapuskae (Hottes &
Frison, 1931) are recorded for the first time.

Macrosiphoniella absinthii (Linnaeus, 1758)

Studied Material: Junin de Cuyo (Mendoza),
Artemisia absinthium Linnaeus, 1753, 14-IX-2004,
apterous and alate viviparous females (J. Ortego
leg.). The species is normally found on Artemisia
absinthium, but has been recorded (Heie 1995) on
C'i.., I ri... i.... zawadzkii. It is widely distrib-
uted in Europe and there are records for North
Africa and Canada (Nieto Nafria et al. 2004).

Macrosiphoniella abrotani (Walker, 1852)

Studied Material: Esperanza (Santa Fe), yel-
low water trap, 29-IX-1999, alate viviparous fe-







Florida Entomologist 89(2)


male (J. Ortego leg.); Malargiie: El Challao (Men-
doza), Artemisia sp., 31-X-1999, apterous and
alate viviparous females and nymphs (J. Ortego
leg.); Maipui (Mendoza), Artemisia sp., 9-II-2002,
apterous viviparous females (J. Ortego leg.);
Rafaela (Santa Fe), Artemisia annua, 26-II-2001,
apterous viviparous females and nymphs (I. Ber-
tolaccini leg.). This species has been recorded on
species of Artemisia and less frequently on some
species of Matricaria and Achillea, in numerous
European countries, Australia, and North Amer-
ica (Nieto Nafria et al. 2004).

Macrosiphoniella pseudoartemisiae Shinji, 1933

Studied Material: Malargiie (Mendoza), Arte-
misia absinthium, 5-IV-1994, apterous viviparous
females (J. Ortego leg.); Trevelin (Chubut), Arte-
misia absinthium, 20-1-2000, apterous viviparous
females (J. M. Nieto Nafria, J. Ortego and M. P.
Mier Durante leg.). This aphid has been recorded
(Lee et al. 2002) on Artemisia annua, mongolica,
princeps, and stolonifera in North and South Ko-
rea, China, Russia (Far East), Japan and India.
Artemisia absinthium is therefore a new host
plant for this species. This is the most surprising
of the four species now included in the South


American aphid fauna catalogue, given its origin
and distribution; but Macrosiphoniella yomogifo-
liae has been recorded in Brazil.

Macrosiphoniella tapuskae (Hottes & Frison, 1931)

Studied Material: Barreal (San Juan), Anthe-
mis cotula Linnaeus, 1753, 23-XI-2002, alate and
apterous viviparous females and nymphs (M. P.
Mier Durante, J. Ortego and J. M. Nieto Nafria
leg.); San Rafael: El Sosneado (Mendoza), Tanace-
tum vulgare Linnaeus, 1753, 29-1-2000, apterous
viviparous females (J. M. Nieto Nafria, J. Ortego
and M. P. Mier Durante leg.). It has been recorded
on different Asteraceae species, in particular
Achillea and Matricaria in most European coun-
tries, and others in the Near East, North Africa,
and North America (Nieto Nafria et al. 2004).
The following key can be used to differentiate
the eight species, but carefully, due to the rich-
ness of species in the genus. Other characters (in
square brackets) are given in order to corroborate
the identification. Should any discrepancies arise
regarding the described characters and those of
the studied specimens another key for differenti-
ating holarctic species must be used (for example,
see Heie 1995).


la. Siphunculi clearly longer than the cauda (Fig. Ig) and only 12-18% of their length reticulate. [Dark grey
color, more or less yellowish or reddish, with ash-colored waxy powder, except for a spinal band and
a strip in front of the insertion of the siphunculi. Without segmental sclerites on dorsum of abdomen
(Fig. Ig). Antennal segment III with 6 to 26 secondary sensoria on proximal 2/3] ............ M. tapuskae
lb. Siphunculi shorter (Figs. la-f, h) or slightly longer than cauda, and at least 40% of their length
reticulate .................................. ..... .......... 2
2a. Siphunculi pigmented, with pale basal area (Fig. la). [Green aphids, with waxy powder.
Siphunculi 50-67% reticulate in length. Presiphuncular sclerites usually absent (Fig. la).
Cauda with 11 to 25 setae. Antennal segment III with 2 to 13 secondary sensoria in
proximal half] ................ ...................................... ........ M. abrotani
2b. Siphunculi wholly pigmented, though not with the same intensity (Figs. Ib-f, h) .......................... 3
3a. Spinal sclerites fully-pigmented on abdominal segments II to VI (Fig. Ib). Antennal segment III
with 29 to 55 secondary sensoria in proximal half. [Dark reddish-grey or purple aphids with
waxy powder. Hind tibiae strongly pigmented (Fig. Ib). Siphunculi reticulate on 48-60% of
its length. Cauda with 12 to 18 setae] ............................................. M. absinthii
3b. Without spinal sclerites on abdominal segments II to VI (Figs. 1c-f, h). Secondary sensoria
of antennal segment III varying in number and distribution ................ .................... 4
4a. Mid part of hind tibiae, at least, pale or scarcely pigmented (Figs. Id, e) ................................. 5
4b. Hind tibiae strongly pigmented (Figs. Ic, f, h) ................ .................................... 6
5a. Antennal segment III with 8 to 32 secondary sensoria, distributed along its entire length. Presiphuncular
sclerites well-pigmented. On Chrysanthemum. [Dark grey or toffee-colored, without waxy
powder. Siphunculi with 63-81% of its length reticulate. Cauda with 9 to 11 setae] ............ M. sanborni
5b. Antennal segment III with 2 to 6 secondary sensoria, spread along proximal half. Presiphuncular
sclerites tenuous (as tenous as setiferous sclerites on dorsum of abdomen) (Fig. Id).
OnArtemisia [Green aphids with some waxy powder. Siphunculi with 62-71% of its
length reticulate. Cauda with 9 to 13 setae. Dorsoabdominal setae blunt or with
slightly expanded tip] ..................................... ................ M. pseudoartemisiae
6a. Processus terminalis 4.8 to 6.8 times ultimate rostral segment, which is 0.7 to 0.9 times
second segment of hind tarsus. Cauda with 26 to 32 setae. Presiphuncular sclerites


June 2006







Ortego et al.: New Aphid Species Recorded from South America


Fig. 1. (a) Macrosiphoniella (M.) abrotani, (b) Macrosiphoniella (M.) absinthii, (c) Macrosiphoniella (M.) artemi-
siae, (d) Macrosiphoniella (M.)pseudoartemisiae, (e) Macrosiphoniella (M.) sanborni, (f) Macrosiphoniella (M.) tan-
acetaria bonariensis, (g) Macrosiphoniella (M.) tapuskae and (h) Macrosiphoniella (M.) yomogifoliae.


absent or present but scarcely pigmented (Fig. If). [Green to grey-brown aphids
with waxy powder. Antennal segment III with 2 to 8 secondary sensoria in
proximal half only].................................................... M tanacetaria bonariensis

6b. Processus terminalis 2.4 to 5.0 times ultimate rostral segment, which is 0.8 to 1.3 times second
segment of hind tarsus. Cauda with 10 to 36 setae (but with 27 setae at most if the processus
terminalis is less than 1.0 times the cauda) .................................................... 7

7a. Ultimate rostral segment 1.0 to 1.3 times second segment of hind tarsus. Processus terminalis
2.4 to 3.5 times ultimate rostral segment. Genital plate usually with 4-6 discal setae
(exceptionally 2 or 8). OnArtemisia and Chrysanthemum. [Green aphids covered with
waxy powder. Presiphuncular sclerites absent or scarcely pigmented (Fig. lh). Cauda with
17 to 24 setae. Antennal segment III with 2 to 8 secondary sensoria] ................... M. yomogifoliae

7b. Ultimate rostral segment 0.8 to 1.1 times second segment of hind tarsus. Processus terminalis
3.6 to 5.0 times ultimate rostral segment. Genital plate with 2-3 discal setae. OnArtemisia
vulgaris andA. absinthium. [More or less greyish-green aphids with waxy powder.
Presiphuncular sclerites present, though scarcely pigmented (Fig. Ic). Cauda with
19 to 27 setae. Antennal segment III with 3 to 14 secondary sensorial .................... M. artemisiae


ACKNOWLEDGMENTS

We express our thanks to Dr. Isabel Bertolaccini
(Universidad Nacional del Litoral, Esperanza, Santa
Fe, Argentina) for lending us a sample of M. abrotani,
Dr. Franz Wolfgang Quednau (Laurentian Forestry
Centre, Sainte-Foy, Qu6bec, Canada) for the informa-
tion on Saltusaphis and Myzocallis; Drs. Roger L.
Blackman and Victor F. Eastop (The Natural History
Museum, London, UK) for help in identifying the
M. pseudoartemisiae specimens and Prof. Georges Re-


maudiere for comments. This study was granted by the
Regional Government of Castilla y Le6n (Spain) [LE-45/
02 and LE034A05].


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June 2006







Maxwell & Fadamiro: Management of Cole Crop Pests with Novel Insecticides 117



EVALUATION OF SEVERAL REDUCED-RISK INSECTICIDES IN
COMBINATION WITH AN ACTION THRESHOLD FOR MANAGING
LEPIDOPTERAN PESTS OF COLE CROPS IN ALABAMA


ELLY M. MAXWELL AND HENRY Y. FADAMIRO
Department of Entomology & Plant Pathology, Auburn University, U.S.A

ABSTRACT

Several reduced-risk insecticides were evaluated for management of three lepidopteran cole
crop pests, Plutella xylostella (L.), Pieris rapae (L.), and Trichoplusia ni (Htibner) in central
Alabama in 2004 (spring and fall plantings) and 2005 (spring only). The following formu-
lated sprays were evaluated: Dipel (Bacillus thuringiensis subspecies kursatki), XenTari
(B. thuringiensis subspecies aizawai), Dipel+XenTari (a premixed test formulation consist-
ing of both subspecies of B. thuringiensis), Entrust (a formulation of spinosad for use in or-
ganic crop production), and Novaluron (insect growth regulator). Variations in the
populations of the three pest species were recorded from season to season with pest pressure
being generally higher in both spring seasons than in the fall season. While moderate to high
populations of P. xylostella and P. rapae were recorded in all three seasons, T ni was detected
only in spring 2005. An action threshold of 0.5 cabbage looper equivalents (CLE) per plant
was used to determine the need for insecticide applications. Insecticide efficacy was deter-
mined by comparing densities of larvae and immatures (larvae + pupae) of each pest species,
crop damage ratings, densities of key non-target arthropods, and number of insecticide ap-
plications in plots treated with each material versus untreated control plots. All five re-
duced-risk insecticide formulations were effective in reducing infestations of the three
lepidopteran pests and in providing marketable cabbage and collards in Alabama. Among
the treatments, Entrust consistently produced the lowest mean damage ratings with the
minimum number of applications per season. No significant effects of insecticide treatments
were recorded in the numbers of spiders and lady beetles found per plant. The results also
suggest that the 0.5 CLE action threshold can be used to produce marketable cabbage and
collards in Alabama with only minimal applications of reduced-risk insecticides.

Key Words: Diamondback moth, Plutella xylostella, imported cabbageworm, Pieris rapae,
cabbage looper, Trichoplusia ni, integrated pest management

RESUME

Varias insecticides de riesgo reducido fueron evaluados para el manejo de tres plagas de le-
pid6pteros del cultivo de col, Plutella xylostella (L.), Pieris rapae (L.) y Trichoplusia ni (Hub-
ner) en el area central de Alabama en el 2004 (las siembras de la primavera y otofio) y el 2005
(solamente la primavera). Las siguientes formulaciones fumigadas fueron evaluadas: Dipel
(Bacillus thuringiensis subespecie kursatki), XenTari (B. thuringiensis subespecie aizawai),
Dipel+XenTari (una formulaci6n de prueba pre-mezclada que consiste de ambas subespecies
de B. thuringiensis), Entrust@ (una formulaci6n de "spinosad" para el uso en la producci6n de
cultivos organicos) y Novaluron (un regulador del crecimiento de insectss. Las variaciones en
la poblaci6n de las tres species de plagas fueron anotadas de una estaci6n a la otra, con la
presi6n de las plagas generalmente mas alta en ambas estaciones de primavera que en la es-
taci6n de otofio. Mientras que poblaciones moderadas y altas de P. xylostella y P. rapae fueron
registradas en las tres estaciones, T ni (fue solamente detectada en la primavera del 2005. El
umbral de acci6n de 0.5 equivalentes del gusano medidor de repollo (EGMR) por plant fue
usado para determinar la necesidad para aplicar el insecticide. La eficacia del insecticide fue
determinada comparando las densidades de las larvas e inmaduros (larvas y pupas) de cada
especie de plaga, la clasificaci6n del daio en el cultivo, las densidades de los artr6podos clave
que no fueron objeto del tratamiento, y el numero de aplicaciones de insecticide en parcelas
tratadas con cada product versus en las parcelas no tratadas (el control). Todas las formula-
ciones de insecticide de riesgo reducido fueron efectivas en reducir infestaciones de las tres
plagas de lepid6pteros y en proveer repollo y col de hoja para la venta en Alabama. Entre los
tratamientos, el Entrust@ de manera consistent produj6 el menor promedio de clasificaci6n
de daio con el numero minimo de aplicaciones por estaci6n. No efectos significativos de los
tratamientos de insecticide fue registrado en el numero de araias y coccinellidos encontrados
por plant. Estos resultados sugieron que el umbral de acci6n de 0.5 EGMR puede ser usado
para producer repollo y col de hoja para la venta en el estado de Alabama con solamente un
minimo numero de aplicaciones de insecticides de riesgo reducido.







Florida Entomologist 89(2)


Cole crops, Brassica oleracea (L.), including
cabbage, collards, broccoli, kale, brussels sprouts,
and cauliflower, are an important component of
diets in many parts of the world.
Cabbage and collards are the key cole crops
grown in Alabama. Growers in the state utilize
both spring and fall plantings for both crops, and
often grow them in rotation with other vegeta-
bles (Kemble 1999). The key lepidopteran pests
of cole crops in Alabama include the diamond-
back moth, Plutella xylostella (L.), imported cab-
bageworm, Pieris rapae (L.), and cabbage looper,
Trichoplusia ni (Hiibner) (Kemble 1999). Plutella
xylostella and P. rapae are often the most abun-
dant pests in many parts of Alabama, while infes-
tations of T ni are sporadic in nature (personal
observation).
Caterpillars of the three lepidopteran species
do direct damage to the marketable part of the
plant by chewing holes in the foliage and produc-
ing frass (Harcourt et al. 1955; Shelton et al.
1982; Talekar & Shelton 1993; Tabashnik 1994),
and are usually managed as a single caterpillar
complex (Mahr et al. 1993). Tolerance of damage
from these caterpillars is extremely low, basically
zero to trace amounts of insect damage or frass in
the final product (Morisak et al. 1984). In order to
avoid significant economic loss, vegetable produc-
ers have typically managed these pests with an
expensive therapeutic approach involving calen-
dar-based applications of conventional insecti-
cides, including various organophosphate, car-
bamate, and pyrethroid formulations. For in-
stance, approximately 30,000 pounds of insecti-
cide active ingredient are used annually for
collard production in Alabama (Williams & Dang-
ler 1992). Excessive and indiscriminate use of
conventional insecticides has resulted in the de-
velopment of pest resistance to insecticides
(Hines & Hutchison 2001; Liu et al. 2002).
Globally, formulated sprays of microbial insec-
ticides such as Bacillus thuringiensis and spi-
nosad have been used widely as an alternative to
chemical insecticides. However, development of
pest resistance to microbial insecticides has been
reported in several locations. For instance, resis-
tance to Bacillus thuringiensis subspecies kur-
satki have been detected in field populations of
P xylostella in various locations in the mainland
U.S. (Mahr et al. 1993; Shelton et al. 1993; Tang et
al. 1997), and in several other locations through-
out the world including Hawaii, Malaysia, the
Philippines, Japan, Central America, and Thai-
land (Talekar & Shelton 1993; Rueda & Shelton,
1995; Tabashnik et al. 1997). Similarly, field pop-
ulations of P xylostella collected in Malaysia have
been reported to show resistance to spinosad
(Sayyed 2004). The problem of insecticide resis-
tance is not limited to P. xylostella. Resistance to
B. thuringiensis has been demonstrated in labo-
ratory populations of T ni (Estada & Ferre 1994)


and in greenhouse populations in British Colum-
bia (Janmaat & Myers 2003).
Traditionally, more attention has been paid
to insecticide-based control programs than bio-
logical control for management of lepidopteran
pests of cole crops (Taleker & Shelton 1993;
Biever et al. 1994; Xu et al. 2004). Although suc-
cessful integrated pest management (IPM) pro-
grams have been developed and implemented in
many parts of the world (Biever et al. 1994), it
appears that insecticide-based control will re-
main the major tactic for managing caterpillar
pests of cole crops for the foreseeable future (Xu
et al. 2004).
Over the past several years, numerous biologi-
cally-based insecticides with novel modes of ac-
tion have been developed and shown to have a
high level of efficacy on lepidopteran pests of cole
crops (Eger & Lindenberry 1998; Liu and Sparks
1999; Hill & Foster 2000; Hines & Hutchison
2001). These include microbial insecticides (e.g.,
several formulations of spinosad and B. thuring-
iensis) and insect growth regulators. These new
materials are termed "reduced-risk insecticides"
because of their narrow spectrum of activity and
low toxicity to humans and non-target organisms,
and are considered IPM-compatible. Although re-
duced-risk insecticides are increasingly being
used by vegetable growers worldwide, little has
been done to evaluate these materials in Ala-
bama. The objective of this study was to evaluate
the efficacy of several reduced-risk insecticides
against lepidopteran pests of cole crops in Ala-
bama. The materials evaluated included three
formulations of B. thuringiensis (Dipel, Xen-
Tari, and Dipel+XenTari mixture) (Valent Bio-
sciences Libertyville, IL), Entrust (Dow Agro-
Sciences, Indianapolis, IN), and Novaluron
(Crompton (now Chemtura), Middlebury, CT).
Dipel is a formulation of B. thuringiensis sub-
species kursatki and is the most commonly used
microbial insecticide on Alabama vegetable crops
(Joseph Kemble, personal communication). Xen-
Tari is a formulated spray of B. thuringiensis
subspecies aizawai, while Dipel+XenTari is a pre-
mixed test formulation consisting of both subspe-
cies ofB. thuringiensis. Entrust is a natural in-
sect control product formulated for the organic
grower. The active ingredient, spinosad, is devel-
oped from a fermentation by-product of the soil-
borne actinomycete bacterium, Saccharopoly-
spora spinosa (Liu et al. 1999). Novaluron is an
insect growth regulator (IGR) that works by in-
hibiting chitin synthesis. It is currently labeled in
the U.S. as Diamond for use on cotton and Ri-
mon for use on apples, potatoes, and sweet po-
tato, and the registrant plans to label Novaluron
for use on cole crops in the near future (K. Grif-
fith, personal communication). These materials
were evaluated over multiple field seasons (2004-
2005) in central Alabama.


June 2006







Maxwell & Fadamiro: Management of Cole Crop Pests with Novel Insecticides 119


MATERIALS AND METHODS

General Methodology

This research was conducted over three grow-
ing seasons; spring 2004, fall 2004, and spring
2005 at the E.V. Smith Research center in
Shorter, AL. Treatments were arranged in a ran-
domized complete block design with three repli-
cates in each spring season and four in the fall
2004 season. All seedlings were obtained from a
nursery in western Georgia (Lewis Taylor Farms;
Ty Ty, Georgia) and were planted bareground fol-
lowing a preseason fire ant (Solenopis invicta)
treatment with Amdro (active ingredient = hy-
dramethylnon, BASF Corporation, Research Tri-
angle Park, NC). Standard field preparation and
crop production practices (i.e., irrigation) were
used to establish cabbage or collard plants in all
three field seasons.
In spring of 2004'Bravo' cabbage was mechan-
ically transplanted on 30-III-2004. Plots were 13.7
m by 9.1 m with plants spaced 45 cm apart within
a row and 90 cm between rows for a total of 300
plants per plot. Plots were separated by a 15.2-m
alley. The following four reduced-risk insecticides
were compared: Dipel, Xentari, Dipel+Xentari
combination, and Entrust@. In fall 2004, 'Top
bunch' collards were mechanically transplanted 2-
X-2004. Plots consisted of two 10-m rows, 100 cm
apart with plants spaced 45 cm apart within a row
and 90 cm between rows for a total of 40 plants per
plot. Five reduced-risk insecticides were com-
pared: Dipel, Xentari, Dipel+Xentari combina-
tion, Entrust@, and Novaluron. In spring 2005,
'Vates' collards were mechanically transplanted at
the E.V. Smith Research Station on 22-IV-2005.
The plot dimensions and treatments evaluated
were as described for fall 2004.
Plots were evaluated weekly for pest infesta-
tion by sampling ten randomly selected plants per
plot for larvae of P. xylostella, P. rapae, and T ni.
Eggs and pupae of the three species also were
sampled. The number of immatures of each spe-
cies was calculated by summing the number of
larvae and pupae. Treatment applications were
made only when larval counts exceeded a thresh-
old of 0.5 cabbage looper equivalents (CLE) per


plant (Shelton et al. 1982, 1983). The CLE
method accounts for the varying levels of feeding
damage caused by the three species. In this
method, 1 CLE = 20 P xylostella larvae = 1.5
P rapae larva = 1 T ni larva (Shelton et al. 1982,
1983). In addition, plants also were sampled for
aphids (number of plants with aphid infestation)
and key non-target predatory insects in our fields,
mainly spiders and lady beetle adults (Coccinel-
idae). Treatment applications were made with a
CO2 pressurized backpack sprayer using a 3-ft
boom with 3 nozzles calibrated to deliver about
25 gpa at 40 psi. Insecticides were applied at the
recommended rates. Dipel, Xentari, and
Dipel+Xentari were applied at the rate of 1 pound
per acre, Entrust@ at 2 oz per acre, and Novalu-
ron applied at the rate of 12 fluid ounces per acre.
Based on the action threshold of 0.5 CLE, the av-
erage number of insecticide applications varied
by treatment and season and ranged from 1.3 to 5
applications per season (Table 1).
At harvest, ten plants were randomly selected
from each plot and rated for caterpillar feeding
damage and marketability was quantified by the
method of Greene et al. (1969). In this method
cabbage plants grown in spring 2004 were rated
based on insect feeding damage on a scale of 1 to
6 as follows: 1 = no apparent insect damage on
head or inner wrapper leaves; 2 = no head dam-
age, but minor feeding on wrapper leaves with 0-
1% leaf area consumed; 3 = no damage on head,
but moderate feeding damage on wrapper leaves
with 2-5% leaf area consumed; 4 = minor feeding
on head (but no feeding through outer head
leaves), but moderate feeding on wrapper or outer
leaves with 6-10% leaf area consumed; 5 = moder-
ate to heavy feeding damage on wrapper and
head leaves and a moderate number of feeding
scars on head with 11-30% leaf area consumed;
and 6 = severe feeding damage to head and wrap-
per leaves with heads having numerous feeding
scars with >30% leaf area consumed (Greene et
al. 1969). A similar method was used to assess
marketability of collards in fall 2004 and spring
2005 with damage rating based solely on the per-
cent of leaf area consumed (since collards is not a
head-producing plant). A damage rating of <3 is
considered marketable under normal conditions,


TABLE 1. MEAN ( SE) NUMBER OF APPLICATIONS OF EACH REDUCED-RISK INSECTICIDE TREATMENT PER PLOT DURING
EACH SEASON. TREATMENT APPLICATIONS WERE MADE ONLY WHEN WEEKLY LARVAL COUNTS EXCEEDED A
THRESHOLD OF 0.5 CABBAGE LOOPER EQUIVALENTS (CLE) PER PLANT.

Treatment/formulation Spring 2004 Fall 2004 Spring 2005

Dipel DF 2.67 0.19 1.50 0.14 4.33 0.19
Xentari DF 2.33 + 0.19 1.25 + 0.13 5.00 0.00
Dipel+Xentari DF 2.67 + 0.19 1.25 + 0.13 4.67 + 0.19
Entrust 80WP 2.33 + 0.19 1.25 + 0.13 3.67 0.29
Novaluron -1.25 0.13 4.00 0.00







Florida Entomologist 89(2)


whereas a damage rating of<4 is marketable only
under exceptional market conditions (Leibee et
al., 1995).
Statistical Analysis. For each season, mean
seasonal larval and immature counts of each lep-
idopteran species, number of plants with aphid
infestation, numbers of key non-target beneficial
arthropods (i.e., spiders and lady beetle adults),
and mean damage rating at harvest were calcu-
lated for each treatment. Data were transformed
by the square-root method I(x + 0.5) and analyzed
for significant treatment effects by analysis of
variance (ANOVA) with the plots considered as
blocks. Means were compared by the Tukey-
Kramer HSD comparison for all pairs (JMPIN
Version 4.0.2, SAS Institute Inc., 1998). Signifi-
cant differences were established at the 95% con-
fidence level (P < 0.05).

RESULTS

Infestation levels of the three lepidopteran
pests varied with growing season. Moderate to
high populations of P xylostella and P rapae were
recorded during all three field seasons, while T ni
population was recorded only in spring 2005. In
general, relatively higher populations of the lepi-
dopteran pests were recorded in both spring sea-
sons compared with the fall season. This was re-
flected also in the number of applications per in-
secticide treatment made during each season
which averaged 2.5, 1.3, and 4.3 for spring 2004,
fall 2004, and spring 2005, respectively (Table 1).
In both spring seasons, caterpillar pest pressure
as measured by CLE per plant per week in un-


treated control plots began two weeks after plant-
ing and moderate caterpillar pressure was ob-
served through harvest in spring 2004 (Fig. 1).
Extremely high caterpillar pressure was recorded
late in spring 2005 with CLEs greater than 3.5
per plant per week recorded in the last two weeks
of the season (Fig. 1). In the lone fall season (fall
2004), however, caterpillar pest infestation did
not begin until six weeks after planting, averag-
ing less than 0.5 CLE per plant per week for the
remainder of the season (Fig. 1). In general, no
significant block (plot) effects were detected (P >
0.05) for any of the dependent variables in any of
the seasons, suggesting that the plots were simi-
lar in pest abundance and treatment efficacy.
In spring 2004, all four reduced-risk insecti-
cides resulted in reductions in the number of
P xylostella larvae (F = 9.5, df = 4, P = <0.0001)
and immatures (F = 8.9, df = 4, P = <0.0001), and
P rapae larvae (F = 3.3, df = 4, P = <0.0001) and
immatures (F = 20.3, df = 4, P = <0.0001) com-
pared with the untreated control (Fig. 2A). How-
ever, significantly higher numbers of P rapae im-
matures were recorded for Dipel compared with
the other insecticide treatments. Higher damage
ratings were recorded in untreated control plots
than in any of the treatments (F = 65.3, df= 4, P =
<0.0001; Fig. 3A). Comparing the treatments,
mean damage ratings were significantly lower in
Entrust than in Dipel+Xentari combination. No
significant effects of insecticide treatments were
recorded in the number of plants with aphids (F =
0.3, df = 4, P = 0.89; Table 2), and in the numbers
of spiders (F = 0.7, df = 4, P = 0.62) or lady beetles
(F = 1.2, df = 4, P = 0.30) found per plant (Fig. 4A).


5-i ----
S -- Spring 2004
S4 -t. Fal 2004
S ---X- Spring 2005








SI I I I I I I /
3 /

2-




1 2 3 4 5 6 7 8 9 10
Weeks after planting

Fig. 1. Caterpillar pressure expressed as mean ( SE) number of cabbage looper equivalents (CLE) per plant re-
corded weekly after planting in untreated control plots during spring 2004, fall 2004, and spring 2005. Planting dates
for spring 2004, fall 2004, and spring 2005 were March 30 2004, October 2 2004, and April 22 2005, respectively.


June 2006







Maxwell & Fadamiro: Management of Cole Crop Pests with Novel Insecticides 121


Spring 2004


* Dipel
* Xentari
* Dipel+Xentari
o Entrust
O Untreated Check


CL larvae CL immatures DBM larvae DBM immatures ICW larvae ICW immatures


Fall 2004


* Dipel
* Xentari
* Dipel+Xentari
D Entrust
B Novaluron
o Untreated Check


a
|bab ha a b b, b

,ik b I bb BB


CL larvae CL immatures


DBM larvae DBM immatures ICW larvae ICW immatures


Spring 2005


* Dipel
N Xentari
B Dipel+Xentari
O Entrust
B Novaluron
o Untreated Check


a
b bab b a b b b b br-
;0014 ffkn


ama

abb bb ab b b b b b bb b
B~if &WsrO I r W


CL larvae CL immatures


DBM larvae DBM immatures ICW larvae ICW immatures


Fig. 2. Seasonal mean ( SE) number of larvae and immatures of lepidopteran species sampled per plant per
week in plots treated with different reduced-risk insecticides during spring 2004 (A), fall 2004 (B), and spring 2005
(C). Key: CL = cabbage looper (Trichoplusia ni); DBM = diamondback moth (Plutella xylostella); ICW = imported
cabbageworm (Pieris rapae). Means followed by the same letter are not significantly different (P > 0.05, Tukey-
Kramer HSD).







Florida Entomologist 89(2)


122



(A)


June 2006


Spring 2004
S6
*g a
5 5

S4
E

bc
be
2

1-


Dipel Xentari DipelaXentari Entrust Untreated Check



B)

6n Fal 2004
S 6-
5-

4-

3-3
2- b b b b b
-H



Dipel Xentari Dipel+Xentari Entst Novaluron Untreated
Check


Spring 2005


r


Xentari Dipef-Xentari Entrust


Novaluron Untreated
Check


Dipel


Fig. 3. Mean ( SE) damage ratings of plants harvested from plots treated with different reduced-risk insecti-
cides during spring 2004 (A), fall 2004 (B), and spring 2005 (C). Line indicates marketability threshold of 3 above
which produce is considered unmarketable. Means followed by the same letter are not significantly different (P >
0.05, Tukey-Kramer HSD).


I







Maxwell & Fadamiro: Management of Cole Crop Pests with Novel Insecticides 123


TABLE 2. SEASONAL MEAN ( SE) NUMBER OF PLANTS WITH APHID INFESTATION IN PLOTS TREATED WITH DIFFERENT
REDUCED-RISK INSECTICIDES.

Treatment/formulation Spring 2004 Fall 2004 Spring 2005

Dipel DF 0.019 0.008 0.025 0.008 0.033 0.01
Xentari DF 0.019 0.008 0.030 0.009 0.043 0.01
Dipel+Xentari DF 0.026 0.01 0.028 0.009 0.043 0.01
Entrust 80WP 0.030 0.01 0.030 0.009 0.033 0.01
Novaluron 0.019 0.007 0.047 0.01
Untreated Check 0.022 0.009 0.056 0.01 0.037 0.01
P 0.89 0.10 0.93
No. plants sampled per treatment (n) 270 360 300


In fall 2004, a treatment effect was recorded
for P. xylostella larvae (F = 2.3, df = 5, P = 0.04).
However, only Entrust resulted in significant
reduction in P xylostella larvae compared with
the untreated control; no significant differences
were recorded for the other treatments (Fig. 2B).
With the exception of Dipel, all treatments re-
duced P. xylostella immatures (F = 4.4, df = 5, P =
0.0006) and P rapae larvae (F = 5.3, df = 5, P <
0.0001). Nonetheless, higher density of P rapae
immatures was recorded in the untreated control
than in any of the treatments (F = 11.3, df= 5, P <
0.0001). All five treatments had lower mean dam-
age ratings in comparison with the untreated con-
trol (F = 38.7, df = 5, P < 0.0001; Fig. 3B). No ef-
fects of treatments were recorded in the number
of plants with aphids (F = 1.8, df = 4, P = 0.10;
Table 2), and in the numbers of spiders (F = 1.5, df
= 4, P = 0.20) or lady beetles (F = 0.7, df = 4, P =
0.62) found per plant (Fig. 4B).
In spring 2005, T ni was collected in the field,
whereas it was not present during the previous
two seasons (Fig. 2C). In general, all treatments
resulted in significant reductions in pest popula-
tions (Fig. 2C). All treatments except Dipel re-
duced densities of T ni larvae (F = 3.3, df= 5, P =
0.006) and immatures (F = 3.7, df = 5, P = 0.003)
compared with the untreated control (Fig. 2C).
For P. xylostella, lower numbers of larvae (F = 8.1,
df = 5, P < 0.0001) and immatures (F = 9.7, df = 5,
P < 0.0001) were recorded for all treatments com-
pared with the untreated control. Similar treat-
ment effects were recorded for P rapae larvae
(F = 3.9, df = 5, P = 0.002) and immatures (F = 4.1,
df = 5, P = 0.001); however, P rapae larval counts
in plots treated with the Dipel+Xentari formula-
tion were not significantly lower than larval
counts in untreated control plots (Fig. 2C). A
mean damage rating of 5.4 was recorded in the
untreated control which was higher (F = 101.4,
df = 5, P < 0.0001) than damage ratings in any of
the five treatments (Fig. 3C). In all three seasons,
mean damage ratings recorded in the treated
plots were never above the marketability thresh-
old of 3 (Green et al. 1969). No differences were
recorded among the treatments in the number of


plants with aphids (F = 0.26, df = 4, P = 0.93;
Table 2), numbers of spiders per plant (F = 1.2,
df = 4, P = 0.30), and numbers of lady beetles per
plant (F = 0.8, df = 4, P = 0.55) (Fig. 4C), suggest-
ing little or no effects of insecticide treatments on
the key non-target predators in our plots.

DISCUSSION

The goal of this study was to evaluate the effi-
cacy of various reduced-risk insecticides in provid-
ing acceptable control of lepidopteran pests of cole
crops in Alabama. In all three seasons, all materi-
als tested resulted in the production of marketable
produce with considerably lower pest pressure and
crop damage ratings compared with untreated
control plots which never yielded marketable pro-
duce. These results indicate that all five reduced-
risk insecticides were effective in controlling lepi-
dopteran pests of cole crops in Alabama. The re-
sults also suggest that the 0.5 CLE action thresh-
old recommended by Shelton et al. (1982, 1983)
can be used to produce marketable cabbage and
collards in Alabama with only minimal applica-
tions of reduced-risk insecticides, particularly in
locations with minor or no endemic populations of
T ni. Although resistance evaluation was not the
primary goal of this study, our results confirming
the high efficacy of the various microbial insecti-
cides tested in this study may suggest that P xy-
lostella resistance to B. thuringiensis is currently
not a major problem in central Alabama, consider-
ing that vegetable growers in this region have
been applying Dipel in their fields for years.
Although we did not always find significant
differences among the reduced-risk insecticides
tested in this study, Entrust consistently pro-
duced the lowest mean damage ratings (although
not always significant) with the least mean num-
ber of applications per season. The relatively
higher efficacy of Entrust recorded in this study
may be due to its broad spectrum activity and
multiple mode of entry. Entrust differs from the
other materials evaluated in this study in that it
successfully kills insects from several orders,
whereas the other treatments are selective to lep-







Florida Entomologist 89(2)


- 0.08 Spring 2004 Dipel
a Xentari
U Dipel+Xentari
0.06- B Etrust
0 Untreated Check
g 0.04


S0.02


0
Spiders Lady beetles

B)

08 Fal 2004 Dipel
0.08 -
0 Xentari
U Dipel+Xentari
a 0.06 Entrust
CO Novaluron
S0.04 0- : D Untreated Check


0.02 -


0
Spiders Lady beetles

C)


SDipel
O Xentari
* Dipel+Xentari
3 Entrust
m Novaluron
0 Untreated Check


S0.08 -


S0.06


0.04 -

-H
S0.02


0-


Spiders


Spring 2005


Lady beetles


Fig. 4. Seasonal mean ( SE) number of non-target spiders and lady beetle adults found per plant per week in
plots treated with different reduced-risk insecticides during spring 2004 (A), fall 2004 (B), and spring 2005 (C).
Means are not significantly different (P > 0.05, Tukey-Kramer HSD).


June 2006







Maxwell & Fadamiro: Management of Cole Crop Pests with Novel Insecticides 125


idopterans only (Cisneros et al. 2002). In addition,
spinosad, the active ingredient in Entrust has
both contact and ingestion activity (Eger & Lin-
denberry 1998; Liu et al. 1999), whereas the other
reduced-risk insecticides must be eaten by the in-
sects in order to be effective. It is thought that the
broad spectrum activity of Entrust will probably
ensure some control of non-lepidopteran pests
such as cruciferous flea beetles, harlequin bugs,
aphids, and other minor pests that the other
chemicals were not effective against. However, we
did not observe in the current study a significant
reduction in aphid-infested plants in Entrust-
treated plots compared to the other treatments or
control. On the other hand, spinosad has been
reported as toxic to beneficial insects such as
Diadegma insulare (Cressons) (Hymenoptera:
Ichneumonidae) (Xu et al. 2004), a very common
and effective parasitoid of P xylostella in North
America (Mahr et al. 1993). Hill & Foster (2000)
showed a 100% D. insulare mortality rate after
8 h of exposure to spinosad-treated brassica leaves,
while Cisneros et al. (2002) recorded up to 98%
mortality of predators exposed to high concentra-
tions of this microbial insecticide. However, we
did not record any significant effect of Entrust
or any of the other treatments on numbers of spi-
ders and lady beetles, the two most important
predators in our fields. Entrust thus appears to
be a promising tool for use in cole crop pest man-
agement and insecticide resistance management
programs, considering that the active ingredient,
spinosad has not been reported to share cross-
resistance mechanisms with any other group of
insecticides (Liu & Yue 2000; Wei et al. 2001). In
general, Xentari was second to Entrust in pro-
ducing acceptable damage ratings. However, the
fact that this material had the highest average
number of applications per season suggests that
it may not provide economically acceptable con-
trol compared to the other treatments.
Significant variations in the populations of the
three lepidopteran pests were recorded from sea-
son to season. In general, lepidopteran pest pres-
sure was higher in both spring seasons than in the
fall. Significant P xylostella pressure was re-
corded in both spring seasons and in the fall,
whereas P rapae pressure was highest in spring
2004 followed by spring 2005. Furthermore, we re-
corded during spring 2004 about 60 flying P. rapae
adults per plot in 5-min visual observations com-
pared to about 8 flying adults in fall 2004, suggest-
ing that this pest may be more severe in the spring
than in the fall. The detection of T ni in spring
2005 may have exacerbated total pest pressure
during this season resulting in above threshold
CLEs and the need to apply insecticides at a much
higher frequency than in the first two seasons.
This is especially likely since T ni is the most vo-
racious and damaging of the three pests (Shelton
et al. 1982; Hines & Hutchison 2001). The reason


for the detection of T ni only in spring 2005 may
be due to later planting date for this season. In
summary, our results confirmed the efficacy of the
tested reduced-risk insecticides in managing di-
rect pests of cole crops in Alabama in a threshold-
based IPM program. These reduced-risk insecti-
cides offer a wide range of pest management op-
tions available to vegetable growers and should be
used wisely or in rotation with one another to min-
imize selection for resistance to any one given ma-
terial. Obviously, the longevity of these new insec-
ticides as effective IPM tools will depend on their
judicious use, compatibility with natural enemies,
and cost effectiveness, among other factors.

