• TABLE OF CONTENTS
HIDE
 Effect of male accessory gland...
 Life history of the red palm weevil,...
 Rise and fall of red oak borer...
 First North American records of...
 Is host size an indicator of quality...
 Releases, distribution and abundance...
 Host specificity of Anthonomus...
 A new genus and new species of...
 Ant tending of Miami blue butterfly...
 Semiochemically based monitoring...
 Development of Spalangia cameroni...
 Parasitism of the brown citrus...
 The whip scorpion, Mastigoproctus...
 A new pest of rice in Missouri:...
 Length of multiple-funnel traps...
 Egg parasitoids of Dalbulus maidis...
 Effects of diet on development...
 Oviposition of the neotropical...
 A new record for Cicindela scabrosa...
 Mitochondrial DNA markers in populations...
 Synonymy of Aleochara (Aleochara)...
 Book reviews
 Errata
 Back Matter














Group Title: Florida Entomologist
Title: The Florida entomologist
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Title: The Florida entomologist
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Subject: Entomology -- Periodicals   ( lcsh )
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periodical   ( marcgt )
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Additional Physical Form: Also issued in print.
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Language: In English; summaries in Spanish.
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 Related Items
Preceded by: Florida buggist (Online)

Table of Contents
    Effect of male accessory gland extracts on female oviposition and sexual receptivity of the Caribbean fruit fly (Diptera: Tephritidae)
        Page 415
        Page 416
        Page 417
        Page 418
        Page 419
        Page 420
    Life history of the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Dryophtoridae), in Southern Japan
        Page 421
        Page 422
        Page 423
        Page 424
        Page 425
    Rise and fall of red oak borer (Coleoptera: Cerambycidae) in the Ozark Mountains of Arkansas, USA.
        Page 426
        Page 427
        Page 428
        Page 429
        Page 430
        Page 431
        Page 432
        Page 433
    First North American records of the east Palearctic seed beetle Bruchidius terrenus (Coleoptera: Chrysomelidae: Bruchinae), a specialist on mimosa (Albizia julibrissin, Fabaceae).
        Page 434
        Page 435
        Page 436
        Page 437
        Page 438
        Page 439
        Page 440
    Is host size an indicator of quality in the mass-reared parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae)?
        Page 441
        Page 442
        Page 443
        Page 444
        Page 445
        Page 446
        Page 447
        Page 448
        Page 449
    Releases, distribution and abundance of Gratiana boliviana (Coleoptera: Chrysomelidae), a biological control agent of tropical soda apple (Solanum viarum, Solanaceae) in Florida.
        Page 450
        Page 451
        Page 452
        Page 453
        Page 454
        Page 455
        Page 456
        Page 457
    Host specificity of Anthonomus elutus (Coleoptera: Curculionidae), a potential biological control agent of wetland nightshade (Solanaceae) in Florida.
        Page 458
        Page 459
        Page 460
        Page 461
        Page 462
        Page 463
        Page 464
        Page 465
        Page 466
        Page 467
        Page 468
        Page 469
    A new genus and new species of the subtribe Cicadina (Hemiptera: Cicadidae: Cicadini).
        Page 470
        Page 471
        Page 472
        Page 473
    Ant tending of Miami blue butterfly larvae (Lepidoptera: Lycaenidae): partner diversity and effects on larval performance.
        Page 474
        Page 475
        Page 476
        Page 477
        Page 478
        Page 479
        Page 480
        Page 481
        Page 482
    Semiochemically based monitoring of the invasion of the brown marmorated stink bug and unexpected attraction of the native green stink bug (Heteroptera: Pentatomidae) in Maryland.
        Page 483
        Page 484
        Page 485
        Page 486
        Page 487
        Page 488
        Page 489
        Page 490
        Page 491
        Page 491a
        Page 491b
    Development of Spalangia cameroni and Muscidifurax raptor (Hymenoptera: Pteromalidae) on live and freeze-killed house fly (Diptera: Muscidae) pupae.
        Page 492
        Page 493
        Page 494
        Page 495
        Page 496
    Parasitism of the brown citrus aphid in Dominica by Lysiphlebus testaceipes and Lipolexis oregmae (Hymenoptera: Aphidiinae).
        Page 497
        Page 498
        Page 499
    The whip scorpion, Mastigoproctus giganteus (Uropygi: Thelyphonidae), preys on the chemically defended Florida scrub millipede, Floridobolus penneri (Spirobolida: Floridobolidae)
        Page 500
        Page 501
        Page 502
    A new pest of rice in Missouri: range expansion of Triops longicaudatus (Crustacea: Notostraca: Triopsidae) into the Northern Mississippi River Alluvial Plains. (Scientific Notes)
        Page 503
        Page 504
        Page 505
    Length of multiple-funnel traps affects catches of some bark and wood boring beetles in a slash pine stand in northern Florida. (Scientific Notes)
        Page 506
        Page 507
    Egg parasitoids of Dalbulus maidis (Hemiptera: Cicadellidae) in Jalisco State, Mexico. (Scientific Notes)
        Page 508
        Page 509
        Page 510
    Effects of diet on development and survivorship of Narnia femorata nymphs (Hemiptera: Coreidae). (Scientific Notes)
        Page 511
        Page 512
    Oviposition of the neotropical brown stink bug Euschistus heros (Heteroptera: Pentatomidae) on artificial and on natural substrates. (Scientific Notes)
        Page 513
        Page 514
        Page 515
    A new record for Cicindela scabrosa (Coleoptera: Carabidae: Cicindelinae) from Hardee County, Florida. (Scientific Notes)
        Page 516
        Page 517
    Mitochondrial DNA markers in populations of Dacus punctatifrons (Diptera: Tephritidae). (Scientific Notes)
        Page 518
        Page 519
        Page 520
    Synonymy of Aleochara (Aleochara) claviger and A. niponensis (Coleoptera: Staphylinidae: Aleocharinae), and first record of A. claviger in Korea.(Scientific Notes)
        Page 521
        Page 522
        Page 523
    Book reviews
        Page 524
        Page 525
        Page 526
        Page 527
        Page 528
        Page 529
        Page 530
    Errata
        Page 531
    Back Matter
        Page 532
        Page 533
Full Text



Lentz et al.: Effect of Male Accessory Glands on Caribbean Fruit Fly Females 415



EFFECT OF MALE ACCESSORY GLAND EXTRACTS ON FEMALE
OVIPOSITION AND SEXUAL RECEPTIVITY OF THE CARIBBEAN FRUIT FLY
(DIPTERA: TEPHRITIDAE)

ANTHONY J. LENTZ', JAMES R. MILLER2, JOSEPH L. SPENCER AND JAMES E. KELLER2
'Department of Biology, Bellarmine University, Louisville, KY 40205

2Department of Entomology, Michigan State University, East Lansing, MI 48824

'Illinois Natural History Survey, University of Illinois at Urbana-Champaign, Champaign, IL 61820

ABSTRACT

Anastrepha suspense (Loew) male accessory glands do not appear to possess a sex peptide,
a factor that induces oviposition or inhibits mating receptivity. Injection of accessory gland
extracts from laboratory-colony males into virgin females stimulated daily deposition of only
4 eggs per female, comparable to injections of whole reproductive tract extract (5 eggs per fe-
male) and negative controls (4 to 5 eggs per female). Mated females laid significantly more
(10 eggs per female per d). Studies of wild-caught males and females yielded the same infor-
mation: injection of an accessory gland/testes extract or saline both elicited 8 eggs per female
per d whereas normally mated females laid 16 eggs per female per d. Female receptivity to
mating following injection of accessory gland or whole reproductive tract extracts was com-
parable to the negative control group, in which 67% to 83% of treated females remated and
63% to 89% of control females remated. In contrast, only 43% of once-mated (positive con-
trol) females remated when placed with males. Once-mated females also took significantly
longer to remate after exposure to males (359 min) than females from both treatment (61 to
169 min) and negative control groups (76 to 122 min). The duration of mating was similar
among all groups (24 to 37 min). These results suggest that oviposition and receptivity in-
hibition in A. suspense are not mediated by male-derived humoral factors.

Key Words: sex peptide, mating inhibition, female mate choice, oviposition

RESUME

Las glandulas accesorias de machos de Anastrepha suspense (Loew) no aparece poseer un
p6ptido sexual que induce la oviposici6n o inhibe la receptividad del apareamiento. La inyec-
ci6n de extractos de la glandula accesorias de machos criados en el laboratorio a hembras
virgenes estimulo la deposici6n diaria de solamente 4 huevos por hembra, comparable a in-
yecciones de un extract del tracto reproductive complete (5 huevos por hembra) y el grupo
de control negative (4 a 5 huevos por hembra). Hembras apareadas pusieron significativa-
mente mas huevos (10 por hembra por dia). Estudios sobre machos salvajes capturados y
hembras rindieron la misma informaci6n: la inyecci6n de un extract de la glandula/testes
accesoria o salina result en 8 huevos por hembra virgen por dia, mientras que las hembras
apareadas normalmente pusieron 16 huevos por hembra por dia. La receptividad de las
hembras hacia el apareamiento despu6s de la inyecci6n de extractos de la glandula accesoria
o del complete tracto reproductive fue comparable con el grupo de control negative: 67% a
83% de las hembras tratadas reaparearon versus 63% a 89% de las hembras del grupo con-
trol que reaparearon. En contrast, solamente 43% de la hembras apareadas solo una vez
(grupo control positive) reaparearon cuando fueron puestas juntas con los machos. Tambi6n,
hembras apareadas solo una vez tomaron significativamente mas tiempo para reaparearse
despu6s de ser expuestas a los machos (359 min.) que las hembras en ambos tratamientos
(61 a 169 minutes) y el grupo de control negative (76 a 122 min.). La duraci6n del aparea-
miento fue similar entire todos los grupos (24 a 37 min.) Estos resultados sugerieron que la
oviposici6n y la inhibici6n de la receptividad enA. suspense no son mediadas por factors hu-
morales derivados del macho.