ACKNOWLEDGMENTS

We thank Jason Burkett and his crew at the E.V.
Smith Extension Research Station for assisting with
crop production and field maintenance; Stinson Ellis
and Akin Morakinyo for assisting with field data collec-
tion; and Micky Eubanks, Wheeler Foshee, and Joseph
Kloepper for reviewing an earlier draft of this manu-
script. The following companies are thanked for provid-
ing insecticide formulations and limited funding for this
study: Valent Biosciences (Libertyville, IL), Dow Agro-
Sciences (Indianapolis, IN), and Crompton (now Chem-
tura), Middlebury, CT).

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June 2006







Santiago et al.: Oviposition Behavior of Leptophobia aripa


HOST SELECTION BEHAVIOR OF LEPTOPHOBIA ARIPA
(LEPIDOPTERA: PIERIDAE)

JOSE A. SANTIAGO LASTRA1, LUIS E. GARCIA BARRIOS1, JULIO C. ROJAS2 AND HUGO PERALES RIVERA1
'Departamento de Agroecologia, Carretera Panamericana y Perif6rico Sur s/n
San Crist6bal de Las Casas, Chiapas. M6xico

2Departamento de Entomologia Tropical, El Colegio de la Frontera Sur
Carretera Antiguo Aeropuerto Km. 2.5, Tapachula, Chiapas, M6xico

ABSTRACT

Host selection and egg laying behavior of wild populations of the mountain white butterfly,
Leptophobia aripa (Boisduval), was observed in the presence of a group of host plants (Bras-
sica oleracea L. var. capitata) of varying quality. Host variation was generated by manipu-
lating three crop management variables: fertilization, water, and light. Leptophobia aripa
was not indifferent to host quality variation, and showed great ability to evaluate and dis-
cern among a group of hosts. A sigmoidal relation was found between egg laying and host
plant size. The latter was probably perceived through the host's diameter, or other physical
and chemical characteristics related to this attribute. More detailed studies are necessary in
order to understand which cues this insect uses to locate its host and which other attributes
it evaluates upon deciding to lay eggs. This understanding could allow for the development
of agro-ecological alternatives in controlling this insect, considered to be a crop pest in some
regions of Mexico and Central America.

Key Words: mountain white butterfly, Brassica oleracea, host plant selection, host quality

RESUME

Se observe el comportamiento de selection y oviposicion de poblaciones silvestres de Lepto-
phobia aripa (Boisduval) ante un conjunto de plants hospederas (Brassica oleracea L. var.
capitata) de distintas calidades, generadas mediante cambios en tres condiciones de manejo
del cultivo: fertilizaci6n, riego y luz. Su comportamiento no fue indistinto a las diferentes ca-
lidades de hospedera, sino que obedeci6 a una compleja selecci6n. Mostrando una gran ca-
pacidad para evaluar y discriminar entire el conjunto de hospederas. Se encontr6 una
relaci6n altamente no lineal entire la oviposici6n y el tamaio de la plant, probablemente
percibida a trav6s del diametro de la hospedera, o por otras caracteristicas fisicas y quimicas
relacionadas con este atributo. Son necesarios studios mas detallados que contribuyan a
entender cudles son las seiales que este insecto usa para localizar su hospedera y que otros
atributos evalha al tomar la decision de ovipositar. Esto permitiria desarrollar alternatives
agroecol6gicas para su control, dado que en algunas regions de M6xico y Centroam6rica se
le considerar como plaga.


Translation provided by the authors.


All herbivorous insects show some degree of
host selectivity. Most adult holometabolous spe-
cies must select an appropriate host for larval
growth and survival (Bernays & Chapman 1994).
Under natural conditions, insects confront many
external stimuli, their own internal physiological
stimuli, and a series of environmental constraints
(Visser 1986; Bernays & Chapman 1994; Badenes
et al. 2004). This makes it very difficult to discern
the relative importance to the insect of chemical,
visual, and mechanical stimuli from host and
non-host plants (Schoonhoven et al. 1998; Hooks
& Johnson 2001). However, it is generally as-
sumed that the host selection process in specialist
insects is governed primarily by volatile chemical
signals, later by visual stimuli, and finally by


non-volatile chemical signals (Hern et al. 1996;
Hooks & Johnson 2001).
Female butterflies reject many potential hosts
when searching for egg laying sites. They demon-
strate a hierarchy in host preferences, discrimi-
nating among plant species, among genotypes,
among individuals with different phenological
and physiological conditions, and even among
plant parts, although not all discriminate at the
finer scales (Thompson & Pellmyr 1991; Bernays
& Chapman 1994). However, this knowledge is
derived from studies of very few insect species
(Bernays & Chapman 1994; Schoonhoven et al.
1998). Furthermore, there may be significant be-
havioral differences within a family, among spe-
cies of the same genus, or even among different







Florida Entomologist 89(2)


populations of the same species (Jones 1977;
Singer & Parmesan 1993; Reich & Downes 2003).
To this date, there are no studies on host selec-
tion behavior of the mountain white butterfly,
Leptophobia aripa (Boisduval). This insect is a
multivoltine species with overlapping genera-
tions. Females lay masses of 15 to 80 eggs (Bau-
tista & Vejar 1999). The mountain white butterfly
specializes in the family Brassicaceae, and it is an
important pest of Brassica crops in Southeastern
Mexico, Central America, and the Caribbean
(CATIE/MIP 1990, Santiago et al. in press). How-
ever, it is not known which plant physiological
stage is best suited for oviposition ofL. aripa. In
the case of cultivated plants, crop management
choices may determine the quality of the plant as
a host (Andow 1991).
The objective of the present study was to ob-
serve the egg laying behavior of L. aripa in host
plant patches (Brassica oleracea L. var. capitata)
of different qualities.

MATERIALS AND METHODS

The experiment was established in the Valley
of San Crist6bal de Las Casas Chiapas, Mexico
(2,113 m.a.s.l.; C(v, 'v. -i. Garcia 1973) within the
cabbage production area of the Highlands of Chi-
apas. Cabbage plants of the variety Copenhagen
Market were started in seed beds. Twenty five
days after germination, each seedling was trans-
planted to a black plastic bag (20 cm high by 15
cm in diameter). The bag contained a 1:1 propor-
tion of clay-loam forest soil and sand.
Sixty four plants were prepared. These were
divided into eight groups of eight plants each, and
placed in a greenhouse. In order to generate dif-
ferent host qualities, each group was submitted to
one of eight treatments for 40 days. These treat-
ments consisted of all possible combinations of
two fertilization levels, two watering levels, and
two photosynthetically active radiation (PAR)
levels (Table 1). Nitrogen fertilization was equiv-
alent to 100 kg Ha-1, the most common dose ap-


plied to cabbage in the study zone (Santiago et al.
in press). Treatments were irrigated with high or
low water treatments every four and eight days,
respectively, from August 1 to September 20,
2002. Accumulated irrigations (326 and 183 mm,
respectively) were roughly equivalent to the high
(320 mm) and low (195 mm) average cumulative
rainfalls during the same period, to be found
within the cabbage production zone where L.
aripa was studied (Cervantes 1997).
Sixty five days after germination, the bagged
plants were moved to an open field 200 m from a
cabbage field to promote visits from wild popula-
tions of L. aripa. The 64 bags were randomly dis-
tributed in a square pattern without contiguous
repetitions (Hurlbert 1984), with 50 cm between
plants. Watering treatments were continued
throughout the time of the plants' exposure to
L. aripa.
For five days, L. aripa's flights during host lo-
cation and egg laying behavior were observed (for
1 h per day between 10 a.m. and 2 p.m.) and this
information was recorded. A total of 28 individu-
als were observed from the time they entered un-
til they left the group of host plants. The behavior
of 8 females (that actually laid eggs during the
five recorded hours) was classified into four types
of acts: linear flight, turning flight, landing and
egg laying. Each behavioral act was recorded on
an experiment layout map.
The cabbage plants were reviewed daily in the
afternoon (5 to 5:30 p.m.) for 11 days, and the
number of eggs laid per plant during 9 h of expo-
sure (8 a.m. to 5 p.m.) was recorded. After being
counted, the eggs were carefully removed with a
damp flannel cloth, in order to avoid hatching and
to minimize visual or chemical stimuli from the
eggs which could inhibit egg laying of conspecific
females (Bernays & Chapman 1994). Hilker &
Meiners (2002) reported for Pieris brassicae (L.)
that egg removal might not completely eliminate
such stimuli. However, in this study, L. aripa laid
eggs repeatedly on most plants from which previ-
ously laid eggs were removed.


TABLE 1. DESCRIPTION OF FACTORS AND LEVELS FOR THE TREATMENTS. EACH LEVEL OF A FACTOR WAS COMBINED
WITH BOTH LEVELS OF THE OTHER TWO FACTORS.

Factors Level 1 Level 2

Nutrient (N) N1: Without fertilizer. N2: Foliar fertilizer (20% N 30% P 10% K -
1.6% micronutrients) at a dose of 12.5 g per
plant. N dose equivalent to 100 kg Ha'. Applied
15 days after transplanting.
Water (W) Wl: Watered with a total of 3,240 ml over a pe- W2: Watered with a total of 5,760 ml over a pe-
riod of 51 days. Equivalent to 183 mm of rainfall riod of 51 days. Equivalent to 326 mm of rainfall
from August 1 to September 20. from August 1 to September 20.
PAR(L) LI: Mesh shade which eliminated 64% of the L2: 100% of the photosynthetically active
photosynthetically active radiation inside the radiation inside the greenhouse.
greenhouse.


June 2006







Santiago et al.: Oviposition Behavior of Leptophobia aripa


Each afternoon after sampling, the group of
plants was enclosed with greenhouse plastic in
order to prevent them from receiving rain water
and additional butterfly visits.
Eighty two days after planting, the height and
diameter of plants were measured, and above
ground biomass was harvested to determine fresh
weight per plant. Also, a 2-cm2 leaf sample was
taken from each plant for determining the foliar
nitrogen and chlorophyll concentrations with
standard methods (AOAC 1999).
The experiment was designed to relate oviposi-
tion to host plant management treatments, assum-
ing that the latter produce variation in host plant
parameters that are relevant for egg-laying behav-
ior (Myers 1985; Hern et al. 1996; Hooks & Johnson
2001). To check this assumption, we also explored
to what extent such variation was actually pro-
duced by treatments. Nutrient, water, and light
treatment effects on plant height, diameter, above-
ground fresh weight, leaf nitrogen concentration,
and leaf chlorophyll concentration were analyzed
with three-factor ANOVAs (Underwood 1997).
Because egg laying counts did not meet as-
sumptions of normality due to numerous zero
counts (Underwood 1997), statistical analysis was
performed by logistical regression (Agresti 1996).
A step-wise multiple linear regression analysis
was carried out between the number of eggs laid
and the five parameters measured for each plant.
A non-linear regression model was fitted between
the number of eggs laid and that factor best ex-
plaining the egg-laying pattern observed in the
linear model. Factors discarded in the linear
model were proven to be non significant for the
non-linear model as well. The non-linear regres-
sion model was fitted and selected with the pro-
gram TableCurveTM 2D (AISN Software, Inc.
1994). The statistical software SPSS version
10.0.5 (1999) was used for the remaining analyses.

RESULTS

When a female L. aripa entered the host plant
patches, on average 64% of behavioral acts were
turning flights over the potential hosts, possibly
for recognition and evaluating purposes. Landing
on the host comprised 12% of behavioral acts. Egg
laying was always preceded by a turning flight.
Linear flights also were observed. The latter alter-
nated with turning flights and landings. Sixty per-
cent of linear flights were over lesser-quality hosts
(e.g., non-fertilized plants). A typical search be-
havior in egg-laying L. aripa females is shown in
Fig. 1, which shows that the butterfly flew over al-
most the entire group of plants and selectively laid
eggs on up to four different highest-quality hosts.
The logistical regression model (maximum
likelihood test: x2 = 14.001, df = 3, P = 0.003)
showed a greater probability of oviposition on fer-
tilized plants (N2) than on non-fertilized plants


(N1) (XWald = 4.163, df= 1, P = 0.041). There was a
marginally greater egg laying probability for
plants which received more watering (W2) than
on those which were watered less (W1) (XWald =
3.212, df = 1, P = 0.073). The probabilities of lay-
ing eggs on plants with a greater (L2) and lesser
(L1) PAR availability were not different (XWald =
0.965, df = 1, P = 0.326) (Fig. 2).
None of the interactions among the three fac-
tors was significant: Nutrient x Watering (X2ald =
0.288, df = 1, P = 0.591). Nutrient x PAR (X2ld =
0.039, df = 1, P = 0.843). Watering x PAR (X2ld =
0.088, df = 1, P = 0.767). Nutrient x Watering x
PAR (X2wa = 0.021, df = 1, P = 0.885).
Nutrient, watering, and PAR caused signifi-
cant variation in physical and chemical plant pa-
rameters evaluated in this study (Tables 2 and 3).
Fertilized plants (N2) were taller, had a greater
diameter, greater fresh weight, greater nitrogen
concentration, and greater chlorophyll concentra-
tion than non-fertilized plants (N1). Plants re-
ceiving more water (W2) had a greater diameter
and greater fresh weight, but similar height, ni-
trogen concentration, and chlorophyll concentra-
tion as compared to less watered plants (Wl).
Plants exposed to greater PAR availability (L2)
were the shortest, had a smaller diameter, less
fresh weight, greater nitrogen concentration, and
similar chlorophyll concentration as compared to
plants with less available PAR (Ll). (Some of
these effects of PAR reduction were possibly
caused by better soil humidity conservation in
shaded bags).
Significant Nutrient x Watering interactions
were found for plant weight and crown diameter.
These plant parameters did not respond to nutri-
ent addition at low watering levels, but responded
strongly at high watering levels (Table 2). Signif-
icant Nutrient x PAR interactions were found for
nitrogen concentration.
The step-wise multiple linear regression anal-
ysis determined that fresh weight is the parame-
ter that best explains variation in the number of
eggs laid per plant (R2 = 0.61, df = 59, F = 90.731,
P < 0.0005). The other four attributes evaluated
proved to be non-significant (diameter, P = 0.248;
height, P = 0.245; chlorophyll, P = 0.615; nitrogen,
P = 0.779). When fresh weight was not included in
the analysis, the only parameter selected as sig-
nificant was diameter (R2 = 0.39, df = 59, F =
36.782, P < 0.0005). Again, the other three pa-
rameters were not significant (height, P = 0.905;
chlorophyll, P = 0.718; nitrogen, P = 0.743).
A non-linear regression model was fitted be-
tween fresh weight and number of eggs per plant.
The best among biologically reasonable models
was a sigmoidal function. This function shows an
abrupt increase in the response variable when
the fresh weight of the plant exceeds a threshold,
estimated for this study to be between 30 and 40 g
(Fig. 3).






Florida Entomologist 89(2)


ENTRANCE


EXIT _










IW NIWIL1
1 HH----





1HIM12 N2WIL1
1NM


1 :N2W2L2:





N2W2L2



Fig. 1. Schematized search behavior in an egg-laying female of L. aripa upon entering the group of mixed-quality
hosts. Shaded cells indicate where eggs were laid, each cell contains a replicate of a determined treatment. For ex-
ample N2W2L2 corresponds to fertilized plants, with more watering and with more PAR availability (see Table 1
for notation; only plants intersected by flight are labeled). Note that 11 out of 15 turning flight occurred over N2W2
plants, and all four cases of egg laying occurred on this same class of plants.

DISCUSSION tion behavior is common among Pieridae, but had
not been previously documented for L. aripa.
In this study, L. aripa was offered heteroge- Many studies have shown that Pieridae larvae
neous patches of hosts. Its egg laying behavior was survive and grow better on well fertilized and well
not arbitrary or indifferent to options presented; watered Brassicaceae plants (e.g., Myers 1985;
rather the butterfly showed a capacity to evaluate Chen et al. 2004). Leptophobia aripa preferred to
and discriminate among the group of hosts. Selec- lay eggs on plants that were fertilized and which


June 2006







Santiago et al.: Oviposition Behavior of Leptophobia aripa


125 **
1 20
S15
ia
0 10
5
0
W N1 N2
al



S25 b
S20
S15
S10
5
0
SLW1 W2



S25 NS C
S20

O
0

0
L1 L2


Fig. 2. Average percentage of cabbage plants on
which L. aripa laid eggs (taken from 11 samples), a) N1:
non fertilized plants, and N2: fertilized plants (**P <
0.05). b) W1: plants with less watering, and W2: plants
with more watering (*P < 0.1). c) LI: plants with lesser
PAR availability, and L2: plants with greater PAR avail-
ability. Error bars: 1 SE.



grew under conditions of greater soil humidity. In
this study, host size, probably perceived as foliar
crown diameter, was the plant parameter factor
associated to host preference by L. aripa. Host size
increased significantly when both nutrient addi-
tion and high watering levels were present. Other
plant parameters commonly modified by manage-
ment (Chen et al. 2004), such as volatiles that act
as cues and/or stimulate oviposition, were not
studied and cannot be ruled out.
No single host management factor or host pa-
rameter has explained selection by Pieridae, and
the importance of different factors varies and re-
mains controversial. One of the species most
closely related to L. aripa is Pieris rapae (L.),
whose egg laying behavior has been widely stud-


ied, but remains controversial. For instance, Root
& Kareiva (1984) reported that P rapae follows a
random flight host search, and lays eggs without
discriminating quality factors. Renwick & Radke
(1983) found that P rapae was not attracted by vol-
atile host cues. They also found that host size and
form were not important in egg laying behavior.
Radcliffe & Chapman (1966) did not find a correla-
tion between plant size and P rapae's egg laying
preference. They concluded that color or chemical
stimuli could be determining factors in host choice.
In contrast, other authors have demonstrated that
P rapae's flight and egg laying patterns are modi-
fied by factors such as plant size, phenology, spe-
cies, humidity content, nutrients, leaf color and
plant chemistry (Jones 1977; Latheef & Irwin
1979; Myers 1985; Andow et al. 1986; Jones et al.
1987; Hern et al. 1996; Hooks & Johnson 2001).
Another related species is Pieris virginiensis
(Edwards). Flight and egg laying patterns of P vir-
giniensis are very similar to those of P rapae. Their
flight is markedly linear; they widely disperse
their eggs, and leave behind apparently attractive
hosts. Their egg laying behavior does not respond
to host-plant size (Cappuccino & Kareiva 1985).
Egg laying behavior observed for L. aripa, un-
like that reported for P rapae and P virginiensis,
did respond to plant size. We found a sigmoidal re-
lation, as would be expected with species that lay
eggs in masses and confront host quality hetero-
geneity (Roitberg et al. 1999). Perhaps L. aripa
perceived size through the host's foliar crown di-
ameter, as this was the second most important
plant parameter explaining host selection.
Host selection by Leptophobia aripa also could
have occurred through other size-related physical
and chemical characteristics not evaluated in this
study. These signals could play an important role in
other ecological interactions. For example, Pieris
napi (L.) uses Arabis gemmifera (Mastum.) as a
plant host. This plant species grows covered by
neighboring vegetation, and for this reason is a
host of inferior quality (in nutritional content and
biomass), but it allows P napi to avoid parasitism
by the Cotesia glomerata (L.) wasp and the Epicam-
pocera succincta (Meigen) fly (Ohsaki & Sato 1999).
Fertilization and watering treatments also
could have modified the plant's chemical composi-
tion; in the case of members of Brassicaceae fam-
ily, it could modify glucosinolate concentrations
(Myers 1985; Mewis et al. 2002; Chen et al. 2004).
These secondary metabolites are produced by the
plants as a chemical defense (Renwick & Radke
1983; Lambdon et al. 2003; Miller et al. 2003).
Specialized insects sometimes use these com-
pounds as chemical cues, and even incorporate
them into their body and use them to defend
against predators and parasitoids (Messchendorp
et al. 2000; Mewis et al. 2002). Several crucifer in-
sects are known to have glucosinolate detoxifica-
tion and sequestration mechanisms (Wadleigh &







Florida Entomologist 89(2)


TABLE 2. SUMMARY OF RESULTS FROM THREE-FACTOR ANOVAS TESTING THE EFFECTS OF NUTRIENT (N), WATER (W),
AND PAR (L) ON PLANT HEIGHT, DIAMETER, ABOVE-GROUND FRESH WEIGHT, LEAF NITROGEN CONCENTRA-
TION, AND LEAF CHLOROPHYLL CONCENTRATION. TEST OF SIGNIFICANT P VALUES < 0.05 ARE IN BOLD.

Height Diameter Weight Nitrogen Chlorophyll

Source df MS F MS F MS F MS F MS F

N 1 1.6 14.9 619.1 38.0 30.7 41.2 0.2 11.9 2.3 13.5
W 1 0.4 3.7 377.5 23.2 10.1 13.6 0.0 0.1 0.2 1.2
L 1 1.6 14.8 691.5 42.5 24.9 33.4 0.2 9.0 0.3 1.8
NXW 1 0.1 0.6 258.6 15.9 5.2 7.0 0.1 2.8 0.0 0.1
NXL 1 0.4 4.0 23.0 1.4 1.6 2.1 0.2 9.0 0.0 0.0
WXL 1 0.3 2.7 30.9 1.9 2.5 3.4 0.0 0.6 0.3 1.5
NXWXL 1 0.3 3.1 55.8 3.4 2.9 3.9 0.0 0.4 0.0 0.2
Error 52 0.1 16.3 0.7 0.0 0.2


TABLE 3. MEAN (+ 1 SE) OF PLANT HEIGHT, DIAMETER, ABOVE-GROUND FRESH WEIGHT, LEAF NITROGEN CONCENTRA-
TION, AND LEAF CHLOROPHYLL CONCENTRATION FOR EACH FACTOR LEVEL.

Factor Height Diameter Weight Nitrogen Chlorophyll

N1 7.9 (0.5) 12.3 (0.9) 8.0 (1.2) 4.0 (0.1) 0.5 (0.1)
N2 11.2 (0.7) 19.6 (1.3) 37.7 (5.5) 4.6 (0.2) 0.9 (0.1)
W1 8.7 (0.6) 13.3 (1.0) 12.5 (2.0) 4.3 (0.2) 0.7 (0.1)
W2 10.2 (0.7) 17.9 (1.4) 30.6 (5.6) 4.3 (0.1) 0.6 (0.1)
L1 10.7 (0.5) 18.9 (1.0) 28.2 (4.8) 4.1(0.1) 0.8 (0.1)
L2 8.1(0.7) 12.0 (1.3) 14.5 (3.9) 4.5 (0.2) 0.6 (0.1)


Yu 1988). Miller et al. (2003) did not find glucos- Another manner in which L. aripa could be at-
inolate sequestration in P. rapae and P brassicae; tracted to larger plants is that observed in R bras-
the case for L. aripa still needs to be studied. sicae. This species, like L. aripa, tends to lay eggs



300


250 *

y=3.96+162.07/(1+EXP(-(x-36.10)/2.85))
Z 200
SRLO.68
CL
4) 150


i 100 -


50



0 20 40 60 80 100

Fresh Weight (g)

Fig. 3. Non-linear regression between fresh weight of cabbage plants and number of eggs laid by L. aripa per
plant throughout 11 days of exposure. (R2= 0.68, df = 59, F = 39.934, P < 0.001).


June 2006







Santiago et al.: Oviposition Behavior of Leptophobia aripa


in large masses when locating large-size hosts
with abundant leaves (Stamp 1980; Le Masurier
1994). The aggregate lifestyle and conspicuous
coloration of its larvae may provide a defense
against predators and parasitoids (Stamp 1980;
Le Masurier 1994).
In many cases, insect egg laying behavior re-
sults from balancing among factors which include
minimizing parasitic and predatory risk, select-
ing the most nutritious host, avoiding intra-spe-
cific competition for food, and maximizing egg lay-
ing (Myers 1985; Ohsaki & Sato 1999). The insect
internally weighs the various stimuli and inhibi-
tors perceived through visual, chemical, and me-
chanical signals (Thompson & Pellmyr 1991;
Hern et al. 1996).
Leptophobia aripa's searching and egg laying
behavior observed in this study demonstrates its
capacity to evaluate and discriminate among a
group of hosts. Egg laying preference associated
to host size has also been found for P brassicae
but not for P. rapae, P virginiensis and P napi.
This confirms that related species may have sig-
nificantly different behavior (Jones 1977; Singer
& Parmesan 1993; Reich & Downes 2003).
Leptophobia aripa is a pest for Brassicaceae
crops in some regions of Mexico and Central
America. Producers in the region have adopted
fertilizers and pesticides rather recently (Santi-
ago et al. in press). Agroecological alternatives to
heavy agrochemical use are desirable. Our find-
ings suggest that nutrient addition to well-wa-
tered plants significantly increases plant weight
(as expected) and, beyond a plant weight thresh-
old, it also increases oviposition. It is important to
study to what extent increased oviposition affects
larval survival and growth, and cabbage head
damage. Other plant parameters such as produc-
tion of cue volatiles need to be investigated and
their relation with plant size established. It is
also important to study tradeoffs between plant
size, cabbage head value, and crop damage
caused by L. aripa, as well as the capacity of al-
ternative management strategies (e.g., intercrop-
ping and moderate organic fertilization) to im-
prove tradeoffs.

ACKNOWLEDGMENTS

We thank Carlos V. P6rez Rodriguez and Juan Col-
lazo L6pez for technical support in the field, and Juan J.
Morales L6pez and Jesis Cannona de La Torre for
chemical analysis. This study was supported by a grant
from Fundacion Produce Chiapas, A. C., and by a grad-
uate scholarship from CONACYT to JASL. We also
thank two anonymous reviewers for useful and con-
structive comments.

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June 2006







Deyrup et al.: Cannibalism in a Male Parasitic Wasp


FEEDING AND SIBLICIDAL CANNIBALISM IN
A MALE PARASITIC WASP (HYMENOPTERA: EULOPHIDAE)

LEIF D. DEYRUP1, ROBERT W. MATTHEWS' AND MARK DEYRUP2
'Department of Entomology, University of Georgia, Athens, GA 30602, USA

2Archbold Biological Station, Florida, P.O. Box 2057, Lake Placid, FL 33862, USA

ABSTRACT

Melittobia digitata Dahms is a small parasitic wasp known for its lethal male combat but
subject to controversy regarding the occurrence of male feeding and cannibalistic feeding in
particular. Here we report our observations supporting siblicidal cannibalism. To test the
ability of a male's capability to feed we smeared sugary dye on the wasps' mouthparts and
observed the dye passing through the digestive system to produce colored feces, confirming
that males have a complete digestive tract. To document siblicidal feeding we injected other
males with water-soluble dye, and paired them with undyed males. Undyed winners that ap-
peared to feed on dyed losers were monitored; dye was evident in their feces. Finally, to de-
termine if males benefit from feeding, we compared the longevity of artificially fed and unfed
males; fed males lived significantly longer than non-fed males (Mann-Whitney Utest = 81.5,
N, = 26, N, = 26, P < 0.001). We discuss possible reasons for the comparative rarity of sibli-
cidal cannibalism and its fitness implications.

Key Words: Melittobia, kin selection, uneven sex ratios, male combat

RESUME

Melittobia digitata Dahms es una avispa parasitoide conocida por sus combates letales entire
machos pero que esta sujeta a controversial respect a la existencia de alimentaci6n por estos
in general, y canibalismo en lo particular. Se reportan aqui nuestros hallazgos en cuanto a
canibalismo. Para probar la habilidad de un macho para comer se le unto una pasta azuca-
rada coloreada en las parties bucales de la avispa. Se observe pasar a trav6s del sistema di-
gestivo para producer heces de color, confirmando asi que los machos tienen un sistema
digestive complete. Para documentary canibalismo entire hermanos se inyect6 a otros machos
un colorante soluble en agua y se colocaron con machos normales. Se monitorearon los ga-
nadores no coloreados parecieron alimentarse sobre los perdedores coloreados; el colorante
era evidence en sus heces. Finalmente, se determine el beneficio de alimentarse, compa-
rando la longevidad de machos alimentados y no alimentados (artificialmente); los machos
alimentados vivieron significativamente mas tiempo que los no alimentados (Prueba U de
Mann-Whitney = 81.5, N1 = 26, N = 26, P < 0.001). Discutimos razones posibles para la ra-
reza del canibalismo entire hermanos y sus implicaciones adaptativas.


Melittobia (Hymenoptera: Eulophidae) are
small, gregarious parasitoids of solitary wasps
and bees, and assorted associates (Edwards &
Pengelly 1966; Krombein 1967; Maeta & Yamane
1974). These parasitoids have intrigued biologists
(e.g. Hamilton 1967) because of their unusual and
highly inbred reproductive strategy. Melittobia
digitata Dahms is also used in educational curric-
ula under the name WOWBug (Matthews et al.
1996, 1997).
Upon finding a suitable host, the female Melit-
tobia stings it, feeds on hemolymph exuding from
the sting wound, and then lays several hundred
eggs, of which over 90% develop into females
(Buckell 1928; Schmieder 1938; Dahms 1984). Fe-
males mate once with a brother, and then cooper-
ate to chew an exit hole and disperse (Deyrup et
al. 2005); their brothers remain behind to die
within their natal host's cocoon (Dahms 1984).


While males' lives may be circumscribed
within their natal cocoon, they are nonetheless
action-filled. Males of most Melittobia species are
highly pugnacious and frequently engage in fatal
fights with their brothers (e.g., Graham-Smith
1919; Malyshev 1968; Matthews 1975; Hamilton
1979; Hartley & Matthews 2003; Abe et al. 2003);
attacks on male pupae are also documented (Her-
mann 1971; Abe et al. 2005) and, because Melit-
tobia are protandrous, male fighting can begin be-
fore the first females emerge. Occasionally, how-
ever, males also will attack females presented to
them (Balfour-Browne 1922; Hermann 1971;
Matthews 1975; Dahms 1984).
There has been speculation as to whether, in
addition to the obvious advantage of dispatching
potential rivals, such attacks might provide an
opportunity for males to feed (Matthews 1975).
Several biologists have gone on record as doubt-







Florida Entomologist 89(2)


ing that Melittobia males feed at all. For example,
while Dahms (1984) observed attacks, he found
no evidence of feeding and pointed out that a
male's gaster grows increasingly thinner until he
dies. Abe et al. (2005) categorically state that
males of M. australica Girault do not feed. Bal-
four-Browne (1922) noted chewing attacks, but
considered them to be an artifact of experimental
conditions. Others disagree, reporting that males
sometimes continue to chew on a defeated male
sibling (Graham-Smith 1919; Matthews 1975) or
on an attacked female (Hermann 1971) for rela-
tively extended periods of time. If they were to in-
gest nutrients during this behavior, such canni-


balism might provide a competitive advantage
(Matthews 1975), enabling a male to live longer or
produce more sperm.
Combat between males of Melittobia digitata
is particularly intense. We noticed that M. digi-
tata males in our laboratory cultures sometimes
spent an extended period of time with their man-
dibles immersed in the tissues and hemolymph of
a defeated male (Fig. 1). In one instance, a male
killed an emerging male by biting through the
emerging male's head capsule, and then inserted
his mandibles deeper into the head capsule. The
victor's palpi were highly active, with motions re-
sembling those of feeding females. As we watched,


Fig. 1. A male of Melittobia digitata Dahms that appears to be feeding on a sibling male (Photo courtesy of Jorge
M. Gonzalez).


June 2006







Deyrup et al.: Cannibalism in a Male Parasitic Wasp


the abdomen of the male began to swell slightly,
as if hemolymph were filling the crop. This obser-
vation lent support to a hypothesis that M. digi-
tata males sometimes feed on a defeated male,
and encouraged our experimental approach to
male feeding with three objectives. The first was
to determine whether male M. digitata have a
functional digestive tract. The second was to re-
solve whether males ingest hemolymph from
other males, and, if so, whether it passes through
their digestive system. The last was to test
whether individual males benefit from feeding.

MATERIALS AND METHODS

Experiment 1: Functional Digestive Tract

We collected 40 M. digitata male pupae (recog-
nizable by the lack of compound eyes) developing in
a single laboratory culture, isolated them individu-
ally in small tightly lidded plastic boxes (50 x 25 x
18 mm, Carolina Biological Supply Co., Cat. No.
ER-14-4584), recorded eclosion dates, and ran-
domly assigned the adults either to the control or
experimental group. The controls were undis-
turbed. When wasps in the experimental group
were 2 days old, we smeared their mouthparts with
either "willow green", "cornflower blue", or "rose
petal pink" cake icing dye (Wilton Enterprises).
After the passage of several hours to allow op-
portunity for treated males to groom, all males
were transferred into clean boxes. We checked the
boxes daily and recorded whether colored fecal
droppings appeared. We also observed the males
under a dissecting microscope to check for dye in
their digestive system, and found that it was
clearly visible through their translucent cuticle. A
?2 test in was used to determine whether individ-
uals in the experimental and control groups dif-
fered significantly in passing colored fecal spots
vs. undyed spots (Statistica 6.0).

Experiment 2: Feeding on Another Male

Because we reasoned that nutritionally
stressed males would be more likely to feed, we
stressed males by isolating individual late male
pupae (+1 d until eclosion) and providing each with
10 newly closed virgin females. Males were al-
lowed to mate ad libitum with these females for up
to 5 days post-eclosion. After 3-5 days the males'
gasters became thin and they appeared emaciated.
To produce weakened males with identifiable
hemolymph as potential losers, we injected them
in the abdomen with blue water-based dye (Mc-
Cormick & Co., Inc.) using a glass pipette (Soda
Lime Glass, 9", J. & H. Berge, Inc.) that had been
stretched while heating it in an alcohol flame.
Typically, the dyed male rapidly weakened, and
was usually dead in 10 to 15 min.


An emaciated undyed male and a "weak"
freshly dyed male were paired in a deep well pro-
jection slide arena (Carolina Biological Supply,
Inc.). Because there was only a short window of
opportunity for combat, we placed them next to
each other to facilitate interaction; even then,
most fighting was non-lethal. Even after lethal
fights, most males did not attempt to feed on their
defeated brother. However, we continued to dye,
expose, and observe the males until we recorded
10 instances of undyed victors that killed their
dyed brother and appeared to feed upon them.
Each of these victors was placed into a separate
observation box and observed for subsequent dye
passage in its fecal droppings.

Experiment 3: Benefit from Feeding

To determine whether males benefit from feed-
ing, we gathered 55 M. digitata male pupae from
five cultures, isolated each pupa in a glass 1 dram
vial, and inspected the vials daily, recording the
date on which each male closed; 52 pupae closed
as adults. Males that closed on the same day
were assigned to an experimental (fed group, n =
26)) or a control (unfed, n = 26) group.
The experimental group was fed insect
hemolymph from a Trypoxylon (Trypargilum) pol-
itum Say prepupa. Using an insect pin to punc-
ture the host prepupal cuticle, we bled one drop of
hemolymph onto a glass slide then gently trans-
ferred a male to the drop with a fine brush. Males
immediately imbibed hemolymph from the drop.
When a male did not drink voluntarily, we coaxed
its head into the drop. The males invariably fed
when their mouthparts touched the hemolymph,
and we allowed males to feed to satiation. The
control group of males was not fed. We did not give
them water or insect saline solution; such re-
sources do not occur in their natural habitat, be-
cause males seldom, if ever, leave the pupa case of
their host.
All individuals in both groups were individu-
ally isolated in 1-dram glass vials and placed in
an incubator at 30C. We recorded how many days
each male survived. The difference between the
treatment and the control groups was analyzed
using a Mann-Whitney U test and a survival
analysis (Statistica 6.0).

RESULTS

In the first experiment, all colors of dye were
immediately visible passing through the upper di-
gestive system into the crop of all 20 treated
males, and color appeared in their droppings
when checked 24 h later. Whereas all males leave
at least some fecal specs, no control males ever
had droppings of a color similar to those of the fed
males. This difference was very highly significant
using a X2 test (x2 = 40.0, P < 0.001) (Statistica 6.0).







Florida Entomologist 89(2)


In experiment 2, each of the males that we had
suspected of feeding on his brother had blue color
moving through the body and into the crop. This
was confirmed when we checked 24 h later that
dye was passed in droppings of all 10 individuals.
In experiment 3, individual male adult life
spans varied, ranging from 12-16 d for unfed
males, and from 13-18 d for fed males (Fig. 2).
However, the lives of fed males werel.5 d longer,
on average, than those of unfed males (unfed SE
= 13.2 + 0.14, x = 13: fed + SE = 14.7 + 0.21, x =
15). Statistically, the difference was very highly
significant (Mann-Whitney U test = 81.5, N, = 26,
N,= 26, P < 0.001) (Statistica 6.0).