The Caribbean fruit fly, Anastrepha suspense try (FDACS-DPI) has developed fly-free zones in
(Loew), is an important quarantine pest of citrus order to ship grapefruit and other fruits to Japan
in Florida. Although its impact on citrus produc- without the use of quarantine treatments. Re-
tion is minor, its presence results in export re- leases of sterile male Caribbean fruit flies that
strictions. The Florida Department of Agriculture would compete for wild females have been pro-
and Consumer Services, Division of Plant Indus- posed as a means of suppressing flies in limited







Florida Entomologist 92(3)


areas to support these zones. The most important
aspect of Sterile Insect Technique (SIT) is the
ability of sterile flies to mate with wild fertile fe-
males and produce a reasonably long female re-
fractory period before remating. Studies on the re-
productive behavior and physiology ofA. suspense
have described the role and use of male phero-
mones (Nation 1972, 1990), lek formation on host
plant leaves (Burk 1983), acoustic courtship sig-
nals (Sivinski et al. 1984; Webb et al. 1984) and
copulation (Nation 1972; Mazomenos et al. 1977).
There have been no studies investigatingA. sus-
pensa for the presence of sex peptide, a male ac-
cessory gland factor that induces oviposition and/
or inhibits sexual receptivity in some Diptera
such as Aedes aegypti (Fuchs et al. 1969), Droso-
phila (Chen et al. 1988), andDelia antiqua (Spen-
cer et al. 1992, 1995), as well as other insects
(Gillott 1988).
Anastrepha suspense males have paired testes
connected to a common duct through the vasa def-
erentia (Dodson 1978). The sex accessory glands
originate where the vasa deferentia converge and
consist of five or six short, tubular glands. Fe-
males typically have three spermathecae and
polytrophic ovarioles (Dodson 1978; Fritz &
Turner 2002); ovaries may contain more than one
flush of mature eggs at a time.
Males court females, forming leks on host
plant leaves in order to mate with females before
dusk (Burk 1983). Stimuli used to attract females
to the leks include pheromones deposited on the
underside of leaves, acoustic signals produced by
wing fanning, and visual cues (Nation 1972; Siv-
inski et al. 1984; Webb et al. 1984). When mating,
flies typically remain in copula for 30 to 37 min
(Nation 1972; Mazomenos et al. 1977), transfer-
ring male-derived substances in addition to
sperm (Sivinski & Smittle, 1987). Oviposition oc-
curs on host plant fruit the next morning follow-
ing copulation (Burk 1983).
Oviposition by A. suspense is strongly influ-
enced by both environmental cues (Landolt & Siv-
inski 1992) and the quality of the oviposition site
(Sivinski & Heath 1988). Mated females with ac-
cess to artificial oviposition devices consisting of a
rolled cloth impregnated with beeswax lay signif-
icantly more eggs per female than those without
access. In addition, the physical features of the
oviposition substrate such as shape and color are
more important oviposition stimulants than
chemical cues for polyphagous species likeA. sus-
pensa (Szentesi et al. 1979; Greany & Szentesi
1979).
Mated females with oviposition devices are
more likely to remate during weekly opportuni-
ties than those without oviposition devices (Sivin-
ski & Heath 1988). Mated females with no oppor-
tunity to oviposit are more likely to mate only
once. In another study (Mazomenos et al. 1977),
none of the once-mated, wild-type females re-


mated within 5 d, even though approximately
50% of the females were not fertilized. By con-
trast Rhagoletis pomonella (Walsh) females are
receptive to nearby males immediately following
copulation, and R. suavis (Loew) may alternate
egg laying and copulation with several males be-
fore leaving an oviposition site (reviewed by
Christenson & Foote 1960).
In support of the sterile insect release pro-
gram, we studied the effect of male accessory
gland secretions on oviposition and mating recep-
tivity in virgin females. Although increasing
doses of gamma irradiation have been correlated
with decreased mating success of sterilized males
(Hooper 1972; Calkins et al. 1988), the means by
which it occurs is not known. Because gamma ir-
radiation of male pupae may conceivably alter
their physiology, including the biosynthesis of
semiochemicals, we studied both laboratory-
strain and wild-caught flies to explore whetherA.
suspense uses a sex peptide.

MATERIALS AND METHODS

Anastrepha suspense were obtained from ei-
ther the mass-rearing facilities of the USDA lab-
oratories at Gainesville, Florida, or collected as
larvae in fruit from central Florida. Larvae-in-
fested fruit were returned to the lab and emerging
larvae were placed on vermiculite to pupate. Fe-
males and males were sexed as newly closed
adults, placed into separate cages at equal den-
sity and provided with an agar-based sugar,
yeast, and wheat germ diet (1:1:1). Flies were
maintained at ambient temperature and humid-
ity under artificial lighting (L:D 14:10). Because
males and females of laboratory strain ofA. sus-
pensa become sexually mature at 10 to 11 d old
(Nation 1972), flies were held at least 14 d after
eclosion before being used in an experiment.

Extract Preparation and Injection

Reproductive tissues consisting of accessory
glands, accessory gland plus testes, or whole re-
productive tracts were removed from sexually
mature (14- to 40-d-old) virgin males (freshly
killed by freezing) and placed into a drop of cold
saline (Spencer et al. 1992) in a dissecting dish.
Each tissue was quickly transferred into a saline-
filled microcentrifuge vial kept on ice. Tissues
were processed into extracts by homogenizing
them for 30 to 60 s in a bath sonicator (Heat Sys-
tems Ultrasonic), then centrifuging at 4500g for
10 min, and removal of the supernatant. The con-
centration of the supernatant (extract) was ad-
justed so that 0.5 mL represented 1 male gland
equivalent. Extracts either were kept on ice and
used within 2 h for injection or frozen before use.
To administer the extract, sexually mature vir-
gin females were anesthetized with nitrogen and


September 2009







Lentz et al.: Effect of Male Accessory Glands on Caribbean Fruit Fly Females 417


1 male equivalent of extract was injected into the
hemocoel as in earlier studies on Diptera, deep to
the lateral mesothorax through a pleuron. Injec-
tion needles consisted of 3-mm glass tubing
drawn into a very fine point (~40 pm at the tip).
Flies were restrained for injection on a Plexiglas@
plate by covering them with Parafilm. Treat-
ment groups not receiving injections also were
anesthetized. Treated flies were placed individu-
ally into 7.5-cm diameter by 15-cm high alumi-
num screen cages.

Effect of Reproductive Tissue Extracts on Oviposition
and Mating Receptivity of Lab-Colony Flies

Experiments were conducted as randomized
block designs. Flies were randomly removed from
the holding cage 1 block at a time. Treatments
within blocks were assigned at random by draw-
ing numbers from a container and all flies within
a block were treated in the random order specified
before proceeding to the next block. Data were
statistically analyzed either with a SAS general
linear models program and Student-Newman-
Keul's multiple range test (SAS Institute, Cary,
NC) or with Fisher's protected least squares dif-
ference test (StatView 4.0, Abacus Concepts Inc.,
Berkeley, CA).
Flies from the FDACS rearing facility were
used for both oviposition and mating receptivity
experiments. An "experimental unit" consisted of
1 female per cage containing 1 oviposition sub-
strate, which was a 2.5-cm agar sphere sur-
rounded with Parafilm and suspended from the
top of the cage. Experiments included up to 7
treatments with 6 to 61 experimental units per
treatment. The control groups included a virgin
control (no injection), female inserted with a nee-
dle only, saline injected females, and mated con-
trols in which 3 males were placed with the fe-
male for 24 h, then removed. Extract-injected
treatment groups consisted of accessory gland ex-
tract injection of 1 male equivalent, accessory
gland extract injection of 5 male equivalents, and
whole reproductive tract extract injection of 1
male equivalent.
Following treatment, approximately half of the
females from each group were monitored for ovi-
position and half for mating receptivity. For the
oviposition group, females were allowed to ovi-
posit for up to 23 d on oviposition spheres re-
placed every few d as egg counts were performed;
short term effects were not examined. For the
mating-receptivity group, 3 male flies were placed
with each treated female on the d following treat-
ment and flies were observed for up to 12 h to de-
termine the time until onset of mating and the du-
ration of mating. At the end of 12 h, males were
removed, females were divided according to
whether or not they had mated, and egg output
was measured as described for the oviposition


group. For each treatment, the percentage of fe-
males mating was determined. Females not mat-
ing were included in the data set for the oviposi-
tion assay.

Effect of Male Reproductive Tract Extract Prepared
from Wild-Caught Flies

Since mass rearing radically alters selection
pressure and may cause changes in mating ability
or mating effectiveness in some Diptera (Bush et
al. 1976), wild Caribbean fruit flies were tested to
determine whether they somehow were different
than lab colony flies. Extracts were prepared from
the accessory gland/testes complex of wildA. sus-
pensa males as described earlier for laboratory
males except that they were collected into saline
containing a mixture of protease inhibitors
(0.0004% w/v each of antipain, leupeptin, pepsta-
tin A, chymostatin). These were added to prevent
the possibility of rapid enzymatic degradation of
extracted proteins. Tissues were sonicated for ~25
s, centrifuged at 4500g for 5 min, and the super-
natant stored in a freezer overnight. There were
25 experimental units per treatment consisting of
3 wild-caught females and 1 oviposition substrate
per experimental unit. In this test, females were
given 1.5-cm diameter blue, cerasin wax domes
for oviposition instead of agar balls. Females were
injected with either a saline control or accessory
gland/tests extract of 1 male equivalent. For the
mated control, males were added to the cage at a
density of 4 males per female for 24 h. Females
were allowed to oviposit for up to 10 d and ovipo-
sition spheres were replaced as counts were per-
formed. Data collected were egg counts per live fe-
male per cage.