DISCUSSION

The results from experiment 1, demonstrating
that the digestive tract of male M. digitata is com-
plete and apparently functional, led to the second
experiment, which established that males that
defeat another male are capable of ingesting
hemolyph from the defeated individual. The com-
bination of these two experiments supports the
assumption that the apparent feeding behavior
that we had previously observed was correctly in-
terpreted because males of M. digitata have a
functional digestive tract and are capable and will
imbibe hemolymph from another male.
We showed that M. digitata can feed, but our
findings may not apply to all species in the genus.
For example, M. femorata Dahms does not appear
to have the same propensity for lethal male com-
bat as M. digitata (R. W. M., unpublished data).
While an M. femorata male conceivably could feed
on a killed female, it would be unlikely to feed on
a brother.
Records of feeding by adult male parasitoids
are rare. Males of few species have access to


Fig. 2. Longevity of fed and unfed males of M. digi-
tata at 30 C (dotted line and V = males that were fed
host hemolymph; solid line and O = males that were un-
fed).


hemolymph, and M. digitata seems to take an ad-
vantage of an unusual situation. Nectar is a more
usual food source for adult Hymenoptera, but nec-
tar-feeding by parasitoids is also rare, and con-
centrated in a few families. At the Archbold Bio-
logical Station (Highlands Co., FL), where flower
visitors have been studied for many years, there
are few records of nectar feeding by male parasi-
toids. Among Ichneumonoidea, nectar feeding oc-
curs in male Agathis longipalpus (Cresson) (Bra-
conidae); among Chalcidoidea nectar feeding oc-
curs in male Leucospis affinis Say, L. robertsoni
Crawford and L. slossonae Weld (Leucospidae). In
contrast, male aculeate Hymenoptera are fre-
quent nectar feeders at the Archbold Biological
Station, including numerous species representing
15 families (M.A.D., unpublished data).
Reports of adult male siblicidal cannibalism in
insects are relatively rare. A situation somewhat
similar to that of Melittobia occurs in ants of the
genus Cardiocondyla; ergatoid males engage in
lethal combat, usually won by an older male that
attacks a recently closed sibling (Stuart 1987;
Heinze et al. 1998). In this genus, however, work-
ers remove the dead male from the nest or feed it
to larvae (Stuart 1987). The situation confronting
Melittobia males differs from that of ants in that
Melittobia males exist in a closed system, without
access to external resources.
In mites, female cannibalism has been re-
ported (Schausberger & Croft 2000; Berndt et al.
2003), but its possible siblicidal nature seems to
require further study. Schausberger & Croft
(2000) reported that Phtoseiulus persimilis Ath-
ias-Henriot preferentially cannibalized non-sib-
lings, but later Schausberger (2003) reported that
if raised without contact with siblings, they pref-
erentially cannibalized siblings. Melittobia digi-
tata males have been reported to occasionally kill
female siblings but whether they also cannibalize
them is not clear (Gonzalez & Matthews 2005).
Cannibalism for its own sake would seem to
have several potential disadvantages. The three
most applicable to M. digitata males are the risk
of being injured or killed in attacking a similarly
capable individual, the risk of contracting a dis-
ease from the consumed individual, and the evo-
lutionary cost to fitness (Elgar & Crespi 1992).
However, like M. digitata attacking male pupae,
some species seem to avoid the problem of attack-
ing a similar organism when early maturing indi-
viduals or individuals of a more advanced devel-
opmental stage kill a less capable immature indi-
vidual (Elgar & Crespi 1992). However, this is not
always the case; for example, cannibalism on
peers has been recorded in intrauterine sharks
(Wourms 1977; Hamlett & Hysell 1998). In M.
digitata violent combat, presumably evolved in
the context of local mate competition, usually
quickly incapacitates the defeated male, thereby
removing the risk of further injury. This would


June 2006







Deyrup et al.: Cannibalism in a Male Parasitic Wasp


leave victorious males free to consume the de-
feated male without further risk. Similarly, can-
nibalism among male Melittobia digitata seems
unlikely to transmit disease, as the combatants
are usually siblings, having fed off the same host,
and lived their entire lives inside a sealed cocoon.
The third potential disadvantage, loss of fitness in
sibling competition, is a complex issue; kin selec-
tion models have endeavored to deal with this
problem (Griffin & West 2002). However, canni-
balism after combat adds yet another advantage
in M. digitata male competition.
The third experiment showed that males who
fed lived significantly longer than unfed controls.
Lengthening one's adult life by the equivalent of
11% is no biologically trivial matter; presumably,
those males that live longer secure more mates,
dispatch more rivals, and have increased fitness
relative to unfed males. Wiltz and Matthews (un-
published) found that males are more likely to die
before exhausting their sperm, which makes lon-
gevity a better indicator of increased fitness than
sperm production. We have observed males feed-
ing on closing males and on pupae that are more
vulnerable. Added longevity in males that emerge
with the first generation of a few short wing fe-
males would benefit greatly in fitness by the ex-
tended overlap with the subsequently emerging
group long wing females. Wiltz and Matthews
(unpublished) study and our observations expose
the possible benefits for males who can extend
their lifespan by feeding.
We conclude that male cannibalism in M. digi-
tata may not be rare when the advantages out-
weigh the disadvantages. The natural history of
M. digitata appears to satisfy this criterion. The
fact that a single male can potentially inseminate
over 200 sisters and is likely to die before exhaust-
ing his sperm (B. Wiltz & R. Matthews, unpub-
lished), as appears to occur routinely in some
Melittobia species, provides a context in which
male feeding and increasing life expectancy would
be advantageous. Male M. digitata that defeat and
then cannibalize brothers may also obtain nutri-
ents needed to maintain sperm production and sex
pheromone production (Cons6li et al. 2002) for an
extended life expectancy, as well as acquire the en-
ergy needed to successfully combat newly closing
brothers (Abe et al. 2005) and repeatedly perform
the relatively elaborate courtship displays that
characterize the genus (Matthews & Matthews
2003, Gonzalez & Matthews 2005).

ACKNOWLEDGMENTS

We thank and appreciate Jan Matthews and Jessica
Beck for reviewing the manuscript, Jorge M. Gonzalez
for the photograph and stimulating discussion, and Stu-
art West for his knowledge and insightful ideas. This
project was supported by an NSF grant (R. W. Mat-
thews, P.I.).


REFERENCES

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June 2006







Davis & Deyrup: New Parasitic Solenopsis


SOLENOPSIS PHORETICA, A NEW SPECIES OF APPARENTLY
PARASITIC ANT FROM FLORIDA (HYMENOPTERA: FORMICIDAE)

LLOYD R. DAVIS'AND MARK DEYRUP2
'3920 NW 36th Place, Gainesville, FL 32606

2Archbold Biological station, P.O. Box 2057, Lake Placid, FL 33862
e-mail: mdeyrup@archbold-station.org

ABSTRACT

A new species of ant, Solenopsis phoretica, is described from a dealate queen found clinging to
the petiole of a nest queen ofPheidole dentata Mayr in Gilchrist County, Florida. The position
of the Solenopsis queen, as well as details of its morphology, strongly suggest that it represents
a parasitic species. It is distinguished from other Solenopsis by its concave clypeal area and
slender, elongate mandibles with an enlarged basal tooth. A single specimen is known.

Key Words: parasitic ant, parasitic Solenopsis, parasitic fire ant

RESUME

Se describe una nueva especie de hormiga, Solenopsis phoretica de una reina dealatada (que
boto las alas) encontrada colgada al peciolo de la reina hormiga de Pheidole dentata Mayr en
el condado de Gilchrist en la Florida. La posici6n de la reina de Solenopsis y los detalles de
su morfologia, sugiere fuertemente que esta represent una especie parasitica. Se distingue
esta especie de otras Solenopsis por tener la area del clipeo concavo y la mandibula elongada
y delgada con un diente basal engrandecido. Un solo specimen es conocido.


Solenopsis is a genus of over 180 described spe-
cies (Bolton 1995). The genus shows variable hab-
its. Many species are polyphagous, above-ground
foragers, such as the notorious pest, Solenopsis in-
victa Buren. Other species, especially those species
formerly placed in the subgenus Diplorhoptrum,
are primarily subterranean foragers. Some of these
subterranean species may issue from small galler-
ies to carry off food and larvae from brood chambers
of other ants (Holldobler & Wilson 1990). A few spe-
cies of Solenopsis are workerless parasites that
were at one time placed in the genera Labauchena
or Paranamyrma (Ettershank 1966). Here, we de-
scribe a new species of Solenopsis based on a single
dealate queen. This species appears to be parasitic
on other ants, but we do not know whether it is
workerless, nor do we know whether it is closely re-
lated to any other parasitic species.
Character states defining Solenopsis are de-
tailed by Ettershank (1966). In the North Ameri-
can fauna the genus can be recognized by the
combination of a few character states: two-seg-
mented petiole; two-segmented antennal club;
propodeum lacking spines or angles; clypeus lon-
gitudinally bicarinate, with a median, apical mar-
ginal seta. The clypeal features are lacking on the
species described below.

Solenopsis phoretica, Davis and Deyrup
new species

Diagnosis of dealate female (Fig. 1): The
dealate female is distinguished from other Sole-


nopsis by the following combination of character
states: mandibles elongate, teeth lacking or vesti-
gial, except for apical point and enlarged basal
angle; clypeus concave, smooth.
Description of holotype dealate female: fea-
tures visible in lateral view described from left
side. Measurements in mm. Total length (length
of head excluding mandibles + length of mesos-
oma + length of petiole + length of postpetiole +
length of gaster): 3.03; head length: 0.55; head
width at rear margins of eyes in frontal view:
0.55; length of mesosoma: 0.88; length of petiole:
0.30; length of postpetiole: 0.20; length of gaster
1.10. Color: yellowish brown, appendages yellow.
Head: smooth, shining, sparsely covered with set-
igerous punctures separated by 2-8 times the
width of a puncture, setae suberect, directed pos-
teriorly in the frontal area, elsewhere directed an-
teriorly; ocelli not enlarged, each ocellus about
the width of antennal scape at base; malar area
long and narrow, slightly shorter than length of
eye; mandibles elongate, over half the length of
head at midline, apical tooth elongate, delimited
proximally by a narrow notch apparently repre-
senting a vestigial tooth, inner profile of mandible
strongly concave, concavity delimited proximally
by strongly produced basal angle with a truncate
apex; clypeus smooth, concave, without carinae,
with four subapical elongate setae; antennae 10-
segmented, scape reaching outer corners of head
in frontal view, antennal club 2-segmented, club
about as long as remainder of funiculus. Mesos-
oma: smooth and shining, with sparse setigerous







Florida Entomologist 89(2)


k ,;


Figure 1. Solenopsis phoretica, new species, dealate queen, lateral view (above) and frontal view of head. Ac-
tual length of insect: 3.03 nunm.


punctures on pronotum, near margins of mesono-
tum, mesopleura, sides of propodeum; disc of me-
sonotum and declivity of propodeum unpunc-
tured; propodeum evenly declivitous in lateral
view, only slightly convex; legs smooth, shining,
with sparse, strong, semidecumbent, distally-di-
rected hairs. Petiole: peduncle short, less than
0.25 length of base of petiole in lateral view; peti-
ole in lateral view triangular, apex broadly and
smoothly rounded; in posterior view apex strongly
convex; ventral process narrowly expanded, with
a small triangular tooth. Postpetiole: low and
rounded above in lateral view, in posterior view
about 1.5 times as wide as long, broadly convex.
Gaster: in dorsal view with prominent, rounded
anterior corners of first tergite; first tergite cov-
ered with sparse, long, posteriorly-directed hairs
that are longer than the distance between them
and emerging from inconspicuous punctures;
tergites 2-4 smooth, with a subapical row of hairs.
Type locality and associated information: col-
lecting data on label of holotype: FL: Gilchrist
Co., Route 47, 2.5 miles north of junction with
Route 232, 9 February 1992, Lloyd R. Davis. Man-
dibles locked around petiole of nest queen of
Pheidole dentata.
We deposited the holotype specimen in the
Museum of Comparative Zoology, Harvard Uni-
versity, Cambridge, MA.


Etymology: species epithet derived fromphore-
tos (Greek), meaning "carried," referring to the
phoretic relationship between the holotype and
the nest queen ofPheidole dentata.

DISCUSSION

It is generally undesirable to describe a spe-
cies of ant on the basis of a single queen. By con-
vention and convenience, ant holotypes are gener-
ally workers. Workers, as well as males, may be
very different from queens. In this case, no addi-
tional specimens have been found since the date
of capture in 1992. Our intent is to alert the
myrmecological community to this unusual spe-
cies, in the hope that this exposure may lead to
the discovery of more specimens and more natu-
ral history information.
Only a limited amount of speculation is justi-
fied, as only a single specimen is available. The
generic placement of S. phoretica is based on its
general resemblance to queens of such small Sole-
nopsis species as S. carolinensis Forel and S. ab-
dita Thompson. Resemblances include the two-
segmented antennal club, smooth and shiny in-
tegument, the type and placement of setigerous
punctures, and the shape of the petiole and post-
petiole. If, however, the antennal club were three-
segmented, rather than two-segmented, the spe-


June 2006







Davis & Deyrup: New Parasitic Solenopsis


cies could be plausibly placed in the genus Mono-
morium. We were also influenced by the prece-
dent of parasitic species of Solenopsis with re-
duced or absent clypeal carinae, such as the South
American S. daguerrei (Santschi). There is no ev-
idence, however, that S. phoretica is closely re-
lated to S. daguerrei and its relatives. The latter
species lacks a number of features found in S.
phoretica: enlarged punctures bearing short setae
on the head and mesosoma; angulate subpetiolar
process; falcate mandibles with a strongly pro-
jecting basal angle. The petiole of S. daguerrei is
sharply angulate above in lateral view, the post-
petiole is narrow in posterior view, the anterior
edge of the mesonotum is slightly protuberant,
overhanging the pronotum, and the inner mar-
gins of the mandibles are oblique with four teeth
(including the apical tooth).
We suspect that S. phoretica is parasitic be-
cause it was found clinging to the petiole of a nest
queen ofPheidole dentata and because the mandi-
bles and concave clypeal area fit exactly around
the petiole. The radical nature of the clypeal and
mandibular modifications suggest a relatively
long phoretic association, although not necessar-
ily with P. dentata. There are other local ants, such
as Pheidole crassicornis Emery, Solenopsis gemi-
nata (Fabricius) and possibly S. pergandei Forel
that have a petiole that might well accommodate
the mandibles of S. phoretica. A phoretic relation-
ship in which the parasite is attached to the peti-
ole of the host queen is, to our knowledge, unique
in ants. Solenopsis daguerrei queens cling to the
neck of their host queen, immobilizing her, and
greatly decreasing her reproductive ability (Sil-
veira-Guido et al. 1973). There is also a highly spe-
cialized parasitic ant, Teleutomyrmex schneideri
Kutter, whose queens ride about unattached on
the host queen (H6lldobler & Wilson 1990).
It is impossible to define, on the basis of our
single observation, the nature of the suspected


parasitic relationship. Solenopsis phoretica seems
equipped for a prolonged period of phoresy on its
host, but it is still possible that S. phoretica dis-
mounts after it is fully imbued with the odor of
the host queen. It is tempting to suggest, by anal-
ogy with known parasitic Solenopsis, that S.
phoretica is a workerless parasite, but there is no
evidence of this, aside from the absence of worker
Solenopsis in the host nest. Whatever relation-
ship S. phoretica may have with its host, it is cer-
tain to be interesting and unusual. We hope that
myrmecologists and other naturalists working in
southeastern North America will be on the look-
out for this species. It might not be necessary to
find nest queens of the host, as at some point in
the life cycle of S. phoretica there should be nu-
merous alate S. phoretica in the host colony.


ACKNOWLEDGMENTS

We thank Stefan Cover for helpful suggestions and
encouragement, and anonymous reviewers for useful
comments. This project was supported by the Archbold
Biological Station.


LITERATURE CITED

BOLTON, B. 1995. A New General Catalog of the Ants of
the World. Harvard University Press, Cambridge,
MA. 504 pp.
ETTERSHANK, G. 1966. A generic revision of the world
Myrmicinae related to Solenopsis and Pheidologeton
(Hymenoptera: Formicidae). Australian J. Zool. 14:
73-171.
HOLLDOBLER, B., AND E. O. WILSON. 1990. The Ants.
Harvard University Press, Cambridge, MA. 732 pp.
SILVEIRA-GUIDO, A., J. CARBONELL, AND C. CRISCI.
1973. Animals associated with the Solenopsis (fire
ants) complex, with special reference to Labauchena
daguerrei. Proc. Tall Timbers Conf. Ecol. Animal
Control Habitat Management 4: 41-52.







Florida Entomologist 89(2)


June 2006


ASSESSMENT OF FEMALE REPRODUCTIVE STATUS
IN ANASTREPHA SUSPENSE (DIPTERA: TEPHRITIDAE)


PAUL E. KENDRA, WAYNE S. MONTGOMERY, NANCY D. EPSKY AND ROBERT R. HEATH
USDA-ARS, Subtropical Horticulture Research Station,13601 Old Cutler Road, Miami, FL 33158-1334

ABSTRACT

Reliable methods are needed for assessing sexual maturity in field-captured tephritid fruit
flies. To provide such a tool for female Caribbean fruit flies, Anastrepha suspense (Loew), this
study documented changes in ovarian development over a four-week period following adult
eclosion. The ovarian maturation process was classified into six developmental stages.
Stages 1-4 described sequential steps in the development of immature ovaries, stage 5 indi-
cated presence of mature oocytes, and stage 6 was the ovipositional phase. For each stage,
four morphometric characters were examined-length of ovary, width of ovary, an ovarian in-
dex (length of ovary multiplied by width of ovary), and length of terminal follicle. Ovarian
characters were compared by stage and correlated with the number of mature oocytes per
ovary (egg load). Ovarian index maximized the differences between sexually mature and im-
mature ovaries, and ovary length provided the best separation of immature stages. All four
characters were positively correlated with egg load, but ovarian index and ovary width were
the two best indicators of mature oocytes. Use of these parameters to assess egg load would
eliminate the need to tease apart ovaries and count mature oocytes, thereby providing an ef-
ficient method for processing large samples of flies. Classification of female sexual maturity
based on an ovary staging system, in conjunction with assessment of egg load in mature
stages, would facilitate evaluation of the physiological age structure of a fly population cap-
tured in field deployed traps.

Key Words: Caribbean fruit fly, ovary development, sexual maturation, oocyte, egg load

RESUME

Se necesitan m6todos confiables para apreciar la madurez sexual de las moscas de la familiar
Tephritidae que han sido capturadas en el campo. Para proveer una media para las hem-
bras de la mosca de fruta del Caribe, Anastrepha suspense (Loew), 6ste studio document
cambios en el desarrollo del ovario sobre un peri6do de cuatro semanas despues de la ecloci6n
del adulto. La madurez del ovario fu6 clasificada en seis etapas de desarrollo. Etapas 1-4 des-
cribieron los pasos en secuencia en el desarrollo del ovario inmaduro, etapa 5 indic6 la pre-
sencia de oocitos maduros, y la etapa 6 fu6 la fase oviposicional. Por cada etapa, cuatro
caricteres morfom6tricos fueron examinados-longitud del ovario, anchura del ovario, un in-
dice del ovario (longitud del ovario multiplicado por la anchura del ovario), y longitud del fo-
liculo terminal. Los caricteres del ovario fueron comparados por etapa y correlacionados con
el numero de oocitos maduros por cada ovario (carga de huevos). El indice del ovario aument6
las diferencias entire los ovarios sexualmente maduros e inmaduros, y la longitud del ovario
provey6 la mejor separaci6n de los estados inmaduros. Todos los caricteres fueron correlacio-
nados positivamente con la carga de huevos, pero el indice y la anchura del ovario fueron los
indicadores mejores de los oocitos maduros. El uso de estos parametros para apreciar la
carga de huevos eliminaria la necesidad de separar los ovarios y contar los oocitos maduros,
proveyendo un m6todo eficiente para procesar una muestra grande de moscas. Clasificaci6n
de la madurez sexual de las hembras basada en un sistema de etapa de ovario, en conjunci6n
con la apreciaci6n de la carga de huevos en etapa de madurez, facilitaria la evaluaci6n de la
estructura de la edad fisiol6gica de una poblaci6n de moscas capturada en mosqueros.

Translation provided by the authors.


Tephritid fruit flies in the genus Anastrepha dian fruit fly, A. obliqua (Macquart), though not
are serious economic pests of fruit crops through- established in Florida, pose additional invasive
out tropical and subtropical regions of the Ameri- threats due to proximity of populations in Mexico
cas (Aluja 1994). The Caribbean fruit fly, A. sus- and the Caribbean (White & Elson-Harris 1992).
pensa (Loew), is a quarantine pest for the citrus Traditionally, monitoring programs for tropical
industry and a production pest of guava and other fruit flies have relied on McPhail traps containing
fruits in Florida (Greany & Riherd 1993). The liquid protein baits, typically hydrolyzed yeast
Mexican fruit fly, A. ludens (Loew) and West In- (Steyskal 1977; Heath et al. 1993). Ammonia was







Kendra et al.: Ovary Development in A. suspense


recognized as the primary fruit fly attractant
emitted from liquid protein baits (Bateman &
Morton 1981), and ammonia-based synthetic
lures have been developed forAnastrepha spp. in-
cluding ammonium acetate and putrescine (Heath
et al. 1995; Thomas et al. 2001) and ammonium bi-
carbonate and putrescine (Robacker 1999).
Relative capture of Anastrepha fruit flies
among traps baited with liquid protein bait for-
mulations and synthetic lures has been highly
variable (Epsky et al. 2004). Field trials ofA. sus-
pensa found that at sites with a high percentage
of mated females, flies made choices among the
liquid protein bait formulations tested while at
sites with lower percentages, flies were less dis-
criminating (Epsky et al. 1993). In laboratory tri-
als, sexually immature females consumed more
protein than sexually mature females (Landolt &
Davis-Hernandez 1993). Using a combination of
electroantennography (EAG) and behavioral bio-
assays, Kendra et al. (2005a, 2005b) evaluated
dose-response of A. suspense to ammonia. EAG
recordings from females 1-14-d old showed that
antennal response to ammonia was not constant,
but varied depending upon the age/sexual matu-
rity of the flies. The antennal response of sexually
mature and immature females correlated with
differences in behavioral response to ammonia in
flight tunnel bioassays (Kendra et al. 2005b).
These laboratory results support the hypothesis
that the variability seen in field captures may be
due, in part, to the physiological age structure of
the fly population during the monitoring period.
Female tephritid fruit flies are sexually imma-
ture at eclosion (anautogenous) and the ovarian
maturation process is dependent upon multiple
factors, including temperature, photoperiod, diet
(especially protein availability), and chemical
cues (Fletcher 1989; Wheeler 1996; Papaj 2000;
Aluja et al. 2001). Therefore chronological age is
not equivalent to physiological age. Dodson
(1982) found that wild A. suspense require at
least 14 d to reach sexual maturity, whereas lab-
oratory-reared strains can mature within 7-8 d
(Mazomenos et al. 1977; Kendra et al. 2005b). In
addition to genetic strain differences, the pres-
ence of males has been shown to affect the rate of
ovarian development inA. suspense (Pereira et al.
2006). With such variability in maturation rate,
reliable methods are needed to ascertain sexual
maturity and mating status in female fruit flies,
particularly field-collected specimens. The most
accurate methods to determine mating status en-
tail examination of the sperm storage organs
(spermathecae and ventral receptacle) for pres-
ence of spermatozoa (Dodson 1982; Fritz &
Turner 2002; Twig & Yuval 2005), and field cage
tests found that 100% of sexually mature A. sus-
pensa females were inseminated within a 72-h pe-
riod (Dodson 1982). To differentiate between sex-
ually mature and immature females, studies on


A. suspense have used measurements of ovary
length (Nation 1972; Dodson 1982), ovary length
and width (Dodson 1978) or ovarian index (ovary
length multiplied by width, Landolt & Davis-Her-
nandez 1993). Nation (1972) also confirmed sex-
ual maturity by the presence of mature terminal
oocytes that are ~1 mm long and opaque. How-
ever, there are decreases in both the percent of
sexually mature females with mature oocytes
once oviposition starts (Dodson 1982) and in the
number of eggs oviposited over the fairly long life
span ofA. suspense females (Sivinski 1993), mak-
ing reliance on single factor determinations unre-
liable for flies trapped in the field. In this report,
we critically examine several ovarian morpho-
metric characters, document changes in these
characters for 28 d following adult eclosion, and
assess how reliably each character serves as an
indicator of sexual maturity inA. suspense.

MATERIALS AND METHODS

Insects

Anastrepha suspense were obtained from a lab-
oratory colony maintained at the USDA-ARS,
Subtropical Horticulture Research Station, Mi-
ami, FL. Rearing conditions consisted of a photo-
period of 12:12 (L:D), 70% RH, and ambient room
temperature (25 + 2C). In preparation for this
laboratory study, pupae (12 d old) were removed
from the colony, placed on weighing trays, and
held in screen cages (30 x 30 x 30 cm). Once adult
flies began to emerge, pupal trays were trans-
ferred to new cages every 24 h until emergence
ceased, typically 4 d. Since females tended to
emerge earlier than males, the first cage often
contained only females and therefore was dis-
carded. The remaining cages were mixed-sex (~1:1
sex ratio) and contained flies of known age, staged
at 1-d intervals. Adult flies were provisioned with
water (agar blocks) and food (refined cane sugar
and yeast hydrolysate, 4:1 mixture) ad libitum.
No oviposition medium was provided since the fe-
males readily laid eggs on the mesh sleeves of the
rearing cages. Known-aged females were collected
and stored in 70% ethanol until dissection.

Morphological Studies

Flies were dissected under a stereomicroscope
(at 25x magnification), their ovaries were re-
moved, and ovarian development was classified
according to the system described for Bactrocera
cacuminata (Hering) (Raghu et al. 2003). This
system identifies six stages in the ovarian matu-
ration process (Fig. 1): previtellogenic phases
(stages 1 and 2), vitellogenic phases (stages 3 and
4), appearance of mature oocytes (stage 5), and an
ovipositional phase (stage 6). After determining
the developmental stage, measurements were







Florida Entomologist 89(2)


June 2006


Fig. 1. Stages of ovarian development in adult Anastrepha suspense, adapted from classification system of Ra-
ghu et al. (2003). Stages 1 (A) and 2 (B) represent follicles in early and late previtellogenic development, respec-
tively. Stage 3 (C) marks initiation of vitellogenesis, accumulation of yolk in terminal follicles; Inset shows enlarged
follicle containing a yolk-filled oocyte (dark lower portion) capped with trophocytes (nurse cells). Stage 4 (D) indi-
cates late vitellogenesis, at which point yolk occupies more than half the follicle. Stage 5 (E) denotes ovaries with
mature oocytes, characterized by an intact chorion (eggshell) with a reflective surface and a reticulated pattern
(pronounced near the micropyle) visible at high magnification (inset). Stage 6 (F) indicates onset of oviposition, con-
firmed by presence of residual follicular bodies corporaa lutea) at base of the ovary (enlarged in inset). All ovary im-
ages at same magnification, scale unit = 0.1 mm.







Kendra et al.: Ovary Development in A. suspense


taken of the ovary length, ovary width, and
length of terminal follicle (i.e., the largest, most
advanced follicle). All measurements were made
with a hand-held micro-scale (to 0.1 mm; Mini-
tool, Inc., Los Gatos, CA) placed beneath the
ovary. Additionally, ovary length was multiplied
by ovary width to obtain ovarian index, a stan-
dard method for assessing sexual maturation
(Landolt & Davis-Hernandez 1993; Kendra et al.
2005b). After the ovaries were measured, they
were teased apart carefully with fine insect pins
(size 00, Elefant brand, Austria) and the number
of mature oocytes (egg load) was counted. To be
considered mature, oocytes had to lack accompa-
nying trophocytes (nurse cells, Fig. 1C) and pos-
sess a fully developed chorion (eggshell, Fig. 1E),
confirmed by the presence of a characteristic re-
ticulated pattern in surface architecture visible
at 100X magnification. Finally, the dorsal length
of thorax (from anterior edge of mesonotum to
posterior end of mesoscutellum) (Sivinski 1993)
and length of forewing (from base of costal vein to
wing apex where vein R,+ terminates at the mar-
gin) were measured as independent indicators of
overall female size. Measurements were recorded
from females that were 1-28 d post-eclosion, and
ten females were dissected for each age class.

Statistical Analysis

Regression analysis was used to describe the
relationship between chronological age and ova-
rian developmental stage using SigmaPlot 8.0
(SPSS Inc., Chicago, IL). Several regression mod-
els were tested including polynomial, hyperbolic,
logarithmic, and sigmoidal. Differences in re-
sponse variables (ovarian characters) among the
developmental stages were analyzed by one-way
analysis of variance (ANOVA) with PROC GLM
(SAS Institute 1985) followed by Tukey's test (P =
0.05) for mean separation. The Box-Cox procedure,
which is a power transformation that regresses


log-transformed standard deviations (y + 1)
against log-transformed means (x + 1), was used to
determine the type of transformation necessary to
stabilize the variance before analysis (Box et al.
1978). Correlations among ovary length, ovary
width, ovarian index, follicle length, and number
of mature oocytes (egg load) within each develop-
mental stage were determined with two-at-a-time
comparisons by PROC CORR. Additional compar-
isons determined correlation between egg load and
the four ovarian characters over the entire 28-d
period (all developmental stages combined).
Finally, analysis of covariance (ANCOVA) with
PROC GLM was used to evaluate effect of differ-
ences in size among the sampled females on com-
parisons among morphometric characters.

RESULTS

Fig. 1 depicts the six stages of ovarian develop-
ment in adult A. suspense, and comparisons of
morphometric characters and egg load among the
different stages are given in Table 1. The relation-
ship between ovarian developmental stage and
female chronological age was best fit by a sigmoi-
dal model, and this is presented in Fig. 2A.
All ovaries from 1-2 d old adults were classified
as stage 1 (Fig. 1A). Stage 1 ovaries were very
small and consisted of parallel, previtellogenic
ovarioles. Ovary length and width were approxi-
mately equal, and these two measurements were
positively correlated (r = 0.64238, P = 0.0023). In
addition, ovarian index was positively correlated
with both ovary length (r = 0.90382, P < 0.0001)
and ovary width (r = 0.90204, P < 0.0001) in stage
1 and all subsequent stages; this was not unex-
pected since ovarian index is a compound charac-
ter derived from ovary length and width. All 3-d-
old and some 4-5-d-old adults had ovaries classi-
fied as stage 2 (Fig. 1B). During this stage, sepa-
rate follicles were first discernible within the ova-
rioles, but they were still previtellogenic. The ter-


TABLE 1. OVARIAN CHARACTERS (MEAN SD) AT EACH DEVELOPMENTAL STAGE IN ADULT A. SUSPENSE.

Age Ovary Ovary Ovarian Follicle
Stage n (d) length (mm) width' (mm) index' (mm2) length (mm) Egg load2

1 20 1-2 0.29 + 0.06 a 0.27 0.05 a 0.08 0.03 a 0.10 0.00 a 0.0 0.00 a
2 20 3-5 0.58 + 0.15 b 0.36 0.09 ab 0.21 0.08 ab 0.11 0.03 a 0.0 0.00 a
3 14 4-6 0.90 + 0.15 c 0.49 0.08 b 0.45 0.12 b 0.26 0.07 b 0.0 0.00 a
4 9 6-7 1.38 0.34 d 0.71 0.12 c 0.97 0.28 c 0.52 0.19 c 0.0 0.00 a
5 21 7-9 1.88 0.22 e 1.29 0.26 e 2.45 0.71 e 1.06 0.12 e 18.2 13.49 c
6 196 9-28 1.56 0.20 d 1.05 0.18 d 1.66 0.43 d 0.96 0.12 d 4.2 3.80 b
F 279.95 205.08 204.32 479.28 64.01
df 5, 274 5, 274 5, 274 5, 274 5, 274
P <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Means within a column followed by the same letter are not significantly different (Tukey's mean separation test [P = 0.05]).
Data were square-root (x + 0.5) transformed prior to analysis; non-transformed means are shown.
Data were log (x + 1) transformed prior to analysis; non-transformed means are shown.







Florida Entomologist 89(2)


6 6 A "--.............
S5

4 6.05
Y=
Y= / -491)
E 3 1 i+e -
0-
0
a 2
SR2 = 0.978

0 5 10 15 20 25 30

>30 B
O
0-
S25
20


0 10 -
-
5 = . -
0o I I
0 5 10 15 20 25 30

0.5 -

x 04

0.3

g 0.2
0
01


0.0 _
0


5 10 15 20 25
Female Age (days post-eclosion)


Fig. 2. Three methods for assessing reproductive sta-
tus in female Anastrepha suspense. (A) Ovarian matu-
ration depicted by developmental stages according to
the system of Raghu et al. (2003). (B) Number of mature
oocytes per ovary (egg load). (C) Ovarian index (length
of ovary multiplied by width of ovary), standardized rel-
ative to length of forewing. All three graphs present
mean values (SE) recorded from ovaries dissected 1-28
d after adult emergence, n = 10 females per day.


minal follicles ranged in length from 0.1-0.2 mm.
Stage 2 ovaries were longer but not wider than
stage 1 ovaries, and there were no correlations
among ovary length, width or follicle length dur-
ing stage 2. Stage 3 (Fig. 1C) was characterized by
the onset of vitellogenesis, the accumulation of
yolk in the terminal follicles, which occurred in fe-
males 4-6 d old. During stage 3 both the ovaries
and the terminal follicles were longer than in
stage 2, and ovary width and ovarian index were
greater than in stage 1. There were positive corre-
lations between ovary length and follicle length (r


= 0.54852, P = 0.0422) and between ovarian index
and follicle length (r = 0.66225, P = 0.0099), but no
correlations between other paired measurements.
Observations of asynchronous gonadotrophic cy-
cles were first noted during stage 3. By stage 4
(Fig. 1D), the yolk content exceeded 50% of the
terminal follicle, and this stage included adults
that were 6-7-d-old. All morphometric characters
were greater in stage 4 ovaries than in previous
stages, and as was observed for stage 3, there
were positive correlations between ovary length
and follicle length (r = 0.92467, P = 0.0004) and
between ovarian index and follicle length (r =
0.91403, P = 0.0006). Stages 1-4 comprised the
classes of sexually immature females, during
which egg load remained at zero (Table 1). At the
first appearance of mature oocytes, found in flies
7-9-d-old, ovaries were classified as stage 5 (Fig.
1E). The largest egg loads were recorded during
stage 5, and accordingly the largest values for all
ovary measurements were obtained from females
in this stage. There were positive correlations be-
tween ovary length and ovary width (r = 0.65385,
P = 0.0013), between ovary length and egg load (r
= 0.67036, P = 0.0009), between ovary width and
egg load (r = 0.91356, P < 0.0001), and between
ovarian index and egg load (r = 0.90050, P <
0.0001) during stage 5. Initiation of oviposition
marked the transition to stage 6 (Fig. 1F), con-
firmed by the presence of at least one residual fol-
licular body (corpus luteum) formed after a termi-
nal follicle releases its oocyte. Based on this cri-
terion, ovaries from the majority of sexually ma-
ture females (9-28-d-old) were classified as stage
6; however, considerable variation was observed
within this age range. Developmental asynchrony
increased with age, and was pronounced by late
stage 6, giving the ovaries of older females an ir-
regular morphology compared with those of
younger mature females. In addition, stage 6 was
characterized by an overall decline in egg load
(Fig. 2B) and ovary size (Fig. 2C) with increasing
age, and mean values of all morphometric charac-
ters decreased in stage 6 compared to stage 5 ova-
ries. Despite this decrease, all measurements ex-
cept for ovary length were significantly greater in
stage 6 than in stage 4. All stage 6 characters
were highly and positively correlated when paired
with the other characters measured (P < 0.0001).
Mature oocytes were first detected in females 7
d old, and were present in some females sampled
each day thereafter up to day 28 (Fig 2B). Mean
egg load fluctuated over this period, with maxi-
mum number of mature eggs on day 8, and second-
ary peaks on days 13, 20, and 26. All four morpho-
metric characters were positively correlated with
egg load, with the highest correlations obtained
with ovarian index (r = 0.78319, P < 0.0001) (Fig.
2C) and ovary width (r = 0.74641, P < 0.0001) from
females 1-14-d-old. Correlations decreased with
increasing female age throughout weeks 3 and 4.


June 2006







Kendra et al.: Ovary Development in A. suspense


Forewing length and thorax length were eval-
uated as characters indicative of overall insect
size. As expected, there was no relationship be-
tween female age and either measurement, nor
were there differences in either measurement
among females from the different developmental
stages. Wing length (mean standard deviation)
was 5.92 0.196 mm and measurements ranged
from 5.0 6.4 mm. Thorax length was 2.44 0.105
mm and ranged from 2.0 2.7 mm. The two mea-
surements were positively correlated (r =
0.50896, P < 0.0001). However, since the wing is a
longer structure, a greater range of length differ-
ences could be measured, giving better resolution
to size differences among female flies. Therefore,
wing length was used to adjust for female size in
ANCOVA. The adjustment was not significant for
any of the morphometric characters; therefore, a
measurement of overall female size did not im-
prove the classification of the females among the
stages. The greatest effect was observed in the
analysis of ovary length (F = 2.06; df = 5, 268; P =
0.0714), indicating that in tests of flies that are
more variable in size, accounting for individual
size may improve use of ovary length measure-
ments as an indicator of sexual maturity.

DISCUSSION

The objective of this study was to identify a re-
liable method by which sexual maturity of female
Caribbean fruit flies can be assessed based on mor-
phological evidence. The photographic documenta-
tion and morphometric analysis presented in this
report indicate that this can be accomplished by
classifying ovarian development into six distinct
stages, adapting the system proposed by Raghu et
al. (2003). As has been reported in B. cacuminata
(Raghu et al. 2003) and other tephritid species
(Fletcher et al. 1978), the gonadotrophic cycles in
A. suspense ovarioles were not synchronous.
Throughout the early stages of oogenesis, most ter-
minal follicles were observed to be developing in
phase; but during the later stages, some follicles
were noticeably delayed. Due to this asynchrony,
consistent assignment of ovaries to a particular de-
velopmental stage was achieved by evaluating the
state of the most advanced ovarioles.
The ovaries ofA. suspense initially increased
in length and then in width during a maturation
phase which spanned the first 8-d post-eclosion in
our laboratory population. Of the four characters
examined, ovary length provided the best separa-
tion of immature stages during this maturation
phase, but ovary length alone did not discriminate
between stage 4 (immature) and stage 6 (mature)
ovaries. Distinguishing between these two stages
required inspection for residual follicular bodies
and assessment of gross ovary morphology. Ova-
rian index, which combined the contributions of
length and width, effectively maximized the dif-


ferences between immature and mature ovaries.
Ovarian index has been used previously for as-
sessment of sexual maturity in this same strain of
A. suspense, and Kendra et al. (2005b) concluded
that peak EAG response to ammonia occurred in
immature flies (4-6-d-old) and peak response to
carbon dioxide occurred in sexually mature flies
(10-12-d-old). Classification by developmental
stage now provides further interpretation of those
results. Maximal antennal response to ammonia
was measured from females with stage 3 ovaries
actively undergoing vitellogenesis (deposition of
yolk proteins), and this coincides with the age of
peak protein consumption reported by Landolt &
Davis-Hernandez (1993). Maximal response to
carbon dioxide was found in stage 6 females dur-
ing the ovipositional phase, which is consistent
with the theory proposed by Stange (1999) that
carbon dioxide serves as a close-range oviposition
attractant for tephritid fruit flies.
The presence of mature oocytes in an ovary is re-
garded as the definitive character for female sexual
maturity (Nation 1972; Aluja et al. 2001). Some 7-
d-old females had mature oocytes, but by 8 d of age
all females had mature oocytes under laboratory
conditions. In a previous study with laboratory
rearedA suspense, indicator variable analysis also
identified day 8 as the breakpoint between sexually
immature and mature females (Kendra et al.
2005b). The transition from maturation phase to
oviposition phase is marked by substantial changes
both physiologically and behaviorally. The 8-d-old
females (stage 5) had the maximum average egg
load, and this was followed by secondary peaks at
5-7-d intervals. Approximately 10% of the 9-28-d
old females (stage 6) had no mature eggs present in
the ovaries. This included 30% of the 18-d-old fe-
males and 50% of the 28-d-old females. Fluctua-
tions in egg load versus age suggest that eggs are
laid in batches initially, when ovarioles are most
synchronous. Over time, the cyclic pattern dimin-
ished apparently due to increasing asynchrony in
oogenesis. Once a female is sexually mature, with
fully developed eggs, she may switch from food-
seeking behaviors, which allow her to obtain pro-
tein for egg development, to oviposition-site seek-
ing behaviors, which enable her to locate suitable
host fruit. Predominance of these two activities
may alternate throughout stage 6 as females un-
dergo successive cycles of oviposition. Although
food-seeking behavior was thought to be primarily
an activity of sexually immature females, the cyclic
fluctuation of egg load indicates that, despite being
sexually mature, a female might not engage in
host-seeking/oviposition behaviors until she pos-
sessed an appropriate egg load. Thus, determina-
tion of egg load of sexually mature females (espe-
cially in stage 6) may provide further discrimina-
tion among females captured in field trials.
The six-stage system is a useful means of eval-
uating female sexual maturity, but its accuracy







Florida Entomologist 89(2)


for stages 4-6 depends upon assessment of mature
oocytes within the ovaries. Without inspection for
the presence of a chorion, it is possible to misiden-
tify a full-sized terminal follicle as a mature oo-
cyte, as supported by the lack of correlation be-
tween follicle length and egg load in stage 5. Also,
stage 6 females may have oviposited all mature
eggs at time of capture or mature oocytes may be
concealed within the ovary (PK, personal observa-
tion). Therefore, the most reliable method for de-
termination of egg load consists of ovary removal,
careful separation of ovarioles and counting of
mature oocytes, which is very time-consuming.
The ideal screening method for field-captured flies
would consist of a quick dissection followed by one
or two simple measurements. Based on compari-
sons of the morphological characters examined in
this study, ovarian index and ovary width are re-
liable indicators correlated with egg load. Use of
these parameters to assess egg load would facili-
tate efficient processing of large samples of flies.
Classification of female sexual maturity by
ovarian developmental stage, in conjunction with
assessment of egg load in the mature stages,
would facilitate evaluation of the age structure of
a fly population responding to specific lures in
field trapping studies. Although a laboratory
strain ofA. suspense was used for this study, the
proposed classification system should have broad
applications since it is based on several ovarian
characters and reflects female physiological age.
In addition, standardization for insect size may
improve resolution of ovary measurements as pa-
rameters for assessing maturity status in more
variable field populations. The utility of this
method for wild populations ofA. suspense and
other tephritid species will need to be addressed
in complementary studies.