RESULTS

Oviposition and Mating Receptivity of Lab-Colony Flies

Injections of male accessory gland or whole re-
productive tract extracts did not increase oviposi-
tion in lab colony, virgin females above that of un-
mated flies (Table 1). Females from the negative
control treatments and the extract treatments
laid a cumulative average of 4.2 eggs per d, signif-
icantly fewer than the average 9.5 eggs per fe-
male laid by normally mated females (P < 0.05,
Student-Newman-Keul's).
Similarly, when virgin females receiving injec-
tions of male reproductive tissue extract were ex-
posed to males, they mated like unadulterated
virgins. Treated females began mating an aver-
age of 61 to 169 min after males were introduced
into the cage, comparable to virgin controls that
began mating 76 to 122 min following introduc-
tion. Mated females waited 6 h before mating
again (Table 2). In addition, 78% of all treated vir-
gin females mated during the 12-h bioassay in







Florida Entomologist 92(3)


September 2009


TABLE 1. OVIPOSITION BY LAB COLONY ANASTREPHA SUSPENSE INJECTED WITH AQUEOUS EXTRACT OF MALE REPRO-
DUCTIVE TISSUE (X S.D.)

Treatment n Eggs/female/d'

Controls 4.8 4.1
Virgin 8 4.3 3.5
Needle insertion only 10 3.5 + 4.6
Saline 10

Male extracts
Accessory gland 13 3.8 2.6
Whole tract 9 5.2 + 2.2

Grand mean unmated treatment females 50 4.2 3.4*
Mated females 61 9.5 6.8*

'The 2 means indicated with an asterisk are significantly different from one another at P < 0.05 by SNK.


TABLE 2. MATING BY LAB COLONY ANASTREPHA SUSPENSE INJECTED WITH AQUEOUS EXTRACT OF MALE REPRODUC-
TIVE TISSUE (X S.D.)

Delay until mating Duration of mating
Treatment n % Mating (min)' (min)1

Controls
Virgin 9 89% 76 48a 25 10a
Needle insertion only 7 86% 114 102a 37 7a
Saline 8 63% 122 69a 24 7a

Male extracts
Accessory gland 1 ME 9 67% 104 66a 28 19a
Accessory gland 5 MD 6 83% 169 100a 35 20a
Whole tract 1 ME 12 83% 61 72a 27 11a

Grand mean unmated treatment females 51 78% 104 82a 29 13a
Mated females 7 43% 359 124b 30 26a

'Treatment means with the same letter are not significantly different from one another within columns atP < 0.05 by SNK.
'ME = male equivalent.


contrast with only 43% of previously mated fe-
males. All groups averaged about 30 min mating
time suggesting that once mating began, previous
treatment had no effect on duration of copulation.

Oviposition by Wild-Caught Flies

Oviposition by wild-caught virgin females in-
jected with accessory gland/testes extract was vir-
tually identical to that of saline-injected virgins,
8.4 and 8.3 eggs/female after 10 d (Fig. 1). Nor-
mally mated wild females laid an average of 16
eggs/female over the same period. Egg deposition
was higher among feral flies than mass-reared
flies, perhaps because blue wax domes were more
amenable to oviposition than agar balls-both
wild-caught and laboratory mated females laid
twice as many eggs as unmated females regard-
less of treatment.


DISCUSSION

Inhibition of mating receptivity and stimula-
tion of oviposition in A. suspense females were
not mediated by transfer of male accessory
gland factors to the female. Sexually mature
laboratory colony females injected with extracts
of male reproductive tissues exhibited no ob-
servable change in egg output or mating pro-
pensity. They behaved as virgins. Protease in-
hibitors used to prevent or minimize digestion
of extracted proteins did not change the results.
Experiments with wild flies yielded virtually
identical results with respect to oviposition in-
duction, suggesting that laboratory selection
had not removed a wild-type sex peptide. It is
possible that delivery of extract into the hemo-
coel, rather than the spermathecae, failed to
stimulate females, despite eliciting positive re-







Lentz et al.: Effect of Male Accessory Glands on Caribbean Fruit Fly Females 419


Cumulate eggs/femalelcage @ 10 days


Saline


Ac geanditestes


Mated


Fig. 1. Oviposition by wild-caught A. suspense fol-
lowing injection with accessory gland/testes extract (X
S.D.). Treated females received 1 male equivalent of ex-
tract and the negative control was saline-injected vir-
gins. For the positive control, males were placed with
females at a density of 4 males per female for 24 h then
removed (n = 25). Treatment means with the same let-
ter were not significantly different (P < 0.05) using
Fisher's protected least squares difference test.


sponses in other Diptera (Fuchs et al. 1969;
Chen et al. 1988; Spencer et al. 1992, 1995).
In mating competition experiments, male A.
suspense rendered sterile by a low dose of gamma
radiation (3 krad) were equally competitive with
normal males when placed with normal females
as measured by egg output (Calkins et al. 1988).
Therefore, sterile male flies are capable of induc-
ing oviposition behavior in females without trans-
ferring viable sperm. Studies on the apple maggot
fly, R. pomonella, with normal and irradiated
males showed similar results (Myers et al. 1976).
Remating in the melon fly, Bactrocera cucurbitae
(Coquillett), was inhibited at the same rate in fe-
males mated to either normal or sterile males as
long as copulation was not terminated prema-
turely (Kuba & Ito 1993). These studies suggest
there is a factors) other than normal sperm that
causes female tephritids to change from virgin to
mated behavior, but we could find no evidence of a
sex peptide extractable by methods amenable to
proteins.
A humoral factor produced by males and
transferred to females may change female be-
havior but in ways not measured by this study.
Jang (1995) demonstrated that female olfactory-
mediated behavior in the Mediterranean fruit
fly, Ceratitis capitata, was altered when virgin
females were injected with 0.2 equivalent of
male accessory gland fluid. In a wind tunnel as-
say, injected, unmated females preferred host
fruit odor (representing post-mating oviposition
site selection) over male pheromone odor, similar
to mated females. Virgin or saline-injected virgin
females preferred male pheromone odor over
fruit odor.
Perhaps mating receptivity and oviposition
are controlled more by some physical aspect of
copulation than by a chemical factor, or by a
chemical factor not originating in the reproduc-


tive tract. Structurally, the spermathecae, asso-
ciated ducts, and the ventral receptacle are in-
nervated with muscle fibers surrounding the
spermathecal capsule, indicating that females
may regulate sperm location and use (Fritz 2002;
Fritz & Turner 2002). Pereira et al. (2006) sug-
gested that exposure to volatile male sex phero-
mones accelerates ovarian development in virgin
A. suspense females as preparation for mating.
In Anastrepha striata (Schiner), females mated
to virgin males lived longer than those mated to
sexually experienced males, presumably because
of a transferred substance (Perez-Staples &
Aluja 2004). However, because A. striata males
provide both orally derived fluid (trophallaxis)
and accessory gland secretions to females during
courtship, additional studies would be needed to
establish the origin of the longevity substance.
Although extensive trophallaxis does not occur
in A. suspense, males lick the female's head dur-
ing copulation, suggesting another route by
which females may acquire semiochemicals. In
the tsetse fly, Glossina morsitans Westwood, ovu-
lation appears to be induced by the physical
stimulation of mating but not by the presence of
a spermatophore in the uterus, or by chemical
factors originating from the accessory gland or
testes (Saunders & Dodd 1972; Gillott & Langley
1981). Inhibition of receptivity is regulated dif-
ferently, being dependent on both chemical and
physical stimuli (Gillott & Langley 1981).
Perhaps the mating system of A. suspense
and other lekking tephritid species is only pe-
ripherally connected to sex peptide communica-
tion or manipulation. In mating systems where
pre-copulatory female choice is highly devel-
oped, as is apparently the case in A. suspense
(e.g., Burk 1983), the arena for post-copulatory
manipulation by either sex may be limited.
That is, males may not benefit by investing in
manipulative chemicals if females are unlikely
to remate until a male's sperm are depleted,
and choosey females may not require the means
to manipulate sperm from multiple males if
they are likely to mate carefully and infre-
quently. If this is the case, one might predict a
greater role for sex peptides and other forms of
post-copulatory sexual conflict in resource-
based mating systems with a high degree of
polyandry, such as occurs in many Rhagoletis
species.

ACKNOWLEDGMENTS

We thank Professor Cedric Gillott for critically re-
viewing an earlier draft of the manuscript and 2 anony-
mous reviewers who made a number of insightful
comments that significantly improved the paper. We
thank Dr. Carrol Calkins for originally providing access
to caribflies, and Richard Guy for his technical assis-
tance in conducting the assays. This work was funded
by a USDA noncompetitive grant to JRM and AJL.











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Florida Entomologist 92(3)







Abe et al.: Life History of Red Palm Weevil


LIFE HISTORY OF THE RED PALM WEEVIL, RHYNCHOPHORUS
FERRUGINEUS (COLEOPTERA: DRYOPHTORIDAE), IN SOUTHERN JAPAN


FUKIKO ABE1, KUNIHIKO HATA2 AND KOICHI SONE2
1United Graduate School of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan

2Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan

ABSTRACT

We surveyed the life history of the red palm weevil, Rhynchophorus ferrugineus (Olivier)
(Coleoptera: Dryophthoridae), in southern Japan, including seasonal changes in the flight
activity of adults and composition of R. ferrugineus in different developmental stages in a to-
tal of 17 infested Phoenix canariensis trees which were cut down in 2003-2005. The flight of
adults began in Mar, showed some peaks in summer and autumn, and ceased in mid-Dec.
Various stages of individuals inhabited infested P. canariensis trees throughout the year.
The composition of individuals at different stages in late fall was dependent on the resource
(white intact tissue) availability in P. canariensis trees. In P. canariensis trees where consid-
erable resource remained, all stages of larvae, pupae, and adults were found, whereas in
palm trees with no resource, few young- and medium-stage larvae were observed. The tem-
perature in the infested part of a palm trunk was 30 C or higher even in winter. From these
results, we view the life history of R. ferrugineus in southern Japan as follows: Adults
emerge from host trees in spring and continue to attack host trees until late fall. Rhyn-
chophorus ferrugineus grows even in winter if intact tissue remains at the peripheral part
of trunks, and there may be 3 or 4 generations per year. A cold winter probably does not have
any negative effects on successful colonization of R. ferrugineus in Japan.