ACKNOWLEDGMENT

The authors are grateful to Monica Schiessl, whose
untiring efforts in maintaining the SHRS fruit fly colony
provided us with a continuous supply of robust insects
for this study. We also acknowledge Don Thomas
(USDA-ARS, Weslaco, TX) and Rui Pereira (University
of Florida, Gainesville, FL) for critical reviews of an ear-
lier version of this manuscript, Micah Gill for assistance
with digital photography, and Pansy Vazquez-Kendra
and Elena Schnell for translation of the abstract. This
article reports the results of research only. Mention of a
proprietary product does not constitute an endorsement
or recommendation for its use by the USDA.

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Florida Entomologist 89(2)


June 2006


CUTEREBRA BOT FLIES (DIPTERA: OESTRIDAE) AND THEIR
INDIGENOUS HOSTS AND POTENTIAL HOSTS IN FLORIDA


FRANK SLANSKY
Department of Entomology & Nematology, University of Florida
Bldg. 970 Natural Area Drive, Gainesville, FL 32611

ABSTRACT

Typical mammal hosts (indigenous rodents and lagomorphs), geographic distributions and
phenologies of the five species of Cuterebra hot flies occurring in Florida are described. This
coverage includes a reevaluation of some previously reported host records and presentation
of unpublished data on larval infestations and captures of adult Cuterebra in Florida. In ad-
dition, indigenous species of Florida rodents for which there appear to be no in-state reports
of larval infestation are listed (both native species of lagomorphs in Florida are hosts of
Cuterebra within the state). Many gaps in our knowledge of the biology of these flies in Flor-
ida are identified, but based on available information, it appears that Florida is not excep-
tional when compared with certain other areas of North America in Cuterebra species
diversity or the species of native rodents that apparently are not used as larval hosts. The
geographic affinities of the Florida Cuterebra are Nearctic. Four of the species (C. americana
(Fabricius), C. buccata (Fabricius), C. emasculator Fitch and C. fontinella Clark) have broad
ranges in North America, whereas C. cuniculi (Clark) appears to be restricted to southern
Georgia and Florida.

Key Words: lagomorph, parasite, phenology, rodent, species diversity

RESUME

Se described los hospederos mamiferos tipicos (roedores y lagomorfos indigenas), la distribu-
ci6n geografica y la fenologia para cinco species de t6rsalo (moscas del g6nero Cuterebra) que
ocurren en la Florida. Algunos de los pasados informes de los hospederos son re-evaluados y
se presentan datos no publicados sobre las infestaciones de larvas y adults de Cuterebra re-
colectados en Florida. Se present una lista de las species de roedores indigenas de Florida
las cuales aparentemente no tienen un registro de infestaci6n de estas larvas dentro del es-
tado (ambas de las species nativas de lagomorfos en Florida son hospederos de Cuterebra).
Muchas incognites en nuestro conocimiento de la biologia de estas mosca en Florida estan
identificadas, pero basadas sobre la informaci6n disponible, parece que Florida no es excep-
cional cuando se compare con ciertas otras areas de America del Norte en cuanto de la diver-
sidad de species de Cuterebra o las species de roedores natives que aparentemente no son
usados como hospederos de las larvas. Las afinidades geograficas de las moscas Cuterebra de
Florida son Nearcticas. Las species C. americana (Fabricius), C. buccata (Fabricius), C. emas-
culator Fitch y C. fontinella Clark tienen un rango geografico amplio en America del Norte
mientras que C. cuniculi (Clark) aparentemente es restringida en el sur de Georgia y Florida.


Cuterebra bot flies (Diptera: Oestridae; often
listed as Cuterebridae) (e.g., Sabrosky 1986; Al-
cock & Kemp 2004; Burns et al. 2005) are obligate
parasites of many native (indigenous) rodents
(mice, rats, tree squirrels, etc.) and lagomorphs
(rabbits, hares, etc.) in the Americas (Sabrosky
1986). Larvae (bots) of these dipterans are subcu-
taneous parasites that live in encapsulated pock-
ets known as warbles. Depending on the species of
Cuterebra and its host, the larvae develop for four
to six weeks, ingesting body fluid and excreting
and respiring through a hole (the warble pore)
they create in the host's skin (Catts 1982; Slansky
& Kenyon 2003). In addition to species they typi-
cally parasitize, these insects occasionally infest
'atypical' hosts, especially non-native (= non-in-
digenous or adventive; Frank & McCoy 1995) ro-


dents and lagomorphs and non-rodent/ non-lago-
morph mammals (including humans) (Sabrosky
1986; Baird et al. 1989; Glass et al. 1998; Harris
et al. 2000; Suedmeyer et al. 2000; Safdar et al.
2003; F. S., unpublished data).
Most of the 30+ species of Cuterebra are tem-
perate zone species, with flies in other cuterebrine
genera (Dermatobia, Metacuterebra, Alouat-
tamyia, Rogenhofera and Pseudogametes) occur-
ring in subtropical and tropical climates (Catts
1982; Sabrosky 1986; Guimaraes 1989; Colwell &
Milton 1998; Bergallo et al. 2000; note, however,
that Pape (2001) suggested that the latter three
genera likely should be included in Cuterebra).
Based on morphological features of the adults and
on larval hosts, Sabrosky (1986) divided Cutere-
bra into four 'groups', defined by a species within







Slansky: Florida Cuterebra and their Mammal Hosts


the group: the rodent-infesting 'americana' and
'fontinella' groups, and the lagomorph-infesting
'buccata' and 'cuniculi' groups.
Diverse biogeographic patterns are exhibited
by various taxa of Florida's indigenous entomo-
fauna and other biota; these may include precinc-
tive species, either depauperate or high species
diversity, declining diversity from north to south
(e.g., peninsula effect), and affinities to different
geographic regions (e.g., Frank 1986; Peck 1989;
Choate 1990; Deyrup 1990; Frank & McCoy
1995). In this paper I address various components
of the biogeography of Cuterebra in Florida, a
topic that has previously not been investigated. I
review literature relevant to the presence in the
state of flies in this genus and of their indigenous
mammal hosts. This coverage includes a reevalu-
ation of some previously reported host records as
well as presentation of unpublished data on lar-
val infestations and captures of adult Cuterebra
in Florida. In addition, I list the indigenous ro-
dents occurring in the state for which there ap-
pear to be no in-state reports of Cuterebra larval
infestation (both native lagomorph species are
hosts for larvae of these flies in Florida). Finally,
I discuss the diversity and geographic affinities of
these flies in Florida and address the question of
whether there are an exceptional number of va-
cant niches (potential host species) for Cuterebra
species in the state.

MATERIALS AND METHODS

Published literature was reviewed to determine
which species of Cuterebra and other cuterebrines
occur in Florida, as well as their typical hosts,
ranges, and phenologies. Unless indicated other-
wise, information before 1986 was obtained from
Sabrosky (1986), who not only compiled and syn-
thesized most of the published information avail-
able at that time on Cuterebra but also reported
numerous unpublished records resulting from his
examination of specimens from many private and
museum collections. Information on mammal spe-
cies in Florida was obtained from the American So-
ciety of Mammalogists (undated), the Florida Fish
and Wildlife Conservation Commission (2004a,b)
and Brown (1997a,b), unless cited otherwise. No-
menclature follows that of the International Taxo-
nomic Information Service (ITIS 2004).

RESULTS

Cuterebra in Florida

Five species of Cuterebra occur in Florida:
C. americana (Fabricius), C. buccata (Fabricius),
C. cuniculi (Clark), C. emasculator Fitch, and
C. fontinella Clark. There apparently are no veri-
fied published records for flies of other Cuterebra
species or in other cuterebrine genera occurring


naturally in Florida. Worth (1950a) listed "Der-
matobia-like" larvae removed from roof (or black)
rats, Rattus rattus (L.) (a non-indigenous, atypi-
cal host species), captured in Hillsborough Co.,
but this appears to be a misidentification of sec-
ond instar Cuterebra larvae, as done previously
(Townsend 1892). In subsequent reports (Worth
1950b,c) in which he thanked a Cuterebra taxono-
mist, C. W. Sabrosky, for identifying the larvae,
Worth no longer mentioned Dermatobia. Below I
discuss the typical hosts, ranges and phenologies
in Florida for these five species.

C. americana

Typical Hosts. There apparently is only one
main typical host species for larvae of C. ameri-
cana, the eastern wood rat Neotoma floridana
(Ord), which ranges throughout the northern two
thirds of peninsular Florida and the Panhandle
(there is also an isolated population on Key
Largo). There appear to be only two published in-
festation reports for this host in Florida. Without
any additional information, Johnson (1930)
stated that he "obtained Cuterebra larvae from
the large wood rat" (presumably N. floridana) in
the state, and Worth (1950b) reported capturing
Cuterebra-infested individuals of this species in
Hillsborough county.
Distribution. County records for captures of
adult C. americana in Florida include Alachua,
Citrus, Duval, Hillsborough, Lake, Orange, Pasco,
and Sarasota. If Worth's (1950a,b) reports of in-
fested R. rattus captured in Dade Co. involved C.
americana, as suspected by Sabrosky (1986), then
this species would appear to occur throughout
peninsular Florida. However, in Worth's papers
the larvae were not described and no mention was
made of obtaining adults for definitive species
identification even though Sabrosky (1986) stated
that Worth "reared" these specimens (in fact,
Worth thanks Sabrosky for identifying the larvae
only to the level of Cuterebra sp.). In addition, the
typical host (N. floridana) of this species appar-
ently does not occur in Dade Co. Finally, larvae of
at least one other Florida Cuterebra species (C.
buccata) have been recorded infesting Rattus spe-
cies as atypical hosts. Taken together, these cave-
ats would appear to call into question the pres-
ence of C. americana in Dade Co. Because this
species has been reported from Georgia and Lou-
isiana (as well as from several other states from
eastern Colorado to Virginia and southward), it
likely also occurs throughout the Florida Panhan-
dle.
Phenology. Sabrosky (1986) provided no dates
for adult captures or host infestations. An adult
female C. americana was collected in Alachua Co.
on 7-X-1992 (P. M. Choate, Dept. Entomology &
Nematology, University of Florida, personal com-
munication). Worth (1950b) capturedR. rattus in-







Florida Entomologist 89(2)


fested with Cuterebra (possibly C. americana; but
see above) in Dade Co. in January and Cuterebra-
infested R. rattus and N. floridana in Hillsbor-
ough Co. in late February through early March
(these were the only times that trapping was
done; see also Worth 1950c). Because the data are
so limited, the phenology of this species in Florida
is uncertain, but it appears to be univoltine out-
side the state (Goertz 1966).

C. buccata

Typical Hosts. Larvae of this species typically
infest eastern cottontails, Sylvilagus floridanus
(J. A. Allen), and probably also individuals of
other Sylvilagus species. Both S. floridanus and
the marsh rabbit S. palustris (Bachman) are
widespread in Florida, but the presence in the
state of the swamp rabbit S. aquaticus (Bach-
man), which might occur in the extreme western
Panhandle, is uncertain. There appear to be no
definitive records of infestation of rabbits of either
of these species by larvae ofC. buccata in Florida.
However, Worth (1950a,b) reported that individu-
als of S. palustris were commonly infested with
larvae of Cuterebra (Sabrosky (1986) does not
mention these records). Although these larvae
were not identified to species, they probably were
either C. buccata or C. cuniculi (see below), the
only Cuterebra species in Florida known to use
rabbits as their typical hosts.
Distribution. This is a very widespread spe-
cies, reported from all states east of the western
mountain states except Maine, Vermont and
Rhode Island. According to Sabrosky (1986), sup-
posed records of this species from St. Johns and
Collier counties (Johnson 1895, 1913) in Florida
presumably involved another species (C. fonti-
nella; see below). Cuterebra-infested S. palustris
collected in Hillsborough Co. (Worth 1950a,b)
may have involved this species, and/or possibly C.
cuniculi (see below). Sabrosky (1986) considered
as valid the claim of Knipling & Bruce (1937) that
a larva of this species was removed from a cow in
September in Sumter Co. However, the involve-
ment of C. buccata (or indeed any species of Cute-
rebra) in this infestation is questionable for a va-
riety of reasons: (1) the larva was a second instar,
and no species identification key for this stage of
the Cuterebra lifecycle was then (nor is now)
available; (2) the authors provide no information
on the characteristics used to identify this larva
either as a species of Cuterebra or as C. buccata in
particular; (3) a cow is a very unusual atypical
host for Cuterebra larvae, and I am aware of no
other reports documenting cattle as hosts; and (4)
cattle are subject to parasitization by larvae of
cattle warble flies (two species of Hypoderma),
both of which occur in Florida (Glick 1976). Lar-
vae of these insects typically form warbles on the
backs of these animals, which was the site of the


supposed Cuterebra larva. Thus, although a Cute-
rebra larva may have infested a cow, as stated by
Knipling & Bruce (1937), I consider this conclu-
sion highly unlikely.
Phenology. There apparently are no definitive
phenological records for this species in Florida, al-
though C. buccata larvae may have infested the S.
palustris trapped by Worth (1950a,b) in late Feb-
ruary to early March (the only time that trapping
was done). Thus, the phenology of this species in
the state cannot presently be determined, but
elsewhere it appears to be at least bivoltine.

C. cuniculi

Typical Hosts. The typical hosts for C. cuniculi
are S. floridanus and S. palustris, with infestation
records for both hosts in the state.
Distribution and Phenology. Cuterebra cuniculi
is very restricted in distribution, apparently occur-
ring only in Florida and southern Georgia. County
and date records for this species in Florida (adults,
unless indicated otherwise) include Alachua (May
and December), Broward (August), Collier (April),
Dade (May), Hamilton (October), Highlands (May
and December), Indian River (a larva from
S. palustris in June; the adult emerged in Octo-
ber), Orange (May), Palm Beach (May and Decem-
ber; also, a larva from an unspecified host in Octo-
ber with the adult emerging in November; and an-
other adult in November from a larva (no date) in-
festing S. palustris), Polk (March), St. Johns
(April) and St. Lucie (a larva from S. floridanus in
December; the adult emerged in February).
Worth's (1950a,b) records of Cuterebra-infested S.
palustris trapped in Hillsborough Co. during late
February through early March (the only time that
trapping was done) likely would have involved this
species and/or C. buccata. Apparently, there are no
records for this species from counties in the Pan-
handle. From the records listed above, it is likely
that this species occurs at least throughout the
peninsular part of the state and that it has two or
more generations during the year. Based on very
limited data, it appears to be bivoltine in Georgia.

C. emasculator

Typical Hosts. The typical hosts for this spe-
cies include tree squirrels (Sciurus sp.), and east-
ern chipmunks, Tamias striatus (L.). There are
Florida infestation records for eastern gray squir-
rels, S. carolinensis Gmelin, and fox squirrels, S.
niger L., both of which are widespread throughout
the state. In contrast, T striatus is restricted to
the northern portions of a few counties in the Pan-
handle (Escambia, Holmes, Okaloosa, Santa
Rosa, and Walton) (Gore, 1990), and there appear
to be no published Cuterebra-infestation records
for individuals of this species in Florida. Southern
flying squirrels, CG.. ....... volans (L.), which are


June 2006







Slansky: Florida Cuterebra and their Mammal Hosts


widely distributed in Florida, have rarely been re-
ported to be parasitized by Cuterebra larvae (pre-
sumably C. emasculator) in the state or else-
where, suggesting that G. volans is an atypical
host species for Cuterebra larvae.
Distribution. Cuterebra emasculator is widely
distributed throughout eastern North America
from just west of the Mississippi River to the At-
lantic coast. Published records for Florida include
Alachua (Sabrosky 1986; Forrester 1992; Slansky
& Kenyon 2000; 2002) and Columbia (Coyner
1994; Coyner et al. 1996) counties, although the
latter record may not have involved C. emascula-
tor A recent study has extended the known range
of this species to over 40 additional counties
throughout the northern and central regions of
the state (including the Panhandle) (F. S., unpub-
lished data). Apparently, C. emasculator is rare in
or absent from the southern counties despite the
presence of potential host squirrels.
Phenology. Sabrosky (1986) does not provide
phenological data for this species in Florida, but in-
fested squirrels typically are observed in the state
from July through October (Slansky & Kenyon
2000; 2002; 2003; F. S., unpublished data). Coyner's
(1994; Coyner et al. 1996) report of finding one in-
dividual of S. niger (out of 123 examined fox squir-
rels) with a larva presumed to be C. emasculator on
21-II-1991 is exceptional. Because no information
was given that the larva was definitively identified
to species, the possibility exists that it was of a dif-
ferent species such as C. cuniculi, which, unlike C.
emasculator, appears to have a winter generation.
Cuterebra emasculator appears to be univoltine in
Florida and throughout its geographic range (Ben-
nett 1972a,b; F. S., unpublished data).

C. fontinella

Typical Hosts. The main typical hosts for
C. fontinella apparently are the white-footed
mouse Peromyscus leucopus (Rafinesque) and the
cotton mouse Peromyscus gossypinus (LeConte)
(records in Sabrosky (1986) and Durden (1995)).
However, adults of this species have been reared
from a variety of other indigenous rodents, includ-
ing the deer mouse Peromyscus maniculatus
(Wagner) (mice of this species apparently are the
main typical hosts for a closely related species,
Cuterebra grisea Coquillett), the golden mouse
Ochrotomys nuttalli (Harlan), the northern grass-
hopper mouse Onychomys leucogaster (Wied-Neu-
wied), the Mexican spiny pocket mouse Liomys
irroratus (Gray), the woodland jumping mouse
Napaeozapus insignis (Miller), the meadow vole
Microtus pennsylvanicus (Ord), and the yellow-
pine chipmunk Tamias amoenus J. A. Allen
(records in Sabrosky (1986); also, Clark & Durden
(2002) for 0. nuttalli). Of these, only P gossypi-
nus, 0. nuttalli, and a subspecies of M. pennsyl-
vanicus occur in Florida. Cuterebra-infested indi-


viduals of P gossypinus, which occurs statewide,
and 0. nuttalli, which is found in the northern
half of peninsular Florida and the Panhandle,
have been captured in the state (Pearson 1954;
Layne 1963; Bigler & Jenkins 1975). In addition,
Layne (1963) trapped Cuterebra-infested Florida
mice, Peromyscus (= Podomys) floridana (Chap-
man), which occur only in Florida (the central por-
tion of the peninsula). It is likely that the mice in
the latter three studies were parasitized by C. fon-
tinella. If so, then 0. nuttalli, P. gossypinus, and
P floridana would apparently constitute the typi-
cal hosts for this Cuterebra species in the state.
Distribution. Cuterebra fontinella is a very
widespread species, occurring throughout most of
the continental US (except Alaska), southern Can-
ada, and northeastern Mexico. Sabrosky (1986)
provides a distribution map for this species, in-
cluding several records for Florida. Because of the
small size of this map and the large symbols used
to mark collection locations, identification of the
counties involved is somewhat tenuous, but these
appear to be Alachua, Broward, Citrus, Collier,
Columbia, Dade, Hillsborough, Lee, Manatee,
Monroe, Orange, Pinellas, Sarasota, St. Lucie,
Union, and Volusia. Pearson's (1954) infestation
records are for Levy Co., and Bigler & Jenkins
(1975) performed their study in Monroe Co. Layne
(1963) did extensive trapping throughout the
northern half of the state (Alachua, Clay, Gil-
christ, Levy, Putnam and St. Johns counties) and
some in Highlands Co. Individual county records
were not presented in the latter study but appar-
ently Cuterebra-infested mice were found in each
of these counties. According to Sabrosky (1986),
Johnson (1895) originally thought a fly captured
in St. Johns Co. was C. buccata but he later cor-
rectly identified it as C. fontinella (Johnson 1913).
However, in the latter publication he provided a
separate record for C. buccata from Collier Co., but
Sabrosky (1986) indicated that Johnson more
likely was again dealing with C. fontinella. Appar-
ently, there are no published records for this spe-
cies from the Panhandle.
Phenology. Sabrosky (1986) provided no phe-
nological data for C. fontinella in Florida. An
adult female C. fontinella was captured in Ala-
chua Co. on 19-IV-2003 (P. M. Choate, Dept. Ento-
mology & Nematology, University of Florida, per-
sonal communication). Pearson (1954) reported
trapping Cuterebra-infested P gossypinus in all
months of the year except February and March,
with almost half of these records in June; he did
not report capture dates for the Cuterebra-in-
fested P nuttalli he trapped. Bigler & Jenkins
(1975) also captured Cuterebra-infested P gos-
sypinus during most months of the year; no trap-
ping was done in December, but parasitized mice
were caught in every other month except October,
with peaks in the prevalence of infestation in Jan-
uary and June. Layne (1963) found Cuterebra-







Florida Entomologist 89(2)


infested P floridana in all quarters of the year. If
these latter three studies involved C. fontinella
(as is likely), then this species probably has two or
more generations per year in Florida. It appears
to be at least bivoltine in other southeastern
states (Durden 1995, Georgia; Clark & Durden
2002, Mississippi) and elsewhere (e.g., Goertz
1966; Wolf & Batzli 2001, Illinois).

Indigenous Rodents not Known to be Parasitized by
Cuterebra Larvae in Florida

Several species of indigenous rodents occur in
Florida for which no published records of parasit-
ization by Cuterebra larvae in this state appar-
ently exist. These are listed below, along with
published reports and a few unpublished records
of Cuterebra infestation (or indication of the ap-
parent lack thereof) from elsewhere in the ranges
of these, and in some cases closely related, taxa.
Castoridae and Aplodontidae. American bea-
vers, Castor canadensis Kuhl, occur in the Pan-
handle and northern third of peninsular Florida.
Apparently, there are no published Cuterebra-in-
festation records for this species in any part of its
range in North America. Sabrosky (1986) listed
only two records of mountain beavers,Aplodontia
rufa (Rafinesque) (note that this species belongs
to a different family (Aplodontidae) than C. ca-
nadensis), parasitized by Cuterebra larvae (Ore-
gon and Washington). These limited records sug-
gest that no Cuterebra species uses beavers of ei-
ther of these two species as typical hosts.
Geomyidae. The southeastern pocket gopher
Geomys pinetis Rafinesque is the only member of
this family in Florida. It is found in the Panhandle
and the northern half to two thirds of the Florida
peninsula. One individual of this species captured
by Worth (1950a; probably in Hillsborough Co.)
was not parasitized by Cuterebra larvae. Sampling
of G. pinetis in Alachua Co. for an entire year and
in Alabama, Florida and Georgia primarily from
December through February (totaling over 150 in-
dividuals trapped) yielded no specimens obviously
infested with Cuterebra larvae (P. E. Skelley,
FDACS/DPI, Gainesville, FL, personal communi-
cation). In the western US, the northern pocket go-
pher Thomomys talpoides (Richardson) is the typi-
cal host of Cuterebra polita Coquillett (a member of
the 'americana' group). There appear to be no Cute-
rebra-infestation records for the several other spe-
cies of Geomys and Thomomys in North America.
Muridae. A number of indigenous murid ro-
dents occur in Florida for which no published
Cuterebra-infestation records in the state appear
to be available. The marsh rice rat Oryzomys
palustris palustris (Harlan) has a statewide dis-
tribution in Florida. There is also a subspecies,
the silver rice rat 0. p. natator Chapman (some-
times listed as the invalid 0. argentatus Spitzer
and Lazell), which is apparently limited to some


of the Lower Keys. There appear to be no Cutere-
bra-infestation records for any members of this
genus in North America; none are listed in Sa-
brosky (1986) and no infested individuals were
captured by Worth (1950a), Pearson (1954), Dur-
den (1995), or Clark & Durden (2002). However,
parasitization of another member of this genus,
0. russatus (Wagner), by Metacuterebra apicalis
(Guerin-Meneville) in South America has been
well documented (Bergallo et al. 2000; Bossi et al.
2002; both Brazil). The hispid cotton rat Sigmo-
don hispidus Say and Ord is distributed state-
wide in Florida. Goertz (1966) reported that indi-
viduals of this species were very rarely parasit-
ized by an unknown species of Cuterebra (possibly
C. americana) in Oklahoma, whereas no such in-
festations were found in Florida (Worth 1950a;
Pearson 1954; Bigler & Jenkins 1975) or else-
where in North America (Clark & Kaufman 1990,
Kansas; Boggs et al. 1991, Oklahoma; Clark &
Durden 2002). Disney (1968) reported infestation
ofSigmodon sp. cotton rats in Honduras by larvae
of Cuterebra (= Metacuterebra) flaviventris (Bau).
Two species of Peromyscus mice occur in Flor-
ida, and Cuterebra-infested individuals of one of
these, P. gossypinus, have been captured in the
state. However, the other species, P polionotus
(Wagner), which is comprised of several subspe-
cies (beach mouse, oldfield mouse, etc.) variously
distributed in Florida, is apparently lacking in
Cuterebra-infestation records. Another indige-
nous mouse species in Florida, the eastern har-
vest mouse Reithrodontomys humulis (Audubon
and Bachman), occurs throughout the northern
two thirds of the peninsula and in the Panhandle.
Little or no parasitization of Reithrodontomys
mice has been reported from elsewhere in North
America (Goertz 1966; Hensley 1976, Virginia;
Sabrosky 1986; Clark & Kaufman 1990; Boggs et
al. 1991; Clark & Durden 2002), which suggests
that members of this genus may serve only occa-
sionally as atypical hosts for Cuterebra larvae.
Two species of Microtus voles occur in Florida:
the pine (or woodland) vole M. pinetorum (Le-
Conte), found in the central part of the northern
one third of the peninsula, and a rare subspecies of
the meadow vole M. pennsylvanicus, namely the
Florida saltmarsh vole M. p. dukecampbelli
Woods, Post & Kilpatrick, which inhabits salt-
marshes in the Cedar Key area (Levy Co.). There
are several records from outside Florida ofindivid-
uals of M. pennsylvanicus and other Microtus
voles parasitized by larvae of various Cuterebra
species (Clough 1965, Wisconsin; Maurer & Ska-
ley 1968, New York, North Dakota and Pennsylva-
nia; Getz 1970, Wisconsin; Hensley 1976,
M. pennsylvanicus but not M. pinetorum; Boon-
stra et al. 1980, British Columbia, Canada), as
well as reports of Cuterebra-infested Clethriono-
mys voles (Sabrosky 1986, Manitoba and Quebec,
Canada; Bowman 2000, New Brunswick, Can-


June 2006







Slansky: Florida Cuterebra and their Mammal Hosts


ada), which do not occur in Florida. However, none
of the Microtus voles captured by Sillman (1955,
Ontario, Canada), Goertz (1966), Shoemaker &
Joy (1967, West Virginia), Hensley (1976, M. pine-
torum), Clark & Kaufman (1990), Boggs et al.
(1991), Bowman (2000), or Clark & Durden (2002),
nor any of the Clethrionomys individuals trapped
by Maurer & Skaley (1968) or Hensley (1976),
were infested with Cuterebra larvae.
Round-tailed muskrats, Neofiber alleni True,
are distributed throughout much of peninsular
Florida, with some isolated populations in the
Panhandle. Sabrosky (1986) provided records of
infestation of an individual of this species (loca-
tion not given) and of the muskrat Ondatra zibe-
thicus (L.) (Michigan). These limited records sug-
gest that no Cuterebra species uses these muskrat
species as typical hosts.
Sciuridae. There are few reports of flying
squirrels (GC/........ species) parasitized by
Cuterebra larvae. Apparently, the only published
North American record is for an individual of G.
volans in Alachua Co., Florida (Forrester 1992),
and I am aware of a few such cases from other
eastern states (F. S., unpublished data). Because
of the rarity of these records, it is likely that Glau-
comys species are atypical hosts of Cuterebra
(presumably C. emasculator). Tamias striatus,
which is restricted in Florida to the northern por-
tions of certain counties in the Panhandle, is a fre-
quent host ofC. emasculator outside the state, es-
pecially in the northern portion of its range.

DISCUSSION

From the above coverage, it is evident that there
are many gaps in our knowledge, specific to Flor-
ida, of the biology of the Cuterebra species occur-
ring in the state. The most complete data on host
species, county distribution, and phenology within
Florida are available for C. cuniculi and C. emascu-
lator. However, if the studies of Pearson (1954),
Layne (1963), and Bigler & Jenkins 1975) involved
C. fontinella (as is likely), then aspects of the biol-
ogy of this species in Florida also are reasonably
well understood. The least amount of information
is available for C. americana and C. buccata.
Obviously, more studies are required to pro-
vide the information needed to better understand
the biology of these five Cuterebra species in Flor-
ida. The mammals that serve as typical and atyp-
ical hosts for these species within the state need
to be determined, or in some cases better docu-
mented. In addition, the distributions and phe-
nologies of these species within the state need to
be established for some of the species or better de-
lineated for the others. A key limitation in the re-
search required to achieve these goals involves
the difficulty of determining Cuterebra species
when only larval specimens are available. Gener-
ally, the larvae of these flies cannot be identified


to species based on their external features; in-
stead, they usually need to be reared to the adult
stage, for which definitive morphologically-based
descriptions are available (Sabrosky 1986). How-
ever, obtaining adults from larvae can be prob-
lematic; second and early third instars removed
from their hosts are unable to pupate, and al-
though more mature third stadium larvae can pu-
pate, they may enter pupal diapause, which can
delay obtaining adults by several months (e.g.,
Bennett 1972a;b). In addition, there can be sub-
stantial mortality of diapausing pupae (F. S., un-
published data). The problem of species identifi-
cation of the larvae will be overcome as compara-
tive DNA sequences become available for more
species of Cuterebra (Otranto et al. 2003; Noel et
al. 2004; F. S., unpublished data). At a broader
level, third stadium Cuterebra larvae can be sep-
arated into species that typically parasitize ro-
dents and those that infest lagomorphs, based on
certain features of their cuticular ornamentation
(Knipling & Brody 1940; Baird & Graham 1973).
Limitations in our knowledge prevent a mean-
ingful biogeographic analysis of the in-state distri-
bution of the Florida species of Cuterebra (Deyrup
1990). However, it is possible to address some
broader patterns for these flies in Florida. Al-
though the biogeography of the genus has not been
studied quantitatively (e.g., species/area relation-
ships), the number of Cuterebra species (five) oc-
curring in Florida appears comparable to that in
certain other states of similar area (Illinois and
Washington; species distributions from Sabrosky
(1986)). In addition, Florida is inhabited by mem-
bers of all four of the Cuterebra groups. The state
contains each of the species chosen by Sabrosky
(1986) to name these groups, as well as C. emascu-
lator, which is in the 'fontinella' group. Thus, Flor-
ida does not appear to be either depauperate or un-
usually rich in its total number of Cuterebra spe-
cies or in representatives of Sabrosky's (1986) four
Cuterebra groups. However, before definitive con-
clusions can be reached regarding Cuterebra spe-
cies diversity within Florida, the effects of habitat
heterogeneity, host species diversity, historical in-
fluences, and other relevant biogeographic factors
must be investigated for the entire genus.
Regional affinities of the indigenous entomo-
fauna of Florida are diverse. In many cases these
reflect relationships to taxa in other areas of the
southeastern US, but for some groups there are
affinities to taxa in southwestern North America
or in the Caribbean region (Frank 1986; Peck
1989; Choate 1990; Deyrup 1990). The five species
of Cuterebra occurring in Florida are all Nearctic
temperate zone species with eastern distribu-
tions, but three (C. americana, C. buccata, and C.
fontinella) range very broadly into western North
America. In contrast, C. emasculator is found
from just west of the Mississippi River eastward
to the Atlantic Ocean, and C. cuniculi is the







Florida Entomologist 89(2)


most narrowly distributed, apparently occurring
only in southern Georgia and in Florida (Sa-
brosky 1986). Only one other species, Cuterebra
abdominalis Swenk, a member of the 'cuniculi'
group, is present in the southeastern US. Al-
though ranging broadly from the Midwest to the
Atlantic coast, this species apparently does not
occur in Florida. Thus, there are no precinctive
species of Cuterebra in Florida (although C. cuni-
culi comes close to being in this category) and
there appear to be no Caribbean ties for the Flor-
ida species of this genus. In addition, Neotropical
species in other cuterebrine genera are absent
from Florida, despite the subtropical climate in
the southern part of the state (Henry et al. 1994).
There appear to be several vacant niches for
Cuterebra species in Florida, in terms of the pres-
ence of indigenous rodent species that apparently
seldom if ever serve as hosts for flies in this genus.
It appears that 11 of the 17 (65%) native rodent
species within Florida fall into this 'vacant niche'
category (note that for these numbers, the various
subspecies are not considered separately): C. ca-
nadensis, G. pinetis, G. volans, M. pennsylvanicus,
M. pinetorum, N. alleni, 0. palustris, P poliono-
tus, R. humulis, S. hispidus, and T striatus. None
of these species are restricted to Florida (although
some of the subspecies are), and most of them ap-
pear to show little or no infestation by Cuterebra
larvae outside the state as well. Of these species,
apparently only M. pennsylvanicus and T striatus
are typical hosts of Cuterebra larvae outside Flor-
ida. It is likely that further study will demon-
strate that individuals of both of these species
serve as hosts for Cuterebra larvae within Florida
because species that typically parasitize these ro-
dents elsewhere (C. fontinella and C. emasculator,
respectively) are present in the state. Thus, al-
though additional research on host use within
Florida, as well as comparative studies of other
areas of North America, are required before a de-
finitive conclusion can be reached, Florida does
not appear to be exceptional in its apparently un-
utilized, potential host species among its indige-
nous rodents. Indeed, apparently the only unique
aspect of the association between Cuterebra spe-
cies and their typical host species in Florida is the
parasitization of individuals of the Florida mouse
(P. floridana), which apparently occurs only in the
state, by larvae of an unidentified species of Cute-
rebra (Layne 1963; probably C. fontinella).
In conclusion, there are many unanswered
questions about Cuterebra/ host species associa-
tions in Florida and elsewhere. In addition to the
need to better understand these flies' biology, such
as their typical and atypical host species, geo-
graphic ranges, and phenologies. Questions such
as what factors determine the suitability of ro-
dents and lagomorphs to serve as hosts, both be-
tween and within these orders, as well as in com-
parison with mammals in other orders, and what


are the effects of larval infestation on the perfor-
mance of individual hosts and host species popula-
tion dynamics, remain to be answered. For exam-
ple, in Florida there are several 'at-risk' (endan-
gered, threatened, or of special concern) species
and subspecies of rodents and a lagomorph (Flor-
ida Fish and Wildlife Conservation Commission
2004b) that might be affected by Cuterebra larval
infestation, but even the most basic data on prev-
alence and intensity of parasitization within these
populations are apparently lacking; similar situa-
tions occur in certain other states as well (Slansky
& Kenyon 2003). Throughout North America, do-
mestic felines with outdoor access can become in-
fested with Cuterebra larvae (F. S., unpublished
manuscript). Unlike with most other hosts, such
occurrences can be fatal to the cats (Glass et al.
1998), and yet information as basic as which spe-
cies of Cuterebra are involved in these cases is not
available. Thus, it would seem that additional re-
search on Cuterebra and host associations both
within and outside Florida is well justified.

ACKNOWLEDGMENTS

I thank P. M. Choate and M. Whaley for reviews of
an early draft of this manuscript, and Janella Abordo,
Paula Kelly, and Maria Riestra for tracking down much
of the literature relevant to this study.

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Mammal. 31: 293-304.


June 2006







Villanueva et al.: Cecidomyiid Species Predacious on Citrus Rust Mite


TWO SPECIES OF CECIDOMYIIDAE PREDACIOUS ON CITRUS RUST MITE,
PHYLLOCOPTRUTA OLEIVORA, ON FLORIDA CITRUS

RAUL T. VILLANUEVA,' RAYMOND GAGNE2 AND CARL C. CHILDERS3
'North Carolina State University, Mountain Horticultural Crops Research and Extension Center
455 Research Drive, Fletcher, NC 28732

2USDA, Systematic Entomology Laboratory, Beltsville, MD

3University of Florida, Citrus Research and Education Center, 700 Experiment Station Rd., Lake Alfred, FL 33850

ABSTRACT

Larvae of two undescribed species of Cecidomyiidae (Diptera) were found preying upon Phyl-
locoptruta oleivora (Ashmead) (Acari: Eriophyidae) on Florida citrus. Identifications to genus
were made from adults reared in the laboratory. The two species had distinctive larval color-
ation. One larval type was completely yellow and was identified as Feltiella n. sp., while the
second larval type had an orange color with a transverse white band close to the mouthparts.
The latter cecidomyiid was identified as belonging to a genus near Lestodiplosis in the broad
sense. Feltiella n. sp. (n = 17) and the species near the genus Lestodiplosis (n = 12) consumed
33.8 4.6 (mean SEM) and 43.0 6.4 citrus rust mite eggs; 14.2 1.4 and 15.0 2.0 citrus
rust mite nymphs, and 3.0 + 0.4 and 5.6 0.9 citrus rust mite adults/10 min., respectively.
There were no significant differences (P > 0.05) in the consumption rates of either predator
on any rust mite life stage. These data indicate that Feltiella n. sp. and the species near the
genus Lestodiplosis are both efficient predators of P. oleivora eggs, larvae, and nymphs.

Key Words: Acari, citrus, Diptera, Eriophyidae, Feltiella, Lestodiplosis, predation

RESUME

Dos species de larvas no descritas de Cecidomyiidae (Diptera) fueron encontradas depre-
dando Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyidae) en citricos de Florida. Las
identificaciones fueron hechas en adults criados en el laboratorio. Los dos cecidomidos tie-
nen distintivas coloraciones larvales. Un tipo es completamente amarillo y es identificado
como Feltiella n. sp., mientras que la otra tiene un collar blanco cerca de las parties bucales.
Este cecidomido fue identificado como una especie en el genero cercano a Lestodiplosis en un
amplio rango. Feltiella n. sp. (n = 17) y la especie cercana al genero Lestodiplosis (n = 12) con-
sumieron 33.8 4.6 (AVG SEM) y 43.0 6.4 huevos del acaro de tostado, 14.2 + 1.4 y 15.0
+ 2.0 ninfas del acaro del tostado, y 3.0 0.4 y 5.6 0.9 adults del acaro del tostado /10 min.,
respectivamente. No hubo diferencias significantes entire las dos species en los rangos de
consume de los diferentes estadios del acaro del tostado. Con los datos presentados aqui es
evidence que Feltiella n. sp. y la especie cercana al genero Lestodiplosis son depredadores efi-
cientes de huevos, larvas y ninfas de P. oleivora.