Key Words: red palm weevil, Rhynchophorus ferrugineus, southern Japan, developmental
stage structure

RESUME

Estudiamos el ciclo de vida del picudo rojo de la palma, Rhynchophorus ferrugineus (Olivier)
(Coleoptera: Dryophthoridae), en el sur del Jap6n, incluyendo los cambios estacionales en la
actividad de vuelo de los adults y la composici6n de R. ferrugineus en diferentes etapas de
desarrollo en un total de 17 arboles infestados de Phoenix canariensis que fueron tumbados
en los anos 2003-2005. Los adults empezaron a volar en marzo, mostraron un incremento
en verano y otoio, y terminaron en el medio de diciembre. Varias estadios de picudo rojo de
la palma habitaron los arboles infestados de P. canariensis por todo el ano. La composici6n
de los individuos en diferentes etapas de la ultima parte del otono fue dependiente de la dis-
ponibilidad de material (tejido blanco intacto) en los arboles de P. canariensis. En los arboles
de P. canariensis donde quedo una cantidad considerable de tejido, todos los estadios de
larva, pupa y adulto fueron encontrados, mientras en las palmeras sin material, pocos esta-
dios j6venes y medios de larvas fueron observados. La temperature en la part infestada del
tronco de la palmer fue 30 C o mas alta aun durante el invierno. De estos resultados, vemos
el ciclo de vida de R. ferrugineus en el sur de Jap6n como la siguiente manera: los adults sa-
len de los arboles hospederos en la primavera y continuan su ataque en los arboles hospede-
ros hasta el fin de otofio. Rhynchophorus ferrugineus crece aun en invierno si hay tejido
intacto en la parte perif6rica de los troncos, y pueden haber 3 o 4 generaciones por aio. Pro-
bablemente un invierno frio no tiene un efecto negative sobre el 6xito en la colonizaci6n de
R. ferrugineus en Jap6n.


The red palm weevil, Rhynchophorus ferrug- palm (P. canariensis), extended to southern Japan
ineus (Olivier) (Coleoptera: Dryophthoridae), is in 1998 and thereafter (Usui et al. 2006). The
widely distributed in southern Asia and Melane- adults of R. ferrugineus are attracted to and de-
sia, and attacks various palm species such as posit eggs in palm sheaths and stems. Larvae de-
Phoenix sylvestris, Cocos nusifera and Metroxylon velop primarily in the crown region and damage a
sago in India, Pakistan, Sri Lanka, Myanmar, In- growing point of palm trees located at the top of a
donesia, the Philippines, and the Gulf states (Ra- trunk. Infestations are problematic because R.
halkar et al. 1985; Murphy & Biscoe 1999). After ferrugineus is not detectable until it has caused
being first recorded in Okinawa in 1975, damage permanent damage (Rahalker et al. 1985). To con-
to palms, almost exclusively Canary Island date trol infestation of palm trees by R. ferrugineus, in-







Florida Entomologist 92(3)


fested palm trees are cut down and crushed or
burned in Japan. However, these mechanical
methods are laborious and costly. Thus, cheaper
alternative methods are desired. Injection of in-
secticides into palm trunks and the use of natural
enemies such as entomopathogenic nematodes
have been tested (Shaseldean 2004; liboshi et al.
2004; Toshima 2006; Yoshimoto 2006).
Knowledge of the life history of R. ferrugineus
would allow us to determine the optimal time to in-
tervene with insecticides and entomopathogenic
nematodes for more effective control. To date, adult
longevity, fecundity, and larval development of R.
ferrugineus have been studied only under labora-
tory conditions (Rahalker et al. 1985). In the field,
oviposition behavior has been observed in Indone-
sia (Kalshoven 1981), and the seasonal patterns of
flight activity have been estimated from adult cap-
tures with pheromone traps in Kagoshima and
Miyazaki Prefecture, southern Japan (Aman et al.
2000; Sato & Irei 2003). However, studies in the de-
velopment and the seasonal changes in the compo-
sition of R. ferrugineus inhabiting P canariensis
trees have not been conducted.
To this end, we examined the composition of in-
habiting R. ferrugineus individuals at different de-
velopmental stages in infested P canariensis trees
cut down in different months of the year. Addition-
ally, we measured the temperature inside the trunk
of infested P canariensis trees during winter to
evaluate the thermal conditions of R. ferrugineus.
We also reaffirmed the flight activity pattern re-
ported by Aman et al. (2000) and Sato & Irei (2003)
by collecting adults and observing adult flight in the
field. Using this information, we inferred the life-
history of R. ferrugineus in southern Japan.

MATERIALS AND METHODS

Seasonal Patterns of Flight Activity of Adults

The seasonal patterns of flight activity of
adults were surveyed in the Korimoto Campus of
Kagoshima University (3134'N, 13032'E), Ka-
goshima, southern Japan, where about 30 P ca-
nariensis were planted in a small area (<1 ha),
and most were dead due to the infestation by R.
ferrugineus since the first record in 2002. On Jun
1, 2004, we set a collision trap (Sankei-shiki Kon-
chu Yuinki, Sankei Chemical Co., Kagoshima) us-
ing pheromones (Biobset Belugium) as lure on the
rooftop of the building of the University (about 15
m above the ground) near the planted P canarien-
sis trees. Checks on the trap were carried out
daily and lure replenished at 2-week to 1-month
intervals until Mar 31, 2005.
Observation of flying adults and collection of
fresh adult carcasses in the Korimoto Campus of
Kagoshima University, as adults flying into our
laboratory, were used to estimate the seasonal
pattern of flight activities of adults.


Composition of R. ferrugineus at Different Developmen-
tal Stages in P. Canariensis Trees

We examined the composition ofR. ferrugineus
at different developmental stages in 17 infestedP.
canariensis trees in Kagoshima that were cut
down at different seasons from Nov 22, 2003 to
Oct 31, 2005. In 11 of the 17 P canariensis trees
which were cut down on Nov 22 in 2003, Jan 10,
Feb 25, Apr 9, May 29, Aug 24, Nov 4, 6, 10, 19 in
2004 and May 28 in 2005, the peripheral part of
the trunks and the base of petioles had been de-
stroyed by the larvae and intact tissue was al-
ready discolored white to brown when they were
cut down. In the 6 P canariensis trees cut down
on Dec 28, 2004 and Oct 9, 20, 31, 2005, infesta-
tion had not so progressed that leaves were only
partly discolored and considerable white intact
tissue remained at the crown part of the trunks
and the base of petioles. We collected eggs, larvae,
pupae, and adults from infested part of the 17 P.
canariensis trees, and recorded their numbers.
We tentatively categorized larvae into 3 stages
based on head capsule widths of 855 larvae;
young (<2.8 mm), medium (2.8-6.0 mm), and ma-
ture (>6.0 mm) stages (Fig. 1). In order to reveal
the effects of resource (white intact tissue) avail-
ability on the composition of larvae of different
stages, the stage was determined for each larva
based on head-capsule width.
We collected adults from the P canariensis
trees cut down and reared them at room temper-
ature with fresh sliced apple to obtain eggs. We
placed each egg in a Petri dish (0 6 cm) with bot-
tom surface covered with a moistened filter paper.
Newly hatched larvae were placed individually in
separate Petri dishes (0 9 cm) and provided fresh
sliced apple as food. The molting of over 100 lar-
vae was checked daily.


young-stage medium-stage mature-stage


-I -


Z -"
3
-SD
*0

zI 4


JL.


S


0 08 l6 24 32 4 48 56 64 72 8 88 96
Head-capsule width (mm)
Fig. 1. Frequency distribution of head-capsule width
of the larvae of Rhynchophorus ferrugineus.


September 2009


r -


I







Abe et al.: Life History of Red Palm Weevil


Temperature Inside the Trunk

Because R. ferrugineus was originally distrib-
uted in tropical and subtropical areas, the estab-
lishment of invading population and the develop-
ment of R. ferrugineus were thought to be im-
peded by a low temperature during winter in Ja-
pan. Thus, using an alcohol-etched stem
thermometer, we measured the temperature in
the infested part of the trunk of P. canariensis
trees on Nov 9, 2004, Oct 9, Oct 20, Oct 20, 2005,
and Jan 5, 2007, to evaluate the thermal condi-
tion of R. ferrugineus during winter.

RESULTS AND DISCUSSION

Seasonal Patterns of Flight Activity of Adults

From Jun 14,2004 to Dec 12,2004, a total of 22
adults (136, 99) were captured by the trap. The
number of captured adults was greatest (6c, 3 )
in Aug. Five adults (26, 3 Y) flew into the labora-
tory in Jun and Jul. We observed the adults flying
around the trap on 4 occasions in Jul and Aug,
and twice in Sep (Table 1). In addition, we col-
lected a fresh carcass on the Korimoto Campus of
Kagoshima University on Mar 3, 2005. This col-
lection of a fresh carcass indicates that this weevil
emerged from a host tree and might fly at the be-
ginning of Mar. Sato & Irei (2003) caught R. fer-
rugineus adults from Mar 10 to Dec 10 in Ka-
goshima with peaks in Jun and Sep to Oct. In
Miyazaki Prefecture, adjacent to Kagoshima Pre-
fecture, Aman et al. (2000) caught adults from
early May to late Nov with 3 peaks in mid-Jun,
late Jul, and early Sep. Taking together the re-
sults of the 3 studies, we can deduce that R. fer-
rugineus adults begin to emerge from their host
plant in Mar and their flight lasts until mid-Dec,


TABLE 1. CAPTURES AND FLIGHT OBSERVATIONS OF
R. FERRUGINEUS ADULTS.