Translation provided by the authors.


The citrus rust mite (CRM), Phyllocoptruta ole-
ivora (Ashmead), and the pink citrus rust miteAc-
ulops pelekassi (Keifer) are pests on Florida citrus
(Acari: Eriophyidae) (Denmark 1963; Childers &
Achor 1999). Both species cause rind blemish in-
juries to developing and mature fruit. Other types
of damage include reduced bonding force of fruit,
premature fruit drop, reduced yields, and lower
juice quality (Allen et al. 1994). The pink citrus
rust mite also can cause leaf distortion, crinkling,
and stunting of new shoot growth (C. C. Childers,
unpublished). A third species, the citrus bud mite,
Aceria sheldoni (Ewing), is frequently found on
citrus but is not considered an economic pest in
Florida (Childers & Achor 1999). Natural enemies


of citrus rust mites include predatory phytoseiid
and stigmaeid mites (Muma 1961; Muma & Sel-
hime 1971; Peia 1992; Childers 1994). Hubbard
(1883) found "a little coral-red maggot and a yel-
low midge larva" (Diptera: Cecidomyiidae, for-
merly Itonididae) feeding on CRM in Florida.
Later, Muma et al. (1961) reported Itonidini spe-
cies feeding on P oleivora and the six-spotted
mite, Eotetranychus sexmaculatus (Riley), on cit-
rus in Lake Alfred, Florida.
The larvae of the cosmopolitan genus Feltiella
(Diptera: Cecidomyiidae) and all described spe-
cies form a group associated exclusively with tet-
ranychid species (Gagn6 1989), and this genus be-
longs in the tribe Lestodiplosini that is composed







Florida Entomologist 89(2)


entirely of predators or parasitoids of insects and
mites (Gagne 1989, 1994, 1995). The identifica-
tion of these mite-eating cecidomyiids is difficult;
for example, in Europe many authors are using
Feltiella acarisuga (Vallot) and Therodiplosis per-
sicae Kieffer as synonyms (i.e., Colombo et al.
1993; Piatkowski 2000; Putte 2002). The larval
stage of F acarisuga (Vallot) is a well-known
predator of the two-spotted spider mite, Tetrany-
chus urticae Koch (Gillespie et al. 1998).
Feltiella acarisuga completes its life cycle in 8
to 10 d in Italy (Roberti 1954) and 29 d on average
in Israel (Sharaf 1984). These differences in de-
velopmental times are likely dependent on tem-
perature and/or humidity differences. The crops
included in studies with F acarisuga were apple
(Roberti 1954), eggplant (Sharaf 1984), cucumber
(Gillespie et al. 1998, 2000), strawberry (Easter-
brook 1998), and plants in greenhouses (Opit et
al. 1997; Enkegaard et al. 2000). Feltiella occiden-
talis (Felt) occurs on strawberry in California
(Oatman et al. 1985) and F minute has been
found on eggplant (Ho & Chen 1998).
There are few examples of eriophyid predation
by larval cecidomyiids. Nijveldt (1969) compiled a
list of cecidomyiid species and their respective
eriophyid prey. The larva of a Medetera species
(Diptera: Dolichopodidae) was reported preying
on Aculus schlechtendali Nalepa on apple in
Washington (Rathman et al. 1988). The eriophyid
Aceria litchii Keifer is a serious pest of lychee
(Litchii chinensis Sonnerat) in Australia and
China, and the larva of Arthrocnodax sp. (Cecid-
omyiidae) was observed preying upon A. litchii
(Waite & Gerson 1994).
The objectives of this study were to identify
two cecidomyiids observed preying upon citrus
rust mites in different citrus orchards in Florida,
as well as to compare and quantify their con-
sumption of different CRM stages.

MATERIALS AND METHODS

Cecidomyiid Collection

Sampling for cecidomyiid larvae was con-
ducted between June and August and again from
October to the first week of December 2001 in a
'Hamlin' orange orchard in Lake Alfred, Florida.
Larvae were collected from citrus leaves and
fruits and transferred individually to Petri dishes
with a 5-0 sable brush. Individual fruits with high
numbers of CRM (>100 cm2) were collected to pre-
pare individual rearing arenas with an adequate
food source for maintaining the two dipteran spe-
cies. Some of the cecidomyiid larvae were allowed
to complete their development so that adults
could be obtained for identification while others
were used for feeding experiments and behavioral
observations. Data obtained on individual prey
experiments were recorded separately.


Rearing of Cecidomyiid Larvae

Individual rectangular, transparent plastic
containers (Pioneer Plastics Inc #295C, Eagan,
MN) with semi-tight lids (31 cm long x 24 cm wide
x 11 cm deep) served as rearing chambers for the
midge larvae. A lightly moistened piece of paper
towel was placed on the bottom of each container
to provide increased humidity. Each CRM-in-
fested orange arena was carefully examined and
all other arthropods removed. Between six to
eight oranges were then placed individually on
PVC rings (3.5 cm diam. x 1 cm high) inside each
plastic rearing container. Two or three cecid-
omyiid larvae of the same type were added to each
fruit in the same container and then covered with
a lid (n > 20 for yellow and orange, respectively).
The containers were held in an environmental
chamber at 25 1C, 60 + 10% RH under fluores-
cent lights set to a photoperiod of 14:10 (L:D) h.
The cecidomyiid larvae were observed daily and
the oranges infested with CRM were replaced as
required. As each cecidomyiid pupa formed, it was
removed and isolated in a 5-cm-diam. Petri dish
held under the same environmental conditions.
However, pupae were difficult to find and often es-
caped detection so that many adults emerged in
the plastic container. Pupae were found attached
to fruit, on or under the paper toweling, or at-
tached to the plastic walls of the rearing units.

Cecidomyiid Predation on Citrus Rust Mites

The predatory behavior of cecidomyiids was
observed on individual Hamlin oranges heavily
infested with all stages of CRM. Individual or-
anges were placed on PVC rings (3.5 cm diam. x 1
cm high) as described earlier. The presence of
high densities of CRM was confirmed on each
fruit by using a dissecting stereomicroscope
(>100 cm2). Since it was not possible to estimate
the age of the larvae used in the experiments, lar-
vae of similar lengths (1.6-1.8 mm) were selected
for each assay. A single cecidomyiid larva was
placed on a fruit within the center field of view by
using a stereomicroscope and monitored for 10
min. rotating the fruit to maintain a constant
focus on the maggot's movements. The number of
eggs, combined larvae and nymphs, and adult
CRM stages consumed per larva during each 10-
min. interval were tallied separately for each ce-
cidomyiid species. In total, 17 yellow and 12 or-
ange cecidomyiid larvae were observed. Data
were analyzed with a single factor analysis of
variance (Zar 1984). Sub-samples of eriophyid
mite populations were collected from fruits into
80% ethanol and then later slide-mounted in a
modified Hoyer's medium and identified to spe-
cies (Baker et al. 1996). Multiple slides were pre-
pared with each containing 10 or more rust mite
motile stages.


June 2006







Villanueva et al.: Cecidomyiid Species Predacious on Citrus Rust Mite


RESULTS

Cecidomyiid Collection and Rearing

Two distinct types of larvae were observed.
One was yellow in color (Fig. la) and the other
was orange with a white collar behind the mouth-
parts (Fig. lb).
Success in rearing cecidomyiids to the adult
stage was obtained with larvae collected during
November and December, but those collected dur-
ing July and August did not complete develop-
ment (Fig. 2). Adult males and females were
reared from yellow larvae whereas only two fe-
males were reared from orange larvae. The two
distinct types of larvae were identified as belong-
ing to two different genera. The yellow larvae
were an undescribed species of Feltiella and the
females obtained from the orange larvae were
identified as a species near the genus Lestodiplo-
sis (in the broad sense). However, they do not fit
well in the genus Lestodiplosis, suggesting that
this species may represent a new genus.

Predation by Cecidomyiids on Citrus Rust Mites

All eriophyids sub-sampled from each 10-min
observation interval were identified as the citrus
rust mite, Phyllocoptruta oleivora. The number of
eggs, combined larvae and nymphs, and adult cit-
rus rust mites consumed during 10-min observa-
tions by both species are shown in Fig. 3. There
were no significant differences (P > 0.05) between
the rust mite stages consumed by either Feltiella
n. sp. or the second dipteran species. Feltiella n.
sp. and the second species consumed 33.8 4.6
(mean SEM) and 43.0 + 6.4 (F = 1.99; 1,27 df; P
= 0.16) CRM eggs; 14.2 1.4 and 15.0 2.0 CRM
larvae and nymphs (F = 1.41; 1,27 df; P = 0.24),
and 3.0 0.4 and 5.6 0.9 CRM adults (F = 0.26;
1,27 df; P = 0.60), respectively.

DISCUSSION

Previous reports of cecidomyiid species feeding
on CRM were based largely on anecdotal informa-
tion. To date, there is no reliable description of
CRM feeding by cecidomyiid predators in Florida
or other citrus growing areas of the world. Mc-
Murtry (1977) and Perring & McMurtry (1996)
cited Muma et al. (1961), who only mentioned a
predatory midge he recovered from P oleivora col-
onies. There was no empirical quantification or
scientific description of cecidomyiid feeding be-
havior. Furthermore, all citations refer to the
original descriptions by Hubbard (1883) who ap-
parently described the two types of larvae (one
yellowish and the other orange with a white col-
lar) reported here. Adults ofFeltiella n. sp. (yellow
larva) and a species near the genus Lestodiplosis
(orange larva with a white collar) were success-


fully collected, reared, and identified to genus in
this study. Voucher specimens are deposited with
the USDA, Systematic Entomology Laboratory,
Beltsville, MD.
Others & Mason (1930) reported that these
midges appeared on occasion but only when high
numbers of CRM were present. This was observed
with Feltiella minute (Felt) when it increased on
Tetranychus kanzawai Kishida (Ho & Chen
1998). Both species examined in this study fed on
P oleivora eggs, larvae, and nymphs of CRM and
completed their development to adults on this
diet. If we extrapolate the average number of all
P oleivora stages consumed in 10 min to 4 min,
then 1.2 adults, 5.7 nymphs and larvae, and 13.5
eggs could be consumed in 4 min by a single
midge larva. This rate of consumption surpasses
the predatory capacity of Iphiseiodes quadripilis
(Banks), one of the most abundant phytoseiids on
Florida citrus (Villanueva & Childers 2005), feed-
ing on A. pelekassi, (Villanueva 2002) in which a
female starved for 24 h fed on 1.8 0.5 A. pele-
kassi in 4 min. A similar comparison was shown
between F minute and Amblyseius womersleyi
Schicha on eggplant (Ho & Chen 1998).
Laboratory observations of the two cecid-
omyiid predators revealed that they search for
CRM eggs by continuously moving the anterior
part of their bodies to the left and to the right
while moving forward and changing direction.
This appeared very similar to the 'questing' be-
havior described for syrphid larvae seeking
aphids (Bargen et al. 1998). Once an egg is de-
tected during this sweeping movement, it is rap-
idly consumed while the larva continues onward.
Consumption of CRM eggs by either cecidomyiid
species was difficult to observe due to the small
size and transparent color of the egg (about 30-40
uim). The presence of P oleivora eggs is essential
for larval development of both cecidomyiids for
two reasons. First, larvae of both species were fre-
quently found on the bottom of the plastic con-
tainers completely separated from the fruit. How-
ever, the arenas the cecidomyiid larvae had aban-
doned were still infested with motile stages of
CRM but lacked CRM eggs that were previously
consumed. When a new fruit with abundant CRM
eggs was provided, the cecidomyiid larvae would
immediately begin feeding and remained on the
fruit. Second, on many occasions, both species of
larvae encountered adult CRM but usually they
were not consumed. Other times, the larva would
raise the adult CRM off the substrate with their
mouthparts and appear to cast the adult aside.
On occasion, when the attacks on CRM adults
were successful, the larval mouthparts were di-
rected to the ventrolateral part of the mite's body,
just behind the second pair of legs. This was not
observed when the attacks were directed to the
immature rust mite stages. Larval and nymphal
CRM stages appeared to be more vulnerable and







Florida Entomologist 89(2)


Fig. 1. Two morphologically distinct larval types of two new species of cecidomyiids surrounded by their citrus
rust mite Phyllocoptruta oleivora prey. (A) Feltiella n. sp. and (B) a species near the genus Lestodiplosis. Note the
color and ring around the anterior end of the larva.


June 2006







Villanueva et al.: Cecidomyiid Species Predacious on Citrus Rust Mite


Fig. 2. Adult Feltiella n. sp., a predator of the citrus rust mite Phyllocoptruta oleivora reared in the laboratory.
The vertical bars beneath the insect are in millimeters.


were usually eaten when grasped on any part of
their bodies.
The relative abundance of these cecidomyiids
was not recorded in this study because the prior-
ity was to identify the species involved and quan-
tify their predation on CRM stages. Most larvae
collected in the field were more abundant on fruit
than on leaves during October and November. The
predominance of midge larvae on fruit surfaces
was consistent with the higher densities of CRM
present on fruits than on leaves.
Observations in both laboratory and field
showed that larvae of both cecidomyiid species
were capable of jumping or springing off a plant
substrate when disturbed. Larvae were observed
to raise the middle part of their bodies into an in-
verted U-shape while keeping both the head and
terminal ends attached to the substrate. The
larva would then rapidly release its hold on the
substrate and leap or spring from the plant sur-
face using tension on the sternal spatula or
breastbone to provide the springing action. Ceci-
domyiid larvae falling to the ground would likely
desiccate and die. From observations in the labo-


ratory rearing arenas, it appears that this move-
ment is directional. When the fruit in the rearing
containers had a few CRM eggs, the larva would


Feltiella n sp.


S20


E
0-


Eggs Ny~phs


Adults Eggs


near Lestodplosis nsp.


-ult
Nyrmphs Adults


Stages
Fig. 3. Mean numbers SEM of different citrus rust
mite (Phyllocoptruta oleivora) stages consumed by lar-
vae ofFeltiella n. sp. and a species near the genus Lesto-
diplosis.


SM







Florida Entomologist 89(2)


abandon the fruit and be found on an adjoining
new fruit that had abundant rust mite eggs.
The dispersal behavior of these midges and
their unnoticed predation on CRM eggs likely
contributed to earlier failed attempts to rear
these species. Similar situations were encoun-
tered by Yothers & Mason (1930) who wrote:
"These larvae are very small and extremely deli-
cate, and all attempts to rear them to maturity
have failed". Few accurate studies on the biology
of predacious cecidomyiids and their effects on
prey mite populations are available. This is the
case for cecidomyiids preying on Eriophyidae in
general, and also on different species of Tetrany-
chidae (Chazeau 1985).
In this study, we demonstrated that these two
undescribed cecidomyiids are capable of feeding
and reproducing on an exclusive diet of CRM.
Both species appeared to have highly specialized
abilities eating CRM eggs and immature stages.

ACKNOWLEDGMENT

We express our thanks for the helpful reviews by
J. P. Michaud and B. Grafton-Cardwell.

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Florida Entomologist 89(2)


June 2006


THE INTRODUCTION OF THE EXOTIC Q BIOTYPE OF
BEMISIA TABACI FROM THE MEDITERRANEAN REGION
INTO CHINA ON ORNAMENTAL CROPS

DONG CHU1'2, YOU-JUN ZHANG1 JUDITH K. BROWN3, BIN CONG4, BAO-YUN XU 1, QING-JUN WU1 AND GUO-REN ZHU1
'Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China

2High-tech Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, P.R. China

3Department of Plant Sciences, The University of Arizona, Tucson, AZ 85721, USA

4College of Plant Protection, Shenyang Agricultural University, Shenyang 110161, P.R. China

ABSTRACT

The Q biotype of Bemisia tabaci (Gennadius), which has been described from the Mediterra-
nean/North African region, was identified for the first time infesting ornamental crop species
in several locations in China. Identification and partial distributions of the exotic B biotype
and the recently introduced Q biotype in China were established by using the mitochondrial
cytochrome oxidase I gene (mtCOI) as a molecular marker. Collections of B. tabaci were
made from representative geographical locations and plant hosts in different provinces of
China. MtCOI sequence analysis revealed that collections from Beijing [AY582872,
AY589499], Yunnan [AY518189, AY587516], and Henan [AY587514] shared >99.6% se-
quence identity with the Q biotype from Spain [AY587513, AY562216, AY596950]. The Q
type from China shared 98.9-99.4% nucleotide sequence identity with Q-like relatives of
B. tabaci described from Israel [AY518191, AY582869]. Phylogenetic analyses indicated that
certain B. tabaci populations that are present in China are the Q biotype, and that the Q bio-
type now in China may have originated from Spain or other nearby locations where the Q
biotype has been identified. This is the first report of the introduction of the Q biotype from
the Mediterranean region into China. The specific outcomes of the Q biotype as an invasive
species in Asia are presently unknown. Certain Q biotype populations from Spain have been
reported to exhibit resistant to neonicotinoid insecticides, which are commonly used for con-
trolling this pest and virus vector in ornamental and field crops. Thus, the close monitoring
of the Q biotype in China and elsewhere, particularly where commercial plants are grown for
export or received for importation, respectively, is essential to avoid the further geographical
expansion of the habitat of the Q biotype.

Key Words: Bemisia tabaci biotype Q, geographical origin, introduced species, mitochondrial
COI gene, phylogenetic analysis

RESUME

El biotipo Q de Bemisia tabaci (Genn.), el cual fue descrito de la region Mediterraneo/Norte
de Africa, fue identificado por primera vez infestando species de cultivos ornamentales en
various lugares de China. La identificaci6n y la distribuci6n parcial del ex6tico biotipo B y el
reci6n introducido biotipo Q en China fueron determinados usando el gen citocromo oxidase
I mitocondrial (mtCOI) como un marcador molecular. Se realizaron colecciones de Bemisia
tabaci de lugares geograficos representatives y hospederos de plants en diferentes provin-
cias de China. El andlisis de la secuencia de mtCOI revel6 que las colecciones de Beijing
[AY582872, AY589499], Yunnan [AY518189, AY587516] y Henan [AY587514] compartieron
>99.6% de la identidad de la secuencia con el biotipo Q de Espana. [AY587513, AY562216,
AY596950]. El tipo Q de China compartieron 98.9-99.4% de la identidad de la secuencia de
nucle6tidos con los relacionados de clase como del tipo Q de B. tabaci descritos de Israel
[AY518191, AY582869]. El andlisis filogen6tico indica que ciertas poblaciones de B. tabaci
que estan presents en China son de biotipo Q biotype, y el biotipo Q que ahora esta present
en China puede haberse originado en Espana u otros lugares cercanos donde el biotipo Q ha
sido identificado. Este es el primer informed de la introducci6n de biotipo Q de la region Me-
diterranea a China. Los resultados especificos de biotipo Q como una especie invasora en
Asia son en estos moments desconocidos. Ciertas poblaciones del biotipo Q de Espana han
sido reportadas que muestran resistencia a los insecticides neonicotinoides que se usa regu-
larmente para controlar esta plaga y vector de virus en cultivos ornamentales y de campo.
Por esto, es esencial realizar un monitoreo extensive del biotipo Q en China y en otros luga-
res, particularmente donde se siembra plants comerciales para exportaci6n o recibidas
para importaci6n para evitar una mayor expansion geografica del habitat del biotipo Q.







Chu et al.: Exotic Q Biotype of Bemesia tabaci in China


The Bemisia tabaci (Genn.) complex (Brown et
al. 1995b) is a hemipteran (Aleyrodidae) pest that
feeds on plant phloem. It also is the most important
vector worldwide of several genera of plant virus
(Brown 2000, 2001; Brown & Bird 1992).
Bemesia tabaci is best described as a species
complex that comprises an unexpectedly large
number of genetically variable populations, some
of which are discernible owing to distinct pheno-
types (Brown et al. 1995a,b). Well-studied B.
tabaci populations that have been differentiated
are referred to as races (Brown & Bird 1992) or
biotypes (Brown et al. 1995a; Costa & Brown
1991). The B biotype (Costa & Brown 1991) is a
particularly aggressive B. tabaci variant. It has
an extremely broad host range, is highly fecund,
and disperses relatively long distances (Brown
2000; Brown et al. 1995b), and has become estab-
lished in many locations beginning approxi-
mately in 1988-present (Costa & Brown 1991;
Costa et al. 1993). Since that time, it has been of
considerable concern as a pest and virus vector in
subtropical and temperate, mild climate zones
where the majority of the world's vegetable and fi-
ber crops are produced (Brown et al. 1995a,b;
Brown 2000). Population genetics studies have
shown that the B biotype probably originated
from the Middle Eastern/North African region
(Frohlich et al. 1999).
In China B. tabaci has become an important ag-
ricultural pest in the late 1990s (Chu et al. 2004;
Zhang 2000), and the introduction of the B biotype
into China was first reported in 2002 (Luo et al.
2002). The B biotype is now known to be wide-
spread in a number of provinces in China where
vegetables, cotton, and ornamentals are produced
(Chu et al. 2004; Wu et al. 2003; this report).
In China and elsewhere, the understanding
that B. tabaci is a polymorphic, cryptic species
that can upsurge without warning and cause
great damage to crop and ornamental species is
lacking. This has often resulted in the delayed
recognition of upsurges in local whitefly popula-
tions and/or of exotic introductions (Reitz &
Trumble 2002), such as the B biotype. This real-
ization has prompted an accelerated interest in
practicing routine monitoring ofB. tabaci popula-
tions to detect early the potentially invasive B.
tabaci, or otherwise upsurgent haplotypes, with
particular emphasis on those that are associated
with the global commercial plant industry.
The purpose of this study was to determine if
the Q biotype (Costa et al. 1993; Guirao et al.
1997) was present in ornamentals and/or annual
flowering or bedding plants in China. Such knowl-
edge is important because the Q biotype has only
recently been recognized as a potentially invasive
pest species in the vegetable and ornamentals in-
dustries in the Mediterranean/Middle Eastern re-
gion (Guirao et al. 1997; Horowitz et al. 2003). The
introduction and establishment of the Q biotype is


anticipated, or was possibly expected to already
have occurred, in at least certain locations, and is
expected to have important and far-reaching eco-
nomic relevance. The potential for damage will
further be exacerbated if Q biotype populations
exhibit resistance to a well-known neonicotinoid
(Nauen et al. 2002; Rauche & Nauen 2003), upon
which the industry presently relies to control
B. tabaci. Resistance to this compound has al-
ready been reported in Spain and Israel (Ebert &
Nauen 2000; Rauche & Nauen 2003).
Recent studies have demonstrated that the mi-
tochondrial cytochrome oxidase I (mtCOI) gene
(Brown 2001; Brown et al. 1995b; Frohlich et al.
1999) is a highly informative coding sequence for
differentiating populations and haplotypes/bio-
types in the B. tabaci complex. In this study, the
mtCOI was used as a population genetics marker
to detect the presence of and identify the Q bio-
type, and subsequently to determine its partial
distribution in a number of provinces in China,
which routinely produce ornamental and bedding
plants. Phylogenetic analysis of the mitochon-
drial COI sequence for B. tabaci collections from
China revealed for the first time that the Q bio-
type is distributed in multiple locations through-
out the country.

MATERIALS AND METHODS

Whitefly Collections

Adult whiteflies (B. tabaci) were collected live
and placed into tubes containing 95% ethanol.
Populations were collected from representative
locations and plant species throughout select
provinces of China (Table 1).

Whiteflies DNA Extraction, the Polymerase Chain Re-
action, and Sequencing

Individual whiteflies were subjected to lysis
and DNA extraction following the procedure of
Frohlich et al. (1999). Polymerase chain reaction
(PCR) (Saiki et al. 1988) primers were employed
to amplify a fragment of the B. tabaci mitochon-
drial COI gene (800-820 bp), using parameters
and PCR primers, as described by Frohlich et al.
(1999).
PCR assays were conducted with 2 pL of each
template DNA in a total reaction volume of 25 pL.
The PCR reaction mix and PCR conditions fol-
lowed Frohlich et al. (1999) with a little modifica-
tion, and 1 unit of Taq DNA polymerase was con-
tained in the PCR reaction mix. PCR products
were separated on 1.0% agarose gels, and bands
were visualized by ethidium bromide staining
and viewed with a UV light source. PCR products
were purified with a kit (EZ Spin Column DNA
Gel Extraction Kit purchased from Sangon Tech-
nology Company, Shanghai) according to the man-







Florida Entomologist 89(2)


June 2006


TABLE 1. BIOTYPE, GEOGRAPHICAL SOURCE, HOST PLANT, AND GENBANK ACCESSION NUMBER FOR B.TABACI MITO-
CHONDRIAL CYTOCHROME OXIDASE DNA (MTDNA COI) SEQUENCES.


Geographical
location and year

Zhengzhou
2003
Zhengzhou
2003
Zhengzhou
2003
Zhengzhou
2003
Zhengzhou
2003
Zhengzhou
2003
Beijing
2003
Beijing
2003
Beijing
2003
Beijing
2003
Beijing
2003
Zaozhuang, Shandong
2003
Taian, Shandong
2003
Nanjing, Jiangsu
2003
Zhejiang
20003
Zhejiang
2003
Shanghai
2003
Shanghai
2003
Kunming, Yunnan
2003
Haikou, Hainan
2003
Tulufan, Xinjiang
2003
Israel
2003
Israel
2003
Spain
2003
Spain
2003

Arizona, USA
2003


Host plant

Brassica oleracea L.

Gossypium hirsutum L.

Cucurbita moschata L.

Brassica oleracea L.
var. capitata L.
Ipomoea batatas L.

Solanun melongena L.

Cucumis sativus L.

Unknown

Capsicum annuum L.

Cucumis sativus L.

Heliantus annuus L.

Cucumis sativus L.

Unknown

Gossypium hirsutum L.

Unknown

Unknown

Euphorbia pulcherrima
Willd.
Brassica albo-glabra Bail.

Euphorbia pulcherrima
Willd.
Solanun melongena L.

Euphorbia pulcherrima
Willd.
Gossypium hirsutum L.

Gossypium hirsutum L.

Solanum lycopersicum L.

Solanum lycopersicum L.


Hibiscus rosa-sinensis L.


GenBank
Accession number

AY582870
AY518186
AY587515

AY596949

AY582873

AY589497

AY587514

AY587519

AY582867

AY596953
AY582871
AY589498

AY582872
AY589499
AY587518

AY587517

AY518185

AY566182

AY596952

AY550274

AY550273

AY587516
AY518189
AY518187

AY582868

AY518190

AY518191
AY582869
AY596951

AY596950
AY587513
AY562216
AY518194


Acronym

HeNanBoleAY582870

Zhengzhou

Zhengzhou

Zhengzhou

Zhengzhou

HeNanSmelAY587514

BJCsatAY587519

BJAY582867

BJ

BJ

BJInilAY582872

SDZZAY587518

SDTAAY587517

JSNJAY518185

ZJAY566182

ZJNBAY596952

ShHaiEpulAY550274

Shanghai

YN2AY587516
YNAY518189
HaiNAY518187

XJAY582868

IBAY518190

IQAY518191

SB
AY596951
SQWAY596950
SQ1AY587513
SQ2
AZAY518194


Whitefly
haplotype
or biotype

B

B

B

B

B

Q

B

B

B

B

Q

B

B

B

B

(non-B/Q)
Asian clade
B

B

Q

B

B

B

Q-like

B

Q


B







Chu et al.: Exotic Q Biotype of Bemesia tabaci in China


TABLE 1. (CONTINUED) BIOTYPE, GEOGRAPHICAL SOURCE, HOST PLANT, AND GENBANK ACCESSION NUMBER FOR
B.TABACI MITOCHONDRIAL CYTOCHROME OXIDASE DNA (MTDNA COI) SEQUENCES.

Whitefly
Geographical GenBank haplotype
location and year Host plant Accession number Acronym or biotype

California, USA Solanun melongena L. AY589496 California B
2003
California, USA Euphorbia pulcherrima AY550272 CLAY550272 B
2003 Willd.
Texas, USA Brassica oleracea L. AY518192 TXAY518192 B
2003 var. capitata L.


ufacturer's instructions. The DNA sequence for
each PCR product was determined from the 5'end
at the Sangon Technology Company, Shanghai.

Phylogenetic Analyses and Identification of B. tabaci
Haplotypes in China

The mtCOI sequences for select, representa-
tive whiteflies (geographical and host or origin)
were determined, and reference mtCOI se-
quences were obtained from the GenBank data-
base. The DNA sequence was obtained for one to
three individuals from each of 30 whitefly collec-
tions (Table 1). The mtCOI sequences were
aligned with the CLUSTAL W algorithm (Thomp-
son et al. 1994). Distances were calculated with
the Kimura 2-parameter model of MEGA2.1 (Ku-
mar et al. 2001). The NJ (Neighbour-Joining) and
UPGMA (Unweighted Pair Group Method with
Arithmatic Mean) algorithms available in
MEGA2.1 (Kumar et al. 2001) were used to infer
phylogenetic relationships, respectively. Two
thousand bootstrap replicates were performed for
each analysis.

RESULTS

Phylogenetic Analysis of the B. tabaci MtCOI

The mtCOI sequence was edited to remove
PCR primer sequences, which yielded a ~470-bp
fragment for each B. tabaci mtCOI sequence. The
mtCOI sequences have been deposited in Gen-
Bank and the Accession Number for each is shown
parenthetically (Table 1). Because the same hap-
lotype was typically observed in field populations,
only one mtCOI sequence was included for each
representative haplotype per field collection.
The mtCOI sequence was used to identify bio-
types and haplotypes, based on phylogenetic rela-
tionships. The mtCOI NJ (Fig. 1) and UPGMA
(not shown) trees revealed similar results and
four main clades were supported, each by robust
bootstrap value. Three distinct clades were re-
vealed with two major nodes strongly supported
by 100% bootstrap values.


One major clade (I) contained sequences for the
B biotype, and this identification was based on a
high degree of shared nucleotide identity with ref-
erence sequences for the B biotype (Costa & Brown
1991). These collections were from Zhengzhou in
Henan Province [AY582870, AY518186,
AY587515, AY596949, AY582873, AY589497],
Beijing [AY587519, AY582867, AY596953,
AY582871, AY589498], Shandong [AY587518,
AY587517], Jiangsu [AY518185], Zhejiang
[AY566182], Shanghai [AY550274, AY550273],
Hainan [AY518187], and Xinjiang [AY582868].
The latter haplotypes grouped closely with the ref-
erence sequences for the B biotype previously
identified in Israel [AY518190], Spain [AY596951],
and the U.S.A., including Arizona [AY518194],
California [AY589496], and Texas [AY518192].
A second major clade (II) contained B. tabaci
identified as the Q biotype (Guirao et al. 1997;
Moya et al. 2001) or variants of the Q haplotype
sequence. The Q-like haplotype was identified in
collections from Yunnan [AY587516,AY518189],
from Zhengzhou in Henan [AY587514], and from
Beijing [AY582872, AY589499]. The Q haplotype
has been identified previously in Spain
[SQ1AY587513, SQWAY596950], where it was
subsequently characterized in biological terms as
the Q biotype (Guirao et al. 1997; Moya et al.
2001), and in Israel [IQAY518191] (Horowitz et
al. 2003). The latter population/haplotype, which
is a very close relative of the Spanish Q biotype, is
probably indigenous to Israel, because B. tabaci is
composed of several genetically distinct groups
with a strong geographical association between
more closely related biotypes (Frohlich et al.
1999; De Barro et al. 2000).
The third clade (III) was represented by a hap-
lotype for a population collected from Zhejiang,
China [ZJNBAY596952], which appears to be of
Asian origin.

Nucleotide Divergence Estimates

Within and between nucleotide sequence di-
vergence were calculated for field collections from
China and reference population sequences for the







Florida Entomologist 89(2)


100


BJAY582867
HeNanBoleAY582870
SDZZAY587518
JSNJAY518185
TXAY518192
SDTAAY587517
55
CLAY550272
AZAY518194
IBAY518190
ShHaiEpulAY550274
ZJAY566182
BJCsatAY587519
XJAY582868
65
HaiNAY518187
SBAY596951


QI AY518191


BJInilAY582872
100 68 YNAY518189
s80 5QWSQWAY596950
YN2AY587516
51
SQ1AY587513
HeNanSmelAY587514
ZJNBAY596952


Clade I
B biotype







I




Clade III
Q and Q-like





Clade III


Figure 1. Phylogenetic tree for B. tabaci based on a fragment (~450 bases) of the mitochondrial cytochrome ox-
idase I gene. The tree was inferred by using the UPGMA method and 2000 bootstrap replicates. Abbreviations for
whitefly collections are shown in Table 1.


B and Q biotypes of B. tabaci. Populations from
Beijing [BJAY582867, BJCAY587519], Xinjiang
[XJAY582868], Shandong [SDZZAY587518,
SDTAAY587517], Henan [HeNanBoleAY582870],
Jiangsu [JSNJAY518185], Shanghai
[ShHaiEpulAY550274], Zhejiang [ZJAY566182],
Hainan [HaiNAY518187] from China shared
more than 99.6% nucleotide sequence identity
with B. tabaci B biotype identified from Spain
[SBAY596951], Israel [IBAY518190], Arizona
[AZAY518194], California [CLAY550272], and
Texas [TXAY518192]. These mtCOI sequences
were 100% identical with B biotype sequences for
populations collected from various other loca-
tions, worldwide.
The nucleotide divergence estimates indicated
that accessions from Beijing [BJlnilAY582872],
Yunnan [YN1YNAY518189, YN2AY587516] and
Henan [HeNanSmelAY587514] shared >99.6%


nucleotide sequence identity with the Q biotype
from Spain [SQ1AY587513, SQWAY596950]. The
mtCOI sequences from the latter collections from
China likewise shared 98.9%-99.4% nucleotide
sequence identity with one population ofB. tabaci
from Israel [IQAY518191].
Sequence comparisons collectively suggest er-
tain collections of B. tabaci from China are the Q
biotype, and the B. tabaci population from Israel
[IQAY518191] also is Q-like. The populations from
Israel and China were slightly more divergent
from one another than collections from China
were from sequences obtained from B. tabaci Q
biotype from Spain. The mtCOI DNA sequence
(~470 bp) for whiteflies identified as the Q biotype
from China was highly invariant (99-100% nucle-
otide identity) (data not shown), suggesting that
they originated recently from a single or a very
few introductions and/or original sourcess.


June 2006







Chu et al.: Exotic Q Biotype of Bemesia tabaci in China


The collection from Zhejiang [ZJNBAY596952]
shared only 82.3%-83.5% nucleotide sequence
identity with reference B. tabaci sequences in-
cluded here, indicating that the Zhejiang haplo-
type was neither B nor Q biotype, and likely rep-
resents a divergent B. tabaci population that orig-
inated 'locally' and is indigenous to Asia.

DISCUSSION

Recently, the Q biotype ofB. tabaci, which had
been a relatively benign pest in the Mediterranean
region (Sim6n et al. 1999), has been recognized as
a serious pest and virus vector, owing to its ability
to reach high population densities (Moya et al.
2001; Sim6n et al. 1999) and to develop resistance
to at least one neonicotinoid (Rauch & Nauen
2003). These characteristics have been noted to-
gether with an increase in Q biotype infestations
in southern Spain, where the B biotype is now al-
most absent, despite its introduction there in the
mid-1990s (Sim6n et al. 1999). What is now recog-
nized as the Q biotype has been identified in the
Iberian Peninsula, in Sardinia and Sicily, and in
Morocco (Brown 2000; Moya et al. 2001), the gen-
eral region (Mediterranean/Middle East/North Af-
rica) to which Q-like haplotypes are thought to be
indigenous (Brown 2000).
Significant differences in host suitability (Mu-
niz 2000) and developmental parameters (Muniz
& Nombela 2001) for the B and Q biotypes, with
respect to four weed species that occur in the win-
ter months, were determined in no-choice assays.
Except for Lactuca serriola L., the mean repro-
ductive parameters for the Q biotype were signif-
icantly greater than those for the B biotype (Mu-
niz 2000). The Q biotype showed higher daily in-
festation rates than the B biotype on most tomato
varieties tested (Nombela et al. 2001). On sweet
pepper, the generation time for the Q biotype was
found to be shorter than that of B biotype at 33
and 17 d, respectively. The number of cumulative
generations of Q biotype also was somewhat
greater than for the B biotype (Muniz & Nombela
2001). Furthermore, the resistance of the Q bio-
type to pesticides has been shown to be more resil-
ient than observed for the B biotype (Anthony et
al. 1995; Costa et al. 1993; Rauch & Nauen 2003).
These collective results are likely linked to the in-
creased pest status of the Q biotype in the Medi-
terranean region during the past several years.
Prior to this study, the Q biotype had not been
reported in China. The high intra-population ho-
mogeneity suggests that a recent introduction of
this biotype has occurred in China. Knowledge of
insecticide resistant-Q haplotype populations in
Spain (Ebert & Nauen 2000) and Israel (Nauen et
al. 2002), and also that Spain and the Canary Is-
lands are important producers of ornamentals
crops that could have made their way to China in
commercial trade, suggests a link between the


movement of ornamentals species from the Medi-
terranean region and the recent presence of the Q
type in China. Importation records have revealed
that poinsettia and other ornamental plants were
imported to China from Spain for the Interna-
tional Horticultural Exposition held in 1999 in
Kunming, Yunnan Province. Such plants could
have provided one possible route of entry into
China for the Q biotype.
Herein, we report that the Q-biotype of B.
tabaci was identified for the first time on infested
ornamentals plants in several different regions of
China. It is now important to closely monitor the
potential establishment and spread of the Q bio-
type in the country to avoid its further dissemina-
tion, which could be devastating to vegetable and
ornamental production. We have identified the Q
biotype in distant locations in China, suggesting
that multiple introductions may have occurred, or
that plants from a single introduction were
moved long distances from the original sources.
Additional studies will be required to test this hy-
pothesis and to determine if the Q biotype is suf-
ficiently adapted to conditions in China to estab-
lish as an invasive pest and vector of plant vi-
ruses in ornamental, vegetable, and fiber crops.

ACKNOWLEDGMENTS
We acknowledge Dr. Matthew A. Ciomperlick (USDA
APHIS PPQ Mission Biological Control Center, Mission,
TX), and Dr. Rami Horowitz (Department of Entomol-
ogyAgricultural Research Organization, Gilat Research
Center M.P. Negev, Israel) for providing whitefly sam-
ples included in the analysis, and two reviewers for in-
sightful comments. This work was funded by National
Basic Research and Development Program, China
(Grant No. 2002CB111400), National Natural Science
Foundation of China (Grant No. 30500331), Beijing Mu-
nicipal Natural Science Foundation (Grant
No.6062024), and Key Laboratory of Vegetable genetics
and Physiology, Ministry of Agriculture.