The number of adults
Flight
captured flew into the observation
Month by a trap laboratory date

Jan -
Feb -
Mar -
Apr -
May -
Jun 16, 19 1d, 29 4
Jul 46 16, 12 -
Aug 66,39 4
Sep 16,19 2
Oct -
Nov 16,3 -
Dec 1 -


with some peaks occurring during summer and
fall in southern Japan. It is also likely that wee-
vils remain stationary within P. canariensis trees
in winter.

Determination of the Number of Instars

The reared larvae molted a maximum of 12
times before pupation. Vaido & Bigornia (1949)
reported that R. ferrugineus molted 9 times be-
fore pupation. These results indicate that R. fer-
rugineus has 10 to 13 instars.

Composition ofR. ferrugineus at Different Developmen-
tal Stages in P. canariensis Trunks and Life History

We collected 6 to 213 R. ferrugineus individu-
als from the 11 P canariensis trees with no white
intact tissue. In these trees, pre-pupae, pupae,
and adults composed 42% or more of the popula-
tion. Adults composed all and 95% of individuals
in the palm trees felled on Feb 25,2004 and Apr 9,
2004 respectively, and about half of the adults
were in cocoons. The adult-biased composition
was also recorded in the P canariensis tree cut
down on May 28, 2005, where most adults were
still in cocoons. However, larvae and pupae pre-
dominated in the others P canariensis trees (Ta-
ble 2).
We collected 36 to 305 individuals from the 6 P.
canariensis trees whose tissue was destroyed only
partly. Larvae composed 67% or more of the indi-
viduals in them (Table 3), and the relative fre-
quency of larvae was higher than that in the 11P.
canariensis trees with no white intact tissue (Ta-
ble 2) (Mann-Whitney U-test; P < 0.05).
The composition of larvae of the 3 stages dif-
fered with resource (white intact tissue) availabil-
ity in infested P canariensis trees. In P. canarien-
sis trees there remained no white intact tissue at
the peripheral part of the trunk and the base of
petioles, all or most of the larvae (more than 90%)
were mature. In the 6 P canariensis trees where
the peripheral part and petioles were destroyed
partly, the larval composition varied. The larval
population consisted of 0-20% of young-, 6-60% of
medium-, and 20-94% of mature-stage of larvae
(Table 4). In P. canariensis trees cut down on Dec
28, 2004, Oct 9, 2005, and Oct 31, 2005, young-
and medium-stage larvae comprised 80, 33, and
43% of the larval population, respectively. These
results suggest that the composition of individu-
als at different developmental stages in infested
trees was dependent on resource availability in
trunks and petioles. A low rate of young- and me-
dium-stage larvae in trees with no resource sug-
gest the possibility that young- and medium-
stage larvae starved to death from the lack of sus-
tenance in the trees with no resource, leaving only
the stronger, more adaptable mature-stage lar-
vae, pupa, and adults.







Florida Entomologist 92(3)


TABLE 2. COMPOSITION OF RHYNCHOPHORUS FERRUGINEUS INDIVIDUALS AT DIFFERENT STAGES IN PALM TREES WITH
NO WHITE INTACT TISSUE.

Relative frequency (%)
Total
Year Date Larvae Prepupa Pupae in cocoon Adults in cocoon Adults weevils

2003 Nov22 14 12 60 9 5 58
2004 Jan 20 55 17 4 4 21 102
Feb 25 0 0 0 50 50 6
Apr9 0 5 0 43 52 21
May 29 56 24 3 0 18 34
Aug24 52 13 17 9 9 23
Nov4 23 8 34 19 16 213
Nov6 22 12 39 20 7 95
Nov 10 28 17 47 8 0 78
Nov 19 58 5 20 8 8 84
2005 May 28 0 0 5 82 13 39


TABLE 3. COMPOSITION OF RHYNCHOPHORUS FERRUGINEUS INDIVIDUALS AT DIFFERENT DEVELOPMENTAL STAGES IN
PALM TREES WHOSE TISSUE WAS PARTIALLY DESTROYED.

Relative frequency (%)
Total
Year Date Larvae Prepupa Pupae in cocoon Adults in cocoon Adults weevils

2004 Dec28 89 3 4 2 2 305
2005 Oct9 97 0 0 0 3 72
Oct 20 78 7 8 3 5 167
Oct 20 67 8 20 1 4 130
Oct 31 83 8 3 6 0 36
Oct 31 78 3 3 5 13 40


TABLE 4. COMPOSITION OF RHYNCHOPHORUS FERRUGINEUS LARVAE AT DIFFERENT DEVELOPMENTAL STAGES IN PALM
TREES WHOSE INTACT TISSUE WAS PARTIALLY DESTROYED.

Relative frequency (%)

Year Date Young-stage Medium-stage Mature-stage Total larvae

2004 Dec 28 20 60 20 272
2005 Oct9 1 32 67 69
Oct 20 3 10 87 129
Oct 20 1 12 87 87
Oct 31 10 33 57 30
Oct31 0 6 94 31


Based on information in the present study and
data on the capture of adults in Kagoshima and
Miyazaki (Sato & Irei 2003;Aman et al. 2000), the
life history of R. ferrugineus in southern Japan
can be summarized as follows. Adults emerge
from host plants and fly in the field from early
Mar to mid-Dec. Because females, if they feed, can
deposit a total of about 300 eggs almost daily for
at most 3 months (unpublished data), the oviposi-
tion season of R. ferrugineus covers a long period


from spring to late fall, and various stages of indi-
viduals are found in an infested P canariensis
tree throughout the year until the intact inner tis-
sue is completely destroyed and sustenance for
the R. ferrugineus runs out.
The monthly mean temperature of the winter
months (Dec to Feb) in southern Japan is about
10C with a minimum temperature falling below
0C (Japan Meteorological Agency). Yoshitake et al.
(2001) reported that the temperature 10 cm deep in-


September 2009







Abe et al.: Life History of Red Palm Weevil


side the trunk of healthy P canariensis tree agrees
with air temperature in winter. However, on Nov 9,
2004, Oct 9, Oct 20, Oct 20, 2005, and Jan 5, 2007,
the temperature in the infested part of the trunk of
P. canariensis trees was 37, 33-35, 30-40, 32 and
32C, respectively During the winter, R. ferrugineus
was feeding in a peripheral part of P canariensis
trees where the temperature was 30C or higher.
These results indicate that a low air temperature
during winter in Japan does not have lethal effects
on R. ferrugineus in host trees and that R. ferrug-
ineus can grow during winter if there remains suffi-
cient intact inner tissue in host trees. The lifetime
from egg to adult of R. ferrugineus reared at 29C
was reported as about 80 d by Rahalkar et al.
(1985), and flight activity showed peaks in summer
and fall (Sato & Irei 2003; Aman et al. 2000). Thus,
this weevil can have at least 3-4 generations a year
in southern Japan.

ACKNOWLEDGMENT

We thank Faculties ofAgriculture, Fisheries, Science
and Engineering of Kagoshima University for the gen-
erous use of Phoenix canarensis trees in this study. We
are grateful to Kagoshima Prefectural Forest Experi-
ment Station for the useful information on infestation of
P. canarensis in Kagoshima Prefecture.

REFERENCES CITED
AMAN, N., KUROGI, S., NAKAMURA, M., AND GOTO, H.
2000. Occurrence of the red palm weevil, Rhyn-
chophorus ferrugineus, in Miyazaki Prefecture. Ky-
ushu P1. Prot. Res. 46: 127-131 (in Japanese with
English summary).
IIBOSHI, H., TOKUHARA, AND T., TAMURA, M. 2004. Con-
trol of the red palm weevil, Rhynchophorus ferrug-
ineus, by Steinernema carpocapsae. Kyushu P1. Prot.
Res. 50: 126 (in Japanese).


KALSHOVEN, L. G. E. 1981. The pest of crops in Indone-
sia (Revised by P. A. Van der Laan), 701 pp.
MURPHY, S. T., AND BISCOE, B. R. 1999. The red palm
weevil as an alien invasive: biology and the pros-
pects for biological control as a component of IPM.
Biocontrol News and Information. 20: 35-46.
RAHALKAR, G. W., HAWALKAR, M. R., RANANAVARE, H.
D., TAMHANKAR, A. J., AND SHANTHRAM, S. 1985.
Rhynchophorus ferrugineus, pp. 279-286 In P. Singh
and R. F. Moore [eds.], Handbook of Insect Rearing,
Elsevier, New York.
SATO, Y., AND IREI, H. 2003. Pest insects of palms invad-
ed into Kyushu and Okinawa. Shinrin Kagaku. 38:
46-51 (in Japanese).
SHAMSELDEAN, M. M. 2004. Laboratory trials and field
applications of Egyptian and foreign entomopatho-
genic nematodes used against the red palm weevil,
Rhynchophorus ferrugineus Oliv. Intl. J. Nematol.
14(1): 44-55.
TOSHIMA, H. 2006. Control of the red palm weevil,
Rhynchophorus ferrugineus, by Steinernema carpoc-
apsae. Noyaku Gaido. 111: 12-17 (in Japanese).
UsuI, Y., MAKIHARA, H., AND GUSHIKEN, M. 2006. Ex-
pansion of infestation of red palm weevil, Rhyn-
chophorus ferrugineus (Oliv.) and its bibliography.
Forest Pests 55: 110-119 (in Japanese).
VAIDO, B. G., AND BEGORNIA, E. A. 1949. A biological
study of the. Asiatic Palm weevil, Rhynchophorus
ferrugineus. (Oliv.) (Curculionidae: Coleoptera). The
Philippine, Agr. 33: 1-27.
YOSHIMOTO, K. 2006. The distribution of Phoenix canar-
iensis (Chabaud) damaged by the red palm weevil,
Rhynchophorus ferrugineus (Olivier), and the pre-
ventive effect by trunk injection in Nagasaki Prefec-
ture. Kyushu J. Forest Res. 59: 201-203.
YOSHITAKE, H., MASAOKA, K., SATO, S., NAKAJIMA, A.,
KAMIYA, S., YUKAWA, J., AND KOJIMA, H. 2001. Oc-
currence of Rhynchophorus ferrugineus (Coleoptera:
Dryophthoridae) on Nokonoshima Island, southern
Japan and its possible invasion further north. Ky-
ushu P1. Prot. Res. 47: 145-150 (in Japanese with
English summary).







Florida Entomologist 92(3)


September 2009


RISE AND FALL OF RED OAK BORER (COLEOPTERA: CERAMBYCIDAE)
IN THE OZARK MOUNTAINS OF ARKANSAS, USA

JOHN J. RIGGINS'2, LARRY D. GALLIGAN' AND FRED M. STEPHEN'
'Department of Entomology, A-319 Agriculture Building, University of Arkansas, Fayetteville, Arkansas 72701

2Present Address: Box 9775, Department of Entomology and Plant Pathology, Mississippi State University,
Mississippi State, MS 39762-9775

ABSTRACT

Oak-hickory forests of the Arkansas Ozarks recently incurred extensive tree mortality due
in part to a native wood-boring beetle, the red oak borer Enaphalodes rufulus (Haldeman)
(Coleoptera: Cerambycidae). Historically, red oak borer has existed throughout southeast-
ern U.S. forests at relatively low population levels, but Arkansas infestation estimates in
2001 and 2003 reported much higher populations. Red oak borer has a two-year generation
with adult emergence occurring synchronously only in odd numbered years. We report here
results of whole-tree estimates of pre-emergent red oak borer population numbers from 7
stands in 2005 and 3 stands in 2003 and 2007 in the Ozark National Forest. Trees were
felled at each sampling site, cut into 0.5 m sections, split on site with hydraulic log splitters,
and a count of live red oak borers was recorded for each tree. In 2001 and 2003, red oak borer
population estimates indicated emerging populations much higher than any previously re-
ported. An exponential decrease during a single cohort between 2003 and 2005, and even
lower populations in 2007 suggest that red oak borer populations have returned to historic
densities.

Key Words: Enaphalodes rufulus, forest entomology, insect outbreak, population dynamics,
population sampling, Quercus rubra

RESUME

Los bosques de roble-nogal de las montanas Ozark del estado de Arkansas recientemente su-
fri6 una mortalidad de arboles extensive debido en parte a un escarabajo native barrenador
de madera, el barrenador de roble rojo Enaphalodes rufulus (Haldeman) (Coleoptera: Ce-
rambycidae). Hist6ricamente, el barrenador de roble rojo ha existido por todos los bosques
del sureste de los Estados Unidos con niveles de poblaci6n relativamente bajos, pero los in-
formes del nivel de infestaci6n en Arkansas en el 2001 y 2003 indica una poblaci6n much
mas alta. El barrenador de roble rojo cumple una generaci6n en dos anos con la emergencia
de los adults, ocurriendo sincr6nicamente durante los anos de numero impar. Reportamos
los resultados de los numerous aproximados de la poblaci6n pre-emergente del barrenador de
roble rojo de los arboles totales de 7 grupos de arboles en 2005 y 3 grupos de arboles en 2003
y 2007 del Bosque Nacional de Ozark. Los arboles en cada uno de los sitios de muestreo fue-
ron tumbados, cortados en secciones del tamaho 0.5 m, partidos con un rajador hidrdulico de
troncos y se noto el numero de barrenadores de roble rojo vivos encontrados por cada arbol.
En el 2001 y 2003, los aproximados de la poblaci6n del barrenador de roble rojo indicaron la
emergencia de poblaciones much mas altas que las reportadas anteriormente. Una dismi-
nuci6n exponencial durante un solo cohorte entire el 2003 y 2005, y aun poblaciones mas ba-
jas en el 2007 sugerieron que la poblaci6n del barrenador de roble rojo ha vuelto a las
densidades hist6ricas.


The Ozark highlands in northern Arkansas en-
compass approximately 6.5 million forested hect-
ares, most of which are made up of oak-hickory
forest type (Guldin et al. 1999). Overall, the red
oak group (Quercus: Section Lobatae), and prima-
rily northern red oak Q. rubra L., comprise ap-
proximately 25% of these forests (Guldin et al.
1999). Widespread oak decline was discovered in
the Arkansas, Missouri, and Oklahoma Ozarks in
1999 (Starkey et al. 2000). Resulting tree mortal-
ity was widespread, with significant impacts on
forest conditions and resources such as economy,


ecology, and aesthetics. During the period of peak
decline, at least one-third of mature northern red
oaks (Quercus rubra L.) died in stands through-
out the Ozarks (Guldin et al. 2006). Red oak borer,
Enaphalodes rufulus (Haldeman) (Coleoptera:
Cerambycidae), was implicated as the primary
contributing agent to this oak mortality event
(Stephen et al. 2001). Red oak borer is native to
eastern North America, and prior to the recent
outbreak in Arkansas, was only reported as a mi-
nor pest of oaks throughout its range (Hay 1974;
Donley & Acciavatti 1980; Galford 1983). Before







Riggins et al.: Red Oak Borer Outbreak


the current outbreak, stands exhibiting 1 emerg-
ing beetle per tree were considered highly in-
fested (Hay 1974).
Despite more than 50 oak decline events in the
eastern United States in the last 150 years (Mill-
ers et al. 1989), neither an associated red oak
borer outbreak nor widespread red oak borer-re-
lated tree mortality has previously been reported
(Stephen et al. 2001). No definitive causative
agents have been identified in the red oak borer
outbreak. However, some evidence indicates that
anthropogenic disturbance in the form of wide-
spread logging from 1890 to 1920 (Faulkner 1997)
and long-term fire suppression (Faulkner 1997)
may have contributed to this outbreak by creating
large areas of densely-stocked, even-aged, over-
mature northern red oak dominated stands (Oak
et al. 1996; Aquino et al. 2008).
During the recent outbreak in Arkansas, esti-
mated density of emerging red oak borers during
2001 sampling peaked as high as ~174 per tree
when data reported by Stephen et al. (2001) are
extrapolated to the whole tree level. Ongoing red
oak borer life-table research and population mon-
itoring in 2004 and 2005 yielded far fewer red oak
borers and indicated a probable decrease in red
oak borer population density at 3 primary moni-
toring sites (F.M.S., unpublished data). The over-
all goal of our research was to document this de-
crease and determine if it was localized or more
widespread. Three primary field locations were
sampled in 2003, 2005, and 2007, and 4 addi-
tional field locations were selected for monitoring
during 2005. Our objective was to estimate pre-
adult red oak borer (late-stage larvae, pupae,
pharate adults) populations before adult emer-
gence by felling trees and immediately dissecting
them to obtain a direct count of live red oak borers
at multiple locations in the Ozark National For-
est. Total counts of all live red oak borers in each
whole-tree sample likely provide the best esti-
mate of red oak borer emerging adults, as mortal-
ity in the short time between our sampling and
adult emergence in the protected environment of
the pupal chamber is likely very low.
Red oak borers exhibit a 2-year life cycle,
emerging synchronously in odd-numbered years
throughout most of their range (Donley & Accia-
vatti 1980). Emergence during even-numbered
years occurs only in the far southern portions of
the range, where a small percentage of the popu-
lation emerges during even years (Hay 1969; Hay
1972). Adults are nocturnal and usually emerge
mid-Jun through mid-Aug (Donley 1978; Donley
& Acciavatti 1980; Fierke & Stephen 2007). Fe-
males deposit an average of 119 eggs singly in
bark crevices and under lichen on boles of host
trees (Donley 1978; Donley & Acciavatti 1980).
After hatching, larvae burrow horizontally
through outer bark into phloem and initiate a
feeding gallery in which they overwinter (Hay


1969; Fierke et al. 2005a). Actively foraging lar-
vae enlarge phloem galleries to approximately 18
cm2 and initiate galleries into xylem tissues
(heartwood) during late spring of the following
year (Hay 1969). Larvae burrow into and up-
wards through the heartwood before their second
winter (Fig. 1A). Larvae enclose themselves in cy-
lindrical pupal chambers approximately 3.75 cm
long at the apex of heartwood galleries, with wood
shavings and frass forming gallery plugs (Hay
1969). Pupae complete metamorphosis during
late spring and early summer of odd-numbered
years (Hay 1969; Fierke et al. 2005a). Adults re-
treat down the heartwood gallery after eclosion
and exit the tree via characteristically-shaped
oval holes in the outer bark, directly over the orig-
inal phloem gallery site. Life histories similar to
those reported previously have been observed
during this outbreak in Arkansas (Stephen et al.
2001; Fierke et al. 2005a; Crook et al. 2007).
Herein, we describe emergence densities of red
oak borers during 3 separate cohorts, and com-
pare these findings with previous reports of red
oak borer population density. This information is
critically important because it is the first de-
tailed, multi-cohort description of red oak borer
density during the first reported outbreak of the
species, and provides evidence that the recent
outbreak has subsided.