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June 2006







Muria et al.: Fall Armyworm Dynamics and Parasitoids in Argentina


POPULATION DYNAMICS OF THE FALL ARMYWORM,
SPODOPTERA FRUGIPERDA (LEPIDOPTERA: NOCTUIDAE)
AND ITS PARASITOIDS IN NORTHWESTERN ARGENTINA

GABRIELA MURiA1, JAIME MOLINA-OCHOA2 AND CARLOS COVIELLA3
'Estaci6n Experimental Agroindustrial Obispo Colombres, William Cross 3150, Las Talitas
4101 Tucuman, Argentina

2Universidad de Colima, Facultad de Ciencias Biol6gicas y Agropecuarias
Km. 40, autopista Colima-Manzanillo, Tecoman, Colima 28100, M6xico

3Universidad Nacional de Lujan, Laboratorio de Ecologia, cruce rutas 5 y 7, CC 221, 6700 Lujan, Argentina

ABSTRACT

In order to know the population dynamics of the fall armyworm (FAW), Spodoptera fru-
giperda (J. E. Smith), and its parasitoids in northwestern Argentina, larvae were weekly col-
lected at two different agrological regions (Tafi Viejo, and Vipos) over four years. The
relationship between larval and parasitoid populations, climatologic factors, percent of in-
fested plants, parasitoid relative importance index, abundance of the parasitoids, and per-
cent parasitism were estimated. FAW attacked cornfields when the plants achieved V1 and
V2 stages. Temperature and rainfall were the climatologic factors that significantly affected
pest density, and temperature affected the parasitoid abundance as well. The FAW parasi-
toids collected were Campoletis grioti (Blanchard), Chelonus insularis (Cresson), Ophion sp.
and Archytas spp. (possibly marmoratus and/or incertus). The average parasitism percent-
age was 39.4% and 15% in T. Viejo and Vipos, respectively. Parasitoid abundance in both re-
gions was similar, but diversity was different possibly relating to the native surrounding
vegetation in Vipos. This is the first report of population dynamics of the fall armyworm and
its parasitoids in northwestern Argentina.

Key Words: Spodoptera frugiperda, parasitoid complex, population fluctuation, perfor-
mance, corn

RESUME

Para conocer la dinamica poblacional del gusano cogollero del maiz (GC), Spodoptera fru-
giperda (J. E. Smith) y la de sus parasitoides en el Noroeste argentino, se colectaron larvas
semanalmente en dos regions agrol6gicas (Tafi Viejo y Vipos) durante cuatro aios. Se de-
termin6 la relaci6n entire la fluctuaci6n poblacional de la plaga y la de sus parasitoides con
los factors climaticos. Se estim6 el porcentaje de plants daiadas, el indice de importancia
relative, la abundancia de los parasitoides, y el porcentaje de parasitismo. El GC ataca los
cultivos de maiz ni bien son implantados, cuando las plants alcanzan las etapas fenol6gicas
V1 y V2. La temperature y la precipitaci6n fueron los factors climatol6gicos que afectaron
significativamente la abundancia de la plaga. La abundancia de los parasitoides tambi6n fue
afectada por la temperature. Los parasitoides colectados fueron Campoletis grioti (Blan-
chard), Chelonus insularis (Cresson), Ophion sp., and Archytas spp. (posiblemente marm-
oratus y/o incertus). Los porcentajes de parasitismo fueron 39.4% y 15% en T. Viejo y Vipos,
respectivamente. La abundancia de los parasitoides en las dos zonas estudiadas fue similar,
pero la diversidad fue diferente posiblemente relacionada a la vegetaci6n circundante en Vi-
pos. Este es el primer studio sobre la dinamica poblacional del GC y la de sus parasitoides
en el Noroeste argentino.

Translation provided by the authors.



INTRODUCTION sects are natural enemies of pest species, and can
be used as biological control agents for reducing
Insects are a dominant component of agricul- pest organisms. Insects are considered indicators
tural ecosystems, and they impact crop yields in of biodiversity, providing a means of determining
many ways. Several species are pests of row and the effects of agricultural practices on whole com-
horticultural crops, reducing yields by the trans- munities or on abundance and dynamics of indi-
mission of diseases or by direct damage. Other in- vidual species (LaSalle 1993).







Florida Entomologist 89(2)


Understanding the factors that influence the
distribution and abundance of an insect is a fun-
damental issue of insect ecology and is a practical
concern with insects that cause economic damage
(Baskauf 2003). Insect population dynamics have
fundamentally different characteristics depend-
ing on the strength and form of exogenous (den-
sity-independent) vs. endogenous (density-depen-
dent) forces. Many factors affect population abun-
dance such as competition, natural enemies, and
resources, but the relative contribution of exoge-
nous and endogenous effects remains an open
question for nearly all biological populations
(Ylioja et al. 1999).
The fall armyworm (FAW), Spodoptera fru-
giperda (J. E. Smith) was recognized as a destruc-
tive pest of many agricultural crops more than
200 years ago (Luginbill 1928). It is an important
economic pest of corn, rice, sorghum, peanut, al-
falfa, cotton, Sudan grass, soybean, tobacco, oat,
wheat, sugarbeet, and diverse pasture grasses
such as Bermuda grass, Johnson grass, and oth-
ers (Sparks 1979; Andrews 1980; Capinera 1999),
and it is widely distributed in America. Its distri-
bution extends eastward into the Caribbean,
southward to northern Argentina and northern
Chile, and northward through Central America,
Mexico, the United States, and southern Canada
(Andrews 1980).
Because the FAW has a wide distribution, it is
subjected to much climatic diversity, namely, tem-
perature, moisture, and soil type. The environ-
mental factors influencing development and sur-
vival, as well as genotype, agricultural practices,
crop phenology, and plant maturity may contrib-
ute to the dynamics of the system in a given locale
(Harrison 1984a; Pair et al. 1986; Barfield & Ash-
ley 1987; Simmons 1992; Riggin et al. 1993).
The FAW is the most important corn pest,
causing yield losses fluctuating from 17% to 72%
in northeastern Argentina (Perdiguero et al.
1967). However, studies related with the popula-
tion dynamics of FAW in Argentina, and how en-
vironmental factors affect this phenomenon were
not previously reported in Argentina. Reports re-
lated to the relationship between date and dam-
age by FAW in commercial corn in northwestern
Argentina have been published by Willink et al.
(1993a, b), and Sosa (2002a, b).
FAW has a diverse complex of natural enemies
in the Americas and the Caribbean basin (Ashley
1979; Ashley et al. 1982; Molina-Ochoa et al.
2003). In Argentina at least thirteen species of hy-
menopteran parasitoids and eight dipteran para-
sitoids are known to attack FAW (Vera et al. 1995;
Virla et al. 1999; Murua et al. 2003; Murua &
Virla, 2004). However, there is a lack of informa-
tion on the natural distribution of FAW and its
parasitoids in northwestern Argentina, as well as
the influence of environmental factors on their
dynamics. We report the population dynamics of


FAW, its parasitoids, and the influence of environ-
mental factors on the dynamics in the northwest-
ern region of Argentina.

MATERIALS AND METHODS

Sampling Sites

Two agrological regions (Zuccardi & Fadda
1985) of northwestern Argentina were systemati-
cally sampled for FAW larvae during the crop-
growing part of the year over a period of four
years. The two regions were located in the prov-
ince of Tucuman. The first region was Tafi Viejo
(Department of Tafi Viejo), located between the
coordinates 26 44' S, 65 14' W, and 609 m alti-
tude. This region is part of the Chaco Pampeana
Plain, and it is characterized by good availability
of soil moisture during the year. The cornfield
used for monitoring was seven ha in size and
planted with the regional corn variety Leales 23.
The second region was Vipos (Department of
Trancas) located at 26 28' S, 65 18'W, and 786 m
altitude, in the Intermontana of Tapia-Trancas
basin. This region is characterized by soil mois-
ture availability only during the rainy part of the
year (December-January), and irrigation is usu-
ally required. The cornfield sampled at Vipos was
ca. 40 ha, and was also planted with the corn va-
riety Leales 23. Other commercial crops grown in
the area included corn, soybean, pumpkins, and
vegetables. All commercial fields routinely ap-
plied insecticides.
Both regions exhibit agrological differences in
their hydrological conditions such as rainfall and
evapotranspiration. These differences determine
the planting date for each region. Corn can be
planted during late October at Tafi Viejo, while
the planting date at Vipos occurs from late De-
cember to early January.
Sampling for FAW larvae was conducted
weekly at Tafi Viejo from October 1999 to January
2000 (Year 1), from October to December in 2000
(Year 2), from October to December 2001 (Year 3),
and November 2002 to January 2003 (Year 4). In
Vipos, the larvae were sampled from January to
March in 2000 (Year 1), from October to December
in 2000 (Year 2), from October to December 2001
(Year 3), and from January to March in 2003 (Year
4). Insecticides were not applied in any of the
fields sampled in this study.

Larval Sampling

FAW larvae were sampled beginning approxi-
mately 10-12 d after the date corn plants exhib-
ited two ligulate leaves, and continued until the
beginning of the reproductive stage (R1) (Ritchie
et al. 1992). The sampling period lasted about five
to seven weeks. Fifty corn plants were randomly
sampled at each sampling date, and divided in


June 2006







Murua et al.: Fall Armyworm Dynamics and Parasitoids in Argentina


five groups of ten plants. The plants were checked
for the presence of FAW eggs, larvae, and/or
adults following the methodologies used by Will-
ink et al. (1993a,b), Garcia Roa et al. (1999), and
Fernandez (2002). The number of corn infested
plants was recorded in order to determine the per-
centage of infested plants (Harrison 1984b).
FAW larvae collected from cornfields were
placed in glass vials (12 cm long x 1.5 cm diame-
ter) containing a piece of fresh corn leaf, and were
kept in a chamber under controlled conditions at
25 + 2C, 70-75% RH, and 14L:10D photoperiod.
FAW larvae were then transferred to similar
tubes containing 1 cm3 of artificial diet (Osores et
al. 1982). The diet vials containing FAW larvae
were maintained in the laboratory until the para-
sitoids had emerged, or until FAW adult emer-
gence (Riggin et al. 1993).

Parasitoid Identification

Parasitoids were recorded and identified by
several specialists. Tachinids were identified by
Lic. Susana Avalos (Facultad de Ciencias
Agropecuarias, Universidad Nacional de C6r-
doba, Argentina), and Campoletis grioti (Blan-
chard) by Dra. Carolina Berta (Fundaci6n Miguel
Lillo, Departamento de Zoologia, Entomologia,
San Miguel de Tucuman, Argentina). The remain-
ing parasitoids were compared to specimens pre-
viously identified by Dr. Luis De Santis (Facultad
de Ciencias Naturales y Museo, Universidad Na-
cional de La Plata, La Plata, Argentina).

Population Variables

The Percent of infested plants (% IP) (Harrison
1984b, Urbaneja Garcia 2000, Diez 2001) was cal-
culated by the following equation:

% IP = Infested plants x 100
total plants

The relative importance index (RII) of the par-
asitoid species allows for an estimation of the spe-
cies not only considering its abundance but also
its occurrence or frequency. In this way, species
poorly represented in individual numbers but fre-
quently recovered over a long period can be bal-
anced with numerous species with sporadic occur-
rence (Remes-Lenicov & Virla 1993; Rueda 1999;
Diez 2001). It was calculated by the following for-
mula:

PRII = No. of individuals of the species No. of samples species occurred x 100
Total No. different species Total No. of samples

Frequency (F) is the percent of individuals of
certain species in relation to total individuals of
all species (Canal Daza 1993; Molina-Ochoa et al.
2001; Molina-Ochoa et al. 2004), and was calcu-
lated by using the following formula:


F = No. individuals of species "i" x 100
No. total collected individuals

Percent of parasitism (%P) was calculated
according to Van Driesche (1983); Pair et al.
(1986), and Cris6stomo-Legaspi et al. (2001), as
follows:.

%P = No. total parasitized individuals x 100
No. total individual observed

Statistical Analysis

Percent data were angularly transformed and
subjected to analysis with the software Statistics
5.5 (2000). In order to determine differences be-
tween and among FAW and parasitoid collections
from the same region and those from different re-
gions, student t tests were performed.
Regression analyses also were performed to
determine the relationship between FAW popu-
lations and parasitoid abundance with temper-
ature and rainfall (Diez 2001; Schliserman
2001) by a stepwise approach. For the analyses,
the mean of low and high temperatures and
mean rainfall during the sampling week, and
the mean of low and high temperatures and
rainfall recorded in the two weeks previous to
the sampling date, were used. From these data
it was possible to estimate the week in which
the environmental factors most affected the
FAW populations, and the abundance of FAW
parasitoids.

RESULTS AND DISCUSSION

The percent of infested plants (%IP) by FAW
larvae was higher at Vipos (_20%) than at Tafi
Viejo (5.5%) (t = 0.0001, P < 0.001, df = 75). The
annual percentage of infested plants at Tafi Viejo
was highest during 2001 (9%), while at Vipos the
highest record was during 2000 (71.3%). The %IP
during the four year study in Tafi Viejo were
5.8%, 0.1%, 8.9%, and 8.9%. However in Vipos
the %IP were 71.3%, 19.7%, 21.3%, and 18.4%.
The total percent of infested plants (%TIP) in
Tafi Viejo, and Vipos were 5.5, and 30%, respec-
tively. ANOVA revealed significant differences in
the number of infested plants among years dur-
ing the 4-year study in both areas (T. Viejo: F =
106.38; P < 0.001; df = 72, and Vipos: F = 91.46;
P < 0.001, df = 74). The results obtained at Tafi
Viejo (early planting) and Vipos (late planting)
agreed with those previously reported by Willink
et al. (1991) for the Tucuman region, and Sosa
(2002a) for the North of Santa Fe province. Ear-
lier plantings had lower levels of FAW infesta-
tion and damage, a response similar to that re-
ported by Mitchell (1978), and Harrison (1984b)
on corn infested by corn earworm and fall army-
worm, respectively.







Florida Entomologist 89(2)


FAW Collection

About 2400 corn plants were examined in Tafi
Viejo, and 132 FAW larvae were collected and 52
parasitoids were recovered. The mean number of
FAW larvae per 10 plants was 0.58, 0.013, 0.89,
and 0.88 during years 1, 2, 3, and 4, respectively.
Larvae were collected as early as late September
because of the early planting date. FAW larvae
were not found in the first phenological stages
(one to three ligulate leaves, V1-V3). One larva
was recorded in year 2 during the late crop-grow-
ing season, when the plants had seven to eight
leaves (V7-V8). Overall, years 1 and 3 produced
higher densities of FAW larvae during the vegeta-
tive stages V3 to V6, similar to what was found by
Hernandez-Mendoza (1989) in Colima, Mexico. In
contrast, higher larval densities were recorded in
years 2 and 4 at the end of the vegetative period.
In Vipos, 2750 plants were examined, 540 lar-
vae were collected, and 82 parasitoids were ob-
tained. The mean number of FAW larvae per 10
plants was 2.59, 2.17, 1.25, and 1.83 during years
1, 2, 3, and 4, respectively. During years 1 and 2,
higher larval numbers were recorded in the V1-V3
stages. Larval populations were consistent
throughout the vegetative plant phase for the
other years. Overall, larval densities diminished
with the age of the cornfield, achieving the lowest
numbers during the beginning of the corn's repro-
ductive stages. Comparing FAW population fluc-
tuations in both locations during this 4-year
study, FAW at Vipos exhibited significantly higher
larval numbers (t = 1.99, P < 0.001, df = 72).

FAW and Corn Phenology

FAW infestations displayed a plant age-depen-
dent response at both localities during the 4-year
study. Reduced mean larval numbers were re-
lated to plant age and development. Mitchell et al.
(1974), and Beserra et al. (2002) found that the
distribution of FAW larvae and eggs varied ac-
cording to the phenological stage of the corn. Dur-
ing the early plant stages (V1-V3), first and sec-
ond instars were predominant, and about one to
six larvae per plant were found. During V4 and
V6, only one larva was usually recovered per
plant. Carvalho & Silveira (1971) found that
small and medium larvae would coexist, but the
number of larvae per plant decreased as larval
size increased. Larval cannibalism, larval mortal-
ity from disease or predators, and larval age are
possible factors that influence distribution.

FAW and Climatic Factors

In Tafi Viejo a temperature-dependent re-
sponse was obtained with respect to FAW popula-
tion abundance [Y = -1.34 + 1.29 log Max 2T
(mean high temperatures recorded in the week


previous to the sampling plus mean high temper-
atures recorded in the second week previous to
the sampling) 0.29 log rainfall 2 (mean rainfall
recorded in the week previous to the sampling
plus mean rainfall recorded in the second week
previous to the sampling)]; n = 30; P < 0.001; R2 =
0.99). Barfield & Ashley (1987) reported that corn
phenology and temperature affected larval devel-
opment, food consumption, and adult female lon-
gevity and fecundity, and that developmental
times were temperature-dependent and were
modified by the stage of corn consumed. However,
at Vipos an associated response was observed
with rainfall [Y = 3.89 + 0.5 log rainfall 0 (mean
rainfall during the sampling week) -0.18 log Max
TO (means of high temperatures during the sam-
pling week)]; n = 30; P < 0.01; R2 = 0.219). Silvain
& Ti-A-Hing (1985) found that the highest popu-
lations of FAW moths and larvae were observed
during the rainy seasons, and lowest during the
dry seasons.
Insect phenology, density, and number of FAW
generations are influenced by the climatic condi-
tions in a given region. Climatologic differences be-
tween and among localities could explain the phe-
nological differences, and climatologic conditions
among years also could explain fluctuations in pest
abundance in each area (Dent 1991; Diez 2001).

Parasitoid Species

Considering the diversity of parasitoids re-
ported from the Tucuman area (Vera et al. 1995;
Virla et al. 1999; Berta et al. 2000; Murua et al.
2002; Murua et al. 2003; Murua & Virla 2003;
Murua & Virla 2004) few species were recovered
in our study. Only two ichneumonids, Campoletis
grioti (Blanchard) and Ophion sp., one braconid,
Chelonus insularis (Cresson), and possibly one or
two species of tachinids, Archytas marmoratus
(Town.) and/orA. incertus (Macquart) were found.
All species were collected at Vipos (Table 1), and
only C. grioti was recovered at Tafi Viejo. Of all
parasitoids collected at Vipos, Archytas spp. com-
prised 38.3%, C. grioti 35.8%, Ch. insularis 22.2%,
and Ophion sp. 3.7%. Seasonally, Ophion sp. was
collected when corn plants were V4, whereas Ar-
chytas spp. and C. grioti were collected at the end
of the crop cycle. These results are in agreement
with those by Virla et al. (1999) and Vera et al.
(1995), who reported C. grioti and Ophion sp., re-
spectively, attacking FAW larvae collected from
corn in Argentina. Our results are also in agree-
ment with Molinari & Avalos (1997), who showed
that the dipteran parasites attacked the last in-
stars of FAW in Argentina. No differences were
determined in the abundance of parasitoids in
both locations during the 4-year study (t = 1.36, P
= 0.19, df = 38). We speculate that differences in
early or late corn planting would not affect the
abundance of FAW parasitoids.


June 2006







Murua et al.: Fall Armyworm Dynamics and Parasitoids in Argentina


TABLE 1. FREQUENCY AND RELATIVE IMPORTANCE OF THE FAW PARASITOID COMPLEX IN VIPOS DURING THE FOUR-
YEAR STUDY.

Frequency during year (%) Relative Importance during year (%)

Species 1 2 3 4 1 2 3 4 Total

C. grioti 52.2 44.4 33.3 5.5 0.26 0.19 0.15 0.004 0.13
Ch. insularis 5.6 33.3 33.3 38.9 0.01 0.007 0.11 0.07 0.056
Ophion sp. 34.8 5.6 4.2 0.0 0.02 0.007 0.146 0.0 0.005
Dipteran spp. 25.0 44.4 29.2 57.9 0.1 0.2 0.1 0.2 0.14


Percent Parasitism

Campoletis grioti was the single parasitoid re-
sponsible for 39.4% parasitism at Tafi Viejo. In
Vipos, overall percent parasitism during the 4-
year study was 15%. The tachinid species, C. gri-
oti, Ch. insularis, and Ophion sp. caused 5.7%,
5.4%, 3.3%, and 0.6% of total FAW parasitism, re-
spectively. The highest record of annual parasit-
ism was recorded during year 2 with 10.5% for C.
grioti and the dipteran species, but during year 3,
C. grioti, and Ch. insularis each caused 8.5% of
parasitism (Table 2).
Kogan et al. (1999) mentioned that cultural
practices developed in a plot can affect in a posi-
tive or negative way the natural enemy popula-
tions, increasing or inhibiting the parasitoid colo-
nization in cultivated fields. These practices could
also have direct or indirect effects, directly
through environment alterations and indirectly
affecting the host plant architecture, lack of food,
or refuge.


The lack of vegetation surrounding the sam-
pling area in the cornfields at Tafi Viejo could be
a reason for the low diversity found. This area
was surrounded by lemon groves where insecti-
cide applications are commonly applied. It is
known that the presence of spontaneous vegeta-
tion associated with the crop results in higher
numbers and diversity of natural enemies related
to this vegetation (Altieri & Whitcomb 1980;
Hoballah et al. 2004).
It is important to consider that C. grioti is a oli-
gophagous parasitoid that attacks different hosts
of several genera in the family Noctuidae. An-
other possible cause for low diversity is early
planting in Tafi Viejo that reduces FAW infesta-
tion, and damage to cornfields (Willink et al.
1991). Conversely, in Vipos corn is planted later
and the fields were surrounded by native vegeta-
tion without significant anthropogenic distur-
bances affecting potential parasitoid refuges.
Higher diversity of parasitoids and higher rates
of parasitism in Vipos also may be related to a


TABLE 2. ABUNDANCE AND PERCENT PARSITISM OF FAW PARASITOIDS OBTAINED IN TAFI VIEJO AND VIPOS DURING THE
FOUR-YEAR STUDY.

Abundance and percent parasitism (%)

Location Tafi Viejo Vipos

Year 1 2 3 4 1 2 3 4

NPS* 500 750 450 700 900 350 750 750
FAWCL 29 1 40 62 233 76 94 137
Species
C. grioti 4 1 18 29 12 8 8 1
(13.8) (100) (45.0) (46.8) (5.2) (10.5) (8.5) (0.7)
Ch. Insularis 2 1 8 7
(0.8) (1.3) (8.5) (5.1)
Ophion sp. -1 1 1 -
(0.8) (1.3) (1.1)
Dipteran spp. -5 8 7 11
(2.1) (10.5) (7.5) (8.0)
Total parasitoids collected (%) 4 1 18 29 20 18 24 19
(13.8) (100) (45.0) (46.8) (8.6) (23.7) (25.5) (13.9)

(*) Number of plants sampled, FAWCL = FAW collected larvae.







Florida Entomologist 89(2)


more diverse habitat with more forest, orchards,
groves, and pastures near to cornfields (Molina-
Ochoa et al. 2001; Hoballah et al. 2004). Overall,
the percent parasitism measured in this study
was similar to other studies. Berta et al. (2000)
reported parasitism ranging between 5.26% and
50% in cornfields with and without insecticide ap-
plication in the province of Tucuman, respec-
tively. Luchini & Almeida (1980) listed FAW par-
asitoids occurring in Brazil and considered C. gri-
oti the most important parasitoid causing about
95% parasitism.
Ashley (1986) and Andrews (1988) listed Ch.
insularis occurring throughout North America
highlighting its role as parasitoid of FAW by
showing parasitism of 63% in southern Florida;
however, Pantoja & Fuxa (1992), Molina-Ochoa et
al. (2001), and Molina-Ochoa et al. (2004) re-
ported lower levels of parasitism by Ch. insularis
of about 5% in Puerto Rico and Mexico, respec-
tively. This braconid has the broadest distribution
in Latin America and South America (Molina-
Ochoa et al. 2003). Lewis & Nordlund (1980) con-
sider this parasitoid an excellent candidate for
augmentative release because it can be intro-
duced throughout its overwintering zone. It is
capable of early-season colonization, and can be
used in direct therapeutic releases on target
crops.
The ichneumonid Ophion sp. caused the lowest
level of FAW parasitism in Vipos, ranging be-
tween 0.8 and 1.3% during the 4-year study. Sim-
ilar results have been reported by Molina-Ochoa
et al. (2001). Gross & Pair (1991) state that
Ophion flavidus (Brulle) parasitized 4th, 5th, and
6th instar FAW with equal success, but were min-
imally successful in completing development on
late 6th instars. This parasitoid caused 19.5% of
parasitism in June in southern Georgia. Ophion
sp. has been reported previously in Argentina
(Vera et al. 1995).
Dipteran parasites played an important role in
Vipos in the 4-year study, providing high parasit-
ization levels.Archytas marmoratus and /or A. in-
certus caused levels of parasitization between
2.1%, and 10.5%. The importance of these species
in Argentina and other South American countries
was emphasized by Molina-Ochoa et al. (2004)
based on reports by Molinari & Avalos (1997) and
Virla et al. (1999).

Parasitoids and Climatic Factors

Temperature was the most important climatic
factor influencing parasitoid populations in both
locations. Similar responses of other parasitoids
have been reported by Diez (2001), and Schliser-
man (2001) for the Tucuman region, such as those
attacking the fruit flies, Ceratitis capitata
(Weid.), Anastrepha fraterculus (Weid.), and cit-
rus leafminer, Phyllocnistis citrella (Stainton).


The maximum temperature in Tafi Viejo was the
most important factor [Y = -1.55 + 1.3 log Max 2T
(mean high temperatures recorded in the week
previous to the sampling plus mean high temper-
atures recorded in the second week previous to
the sampling) 0.3 log rainfall 0 (mean rainfall
during the sampling week)]; n = 38; P < 0.001, R2
= 0.99) affecting the parasitoid population fluctu-
ation.
In Vipos minimum temperature was impor-
tant factor but no climatic factor was a significant
variable describing parasitoid populations [Y =
2.62 + 0.387 log T Min 0 (mean low temperatures
during the sampling web) 0.18 log rainfall
1(mean rainfall recorded in the week previous to
the sampling)]; n = 30; P = 0.11; R2 = 0.085). Fac-
tors, such as insecticides, farming and cultural
practices, and other natural enemies, may be in-
fluencing parasitoid populations.

ACKNOWLEDGMENTS

The authors thank MSc. Patricia Diez (CRILAR-
CONICET, Tucuman, Argentina) and Dr. John J. Hamm
(USDA-ARS Crop Protection & Mangement Research
Laboratory, P.O. Box 748, Tifton, GA 31793-0748, USA)
for critical review of the manuscript. We thank Mr.
Ruben Pedraza (Servicio meteorol6gico del INTA-EEA
Famailla-Tucuman, Argentina) and Ing. C6sar Lamelas
(Secci6n Agrometeorologia EEAOC-Tucuman, Argen-
tina) for meteorological data and advice.

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June 2006







Austin et al.: Coptotermes formosanus Genetics


GENETIC EVIDENCE FOR TWO INTRODUCTIONS OF THE
FORMOSAN SUBTERRANEAN TERMITE, COPTOTERMES FORMOSANUS
(ISOPTERA: RHINOTERMITIDAE), TO THE UNITED STATES

JAMES W. AUSTIN', ALLEN L. SZALANSKI2, RUDOLF H. SCHEFFRAHN3, MATT T. MESSENGER4,
JACKIE A. MCKERN2 AND ROGER E. GOLD'
'Center for Urban and Structural Entomology, Department of Entomology
Texas A&M University, College Station, TX 77843-2143

2Department of Entomology, University of Arkansas, Fayetteville, AR 72701

3Department of Entomology, University of Florida-Ft. Lauderdale Research and Education Center
3205 College Avenue, Ft. Lauderdale, FL 33314

4Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268

ABSTRACT

Exotic introductions of Formosan Subterranean Termite (FST) to the United States from
Asia have had significant economic consequences. Multiple introductions through marine
transport have been proposed, but identification of these routes has yet to reveal more than
one lineage in the continental U.S. DNA sequencing ofa 640-bp cytochrome oxidase II (COII)
mitochondrial DNA (mtDNA) marker to 60 disjunct populations, revealed two independent
lineages spanning the continental U.S., Hawaii, Japan, and China. Limited genetic variation
was observed with this marker. Group I constitutes a largely Asian clade, while Group II is
comprised of both Asian and southern U.S. populations. This is the first study which has doc-
umented 2 distinct lineages to continental United States and Hawaii.

Key Words: invasive species, DNA sequence, genetic variation, molecular diagnostics, termite

RESUME

Las introducciones ex6ticas de la termita subterranea de Formosa (TSF) de Asia a los Esta-
dos Unidos han tenido consecuencias econ6micas significativas. Introducciones multiples
por medio del transport marino han sido propuestas, pero la identificaci6n de estas rutas to-
davia no ha revelada mas que un linaje en los Estados Unidos continental. La secuenciaci6n
de un marcador de 640-bp del citocromo-c-oxidasa II de ADN mitochondrial (mtADN) a 60
poblaciones separadas, revelo dos linajes independientes atravesando los Estados Unidos
continental, Hawaii, Japan y China. El marcador mostr6 una variaci6n gen6tica limitada. El
grupo I constitute un clado principalmente asidtico, mientras el grupo II consiste de pobla-
ciones asidticas y del sur de los Estados Unidos. Este es el primer studio que document los
dos linajes distintas en los Estados Unidos y Hawaii.


Formosan subterranean termite (FST) Copto-
termes formosanus Shiraki (Isoptera: Rhinoter-
mitidae), has long been suspected to have origi-
nated from Formosa (the Island of Taiwan), but
endemic to mainland China due to the identifica-
tion of a termitophile from there (Kistner 1985).
FST has been reported from 14 southern prov-
inces in China with a northern limit of 33028' N
and a western limit of 10435'E (Gao et al. 1982;
He & Chen 1981; Lin 1986) (Fig. 1). Introductions
of this exotic pest have been documented around
the world following closely with trade routes ex-
tending to the United States and beyond (Chho-
tani 1985). Historical shipping trade between the
east and west over the past 450 years (Welsh
1996; Lim 1997), and the likely introductions) of
FST to the continental U.S. after World War II (La


Fage 1987), have made tracking introduction
points difficult. Trading centers in Guangdong
Province (e.g., Macau, Guangzhou, Shenzhen,
and Hong Kong), Fujian Province (e.g., Puyuan)
and Shanghai Province, China, and Taiwan have
provided likely ports of origin for FST (See Prov-
ince Map, Fig 1). Gay (1967) suggests that intro-
ductions of FST into Guam, Midway Island, the
Marshall Islands, and the Hawaiian islands are
most likely due to shipping trade.
FST is believed to have been introduced to
Japan almost 300 years ago (Mori 1987; Su &
Tamashiro 1987; Wang & Grace 1999; Vargo et al.
2003), and has been hypothesized to have been in-
troduced to Hawaii almost 100 years ago (Su &
Tamishiro 1987). The history of FST introduc-
tions to the continental United States is more am-







Florida Entomologist 89(2)


120E


... 20N


Fig. 1. Provincial Map of China based on Wang et al. (2
terms formosanus infestations.



biguous because of likely misidentifications. For
example, early samples of Coptotermes in Hous-
ton, Texas, during the 1950s were identified as
C. crassus Snyder, but were later positively iden-
tified as C. formosanus.
Presently, FST is distributed across the south-
east United States (Spink 1967; Howell et al.
1987; La Fage 1987; Su & Tamashiro 1987; Appel
& Sponsler 1989; Chambers et al. 1998; Su &
Scheffrahn 1998a; Cabrera et al. 2000; Haw-
thorne et al. 2000; Howell et al. 2000; Su & Schef-
frahn 2000; Hu et al. 2001; Scheffrahn et al. 2001;
Jenkins et al. 2002), and disjunct populations in
southern California (Atkinson et al. 1993;
Haagsma et al. 1995) are thought to have origi-
nated from Hawaii. Without doubt, their contin-
ued presence and growing distributions) have
been exacerbated by commerce and trade prac-
tices within the United States (Cabrera 2000;
Jenkins et al. 2002; Glenn et al. 2003), and by the
general lack of education and research funding di-
rected towards this problem until recently (Oper-


!002). Shaded provinces reflect areas with known Copto-




ation Full Stop, a FST interdiction research unit
located in New Orleans, Louisiana was initiated
by the United States Department of Agriculture,
Agricultural Research Service in 1998).
Several studies applying genetic or biochemi-
cal interpretations of FST populations have at-
tempted to identify introduction routes of FST.
However, while multiple entry points appear
likely, the lack of genetic variation in this inva-
sive species has made identification of these
routes difficult to achieve. Studies applying cutic-
ular hydrocarbons (Haverty et al. 1990), alloz-
ymes (Korman & Pashley 1991; Strong & Grace
1993; Broughton & Grace 1994; Wang & Grace
2000), mitochondrial DNA (mtDNA) (Jenkins et
al. 2002), and microsatellite DNA (Vargo & Hend-
erson 2000; Husseneder & Grace 2000; 2001a, b;
Husseneder et al. 2002) have been reported, but
current literature has not conclusively estab-
lished the origins of alternative routes to the
United States. These studies have implicated that
more than one introduction route existed, but


June 2006


.---------







Austin et al.: Coptotermes formosanus Genetics


they have not corroborated their suppositions
with the inclusion of additional FST populations
which might elucidate this observation.
Presumably, this could be attributed to the
overall lack of genetic diversity of FST globally. In
introduced populations, the lack of clear colony
boundaries and the potential for considerable
mixing of individuals among colonies may lead to
the formation of colonies which could extend over
large areas making colonial identity difficult, an
observation observed in unicolonial ant species
(Argentine ant Linepithema humile) (Tsutsui et
al. 2000, 2001). Alternatively, it may be that the
natural dispersal of FST alates is more signifi-
cant than previous recorded distances (Messen-
ger & Mullins 2005), an explanation proposed for
the low mitochondrial DNA (mtDNA) divergence
among sites spanning across states such as Geor-
gia (Jenkins et al. 2002). However, human-aided
dispersal of FST would be equally plausible as a
contribution to low mtDNA divergence. Some ar-
gue that the lack of genetic diversity in FST could
be due to genetic bottlenecks (Strong & Grace
1993; Broughton & Grace 1994) with limited
founder effect. Others suggest the possibility of
significant inbreeding due to neotenic involve-
ment (Wang & Grace 1995). For this to be accept-
able, one must assume that there would be some
inbreeding depression or fixation.
Herein, we report that while multiple intro-
ductions of FST (to the United States) are pre-
sumed, limited genetic variation in this species
restricts the clarification of exactly where these
exotic introductions originated from when using
some molecular markers. We provide evidence of
2 distinct lineages, occurring in the continental
United States and in the Hawaiian Islands, with
identical lineages from China.

MATERIALS AND METHODS

Coptotermes formosanus were collected from
all known continental United States where FST
has been reported, the Hawaiian Islands, Japan,
Hong Kong, and China (Table 1). Morphological
identification of specimens used in this study
were performed by applying the keys of Schef-
frahn et al. (1994), and verified with a FST molec-
ular diagnostic method (Szalanski et al. 2004).
Voucher specimens, preserved in 100% ethanol,
are maintained at the Arthropod Museum, De-
partment of Entomology, University of Arkansas,
Fayetteville, AR, the University of Florida-Ft.
Lauderdale Research and Education Center, Ft.
Lauderdale, FL, and the Center for Urban and
Structural Entomology, Department of Entomol-
ogy, Texas A&M University, College Station, TX.
Alcohol preserved specimens were allowed to
dry on filter paper, and DNA was extracted from
individual worker, or soldier heads by using the
Puregene DNA isolation kit D-5000A (Gentra,


Minneapolis, MN). Extracted DNA was resus-
pended in 50 pL of Tris:EDTA and stored at
-20C. Polymerase chain reaction (PCR) was con-
ducted with the primers TL2-J-3037 (5-ATGGCA-
GATTAGTGCAATGG-3) designed by Liu and
Beckenbach (1992) and described by Simon et al.
(1994) and Miura et al. (1998), and primer TK-N-
3785 (5-GTTTAAGAGACCAGTACTTG-3) from
Simon et al. (1994). These primers amplify a 3'
portion of the mtDNA COI gene, tRNA-Leu, and a
5' section of the COII gene. PCR reactions were
conducted with 1 pL of the extracted DNA (Sza-
lanski et al. 2000), with a profile consisting of 35
cycles of 94C for 45 s, 46C for 45 s, and 72C for
60 s. Amplified DNA from individual termites
was purified and concentrated by using Microcon-
PCR Filter Units (Millipore, Bedford, MA).
Samples were sent to The University of Arkan-
sas Medical School DNA Sequencing Facility (Lit-
tle Rock, AR) for direct sequencing in both direc-
tions with an ABI Prism 377 DNA sequencer (Fos-
ter City, CA). To facilitate genetic comparison
with existing GenBank DNA sequences, 113 bp
from the 5' end of the sequence was removed, and
the remaining 667 bp was used. GenBank acces-
sion numbers for the FST haplotypes found in
this study are AY453588 and DQ386170. DNA se-
quences were aligned with BioEdit version 5.09
(Hall 1999) and Clustal W (Thompson et al. 1994).
The distance matrix option of PAUP* 4.0b10
(Swofford 2001) was used to calculate genetic dis-
tances according to the Kimura 2-parameter
model (Kimura 1980) of sequence evolution.

RESULTS AND DISCUSSION

Introduction of exotic termites to the United
States is an ongoing problem that is invariably
sustained by modern trade and limited or non-ex-
istent quarantine regulations.
Native populations (in China) of FST should
possess greater genetic diversity. For this reason,
focusing on the nature of genetic variation in pop-
ulations from China and neighboring Asian coun-
tries (Vargo et al. 2003) is a logical starting point
when evaluating the nature of introduced popula-
tions to the United States (Husseneder et al.
2002) and its territories. In the present study we
evaluated native populations of FST from Guang-
dong, Shanghai, and Fujian provinces (Hong
Kong, Puyuan, Guangzhou, and Xhinhui) in
China. However, only two distinct COII haplo-
types were observed.
Applying C. acinaciformis (Froggatt), C. lacteus
(Froggatt), and Heterotermes cardini (Snyder) as
outgroups, Haplotype group I contains locations
from Hong Kong, Japan AB109529, Hsin-Hui
(presently known as Xhinhui), China (from Jen-
kins et al. 2002), Puyuan and Guangzhou, China,
Oahu, HI, Nagasaki, Japan, and Ft. Worth, TX
[presumably this sample was collected from







Florida Entomologist 89(2)


TABLE 1. COPTOTERMES FORMOSANUS COLLECTION DATA.

Location Country N Hap Source


Hong Kong

Ft. Worth, TX
Oahu, Hawaii
Hong Kong
HI
Maui, HI
GA
Cairo, GA
Lawrenceville, GA
Tucker, GA
Dallas, GA
Savannah, GA
GA
Spindale, NC
Forest City, NC
Rutherfordton, NC
Marco Island, FL
Trinity, FL
Niceville, FL
Florida City, FL
Temple Terrace, FL
Palm Beach, FL
Pompano Beach, FL
Galveston, TX
San Antonio, TX
Garland, TX
Rockwall, TX
Stennis Sp Ctr, MS
New Orleans, LA
Lake Charles, LA
New Orleans, LA
St. Rose, LA
New Orleans, LA
SC
Nagasaki
Puyuan
Guangzhou
Mobile, AL
GA
Hsin-hui (Xhinhui)
Hsin-hui (Xhinhui)
Oahu, HI


China
Asia
USA
USA
China
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
Japan
China
China
USA
USA
China
China
USA


This study
AB109529
Jenkins et al. 2002
This study
This study
AY536406
This study
AY536405, AY027489
AY683220
AY683213
AY683214
AY683214-15
AY683217-219
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
AY536407,AY683217
This study
This study
This study
Jenkins et al. 2002
Jenkins et al. 2002
This study
AY536403
AY536404
Jenkins et al. 2002
Jenkins et al. 2002
Jenkins et al. 2002
Jenkins et al. 2002
Jenkins et al. 2002


Grapevine, TX, because the only known occur-
rences of FST in Tarrant County, TX, occur in the
Northeast portion of this county (pers. Comm.
Mike Merchant)]. Group II contains several FST
populations from disjunct locations: Hong Kong,
North Carolina, South Carolina (Jenkins et al.
2002), Georgia, Florida, Alabama (Jenkins et al.
2002), Mississippi, Louisiana, Texas, Oahu and
Maui, HI (Figs. 2 and 3). Representative taxa from
group I were slightly more divergent based on
Maximum likelihood analysis (Fig. 3). Inclusion of
FST sequence data from Jenkins et al. (2002), des-


ignated by their respective haplotype descriptions
(A through H), also fall within the two groups pre-
sented herein (Table 2, Figs. 2 and 4).
Fei and Henderson (2003) noted that incipient
colony establishment was somewhat more restric-
tive for outbred primary reproductive, owing dis-
crepancies to environmental adaptive resource
differences from two disjunct populations from
Louisiana. Furthermore, Coaton & Sheasby
(1976), and Lenz & Barrett (1982) suggest that
dominant use of neotenics for colony growth in
C. formosanus may be a successful strategy to in-


June 2006









Austin et al.: Coptotermes formosanus Genetics


Hong Kong, China
72 2 Puyuan, China
3 Guangzhou, China
------------4 Nagasaki, Japan
5 Nagasaki, Japan
6 Hong Kong, China
7 Oahu Hawaii, USA
8 Oahu Hawaii. USA HaD F


Fig. 2. Maximum Parsimony Analysis of Coptotermes formosanus lineages in North America. For consistency,
open and closed circles reflect the different mtDNA COII lineages of C. formosanus, while the numbers are used for
comparison and clarification of geographic location in Figures 3 and 4.


Maximum Parsimony


100


56


J) Nagasaki, Japan
Ft Worth Texas, USA Hap E
Asia AB109529
Hong Kong, China
3Hsn hui, China Hap G
Hsin hui, China Hap H
Hawaii, USA AY536406
New Orleans, USA AY53640
Georgia, USA AY536405
Cairo Georgia, USA AY683220
Cairo Georgia, USA AY683221
Lawrenceville Georgia, USA
Tucker Georgia, USA AY683214
Dallas Georgia, USA AY683216
Dallas Georgia, USA AY683215
Savannah Georgia, USA AY683219
Savannah Georgia, USA AY683218
Suwannee Georgia, USA AY683212
New Orleans Louisiana, USA AY683217
Hong Kong, China
Hong Kong, China
* Spindale North Carolina, USA
Forest City North Carolina, USA
Rutherfordton North Carolina, USA
* Marco Island Florida, USA
Trinity Florida, USA
Niceville Florida, USA
S Florida City Florida, USA
STemple Terrace Florida, USA
* Palm Beach Flonda, USA
* Pompano Beach Florida, USA
* Maui Hawaii, USA
S Maui Hawaii, USA
S Galveston Island Texas, USA AF525317
S Galveston Island Texas, USA
San Antonio Texas, USA
S Garland Texas, USA
Rockwall Texas, USA
S Georgia, USA AY027489
S Stennis Space Center Mississippi, USA
* Stennis Space Center Mississippi, USA
- Stennis Space Center Mississippi, USA
* Stennis Space Center Mississippi, USA
S Lake Charles Louisiana, USA
* New Orleans Louisiana, USA AF107488
- Lake Charles Louisiana, USA
S Lake Charles Louisiana, USA
S St. Rose Louisiana, USA
- New Orleans Louisiana, USA Hap B
S South Carolina, USA Hap C
- Mobile Alabama, USA Hap D
S Georgia, USA Hap A


9-69610







Florida Entomologist 89(2)


Fig. 3. Introduction routes of Coptotermes formosanus from Asia to North America. Dashed arrow pointing to-
wards Southern California suggests the introduction from Hawaii based on anecdotal information that has not been
corroborated in genetic studies to date.


vade new environments. If this adaptive strategy
is true for C. formosanus, reduced genetic varia-
tion may be the result and would account for some
of the limited population viscosity observed to
date. Habitat fragmentation and anthropogenic
disturbances significantly reduce population vis-
cosity. More comprehensive studies of FST may
not reveal significant genetic diversity. For FST,
reduced genetic variation does not necessarily
mean reduced fitness or vigor, but may simply im-
ply that there is greater reproductive plasticity.
For example, Hyashi et al. (2004) demonstrated
that Reticulitermes speratus (in Japan) can utilize
facultative parthenogenic reproduction. This
would be a significant establishment capability
for termites like FST when introduced to non-en-
demic locations such as the United States.
There have been numerous emigrations of peo-
ple to Hong Kong throughout history. Major migra-
tions of Chinese settlers from mainland China to
Hong Kong have been recorded as early as the
Song Dynasty (960-1279) (Welsh 1996). After the
end of World War II and the communist takeover of
mainland China in 1949, hundreds of thousands of
people emigrated from China to Hong Kong (Welsh
1996). In fact, locations such as Xhinhui, a treaty
port in 1904, was an important outlet for Chinese
emigrants to the United States (Anonymous 2004).


The introduction of FST to the U.S. likely occurred
several times, perhaps more than ten different oc-
casions (RHS, personal communication). Given
this fact, it is remarkable that the established link
between the U.S. and China has never been sub-
stantiated for more than one FST lineage.
Populations of FST from Japan appear only in
one of the presented clades (Group I, Fig. 2), and
further sampling from more locations (in Japan)
may provide additional information on whether
Japan could have contributed more significantly
to FST introductions to Hawaii or the continental
United States. Group I (Fig. 2) is largely com-
prised of samples from Asian/Pacific locations but
has one sample (Ft. Worth, TX) that was collected
in the continental U.S. (Fig. 3). This is significant
because it implicates a second introduction route
to the continental U.S. that has never been iden-
tified in previous studies. Group II, is comprised
of FST samples from nearly all known southeast-
ern states (Alabama, Florida, Georgia, Louisiana,
Mississippi, North Carolina, South Carolina),
Texas, Hawaii, and several FST from China. Both
clades are well-supported by strong bootstrap
support (>80%) by both parsimony and Liklihood
analyses (Figs. 1 and 3).
Although FST distributions have been more
recently updated (Wang et al. 2002), the lack of a


June 2006







Austin et al.: Coptotermes formosanus Genetics


TABLE 2. HAPLOTYPE VARIATION AT 15 NUCLEOTIDE SITES FOR NINE COPTOTERMES FORMOSANUS HAPLOTYPES.

Hap 8 11 19 32 33 46 176 211 222 297 333 427 643 653 662

1 C G A A T A A A A T A G A T A
2 .. G A
3 G
4 A G
A G G A C
D .G A C
F A
G G T T A
H .T A T T T

*Jenkins et al. (2002).


geographic explanation for a second lineage intro-
duced to the United States remains unclear
(Wang & Grace 2000). Sequence data obtained
from GenBank, from Jenkins et al. (2002), pro-
vides a second haplotype match in the continental
United States (haplotype E from Ft. Worth, TX)
that represents the first documented case corrob-
orating multiple lineages from presumably multi-
ple introductions (at least two in the present
study). These two distinct haplotypes share one
commonality-both groups have representatives
with identical haplotypes (lineages) from Hong
Kong, Japan, Hawaii, and the continental United
States (Fig 3).
There were numerous FST samples where re-
peated attempts to amplify sufficient DNA for se-
quencing of the mtDNA COII gene were not suc-
cessful (e.g., FST from San Diego, California and
Tai Chuong, Taiwan). These results were not sur-
prising, as we have routinely observed ~60% effi-
ciency when using the COII marker with FST.
However, amplification of the 16S rRNA for these
samples was successful. We routinely observe
>90% efficiency for this marker with FST. While
the utility of the 16S marker is excellent for phy-
logenetic studies of the genus Coptotermes (JWA,
unpublished), for molecular diagnostic methods
(Szalanski et al. 2004), or other rhinotermitids
(Szalanski et al. 2004; Austin 2004a; 2004b), it
does not provide the degree of genetic variation
suitable to discern the two distinct FST haplo-
types observed in this study. The slightly larger
COII amplicon (640 bp versus 428 bp of 16S
rRNA) provides only a small increase in resolu-
tion between FST populations, even though it
works well for other Rhinotermitidae (Austin et
al. 2002, 2004c). Our laboratory experience with
FST suggests that in general, it is more difficult
to extract high quality DNA from Coptotermes for
genetic studies when compared to other rhinoter-
mitids, a problem that may be more common than
reported. Additional problems may include the
presence of unknown inhibitors, method of sam-
ple preservation (some preservation methods are


known to provide poorer quality DNA for genetic
studies (Post et al. 1993; Reiss et al. 1995; Dillon
et al. 1996) or the age of samples provided.
While the idea that multiple introductions to
the United States have been proposed, alternate
introduction routes have never been substanti-
ated in literature. This study provides a glimpse
of some of the difficulties encountered working
with FST. Most notably, it would appear that the
low genetic variation detected with our COII
marker in this species does not equate to reduced
fitness or establishment capability.
Populations of nearly all species, social or other-
wise, exhibit at least some degree of genetic differ-
entiation among geographic locales (Ehrlich &
Raven 1969). Herein, we present two distinct COII
haplotypes of FST in the continental United States
(one based on our own samples evaluated, and a
second from Jenkins et al. (2002)). However, our
results appear to contradict the degree of variation
described by Jenkins et al. (2002). They describe 8
different COII haplotypes (maternal lineages)
from 14 geographic locations across the southeast
United States, Hawaii, and China. Applying the
COII marker to 60 geographic locations (Table 1)
we only identified 2 haplotypes-one in Japan, two
in Hawaii, the continental United States, and
China, respectively. Noting that many of the vari-
able sites in Jenkins et al. (2002) occur at positions
651 through 685 of their slightly larger COII am-
plicon (total size of the amplicon was 685), it is un-
clear where the discrepancies occurred. One possi-
bility may be due to sequence error that could only
be detected by comparison with greater taxon sam-
pling. Other possibilities may be due from im-
proper sequence alignment or mispriming of tem-
plate DNA during PCR. We elected to include all
taxa from Jenkins et al. (2002) into our sequence
dataset (COII lineages A through H), which may
have provided an advantage due to our larger
number of locations sampled. As with animal pop-
ulations, additional genetic structure normally is
to be expected over increasing spatial scales,
where populations can show additional differenti-







Florida Entomologist 89(2)


SHong Kon, China


O() Gunghou. China


( Oahu Hawaii. USA


SOahu Hawaii. USA Hap F


) Hsin-ui. China Hap G


Ft Worth Texas, USA Hap E


SHong Kong China


-- @. Hain4r, China Hap H


SSavannah Georga, USA


Florida City Florida, USA


Maui Hawaii, USA


Garland Texas, USA


New Orkean Louisiana, USA Hap B


South Carolina. USA Hap C


Mobile Alabn,. USA Hap D


Gaoria, USA Hap A


CoptofenesaosnaetomisAF262610


Coptofnrmes lecus AF22060C


I Hewtemm cardni Bahamas.
- 0.005 substitutions/site

Fig. 4. Maximum Likelihood analysis Coptotermes formosanus lineages in North America.


June 2006


Maximum Likelihood







Austin et al.: Coptotermes formosanus Genetics


ation due to spatial habitat structure and isolation
by distance (Avise 2004). However, our results
seem to refute this generalization for FST, a fact
probably attributed to its establishment ability in
fragmented urban ecosystems and their indirect
interactions with humans.
The preponderance of FST research appears to
support our findings. Haverty et al. (1990) found
no differences in qualitative cuticular hydrocar-
bon profiles among four FST populations in the
U.S. Korman & Pashley (1991) concluded that
populations from Florida and New Orleans are in
the same group and are very closely related to
each other, a finding also corroborated within the
present study (Fig. 3). Strong & Grace (1993) con-
cluded that low genetic and phenotypic variabil-
ity in introduced FST populations to Hawaii could
have been from a single event. Broughton &
Grace (1994) observed that only 9 of 16 different
restriction enzymes cut mtDNA zero or once.
Vargo et al. (2003) was unable to detect signifi-
cant isolation by distance among colonies at the
spatial scale studied (0.7-70 km) from 2 disjunct
populations of FST in Japan, nor from popula-
tions in New Orleans, LA and Oahu, HI. This sug-
gests a general lack of strong population viscosity
in introduced populations of FST. The finding also
seems to be contrary to Jenkins et al. (2002),
whose FST samples ranged in distance from 6-37
km in Atlanta, GA. Wang & Grace (2000), apply-
ing enzymatic polymorphisms, concluded that at
least two introductions to the United States have
occurred, but the second clade in their study
lacked sufficient samples from China to deter-
mine the origin of a second route.
More recently, the utility of mtDNA markers
for identifying where exotically introduced Het-
erotermes (Szalanski et al. 2004), Nasutitermes
(Scheffrahn et al. 2004) and Cryptotermes/Proc-
ryptotermes (RHS, unpublished) to the United
States is being investigated. The principal caveat
with studies of this nature is that significant rep-
resentation oftaxa is essential, particularly when
dealing with species of limited genetic variation
like FST. A secondary caveat is that tremendous
skill in identifying termites morphologically is es-
sential to ensure the validity of a genetic study
based on known, identified samples. Because FST
was likely misidentified when it was first ob-
served in the continental United States, little at-
tention was given, and subsequent populations
have developed over the years. This was one of the
reasons behind developing molecular diagnostics
for this species (Szalanski et al. 2004), and a need
to genetically review some species to corroborate
their original identifications (Scheffrahn et al.
2004). As population-level studies for FST from
various locations across the world continue to ac-
cumulate (see Vargo et al. 2003), perhaps a better
understanding of local factors which contribute to
the low genetic diversity observed in FST will be-


come more apparent. Given the 300 years of
known occurrence in Japan (Vargo 2003) and the
lack of genetic variation in China, it is unlikely
we will observe significant variation in this spe-
cies within the U.S. Random genetic drift is un-
likely to occur at a rate that we will detect any-
time soon. Perhaps more intuitively, we should
not assert our scientific prejudices about the na-
ture of reduced genetic variation in FST (causing
some reduction in fitness), or Isoptera in general,
until we more exhaustively investigate their ba-
sic biology and reproductive systems.

ACKNOWLEDGMENT

We thank R. Davis, M. Merchant, G. Henderson,
K. Grace, J. Nixon, L. Yudin, J. Lopez, K. L. Mosg,
J. Chapman, S. Cabellero, J. Chase, B. McCullock,
O. Miyashita, E. Phillips, P. Ban, M. Weinberg, J. Stotts,
N.-Y. Su, E. Vargo, P. Fitzgerald, M. K. Rust, D. Mura-
vanda, J. Darlington, L. Ethridge, and J. Woodrow for
collecting termite samples. Research was supported in
part by the University of Arkansas, Arkansas Agricul-
tural Experiment Station, the University of Florida
Research Foundation, the Center for Urban and Struc-
tural Entomology, Texas A&M University, and a grant
from USDA-ARS Agreement No. 58-6435-3-0045.

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Florida Entomologist 89(2)


SIZE, FECUNDITY, AND GONADIC MATURATION
OF TOXOTRYPANA CURVICAUDA (DIPTERA: TEPHRITIDAE)

ALFREDO JIMENEZ-PEREZ AND PATRICIA VILLA-AYALA
Laboratorio de Ecologia Quimica, Centro de Desarrollo de Productos Bi6ticos, Apartado Postal 24
Yautepec, Morelos, M6xico Instituto Polit6cnico Nacional, M6xico

ABSTRACT
The papaya fruit fly, Toxotrypana curvicauda Gerstaecker, is an important pest of papaya. It
is distributed from Florida, USA, to northern South America. We studied aspects of its biol-
ogy on papaya, Carica papaya. Females and males emerged within a 3-d period with similar
numbers emerging daily. Females are heavier than males but had similar longevity. Pupar-
ial length, puparial weight, and adult weight did not correlate with adult longevity. First
chorionated eggs were recorded 4 d after emergence. Females 6 d old had an average of 44
2.2 (sem) chorionated eggs. Heavier females have a reproductive advantage as they have
more chorionated eggs than light females. More than 85% of females lived at least 6 d.

Key Words: egg maturation, longevity, emergence rhythm, Carica papaya, Mexico

RESUME

La mosca de la fruta de la papaya, Toxotrypana curvicauda Gerstaecker, es un insecto plaga
de la papaya, Carica papaya, que se distribuye desde la Florida en los EUA hasta la part
norte de Sudam6rica. En esta ocasi6n, estudiamos aspects de su biologia en Carica papaya.
Ambos sexos emergen en un lapso de 3 dias con similar numero de hembras y machos emer-
giendo diariamente. Las hembras son mas pesadas que los machos pero presentan similar lon-
gevidad. No existe relaci6n entire longevidad del adulto y el largo o ancho de la pupa o por el
peso del adulto. Los primeros huevos corionados se observaron en hembras de 4 d de edad. En
promedio cada hembra madura sexualmente (6 d de edad) present 44 2.2 huevos coriona-
dos. Las hembras pesadas tienen una ventaja reproductive ya que presentan mas huevos co-
rionados que hembras ligeras. La mayoria de las hembras (> 85%) vivieron al menos 6 dias.


Translation provided by the authors.


The papaya fruit fly, Toxotrypana curvicauda
Gerstaecker (Diptera: Tephritidae), is one of
seven species of the genus Toxotrypana (White &
Elson-Harris 1992). Toxotrypana curvicauda has
been reported from south Florida and south
Texas, through much of Central America, to
northern South America including some Carib-
bean islands (Knab & Yothers 1914; Eskafi &
Cunninghan 1987; O'Doherty & Link 1993).
Several authors have reported on the biology
of this species. Knap & Yothers (1914) and Mason
(1922) produced the first reports on the life cycle.
Landolt (1984) studied ovary and egg develop-
ment and reported that males are ready to mate
on the day they emerge while females have a 6-d
premating period. Aluja et al. (1994) evaluated
preference for papaya varieties and Landolt &
Hendrichs (1983) and Aluja et al. (1997a, b) re-
ported on spatial and temporal distribution of
this fly. The sex pheromone of T curvicauda was
identified by Chuman et al. (1987). Improvements
in trapping techniques with sex pheromone were
done by Landolt et al. (1988), Landolt & Heath
(1990), and Heath et al. (1996). Castrej6n-G6mez
et al. (2004) reported the use of food attractants
for capturing the papaya fruit fly.


Body size and/or weight have been tradition-
ally considered key determinants of an organism's
ecological and physiological properties (Thornill &
Alcock 1983; Honek 1993), because it is strongly
correlated with many physiological and fitness
characteristics (Reiss 1989; Roff 1992). Female
weight is generally accepted as an index of poten-
tial fecundity, assuming a positive relationship be-
tween the number of oocytes in the ovarioles and
the weight of the female. Thus, heavy females
have more eggs available for fertilization (Tza-
nakakis 1989; Fay 1989; Sivinski 1993). Similarly,
large size or weight has been associated with
greater longevity (Bloem et al. 1994). In mass
rearing facilities, weight is an important parame-
ter when evaluating the quality of laboratory pop-
ulations (Chambers & Ashley 1984).
There is no published information available
on puparial morphometry and adult emergence
rhythm of the papaya fruit fly. Also, the relation-
ships among puparial weight and number of mature
eggs, and gonadic maturation and female age have
not been explored. This information is important for
understanding the biology of T curvicauda on C. pa-
paya and for planning research on the mating sys-
tem as a tool for management of this papaya pest.


June 2006







Jim6nez-P6rez & Villa-Ayala: Size and Fecundity of T. curvicauda


MATERIALS AND METHODS

Insects
Insects were collected as larvae from infested
papaya fruits obtained from an experimental pa-
paya plantation at the CEPROBI (Centro de De-
sarrollo de Productos Bi6ticos) grounds at Yaute-
pec, Morelos, Mexico. Detailed information on lo-
calization, native vegetation, and climatic infor-
mation of the CEPROBI grounds can be found in
Aluja et al. (1997a). Mature larvae (100-200) were
placed for pupation into a plastic cylindrical con-
tainer (11 cm high and 8.5 cm diameter) with
sterile soil (6 cm deep) and covered with mesh se-
cured with a rubber band. Containers were wa-
tered as necessary to keep soil moist. One week
after pupation, puparia were recovered and
washed under running water and dried on paper
toweling. An Ohaus balance (Explorer, 0.0001 g
accuracy, made in Switzerland) was used to weigh
the puparia. Each puparium was numbered and
kept individually in a plastic container (9.5 cm
high and 3 cm diameter) with sterile soil until
adult emergence. Each puparium was photo-
graphed under a Nikon SMZ 1500 stereoscope
with a Nikon Coolpix 4500 digital camera. Sig-
maScan v5 (Systat Software Corporation) was
used to measure puparial length and width.
Adults were weighed on their emergence day with
the same balance noted above. Adults were main-
tained individually in plastic containers, and fed
water and sugar (Sharp & Landolt 1984). Puparia
and adults were kept under natural humidity (50-
60%), temperature (27 2C) and light regime
(12L:12D). This experiment was carried out from
August to September, 2004. For each insect, we
recorded puparial weight, length, width; and
adult emergence, weight, longevity and sex.

Relationships among Puparial and Adult Weight and
Number of Chorionated Eggs
To determine whether puparial and adult
weight correlated with the number of chorionated
eggs, 40 females (6-8-d-old) were killed by freez-
ing (8 min in a domestic freezer, -20C). The abdo-


men was removed from the body and dissected
under the stereoscope (described above). Both
ovaries were removed, immersed in 1% acetocar-
min for 60 s and transferred to clean saline solu-
tion. Non-chorionated eggs retain the stain but
the presence of the chorion prevents stain absorp-
tion (e.g., Fernando & Walter 1999). Unstained
eggs were classified as mature and were pre-
sumed to be available for oviposition, while
stained eggs were classified as immature eggs.

Gonadic Maturation

To determine if there was a relationship be-
tween adult age and the number of chorionated
eggs, we compared the number of mature eggs
contained in one ovary of females at 0, 2, 4, 6, 8,
10, and 12-d of age. We dissected 20 females for
each age. Dissection, egg staining, and egg count-
ing followed the methodology described above.

Statistical Analysis

Differences in puparial weight, length and
width, as well as adult weight and longevity be-
tween males and females were determined with a
t test. Regression analysis was used to determine
the influence of puparial and adult weight on
adult longevity and on the number of mature eggs
of 6- to 8-d-old females. Analysis of covariance
(ANCOVA) followed by a least squared means
(LSM) test was used to determine if the number of
chorionated eggs was affected by female age. Pu-
parial weight was used as a concomitant variable.
All statistical analyses were carried out with SAS
(SAS Institute 1996). All data are reported as
mean standard error.

RESULTS

Female puparia were significantly heavier and
longer than male puparia (Table 1). Similarly
adult females were significantly heavier than
adult males (Table 1). In contrast, puparial width,
puparial period and adult longevity were similar
for both sexes (Table 1). More than 85% of females


TABLE 1. AVERAGE ( STANDARD ERROR) PUPAL AND ADULT CHARACTERISTICS OF TOXOTRYPANA CURVICAUDA ON C.
PAPAYA.

Females Males

Variable (n = 226) (n = 175) t P

Pupal weight (mg) 75.0 0.90 70.2 1.00 3.58 <0.001
Pupal length (mm) 10.3 0.04 9.9 0.05 6.165 <0.001
Pupal width (mm) 2.4 0.05 2.3 0.03 1.75 >0.05
Pupal period (d) 21.6 0.06 21.6 0.07 0.01 >0.05
Adult weight (mg) 48.8 0.60 42.7 0.70 6.639 <0.001
Adult longevity (d) 19.9 1.14 20.0 0.93 0.05 >0.05







Florida Entomologist 89(2)


lived at least 6 d. However, female and male min-
imum and maximum longevity were 2-59 and 2-
55 d, respectively (Fig. 1).
Puparial length increased as puparial weight
increased, for both sexes (males: F = 369, df =
1,172, P > 0.0001; females: R = 0.68 and F = 423,
df = 1,223, P > 0.0001, R2 = 0.65). There were no
relationships between puparial weight and lon-
gevity or for adult weight and longevity for either
females (F = 0.007, df = 1,92, P > 0.05; F = 0.25, df
= 1,92, P > 0.05) or males (F = 1.1, df = 1,141, P >
0.05 and adult F = 2.57, df = 1,141, P > 0.05), re-
spectively.

Sex Ratio and Emergence Rhythm

Most insects emerged during the first two days
at the start of adult emergence (Fig. 2). Similar
numbers of males and females emerged in the
first (t = 0.452, P = 0.66), second (t = 0.31, P =
0.76) and third day (t = 1.27, P = 0.23). However,
significantly more females emerged overall (t =
2.96, P = 0.003), resulting in a 1:1.26 male:female
sex ratio.

Relationship between Puparial and Adult Weight, and
Number of Chorionated Eggs

Heavier females have a reproductive advantage


over 1
chorio

Gonad

No
2-d-ol
from
onate
as fer
had n


100


75


50
E 50

E
E
I 25






Fig
curvic
tepec,


35

S 30
SMales
o 25 Females

S20 -2

15
E
10

o


0.0 1 0 20
Days after the first emergence

Fig. 2. Mean ( standard error) number of T cur-
vicauda adults that emerged per day starting with first
adult emergence; n = 226 and 175 for females and
males, respectively.


eggs of 4-d-old females (Fig. 4). There was no dif-
ference in number of chorionated eggs produced
by 10- and 12-d-old females.

DISCUSSION


ight females as heavier females had more Toxotrypana curvicauda females had a 6-d pre-
nated eggs than light ones (Fig. 3A, B). copulatory period. During the female's precopula-
tory period, ovaries and eggs increase in length
ic Maturation and width (Landolt 1984). Our results show that
the first chorionated eggs appeared later than 2 d
Schorionated eggs were obtained from 0- and after emergence, indicating that several days are
d females. Thus, these data were removed required to produce mature eggs, as in many te-
;he statistical analysis. The number of chori- phritids (Williamson 1989). Females that were 6-
d eggs significantly increased up to 100 eggs d-old had more than 45 chorionated eggs per
nale age increased (Fig. 4). Females at 10 d ovary, and 10-d-old females had more than 70
ore than double the number of chorionated chorionated eggs per ovary. In contrast, Knab &
Others (1914) indicated that females had around
100 eggs and Rojas (1992) reported 67.8 ovarioles
per females. However, none of the preceding au-
thors related the number of eggs or ovarioles with
female puparial or female adult weight. Our re-
o 0 Females sults indicate that research addressing female fe-
oO o Males cundity also should consider the positive and lin-
ear relationships between puparial and adult
V* weight as well as age and the number of chorion-
e ated eggs.
o According to Mason (1922), T curvicauda fe-
males may lay 2 to 30 eggs during each oviposi-
,? tion. However, Rojas (1992) and Landolt & Reed
(1990) reported that on average each female laid
oC 5.4 and 29 eggs, respectively. This indicates that
.-9 for females to lay their full egg-load, gravid 6-d-
S 10 20 30 40 50 60 old females need to oviposit more than three
Days times a day. According to Landolt and Hendrichs

. 1. Cumulative mortality of male and female T (1983), females may oviposite from 1 to 13 times
auda at 27 + 2 C, 50-60 RH and, 12L:12D at Yau- each. Under laboratory conditions, gravid females
Morelos, Mexico. may oviposit more than three times in a day


June 2006







Jim6nez-P6rez & Villa-Ayala: Size and Fecundity of T. curvicauda


y= 4.8+1.421x
F = 40.93, df=1,48, P <0.001
R'= 0.48


66 *


200-



4) 10 -



c 120-
o
-c
0


80-
E
z


,, Fig. 4. Mean ( standard error) number of chorion-
40 0 80 100 120 ated eggs per ovary of T curvicauda females of different
ages. Bars topped with the same letter are not signifi-
Female puparium weight (mg) cantly different (LSM; P = 0.05).


200 -
216-








c 120 -
-s
*2


| 80-
E
z


y= 21.8+ 1.820x
F = 41.90, df 1,48, P < 0.001
R = 0.46


.,


.0
e4 *


20 40 00 80
Female adult weight (mg)

Fig. 3. Relationship between (A) pupal weight and
number of chorionated eggs and (B) female adult weight
and number of chorionated eggs in T curvicauda.


(A. Jimenez-Perez, personal observation). This
suggests that females may find at least three suit-
able oviposition substrates daily indicating its
importance as a pest.
Knab & Yothers (1914) failed to find immature
eggs in mature females and indicated that the
oviposition period should be short. However, our
observations indicate that mature and immature
eggs coexist within the ovary, as indicated by Wil-
liamson (1989) for most tephritid species.
A precopulatory period has important implica-
tions for populations. It increases pre-reproduc-
tive mortality and decreases population growth.
A female biased sex ratio may be a mechanism to


diminish the effects of pre-reproductive female
mortality. Puparial weight can be an additional
factor used to assess the number of chorionated
eggs. Our results will aid us in planning future re-
search programs on T curvicauda.

ACKNOWLEDGMENTS

We thank R. Arzuffi-Barrera for comments on the
manuscript, and anonymous reviewers for constructive
suggestions. This study was funded by grant CGPI-
20040736 from Instituto Polit6cnico Nacional, M6xico to
A. Jim6nez-P6rez. The authors are COFAA fellows.

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June 2006







Bento et al.: Biology and Mating Behavior ofAtheloca subrufella



BIOLOGY AND MATING BEHAVIOR OF THE COCONUT MOTH
ATHELOCA SUBRUFELLA (LEPIDOPTERA: PHYCITIDAE)


JosE MAURICIO S. BENTO1 DORI E. NAVA1, MARCONE C.M. CHAGAS2, ANDRE H. COSTA1,
DANILO J. LIBARDI1 AND JOSE ROBERTO P. PARRA1
1Departamento de Entomologia, Fitopatologia e Zoologia Agricola, ESALQ-USP
Caixa Postal 09, 13418-900, Piracicaba, SP, Brazil

2Embrapa/Emparn, Caixa Postal 188, 59.020-390, Natal, RN, Brazil


ABSTRACT

The coconut moth, Atheloca subrufella, is responsible for most of the flower and fruit shed-
ding in coconut cropping systems. Despite this, little is known with regard to its biology and
behavior. In order to understand its biology, the duration and viability of the egg, larval, and
pupal stages, number of instars, pupal weight of males and females, sex ratio, adult longev-
ity, and fecundity were determined. In the mating behavior study, observations included
mating time and duration. Duration and viability of the egg, larval, and pupal stages were
3.0 and 93.0, 14.3 and 85.0, and 11.2 d and 91.0%, respectively, totaling 28.5 d (egg-adult)
with 72.0% viability. There were four instars, with head capsule means of 0.27, 0.45, 0.80,
and 1.33 mm for the 1st, 2nd, 3rd, and 4th instars, respectively. The sex ratio was 0.55, and
the mean pupal weight was 22.2 mg for males and 25.2 mg for females. The pre-oviposition,
oviposition, and post-oviposition periods averaged 2.4, 7.5, and 5.5 d, respectively. The lon-
gevity of males and females was 17.5 and 15.2 d, with a mean fecundity of 216 eggs. With re-
gard to mating behavior, 91.0 and 9.0% of the tested pairs mated on the first and second day
of adult life, respectively. Mating always began between 1900 and 2300 h, corresponding to
an interval between 45 and 285 min after dusk, with a mean mating duration of 95 min.

Key Words: Insect, Aracaceae, palms, coconut palm pest

RESUMO

A traca-do-coqueiroAtheloca subrufella 6 responsavel por boa parte da queda de flores e fru-
tos na cultural do coqueiro. Apesar disso, ainda pouco se conhece sobre sua biologia e com-
portamento. Para o estudo da biologia deste inseto, determinaram-se a duracgo e viabilidade
das fases de ovo, larva e pupa, o numero de instares, peso de pupas de machos e f6meas, ra-
zao sexual, longevidade dos adults e fecundidade. Para o estudo do comportamento sexual
observou-se o horario e a duracgo do acasalamento. A duracgo e a viabilidade das fases de
ovo, lagarta e pupa foram de 3,0 e 93,0; 14,3 e 85,0; e 11,2 dias e 91,0%, respectivamente, to-
talizando 28,5 dias (ovo-adulto) e 72% de viabilidade. O numero de instares foi 4, com m6dias
de capsula cefalica de 0,27; 0,45; 0,80 e 1,33mm, para o 1, 2, 3 e 4 instares, respectiva-
mente. A razao sexual foi 0,55 e o peso m6dio de pupas 22,2 mg para machos e 25,2 mg para
f6meas. As duracoes dos periods de pr6-oviposicio, oviposicio e p6s-oviposicio foram de 2,4;
7,5 e 5,5 dias, respectivamente. A longevidade de machos e f6meas foi de 17,5 e 15,2 dias, com
fecundidade m6dia de 216 ovos. Em relacio ao comportamento sexual, 91,0 e 9,0% dos ca-
sais, copularam no primeiro e segundo dia de vida, respectivamente. O inicio da c6pula ocor-
reu sempre entire as 19 e 23 horas, correspondendo ao intervalo de 45 a 285 min, ap6s o
entardecer, com uma duracgo m6dia da c6pula de 95 min.


The coconut moth Atheloca subrufella (Hulst,
1887) [= Hyalospila ptychis (Dyar, 1919)] (Lepi-
doptera: Phycitidae) is one of the most important
coconut pests (Ferreira et al. 2002). In Brazil, the
first reports on the occurrence of this insect are
dated to the beginning of the 20th century in Ba-
hia and Pernambuco (Bondar 1940; Costa Lima
1949). It has been reported in the states of Ama-
zonas (Sefer 1963), Sergipe, and Rio de Janeiro
(Silva et al. 1968). More recently, with the expan-
sion of coconut production in several regions of
the country, its now occurs in all of the Brazilian


states where coconuts are grown (Ferreira et al.
2002). The coconut moth also occurs in the south
of the USA (Georgia and Florida), the north of
Mexico (Habeck & Nickerson 1982; Hodges et al.
1983; Adams 2004), Cuba, and the Virgin Islands
(Bondar 1940; Heinrich 1956; Kimbal 1965;
Moore 2001). Palm trees in the family Arecaceae
are the most important hosts, with reports mainly
in the genera Cocos, Attalea, Syagrus, Sabal, and
Serenoa (Bondar 1940; Costa Lima 1949; Kimbal
1965; Silva et al. 1968; Moura & Vilela 1998;
Moore 2001; Ferreira et al. 2002).







Florida Entomologist 89(2)


Although significant damage occurs in differ-
ent regions, the bioecology ofA. subrufella is still
unclear and little known. The adult is a microlepi-
dopteran with a wingspan from 14 to 18 mm; the
moth is brownish and lives protected by open co-
conut spathes during the day (Bondar 1940). As
soon as the inflorescence opens, the moth lays its
eggs on female flowers (Moura & Vilela 1998). The
newly-hatched caterpillars feed on the carpels of
still-tender flowers or, if the flower has already
been fertilized, they penetrate the developing co-
conut through the lower part of the bracts
(Bondar 1940; Ferreira et al. 2002). Attacked
flowers are aborted and fall off; the presence of
the pest can be recognized by the feces at the site
and by a change in color in female flowers, which
become dark brown (Bondar 1940; Moura & Vilela
1998). In young coconuts, the caterpillar feeds on
the mesocarp, opening a series of galleries and
causing premature shedding of fruits. Feces and
small fecal pellets bound by silk strands can be vi-
sualized on the external surface of young coco-
nuts, facilitating recognition of the pest. Attacked
coconuts that do not fall off become deformed and
have reduced commercial value (Bondar 1940;
Ferreira et al. 2002).
Controlling A. subrufella is particularly diffi-
cult because of the continuous development of in-
florescences in the coconut palm, which makes
agrochemical spraying not viable. In addition,
spraying insecticides may affect a number of ben-
eficial and pollinating insects, such as bees
(Moura & Vilela 1998); moreover, the effectiveness
of insecticides is low, because the caterpillars are
protected between the bracts of young coconuts.
Thus, the purpose of this work was to study in
detail the biology and mating behavior of A. sub-
rufella, aimed at future studies for the integrated
management of this pest, with emphasis on the
production of sex pheromone.

MATERIALS AND METHODS

Starting a Stock Rearing

The insects used in the experiments were col-
lected from infested young coconuts in commer-
cial dwarf coconut plantations in the municipal
district of Touros-RN, Brazil. Infested coconuts
were taken to Emparn's (Empresa de Pesquisa
Agropecuaria do Rio Grande do Norte) Labora-
tory of Entomology, where they were arranged on
plastic trays containing sterilized sand for pupa-
tion. The pupae obtained were separated and sent
to the Insect Biology Laboratory of Departamento
de Entomologia, Fitopatologia e Zoologia Agricola
of Escola Superior de Agricultura "Luiz de
Queiroz" (ESALQ), Universidade de Sao Paulo
(USP), in Piracicaba, SP, Brazil, where a stock
rearing in young coconuts was initiated to sup-
port the biology and behavioral studies. The biol-


ogy experiments were carried out with labora-
tory-reared second-generation insects, while the
behavioral studies were conducted with third-
generation insects.
Adults were maintained in cages made from
PVC tubes 10 cm in height x 10 cm in diameter
and lined with paper towel to provide an egg-lay-
ing substrate. The ends of the tube were covered
with a 12-cm diameter Petri dish to prevent the
insects from escaping. Adults were fed a 10%
honey solution; the food and paper towel contain-
ing eggs were replaced every two days.

Coconut Moth Biology

Upon hatching, 150 larvae were placed in clear
plastic cups 7 cm height and 6 and 5 cm diameter
at the base and top, respectively, to determine the
duration and viability of the egg, larval, and pu-
pal stages, pupal weight of males and females, sex
ratio, longevity of males and females, and fecun-
dity, as recommended by Parra (2001). Young co-
conut fruits (2.5 cm 0.5) were placed inside the
cups, maintained on filter paper (same diameter
as the cup), in order to absorb the excess moisture
released by the fruit and to avoid or reduce con-
tamination by saprophytic microorganisms. After
ten days of larval development, each fruit was re-
placed with a fresh one. Pupae were later individ-
ually placed in plastic cups 3.0 cm height x 1.5 cm
diameter, and arranged on a perforated metal
tray and containing filter paper inside, which was
moistened daily in order to maintain adequate
humidity during that stage of development. Sexes
were separation during the pupal stage, accord-
ing to the methodology by Butt & Cantu (1962).
The number of instars was determined by
daily measuring the head capsule width of 15 cat-
erpillars with an ocular micrometer attached to a
stereoscopic microscope, according to Parra &
Haddad (1989).
Longevity and fecundity determinations were
made on each of 25 pairs in PVC cages, as previ-
ously described. Food was replaced daily, and the
number of eggs and mortality of males and fe-
males were recorded. Rearing was conducted in
an air-conditioned room at a temperature of 25
1C, relative humidity of 70 10%, and 14h pho-
tophase.
The x2 test was used to determine the possible
occurrence of protogyny, considering a daily sex
ratio of 0.5 as the expected values. Regression
analysis was used to determine a mathematical
model that would best explain the daily emer-
gence rhythm of males and females.