MATERIALS AND METHODS

Research areas were initially chosen in 2001
during studies to develop sampling methods
(Fierke et al. 2005a; Fierke et al. 2005b). Trees
were selected from those locations (White Rock,
Fly Gap and Oark) during 2003, 2005, and 2007.
Trees were also selected at 4 additional widely-
distributed stands (Pilot Rock, Pedestal Rocks,
Dickie Junction, and Ozone) to expand the geo-
graphic area of population estimates for the 2005
cohort. The geographic location and topography of
each sampling location are illustrated in Fig. 2.
Observations based on additional field sampling
conducted in the same locations following 2005
adult emergence confirmed population decline in
2005. Only the highest density stands originally
chosen in 2003 (White Rock, Fly Gap and Oark)
were selected for the 2007 sampling effort. Focus-
ing on these most severely affected areas allowed
a "worst-case" estimate of red oak borer popula-
tions. These locations were chosen based on spe-
cific site and stand characteristics (ridges, high
northern red oak stocking, tree age greater than
50 years, and visual evidence of red oak borer ac-
tivity) because prior studies suggested associa-
tion of these characteristics with increased red
oak borer hazard (Fierke et al. 2007; Aquino et al.
2008). Specific descriptions of vegetation commu-
nities and stand variables have been previously
described by Fierke & Stephen (2007).







Florida Entomologist 92(3)


Fig. 1. Early-instar red oak borer larva phloem gallery (A) and cross-section of red oak borer xylem galleries (B).


All areas were systematically visited before red
oak borer adult emergence, and potential sample
trees were categorized into 1 of 3 infestation-his-
tory classes based on a rapid estimation procedure
(Fierke et al. 2005b). This procedure was designed
to estimate red oak borer infestation severity as a
function of red oak borer emergence holes in the
basal 2 m of the tree and visual estimates of per-
cent crown dieback. Trees were felled at each site,
cut into 0.5-m long bolts, beginning 0.5 m above


the ground and terminating at the highest sign of
red oak borer infestation neonatee attack holes
and/ or emergence holes). Total tree height, diam-
eter at breast height (DBH), and diameter at mid-
point of each 0.5-m sample bolt were recorded for
each tree. All bolts were dissected on site with gas-
oline-powered hydraulic log splitters. A total count
of live red oak borer larvae, pupae, and pharate
(pre-emergent) adults (hereafter collectively re-
ferred to as "live red oak borer") was recorded.


September 2009







Riggins et al.: Red Oak Borer Outbreak


nickie Juinrtinn


Fig. 2. Geographic location and topography of sampling sites in Ozark National Forest of Arkansas, U.S.A.


Logistical challenges during preliminary sam-
pling in 2003 resulted in a slightly late start, with
some samples being taken after earliest emer-
gence had begun in Jun. As a result, in 2003 a
number of current generation emergence galler-
ies were counted in lieu of their recently departed
inhabitants. These recently-vacated galleries
were easily distinguished from previous genera-
tion emergence galleries by absence of fungus or
any appearance of necrotic tissue in the heart-
wood gallery lining. They were also easily distin-
guished from unsuccessful heartwood galleries by
the presence of frass plug remnants and the ab-
sence of cadavers. While sample timing may have
introduced small error into 2003 counts, the esti-
mates of red oak borer density form an important
baseline for subsequent sampling years, and the
aforementioned absence of fungus or necrotic tis-
sue allowed us to differentiate between empty
current generation emergence galleries and gal-
leries from previous red oak borer cohorts. We as-
sume that their inclusion in the total counts for
2003 provides a more accurate estimate of emerg-
ing red oak borer in 2003 than would be possible
if those data were excluded. We cannot exclude
the possibility that some of these galleries were
formed by other closely related species such as E.
atomarius, which could have slightly skewed


2003 counts. However, no other large pharate ce-
rambycids were recorded during data collection,
suggesting that effects due to this were minimal.
During all tree dissections, care was taken to
completely uncover and examine all signs of red
oak borer presence, whether in the form of phloem
galleries with entrances to heartwood galleries, or
heartwood galleries on the basal cross-section of
each sample bolt (Fig. 1B). Bolt dissections began
with complete removal of all outer bark to expose
any potentially occupied heartwood galleries.
Each visible heartwood gallery, including those
continually discovered during successive split-
ting, was then fully exposed to its apical terminus
to determine if a living red oak borer was present.
Second-year larvae are approximately 2.5-4.0 cm
in length (F.M.S., unpublished data) and are
readily visible once trees are dissected. Bolts
without externally obvious heartwood galleries
were thoroughly dissected to minimize omitting
any red oak borers. Because of our previous expe-
rience and the robust nature of late-stage larvae
and their correspondingly large and obvious
heartwood galleries (Figs. 1A and 1B), we assume
that very few red oak borer were omitted from
counts.
A count of live red oak borers in each tree was
divided by total bark surface area for that tree.


S"Arkansas

Vo m





Fly Gap
m+AAL.


White Rock


Pilot Rrwck







Florida Entomologist 92(3)


Bark surface area was calculated from diameter
measurements of each sample bolt. Densities of
red oak borers were expressed per square meter
of bark surface area to provide a standardized
unit that could be compared among locations and
years despite variation in the number and size of
trees sampled. Analysis of variance with JMP
7.0 (SAS Institute, 2007) was conducted, and
significance was judged at a = 0.05 with Tukey-
HSD used for mean separation when appropri-
ate.

RESULTS

DBH for all 98 trees sampled ranged from
14.5-42 cm, and mean DBH was 29.8 cm (+0.54
SE). Mean total tree height was 18.9 m (+0.26,
range 12.5-24.5 m). Mean total height of infesta-
tion was 13.6 m (+0.24, range 7.5-19.5 m). The av-
erage total bark surface area of the 98 trees sam-
pled in this study was 9.4 m2 (+0.32, range 2.3-
17.2). There were no significant differences in
mean DBH, total tree height, height of infesta-
tion, or bark surface area among different years of
the study (P > 0.1, F < 2.4).
Overall mean density of live red oak borer was
significantly higher in 2003 than in either 2005 or
2007 (F297 = 45.37, P = <0.0001) at 4.11, 0.18, and
0.10 per sq. m, respectively, (Table 1). All loca-
tions exhibited red oak borer population declines
from the 2003 to 2005 cohort, but there was no
statistically significant decrease from the 2005 to
2007 cohort (Table 1). Red oak borers were en-
countered much more frequently and in greater
numbers per tree in 2003 than in subsequent
sampling years (Fig. 3). In 2003, mean number of
red oak borer per tree was 31.7 (7.9 SE). Mean
number of red oak borer per tree was 1.6 (+0.37)
in 2005 and 1.0 (+0.25) in 2007.


DISCUSSIoN

Estimates of the population density of live red
oak borers immediately before adult emergence
in the Arkansas Ozarks were derived from 3 sep-
arate cohorts (2003-2007) inhabiting 98 northern
red oaks that were felled and completely dis-
sected. The earliest estimates of red oak borer
within-tree population density were made during
the 1999-2001 cohort by Stephen et al. (2001).
Their sample size was small; 15.1-m long bolts
collected from 2 trees, and resulted in an esti-
mated average emergence density of 18.5 (4.3)
red oak borers per sq. m. This estimate is more
than 4 times higher than the average density of
the 2003 cohort, and when extrapolated to aver-
age whole tree size calculated for the present
study (9.38 sq. m), would result in an approxi-
mate density of 174 emerging adults per tree dur-
ing the 2001 cohort. Populations of red oak borers
may have declined approximately 88% from the
2001 to 2003 cohort and declined again (96%) be-
tween the 2003 and 2005 cohort. The extent of
this reduction in density can be visualized by ex-
amining frequency distributions of total numbers
of red oak borers per tree, which show that ~ 53%
of trees sampled in 2003 contained more than 20
live red oak borers per tree (Fig. 3A). In 2005 and
2007 combined, only 2 of the 81 trees sampled
contained 10 or more red oak borers, and 38 trees
contained no red oak borers (Figs. 3B and C).
Hay (1974) described endemic red oak borer
populations as averaging less than 1 larva per
tree. Given our average tree size and bark surface
area, Hay's (1974) description of endemic popula-
tion densities can be estimated at approximately
0.11 larvae per sq. m of bark surface area. In 2003
the average density of 4.11 larvae emerging per
sq. m of surface area from 17 sample trees was


TABLE 1. MEAN DENSITY (NUMBER PER SQUARE METER OF BARK SURFACE AREA) OF LIVE RED OAK BORERS RECORDED
FROM NORTHERN RED OAK TREES AT LOCATIONS IN OZARK NATIONAL FOREST DURING THE 2001-2003, 2003-
2005, AND 2005-2007 COHORTS.

2003 2005 2007

Location Density n Density n Density n F df P

Fly Gap 5.74 a 10 0.30 b 8 0.15 b 8 14.13 2, 25 <0.0001
White Rock 2.35 a 3 0.19 b 9 0.06 b 8 42.50 2, 20 <0.0001
Oark 1.01 a 4 0.35 ab 8 0.09 b 8 3.56 2, 18 0.0525
Pedestal Rocks n/a 0 0.01 8 n/a 0 n/a n/a n/a
Pilot Rock n/a 0 0.03 8 n/a 0 n/a n/a n/a
Dickie Junction n/a 0 0.39 8 n/a 0 n/a n/a n/a
Ozone n/a 0 0.05 8 n/a 0 n/a n/a n/a
Overall 4.11a 17 0.18b 57 0.10b 24 45.37 2, 97 <0.0001

*The column labeled n indicates number of trees dissected during each location-year combination.
Means were considered significantly different at P d 0.05. Rows labeled with different lower-case letters represent significantly
different mean cohort densities (a = 0.05).