Mating Behavior of Coconut Moth

Preliminary visual observation studies with
A. subrufella pairs demonstrated that mating in
this species always occurred after dusk. Therefore,


June 2006







Bento et al.: Biology and Mating Behavior ofAtheloca subrufella


5 groups of 9 pairs in their first day of life were
formed, in clear plastic cups 13.0 cm in height and
8.5 6.0 cm in diameter at the base and top, respec-
tively, inverted on a Petri dish 9.0 cm in diameter
x 0.8 cm in height, containing filter paper moist-
ened with distilled water at the base. These pairs
were arranged in a greenhouse under natural
light, temperature of 25 3C, and relative humid-
ity of 70 10%. Visual observations were made ev-
ery 10 min, from the beginning of dusk until the
mating activities ceased; the age, time at the start
and end of copulation, duration, and a description
for the courtship and copulation activity were re-
corded. Observations were made with a hand
flashlight (Maglite with a red filter). The light
source was maintained at a ca. 60 cm from the
arena so as to prevent possible interference with
insect behavior. This experiment was conducted
until the fifth day of life of those adults, and only
pairs that performed at least one copulation dur-
ing that period were considered for the analysis.
The sunset time for Piracicaba-SP was ob-
tained from Observat6rio Nacional in Rio de Jan-
eiro-RJ, Brasil, and occurred on average at 1830 h
in March 2004 during the bioassays (Moreira
2004).

RESULTS

Biology

The means ( SEM) for duration of the egg,
caterpillar, and pupal stages was 3.0 + 0.01; 14.3
+ 0.09, and 11.2 0.09 d, and viability was 93.0 +
0.04; 85.0 + 0.30, and 91.0 + 0.29%, respectively.
The complete cycle (egg-adult) lasted 28.5 0.96 d
and total viability was 72.0 0.34%.
Four instars were determined forA. subrufella,
with mean head capsule width of larvae of 0.27,
0.45, 0.80, and 1.33 mm, for the 1st, 2nd, 3rd, and
4th instars, respectively. The mean pupal weight
was 22.2 5.0 mg for males and 25.2 4.1 mg for
females. The sex ratio was 0.55 and the longevity
of males and females was 17.5 1.19 and 15.2 +
0.95 d, respectively.
The durations of the pre-oviposition, oviposi-
tion, and post-oviposition periods were 2.4 0.20,
7.5 + 0.68, and 5.5 0.84 d, respectively. In aver-
age terms, the oviposition rate was 29 eggs per d,
and fecundity was 216.4 20.86 eggs per female.
Egg-laying decreased through the oviposition pe-
riod, but oviposition until the 14th day was ob-
served for some females (Fig. 1).
Females emerged in higher numbers than
males during the first two d of emergence, indicat-
ing that this species presents the protogyny phe-
nomenon (Fig. 2). Significant values were deter-
mined by the X2 test for the first two d (higher
number of emerged females) and for the last two d
(higher number of emerged males). There were no
significant differences for the third and fourth d.


I ; 1 5 C 7 ( IC II I) I)
0*
3.Lj,.








Figure. 1. Daily oviposition of A. subrufella reared
during the larval stage on young coconut fruits. Temp.:
25 1C; RH: 70 + 10%, photophase: 14h. Bars indicate
the standard error of the mean (SEM).


The regression analysis factor for the emer-
gence rhythm of males factor was significant (y05
= -4.55 + 4.92x 0.59x2;R2 = 0.86; F = 10.02; P =
0.01; df = 2), where the coefficients were different
from zero by Student's "t" test (P 0.05). The
emergence rhythm of females was significant (y =
- 36.99 + 262.05/x 214.64x2;R2 = 0.97; F = 20.99;
P = 0.01; df= 2), indicating that the equations fit
the data well.

Mating Behavior

From 45 A. subrufella pairs observed, 29
mated (64.4%). Of these mating pairs, 91.0 and
9.0% copulated on the first and second days of life,
respectively.
The beginning of copulation always occurred
between 1900 and 2300 h, corresponding to the
interval from 45 to 285 min after dusk. The mat-
ing frequency from 2100 to 2200 h was statisti-
cally different from 1900 to 2000, but it was not
different from 2000 to 2100 h, and from 2200 to
2300 h (Fig. 3). The mean copulation duration was
95 min (43-149 min).

DISCUSSION

There have been few reports on the biology of
A. subrufella. Data presented in this paper sug-
gest high biotic potential, although in the field cli-
matic factors and the action of natural enemies
must contribute to reducing this potential. Ac-
cording to Bondar (1940), the life cycle of this
moth is approximately 25 to 30 d, while Moura &
Vilela (1998) mentioned 40 d; these authors did
not present durations for the different stages. In
the present work, the life cycle lasted 28.5 d, on
average, for a temperature of 25C. The females
normally begin laying eggs about 2 d after mat-
ing, laying a higher number of eggs in the very







Florida Entomologist 89(2)


50
B Male l Female

40-


130


20




0
23 24 25 26 27 28
Day after emergence

Figure. 2. Emergence of A. subrufella females and males. The solid and dashed lines indicate the emergence
trend for males and females, respectively. Temp.: 25 1C; RH: 70 10%, photophase: 14h.


first days. Even though the females have a lon-
gevity of about 2 weeks, the oviposition period is
short, around 7 d, with a mean fecundity of 216
eggs per female. The post-oviposition period was
5.5 d. Eggs were laid individually, initially show-


b





19-00-20:00


2000-21:00


ing a pale-yellow color, becoming slightly reddish
later, and acquiring a dark hue on the last day of
embryonic development. The caterpillar stage
lasted 2 weeks on average, and caterpillars may
reach 15 mm in length. The pupal stage lasted 11


a


21:00-22:00


22:00-23:00


Time (h)

Figure. 3. Mating time ofA. subrufella. Means followed by the same letter are not statistically different by Tukey
test at the 5% probability level. Sunset time for Piracicaba-SP, Brazil, at 1830 h (March 2004).


June 2006







Bento et al.: Biology and Mating Behavior ofAtheloca subrufella


d, differing from findings by Bondar (1940), who
found a duration from 6 to 8 d. These differences
may be associated with abiotic factors, not men-
tioned by Bondar (1940).
The emergence rhythm ofA. subrufella adults
indicates that females begin emerging before
males (protogyny). The percentage of emerged fe-
males was significantly higher than that of males
in the first two d (Fig. 2). The biological reason for
this emergence pattern is still little understood in
insects (Thornhill & Alcock 1983), but it has been
demonstrated in other lepidopteran species and
could represent an evolutionary strategy to pro-
mote mating between individuals from distinct
populations (Uematsu & Morikawa 1997).
Mating always started during the scotophase,
and were mainly concentrated in a period of two
to four h after dusk. After the beginning of dusk,
females and males became very agitated, espe-
cially the latter. With time, the females remained
almost motionless, possibly in a "calling" position,
even though no exposure of any exogenous gland
or abdomen movements could be observed. A male
near a female showed frenetic antennal and wing
movements, walking in a semi-circular fashion
around the female's body. Once the female was re-
ceptive, the male assumed a (contrary position) in
relation to her, at a 180 angle, then walked back-
wards, with light wing and abdomen movements
until copulation was achieved; this could last 1 h
and 30 min on average (P < 0.05). If for some rea-
son the female rejected the male's lunge before
copulation was accomplished, the male restarted
the whole procedure. In general, more than 90%
of females mated on the first day of life.
More than 12 annual generations with high bi-
otic potential may develop, because, in addition to
laying more than 200 eggs, the insect shows high
viability in all developmental stages. Develop-
ment of an artificial diet forA. subrufella, which is
available for other insects in the same family
(Singh 1983), would facilitate the development of
future management strategies for this pest.

ACKNOWLEDGMENTS

Thanks to Fundacao de Amparo a Pesquisa do Es-
tado de Sao Paulo-FAPESP (Project 01/06587-6) and
Banco do Nordeste for financial support to the research,
to Dr. M.L. Haddad for help with the statistical analy-
ses, and to Dr. A. Solis, from the National Museum of
Natural History, Washington DC, USA, for clarifications
on scientific nomenclature.

REFERENCES CITED

ADAMS, J. K. 2004. Moths and Butterflies of Georgia
and the Southeastern United States. http://www.dal-
tonstate.edu/galeps/index.htm (22 Jul. 2004).


BONDAR, G. 1940. Insetos nocivos e mol6stias do co-
queiro (Cocos nucifera L.) no Brasil. Tipografia Na-
val, Salvador. 156 pp.
BUTT, B. A., AND E. CANTU. 1962. Sex determination of
lepidopterous pupae, ARS. United States Departa-
ment of Agriculture, Washington. 7: 33-75.
COSTA LIMA, A. C. 1949. Insetos do Brasil, 6 tomo,
capitulo 28, Lepid6pteros (2o parte). S6rie Didatica
Num. 8. Escola Nacional de Agronomia, Rio de Jan-
eiro. 420 pp.
FERREIRA, J. M. S., R. P. C. ARAiJO, AND F. B. SARRO.
2002. Insetos e acaros, In J. M. S. Ferreira [ed.],
Coco, Fitossanidade. Embrapa Tabuleiros Costeiros,
Brasilia, Embrapa Informacio Tecnol6gica (Frutas
do Brasil; 28). 136 pp.
HABECK, D. H., AND J. C. NICKERSON. 1982. Atheloca
subrufella (Hulst.), A Pest of Coconuts (Lepidoptera:
Pyralidae: Phycitinae). Florida Dept. Agr., Div. Plant
Ind. 241. 2 pp.
HEINRICH, C. 1956. American Moths of the Subfamily
Phycitinae. U.S. Nat'l. Mus. Bull. 207: 1-581.
HODGES, R. W., T. DOMINICK, D. R. DAVIS, D. C. FERGU-
SON, J. G. FRANCLEMONT, E. G. MONROE, AND J. A.
POWELL. 1983. Check List of the Lepidoptera ofAmer-
ica North of Mexico. E. W. Classey Ltd. & The Wedge
Entomological Research Foundation, London. 284 pp.
KIMBALL, C. P. 1965. The Lepidoptera of Florida. Flor-
ida Dept. Agr., Div. Plant Ind., Arthropods of Florida
and Neighboring Land Areas 1: 1-363.
MOORE, D. 2001. Insects of palm flowers and fruits, In F.
W. Howard, D. Moore, R. M. Giblin-Davis, and R. G.
Abad [eds.], Insects on Palms. CAB International,
Wallingford, Oxon. 400 pp.
MOREIRA, J. L. K. 2004. Anudrio Interativo do Obser-
vat6rio Nacional. http://euler.on.br/ephemeris/index.
php, (27 Jul, 2004).
MOURA, J. I. L., AND E. F. VILELA. 1998. Pragas do Co-
queiro e Dendezeiro. 2. ed. Aprenda Facil, Vicosa.
124 pp.
PARRA, J. R. P., AND M. L. HADDAD. 1989. Determinacio
do Numero de Instares de Insetos. Piracicaba: Fealq,
49 pp.
PARRA, J. R. P. 2001. T6cnicas de Criacgo de Insetos
para Programas de Controle Biol6gico. 6. ed., Piraci-
caba: Fealq. 134 pp.
RHAINDS, M., G. GRIES, AND C. CHINCHILLA. 1995. Pu-
pation site and emergence time influence the mating
success of bagworm females, Oiketicus-kirbyi. Ento-
mol. Exp. Appl. 77: 183-187.
SEFER, E. 1963. Catalogo dos insetos que atacam as
plants cultivadas da Amazonia. Bol. T6c. Inst.
Agron. Norte 43: 23-53.
SILVA, A. G., C. R. GONQALVES, D. M. GALVAO, A. J. L.
GONQALVES, J. GOMES, M. N. SILVA, AND L. SIMONI.
1968. Quarto Catalogo dos Insetos que Vivem nas
Plantas do Brasil: Seus Parasitas e Predadores.
Servico de Defesa Sanitaria Vegetal, Rio de Janeiro.
Parte II, Tomo 1. 622 pp.
SINGH, P. 1983. A general purpose laboratory diet mix-
ture for rearing insects. Insect Science and its Appli-
cation, Elmsford 4: 357-362.
UEMATSU, H., AND R. MORIKAWA. 1997. Protogyny in
diamondback moth, Plutella xylostella (Lepidoptera:
Yponomeutidae). Japan. J. Appl. Entomol. Zool. 41:
217-223.







Florida Entomologist 89(2)


June 2006


EVALUATION OF FRANKLINIELLA BISPINOSA
(THYSANOPTERA: THRIPIDAE) AS A VECTOR OF THE
TOMATO SPOTTED WILT VIRUS IN PEPPER


YOLANDA AVILA1, JULIANNE STAVISKY', SARA HAGUE', JOE FUNDERBURK1, STUART REITZ2 AND TIM MOMOL1
1North Florida Research and Education Center, University of Florida, 155 Research Road, Quincy, FL 32351

2USDA ARS CMAVE, 6383 Mahan Drive, Tallahassee, FL 32308

ABSTRACT

Frankliniella occidentalis is the key vector responsible for the emergence of Tomato spotted
wilt virus as a global threat to agriculture. Frankliniella bispinosa is a common thrips in
Florida, the Bahamas, and Bermuda, but the role of F bispinosa in the epidemiology of the
virus is not known. The purpose of this study was to determine the ability ofF. bispinosa to
acquire and transmit Tomato spotted wilt virus in pepper. In laboratory experiments, the
number of larvae produced per F bispinosa female was less than the number of larvae pro-
duced per F occidentalis female. The larvae of F bispinosa successfully acquired Tomato
spotted wilt virus, although at a lower percentage than F. occidentalis. Viruliferous adults of
both species transmitted the virus to pepper. Our results confirm the competence of F bispi-
nosa as a vector of Tomato spotted wilt virus.

Key Words: Frankliniella occidentalis, Tospovirus, vector competence, viral acquisition, viral
transmission, Capsicum annuum

RESUME

El trips, Frankliniella occidentales, es un vector clave y responsible para la emergencia del
virus de la marchitez manchada del tomate como una amenaza global para la agriculture.
Un otro especie de trips comun en Florida, Bahamas y Bermuda es Frankliniella bispinosa,
pero su papel en la epidemiologia del virus de la marchitez manchada del tomate no es co-
nocido. El prop6sito de este studio fue para evaluar la habilidad de F bispinosa para adqui-
rir y transmitir el virus de la marchitez manchada del tomate al chile. Las larvas de
F bispinosa adquirieron con buen 6xito el virus de la marchitez manchada del tomate, aun-
que a un porcentaje menor que en F occidentalis. Adultos viruliferos de las dos species
transmitieron el virus a chile. En experiments del laboratorio, el numero de larvas produ-
cidas por hembra de F bispinosa fue menor que el numero de larvas producidas por la hem-
bra de F occidentalis. Nuestros resultados confirman la capacidad de F bispinosa como un
vector del virus de la marchitez manchada del tomate.


In addition to damaging plant tissues while
feeding, some species of thrips vector Tomato spot-
ted wilt virus (TSWV), a tospovirus transmitted
through the saliva ofthrips during feeding (Hunter
& Ullman 1992; Ullman et al. 1997). Tomato spot-
ted wilt was first observed in 1915 in Australia and
described as the "spotted wilt" of tomatoes (Brittle-
bank 1919) that later was associated with trans-
mission by thrips (Pittman 1927). The viral etiol-
ogy was reported by Samuel et al. (1930).
TSWV causes economic loss in many agricul-
tural crops. The virus has a broad host range, in-
fecting over 1000 plant species, and causing an
estimated crop loss of one billion US dollars per
year throughout its host range (Prins & Goldbach
1998). Tomato, tobacco, lettuce, pepper, papaya,
eggplant, green beans, artichokes, broad beans,
celery, some ornamental plants, and other plants
experience severe losses due to the virus (Rosella
et al. 1996).


Ullman et al. (1997) reviewed the relevant sci-
entific literature involving the relationship be-
tween TSWV and its thrips vectors. Only first and
second instars of vector thrips species acquire
TSWV during feeding upon an infected host, and
the virus survives molting, pupation, and the re-
placement of tissues during the prepupal and pu-
pal stages of thrips development. Adults are un-
able to acquire TSWV. Infected adults are respon-
sible for transmission and spread.
Outbreaks of tomato spotted wilt are difficult
to manage. Growing seedlings under cover, avoid-
ing sequential plantings, removing acquisition
hosts for the larvae, rotating with non-susceptible
crops, and use of UV-reflective mulch are some-
times useful as management tactics (Cho et al.
1998; Kucharek 1990; Momol et al. 2004; Rosella
et al. 1996). Tomato hybrids were developed with
a single-gene dominant resistance trait, but this
resistance was overcome by strains of the virus







Avila et al.: Frankliniella bispinosa vectors Tospovirus


(Rosella et al. 1996). Attempts to regulate vector
populations with insecticides have not been suc-
cessful, and populations ofthrips developed resis-
tance to broad-spectrum insecticides (Brodsgaard
1994; Immaraju et al. 1992). Further, primary
spread of TSWV is not prevented by insecticides
because insecticide-exposed viruliferous adults
successfully transmitted the virus before death
(Momol et al. 2004).
Thrips species known to transmit TSWV are
Thrips tabaci (Lindeman), Thrips setosus (Moul-
ton), Frankliniella occidentalis (Pergande), Fran-
kliniella schultzei (Trybom), Frankliniella fusca
(Hinds), and Frankliniella intonsa (Trybom)
(Sherwood et al. 2001). Frankliniella occidentalis
is the primary vector of TSWV due to its increas-
ingly global distribution (Wijkamp et al. 1995).
Frankliniella bispinosa (Morgan), which is dis-
tributed in parts of the southeastern US, Ber-
muda, and the Bahamas (Nakahara 1997), has
been suspected, but not proven, as a vector of
TSWV (Tsai et al. 1996; Webb et al. 1998).
The plants on which adult thrips can be col-
lected have been cited in the literature as host
plants (Mound and Teulon 1995), but adults fre-
quently inhabit flowers that are not reproductive
hosts. Adults of F bispinosa are abundant in the
flowers of bell pepper, Capsicum annuum L., in
Florida along with adults of other species, includ-
ing F occidentalis (Funderburk et al. 2000;
Hansen et al. 2003; Reitz et al. 2003). The suit-
ability of pepper as a reproductive host ofF. bis-
pinosa has not been determined, and the possible
role of F bispinosa in TSWV epidemics is un-
known. The purpose of our research was to deter-
mine the competence ofF. bispinosa as a vector of
TSWV in pepper. An experiment was conducted
to determine the ability of F bispinosa to repro-
duce and acquire the virus on pepper compared to
the key vector, F occidentalis. Another experi-
ment was conducted to verify that F bispinosa
adults are able to transmit the TSWV to un-
infected pepper.


MATERIALS AND METHODS

Pepper Establishment and Maintenance

Individual 'Camelot X3R' bell peppers were
transplanted into a 16 x 16 cm pot containing soil
mixture (Fafard 3B Mix, Agawam, MA) and about
100 were maintained under greenhouse condi-
tions. Plants were fertilized with Peat-Lite spe-
cial 15-16-17 fertilizer (Scotts-Sierra Horticul-
tural Products Company, Marysville, OH) and
Miracle-Gro Bloom Booster 10-52-10 fertilizer
(Miracle-Gro, Marysville, OH). Virus acquisition
and transmission experiments were conducted in
growth rooms at 23 to 25C under a photoperiod
of 14 h light, 10 h darkness.


Virus Acquisition Experiment

Plants for use in TSWV acquisition trials were
mechanically inoculated with TSWV isolates col-
lected from naturally infected pepper plants at
the North Florida Research and Education Cen-
ter. Using a mortar and pestle, we homogenized
TSWV infected leaf tissue in 5% sodium sulfite so-
lution containing diatomaceous earth in order to
prepare an inoculum. Cheesecloth was used to ap-
ply inoculum to 3 or 4 leaves per experimental
plant. Seven to 10 days after mechanical inocu-
lum, experimental plants were tested for TSWV
infection by a commercially available double anti-
body sandwich enzyme-linked immunosorbent as-
say (ELISA) kit (Agdia, Elkhart, IN). Glass tubes
and polystyrene balls (Precision Plastic Ball Co.,
Chicago, IL) were used in place of a microplate.
Samples were scored for the presence of a colori-
metric reaction indicating TSWV infection.
Individual TSWV infected pepper plants be-
tween 8 and 10 weeks of age were covered by a
polyethylene cylindrical cage (35 cm x 15 cm) (n =
23 and 25 for F bispinosa and F occidentalis, re-
spectively). The opening at the top was covered
with fine mesh to prevent thrips escape, and there
were two side openings (2.5 cm x 2.5 cm) covered
with mesh. Ten females ofF occidentalis or F bis-
pinosa were introduced into the cage containing
one infected plant. The adults were removed after
6 d. The plants were visually inspected for larvae
at 6, 8, and 10 d after initial infestation with
adult thrips. Each larva when found was trans-
ferred to green bean pods. After developing into
adult, each was tested with an indirect ELISA to
detect for the presence of the NSs protein encoded
by TSWV RNA (Bandla et al. 1994). The NSs pro-
tein is present in thrips cells as a result of TSWV
replication, demonstrating that the thrips is a
host for TSWV.
The mean numbers of F occidentalis larvae
and F bispinosa larvae recovered per cage after
10 days were compared with a two sample t-test
[PROC TTEST in SAS System Software (SAS In-
stitute 1999)]. The percent virus acquisition of
F occidentalis and F bispinosa larvae also was
compared by a two-sample t-test.

Virus Transmission Experiment

Transmission trials were conducted to confirm
that the adults ofF. bispinosa and F occidentalis
adults transmit TSWV to pepper. Cohorts of
about 50 F bispinosa and F occidentalis larvae
were allowed to feed on infected tomato fruit until
developing into pupae. After developing into an
adult, 5 to 10 of each species from each cohort
were tested to verify TSWV acquisition with the
indirect ELISA method described previously
(Bandla et al. 1994). Twenty putatively virulifer-
ous F occidentalis or F bispinosa adults from co-







Florida Entomologist 89(2)


horts that tested positive were introduced into a
polyethylene cage (55-cm-long xx 30-cm-wide x
48-cm-high) containing 4 healthy pepper plants of
8 weeks old. There were 19 and 8 cages estab-
lished for F occidentalis and F bispinosa, respec-
tively. The peppers were tested after 21 days for
TSWV by ELISA as described above. Transmis-
sion by F occidentalis and F bispinosa was com-
pared by a chi-square test.

RESULTS

Pepper was a suitable reproductive host for
F occidentalis and F bispinosa in our study. The
mean total number (+ SEM) of larvae recovered
over 10 d when introducing 10 F occidentalis or F
bispinosa adult females on individually caged
pepper plants infected with TSWV was 47.7 (7.2)
and 15.3 (2.5), respectively. The difference was
significant (t = -4.08, df = 58, P < 0.0001).
A higher percentage of F occidentalis acquired
the virus versus F bispinosa (t = -2.07, df = 53,
P < 0.05).The mean percent acquisition (SEM) of
TSWV by F occidentalis and F bispinosa larvae
feeding on infected pepper plants, as determined
by an indirect ELISA to detect for the presence of
the NSs protein encoded by the virus RNA, was
21.9 (3.1) and 14.6 (2.9), respectively.
The adults of F bispinosa and F occidentalis
successfully transmitted TSWV to pepper. In the
virus transmission experiments, pepper plants ex-
posed to TSWV-infected adults of F bispinosa
were ELISA positive in 4 out of 8 replicates. Pep-
per plants exposed to viruliferous adults ofF occi-
dentalis were ELISA positive in 6 out of 19 repli-
cates. The difference in transmission between the
two species was not significant (X2 = 0.8; df= 3).

DISCUSSION

The results from the acquisition experiment
indicate that under laboratory conditions F occi-
dentalis is more likely to acquire the virus, and
thus may be a more effective vector in pepper
than F bispinosa. The number of larvae of F occi-
dentalis produced per female was 3.1-fold greater,
indicating that F occidentalis has a greater in-
trinsic capacity than F bispinosa to increase on
pepper. The greater the intrinsic capacity of in-
crease of a vector species on a host plant the
greater the potential for acquisition and spread of
TSWV (Peters et al. 1996). Differences in feeding
preferences and host suitability between F occi-
dentalis and F bispinosa may result in varying
abilities of each species to acquire TSWV depend-
ing on the host plant. Webb et al. (1998) observed
in laboratory studies higher rates of acquisition of
TSWV for F bispinosa than for F occidentalis
when the larvae fed on Datura stramonium L.
Van de Wetering et al. (1999) analyzed 14 pop-
ulations of F occidentalis. Each population ac-


quired and transmitted TSWV, but there were
marked differences in their efficiency, expressed
as the percentage of transmitting adults. Labora-
tory experiments also do not account for ecologi-
cal factors that influence thrips populations un-
der field conditions, such as parasitism or preda-
tion that may reduce thrips populations, nor does
it account for differences in mobility and other be-
haviors between the two thrips species that might
affect the spread of TSWV in field peppers. Ram-
achandran et al. (2001) showed that the adults of
F bispinosa moved more rapidly in field peppers
than the adults of F occidentalis. This behavior
allowed the adults of F bispinosa to more fre-
quently escape predation of Orius insidiosus.
The abundance of F bispinosa and F occiden-
talis in certain geographical regions also should
be considered when assessing these species as a
potential threat as a vector of TSWV in field pep-
per. Hansen et al. (2003) found F bispinosa in
much greater abundance than F occidentalis in
central Florida, while both species were abun-
dant in northern Florida.
In this study, we have shown that under labo-
ratory conditions F bispinosa is a competent vec-
tor of TSWV in pepper. The species reproduced
and acquired TSWV from infected pepper plants.
Viruliferous adults of F bispinosa transmitted
TSWV to pepper. Reproduction of F occidentalis
on pepper and virus acquisition by the larvae
feeding on pepper was greater than that by F bis-
pinosa; however, species-specific attributes may
play a role in the ability of both vectors to vector
TSWV in field conditions. The adults of F bispi-
nosa are more mobile within pepper fields than
the adults ofF occidentalis (Ramachandran et al.
2001). TSWV epidemics occur on field pepper in
our region (Gataitis et al. 1998), but the role of
each species in disease epidemiology under field
conditions is not understood.

ACKNOWLEDGMENT

We thank Hank Dankers, Kristen Bowers, and Steve
Olson for technical assistance. Heather McAuslane and
Michelle Stuckey made comments on the manuscript.
The research was supported by a USDA-CSREES grant
in Tropical/Subtropical Agriculture.

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Florida Entomologist 89(2)


METAMASIUS CALLIZONA (COLEOPTERA: DRYOPHTHORIDAE):
LONGEVITY AND FECUNDITY IN THE LABORATORY


J. H. FRANK', T. M. COOPER1 AND B. C. LARSON2
1Entomology & Nematology Dept., University of Florida, Gainesville, FL 32611-0620

2Florida Yards and Neighborhoods Program, Environmental Horticulture Dept.
111 Mehrhof Hall, University of Florida, Gainesville, FL 32611-0675

ABSTRACT

Metamasius callizona (Chevrolat) is native to southern Mexico and Guatemala. It was de-
tected in Broward County, Florida, in 1989 and has spread to 20 counties in southern Flor-
ida, where it devastates populations of native epiphytic bromeliads and also attacks
cultivated bromeliads. Larvae mine into stems of larger bromeliads, killing them. New data
were obtained at =25 C and a photoperiod of 14:10 L:D to optimize cultures of this insect to
serve as hosts for the production of biological control agents. After pairing with males, it took
an average of 28.9 d (17.8, range 8-89 d) for females to begin laying eggs; thereafter, each
female laid eggs for the remainder of her life, or within just a few days of her death. The total
duration of life of 75 ovipositing females averaged 156.4 d (96.7, range 26-387 d); their life-
time egg production averaged 39.6 eggs (40.0, range 2-188 eggs).

Key Words: Mexican bromeliad weevil, biological control, Florida, invasive species, adven-
tive insects

RESUME

Metamasius callizona (Chevrolat) es native del sur de M6xico y Guatemala. Fue detectado
en el condado Broward de Florida en 1989. Ahora, ocupa 20 condados del sur de Florida
donde destruye poblaciones nativas de bromeliaceas epifitas y ataca a bromeliaceas cultiva-
das. Las larvas minan los tallos de las bromeliaceas grandes causandoles la muerte. La
nueva informaci6n presentada aqui es para mejorar las colonies de este insecto como hu6s-
ped para agents de control biol6gico. Despu6s de aparearse, las 75 hembras bajo investiga-
ci6n mantenidas bajo 25 C yun fotoperiodo 14:10 luz:obscura iniciaron la oviposici6n en 28,9
dias (17,8 con un rango de 8-89) y continuaron oviposici6n durante toda la vida. La dura-
ci6n de vida de las 75 hembras fue de 156,4 dias (96.7 con un rango de 26-387); y la produc-
ci6n total de huevos por cada una fue de 39,6 (40,0 con un rango de 2-188).
Translation provided by the authors.


Two Neotropical species of Metamasius ar-
rived and became established in Florida in the
1980s. The first was M. hemipterus (L.), which is
a secondary pest of sugarcane and some ornamen-
tal palms (Weissling & Giblin-Davis 1998). The
second, with which we are concerned here, is
M. callizona (Chevrolat), a pest of bromeliads
(Larson & Frank 2004).
Metamasius callizona is one of many invasive
insects in Florida (Frank & Thomas 2004). It was
first detected in Florida in 1989, and is a pest of
cultivated bromeliads such as Ananas comosus
(L.) (pineapple) and numerous genera, species,
and hybrids of ornamental bromeliads (Frank &
Thomas 1994). It can be managed in plantings of
cultivated bromeliads by applications of chemical
insecticides. However, it is also a devastating pest
of Florida's native bromeliad populations, and
has spread to 20 counties in southern and central
Florida. These 20 counties contain habitats for
virtually all of the range of 11 of the 12 at-risk


bromeliad species, and part of the range of the
12th species. Insecticides are impracticable for the
control of M. callizona because of the epiphytic
growth of all native Florida bromeliads, their oc-
currence in nearly all of south Florida including
Federal, state, and county parks, and the poten-
tial environmental damage to non-target organ-
isms on land and in water bodies from widespread
spraying (Frank 2002). The weevil is destroying
'naive' populations of 'protected' endangered and
threatened native bromeliad species (Frank &
Cave 2005).
Metamasius callizona arrived in Florida as a
contaminant of ornamental bromeliads imported
from Mexico (Frank & Thomas 1994). We believe
that its large distribution within Florida occurred
by movement of weevil-contaminated ornamental
bromeliads. Thus, there is great risk in places
that import ornamental bromeliads from Mexico
and, now, from Florida. These include Hawaii,
with its pineapple industry, and Puerto Rico, with


June 2006







Frank et al.: Oviposition by Metamasius callizona


not only a pineapple industry but also a rich na-
tive bromeliad flora. Constant vigilance is needed
to guard against this.
As part of a biological control program aimed
at M. callizona, the development of eggs, larvae,
and pupae was investigated in the laboratory
(Salas & Frank 2001). To complement those stud-
ies, we report here on laboratory longevity and fe-
cundity of adult females.
The weevil genus Metamasius was tradition-
ally placed in the family Curculionidae (e.g.,
Anderson 2002). However, Anderson (2003) and
others reassigned it and related genera to a fam-
ily named Dryophthoridae, which previously was
the subfamily Dryophthorinae (= Rhynchophori-
nae) of Curculionidae. Because of this and other
changes in classification of Curculionoidea, the
name 'weevil' seems to apply to insects of several
families.

MATERIALS AND METHODS

A greenhouse culture of M. callizona had been
maintained since the early 1990s at the Entomol-
ogy and Nematology Department, University of
Florida. The original stock was collected in vari-
ous Broward County parks in southern Florida. It
was augmented from time to time with freshly-
collected specimens to promote genetic diversity.
The greenhouse was heated in winter and cooled
in summer to eliminate temperature extremes.
By 1995, pineapple crowns, discarded by grocery
stores, were adopted as the sole food for adults,
ovipositional substrate, and site for development
of the immature stages. By 2001, the rearing was
concentrated within cages of various sizes in the
greenhouse to reduce escape from the greenhouse
by adults, and predation by frogs (Hyla sp.) and
lizards (Anolis sp.). Provided that air humidity
was high (natural air humidity supplemented by
watering from a garden hose with sprinkler head
once every 2 d), this system was adequate for
maintenance of the weevil culture. It eliminated
need for culture of potted bromeliads and it mini-
mized labor. The most laborious aspect was to ex-
tract weevil pupae from cocoons, and these as well
as adults and larvae, from pineapple crowns, once
development of most of each cohort within a cage
had reached the pupal stage.
Beginning in August 2004, weevil pupae, ex-
tracted from cocoons in the greenhouse culture,
were brought indoors to a rearing room and
housed individually in plastic vials. The rearing
room was maintained at 25C (high 25.4 0.3,
low 24.3 0.3, n = 449 d). Air humidity was sup-
plemented by two electrical humidifiers (RH high
48.1 + 5.8%, low 40.2 5.8%, n = 449 d) although
it was perhaps of little consequence to the weevils
within the closed vials with moist pineapple
leaves. A photoperiod of L:D 14:10 was main-
tained with overhead fluorescent lighting (495


lux) in the windowless room. This allowed the ex-
act date of emergence of the resultant adult wee-
vils to be recorded. Within 3-5 d of its emergence,
each female was assigned a code number and
paired with a coded male of similar age and
placed in a transparent plastic vial (7 cm h, 3.8
cm internal diam.) with snap cap. Immediately,
four lengths of pineapple leaf(= 5 cm) were added
as food and ovipositional substrate. Those leaves
had been kept chilled since their collection from
grocery stores, and within each vial they provided
moisture. Pineapple leaf lengths were replaced in
each vial (with a living weevil) once every 2 d.
We examined each vial daily for survival of
adult weevils and, using a dissecting microscope,
for presence of eggs. As soon as the first egg was
detected within each vial, the male weevil was re-
moved and placed in a separate vial. Most eggs
were oviposited singly in pockets cut by females
in pineapple leaf lengths, but some were detected
being held against the floor or the walls of the vi-
als by moisture. Every egg observed was recorded
and removed. Removal often resulted in destruc-
tion of the egg; therefore, fertility was not re-
corded. Female weevils were initiated to this re-
gime until 75 of them had begun to oviposit. Data
were recorded daily until all 75 females had died,
and then were analyzed statistically.
The question of whether oviposition declines
within the lifetime of a female was addressed by
comparison of the number of eggs laid during the
initial and final halves of the reproductive period.
To achieve this, we recorded on spreadsheets an
absolute scale (day of emergence to day of death)
a (first egg laid) and b (death). We subtracted a
from b to calculate midpoint (x) for each female
that oviposited. Thus, we defined the initial and
final halves of the reproductive period, then noted
the number of eggs laid in each of those two peri-
ods, to present descriptive statistics. For this
analysis, the two periods had to be of equal dura-
tion in whole days; to equalize them when there
was a midpoint day, we ignored any data for that
midpoint day; thus the total number of eggs re-
corded in this analysis is very slightly less than
the actual total number recorded. For conve-
nience, we considered these to be the two halves
of the reproductive period although we acknowl-
edge that the reproductive period could be
deemed to end on the day the last egg was laid
(which varied from 2 to several days earlier).

RESULTS AND DISCUSSION

The total duration of life of the 75 ovipositing
females studied averaged 156.4 d ( 96.7 [SD],
range 26-387 d); their lifetime egg production av-
eraged 39.6 eggs ( 40.0, range 2-188 eggs). After
pairing females with males, it took an average of
28.9 d (+ 17.8, range 8-89 d) for females to begin
laying eggs.







Florida Entomologist 89(2)


The daily oviposition by the 75 females that
oviposited (after pairing) is shown in Fig. 1A. In
the first 14 d after pairing, only one egg had been
laid because almost all females were still in the
preoviposition period. Not until d 89 were all sur-
viving females in the group contributing eggs.
However, by then, considerable mortality had oc-
curred (Fig 1B). A graph of ovipositing females
that survived 100 d (plotted but not shown) indi-
cates a gradual build-up in daily oviposition until
about d 89 cf. a rapid build-up after = d 14.
Oviposition does not decline within the life of a
female. We compared the number of eggs that fe-
males laid during the initial and final halves of
the reproductive period. Thirty females laid more
eggs in the initial half of their reproductive pe-
riod, 39 in the final half, and six equally. Seven of
eleven females that survived > 300 d laid more
eggs in the final half of their reproductive period
than in the initial half. Despite halving of the
number of ovipositing females by d 140 (Fig. 1)
there was no evidence of a decline in numbers of
eggs laid daily. There was thus no evidence that
fecundity declined as females aged. Rather, the
evidence suggests that each female continued ovi-
positing until shortly before death.
The median interval between last oviposition
and death was 5 d (mean 8.3 8.8 d). If we accept
that the oviposition rate was either 0.32 eggs/fe-
male/day (from first egg laid until death), or one
egg every 3 d (see below), then there is virtually


no room for an explanation other than senescence
for cessation of oviposition.
No regular periodicity in oviposition by any fe-
male was detected, so the irregularities of the
data presented in Fig. 1A are the result of random
variation. Variation in numbers of eggs laid daily
by any female was 0-4, with 0 (76.95%) followed
by 1 (20.47%), 2 (2.34%), 3 (0.22%), and 4 (0.02%)
calculated from day of pairing. The total fecundity
of each female was highly correlated (n = 75, r =
0.7693, P < 0.001) with longevity, but yet 23%
(1.00-0.77) of the variation was not explained by
longevity. The longevity of males, held separately,
appeared to match that of females.
The 75 females laid a total of 2,973 eggs. The
sum of oviposition days, if calculated (a) from
pairing to death of each female, was 11,505, but if
calculated (b) from first egg to death was 9,392.
An oviposition rate (eggs/female/day) might be
calculated as (a) 2973/11505 = 0.26 or (b) 2973/
9392 = 0.32. On the above evidence, we might ex-
pect = 3,194 eggs from every 100 females treated
similarly, assuming that 80.6% of them oviposit.
In attempts to mass-produce weevils as hosts for
a laboratory-reared biological control agent, it
should be remembered that older females con-
tinue to oviposit at an unreduced rate.
The proportion of female M. callizona that laid
eggs (80.6%) was very similar to that of M. hemi-
pterus (76%) as was their preovipositional period
(28.9 d) compared with that of M. hemipterus (27.0


I IJ ., I I I No. of eggs laid daily by 75 M. callzona


r I Ne ^ ) R- B o co I
-r NNNN4N C)05 ~o)
Daysfrom pairing


Survival of 75 M. callizona B


0 -t N o W
CM V- iB a, ;:'-C


D som pai ri
Days from pairing


Fig. 1. A. Number of eggs laid daily by 75 Metamasius callizona females from day of pairing (3-5 d after emer-
gence from the pupal stage) until the last died. B. Survival of those same 75 females over the same time period.


June 2006




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