September 2009







Riggins et al.: Red Oak Borer Outbreak


2003



Wr

o
'1.. I.iI
0-4 s5 912 13-16 1720 21-24 2S 28 23
# Red oak Borers per Tree
2005
30

-20S
1

I-

0 1 2 J* 6 >9
# Red oak Borers per Tree
2007

1U






0 1 2 3 4 5
# Red oak Borers per Tree

Fig. 3. Red oak borer frequency distribution based on
total counts of live red oak borer per northern red oak
tree dissected in the field during three successive co-
horts in Ozark National Forest of Arkansas, U.S.A.


about 37 times greater than endemic levels. After
the abrupt decline of red oak borer populations
during the 2003-2005 cohort, overall average den-
sity of red oak borers (0.18/ sq. m) was only
slightly higher than expected at endemic levels
(0.11/ sq. m). Live red oak borer density immedi-
ately before emergence in 2007 (0.10 /sq. m) was
not statistically lower than in 2005. This density
estimate indicates that red oak borer populations
at our sampling locations in Ozark National For-
est in 2007 have returned to population levels
consistent with those reported by Hay (1974), in-
dicating that the recent outbreak event is over.
This insect outbreak is unusual because red
oak borer is a widely-distributed native species,
but has never been reported in high numbers any-


where in its range throughout hardwood forests of
the eastern United States and southeastern Can-
ada. More than 57 oak decline events have been
documented in the eastern United States between
1856 and 1986 (Millers et al. 1989), including
1959 and 1980 in Arkansas (Toole, 1960; (Toole
1960; Bassett et al. 1982). In none of these decline
events has red oak borer been described as a ma-
jor contributing factor to tree mortality.
Cohort senescence theory (Mueller-Dombois
1987) predicts this type of insect and pathogen ac-
tivity in a mosaic across the landscape as hosts
begin to senesce due to the accumulation of stress
over their lifetimes. Physiological age is the ulti-
mate "score-keeper" of sorts, as the effects of a
myriad of predisposing, inciting, and contributing
factors (e.g., topographic position, acute drought,
and insects, respectively) accumulate throughout
trees' lives. The decline-disease spiral eventually
culminates with tree death (Manion 1991). Condi-
tions associated with oak decline are high red oak
stocking density, ridge topographic position, poor
site quality, older age (>60 years), advanced phys-
iologic age (as measured by the ratio of site index/
stand age), and xeric soils (Starkey et al. 1989;
Oak et al. 1996; Starkey et al. 2000; Heitzman &
Guldin 2004; Kabrick et al. 2008). Red oak borer
populations could potentially resurge if condi-
tions favorable to oak decline are eventually rep-
licated. Berryman (1986, 1987) characterized in-
sect outbreaks in 2 general forms: (1) gradient re-
sponses to changes in environmental favorability
(e.g., abundance of resources, predators, and par-
asitoids); and (2) eruptive outbreaks that begin in
epicenters of highly favorable conditions and have
the ability to spread into less susceptible areas
and eventually infest vast areas of hosts (Valenti
et al. 1999). Classifying red oak borers into 1 of
these categories of outbreak pest is complicated
because studies outlining its long-term popula-
tion dynamics are lacking. However, the speed of
onset, severity, and size of the outbreak suggests
an eruptive pest, as is the apparent ability to suc-
cessfully attack healthier host trees. However, it
is more likely this outbreak fits into the frame-
work of a graded response to a landscape-level su-
perabundance of host trees beginning to senesce
at the same time.

CONCLUSION
The dramatic outbreak of red oak borer first re-
ported in northern Arkansas in 1999 appears to
have subsided in 2005 and populations remain low
in 2007. Red oak borer numbers increased expo-
nentially, and then abruptly returned to endemic
levels almost entirely during the course of 2 gener-
ations. The oak decline event that accompanied the
outbreak (Starkey et al. 2004) was unique given
the role of red oak borer as a major contributing
factor (Manion 1991). At this time we remain un-











certain of the cause of the outbreak and subse-
quent red oak borer population decline. It is likely,
based on current understanding of oak decline eti-
ology, that direct (forest management practices)
and indirect anthropogenic disturbance (e.g., cli-
mate change) were both involved with this event.
Continued monitoring of red oak borer popula-
tion abundance and dynamics may help antici-
pate future outbreaks. Ongoing remote sensing
and spatial modeling may provide near real-time
assessment of relative forest health and predic-
tive hazard assessment at individual tree, stand,
and landscape levels (Franklin 2001; Wang et al.
2007; Aquino et al. 2008; Riggins et al. 2009). Sil-
vicultural treatments such as alteration of spe-
cies composition, thinning, selective removal of
infested trees, and prescribed fire are manage-
ment practices which have been prescribed by for-
est managers in an attempt to improve the spe-
cies diversity and age structure of Ozark National
Forest (Donley 1981; Guldin et al. 2006). Removal
of infested oaks in Ohio resulted in a 50% reduc-
tion of red oak borer population within the follow-
ing generation, and about 90% during the second
generation after silvicultural control was applied
(Donley 1981). Prescribed fire and selective thin-
ning both serve to remove poor specimens from
the stand and should provide similar results.
These practices may help to lessen future wide-
spread red oak borer activity in the Arkansas
Ozarks.

ACKNOWLEDGMENTS

Many thanks to Dr. James M. Guldin for assistance
throughout this project. Thanks to D. Crook, D. Kinney,
V. Salisbury, R. Barnhill, J. Jones, L. Aquino, T. Dahl, C.
Abbot, V. Ware, M. Stephen, J. Bates, M. McCall, R.
Corder, M. Fierke, R. Verble, and L. Haavik for helping
with field work, and to J. Jones for assisting in database
management. Funding for this research was provided
by the University of Arkansas Agricultural Experiment
Station and grants from the USDA Forest Service
Southern Research Station and USDA Forest Service
Forest Health Protection STDP and FHM programs.

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Florida Entomologist 92(3)


September 2009


FIRST NORTH AMERICAN RECORDS OF THE EAST
PALEARCTIC SEED BEETLE BRUCHIDIUS TERRENUS
(COLEOPTERA: CHRYSOMELIDAE: BRUCHINAE), A SPECIALIST ON
MIMOSA (ALBIZIA JULIBRISSIN, FABACEAE)

E. RICHARD HOEBEKE1, A. G. WHEELER, JR.2, JOHN M. KINGSOLVER3AND DAVID L. STEPHAN4
'Department of Entomology, Cornell University, Ithaca, NY 14853

2Department of Entomology, Soils, and Plant Sciences, Clemson University, Clemson, SC 29634

3Florida State Collection of Arthropods, c/o P.O. Box 147100, Gainesville, FL 32614

4Department of Entomology, North Carolina State University, Raleigh, NC 27695

ABSTRACT
The eastern Palearctic bruchine seed beetle Bruchidius terrenus (Sharp) (Coleoptera: Chry-
somelidae: Bruchinae), a specialist seed predator of mimosa or silk tree (Albiziajulibrissin),
is reported for the first time in North America based on collections from seven southeastern
states. This is the third Asian insect species recently reported from A. julibrissin in the
United States. A diagnosis, description, photographs of the adult, a summary of the known
U.S. distribution, notes on seasonal history, and a revision to an existing key to North Amer-
ican Bruchidius are presented.

Key Words: Coleoptera, Chrysomelidae, Bruchinae, Bruchidius terrenus, adventive, inva-
sive species, new records

RESUME

Se report la presencia del escarabajo bruquido Palearctico oriental de la semilla, Bruchi-
dius terrenus (Sharp) (Coleoptera: Chrysomelidae: Bruchinae), un depredador especialista
en semilla de mimosa o el arbol de seda (Albizia julibrissin), por primera vez en Norteam6-
rica basado en colecciones hechas en siete estados de sureste de los Estados Unidos. Este es
la tercera especie de insecto asidtico reportado de A. julibrissin en los Estados Unidos. Se
present una diagnosis, descripci6n, fotos del adulto, un resume de la distribuci6n conocida
en los EE.UU., notas sobre su ciclo estacional y una revision de una clave existente de los
Bruchidius de Norteam6rica.


The Old World genus Bruchidius, with about
300 described species (Kingsolver 2004), is repre-
sented in North America by 2 adventive species,
B. villosus (F.) and B. cisti (F.). Both species ap-
parently were accidentally introduced with seeds
of their leguminous host plants (Bottimer 1968).
The European B. illosus, first recorded in North
America from Massachusetts (Olsen 1918), has
been reported from Ontario and Quebec south to
North Carolina (Chantal 1972; Redmon et al. 2000;
Kingsolver 2004). This specialist of genistoid le-
gumes has been released for the biological control of
Scotch broom, Cytisus scoparius (L.) Link, in the Pa-
cific Northwest (Coombs et al. 2004; Hulting et al.
2008). The Eurasian species Bruchidius cisti was
first found in North America in British Columbia in
1922 at Nicola and again in 1965 at Lillooet (Bot-
timer 1968) and has been reported as a pest of the
forage legume sanfoin, Onobrychis viciifolia Scopoli,
in Montana (Hewitt & Burleson 1976).
An unidentified beetle, which would become the
third adventive species of Bruchidius in North


America, came to our attention in Aug 2004. A ho-
meowner in Elizabeth City, North Carolina (Tony
Barefoot), by happenstance, examined developing
pods of mimosa and discovered nearly all were in-
fested with small yellowish larvae. Wanting to know
more about the insect, he contacted an extension en-
tomologist at North Carolina State University
(Stephen Bambara). It was first thought that the
larvae might represent a seed chalcid (Eurytomidae
or Torymidae). Additional infested pods of A. juli-
brissin, collected on Sep 1, 2004 (by D.L.S.), about 5
miles NNW of Raleigh (Wake Co., NC), were
brought indoors for rearing. Adults of an unfamiliar
bruchine seed beetle emerged in mid- to late Sep.
Specimens eventually sent to E.R.H. in May 2007
were identified as the eastern Palearctic Bruchidius
terrenus (Sharp). The identification was confirmed
after specimens were compared with identified ma-
terial housed in the collection of the National Mu-
seum of Natural History (Washington, DC).
In this paper, we give the first North American
records ofB. terrenus, an Asian seed specialist of







Hoebeke et al.: First North American Records of Bruchidius terrenus


mimosa or silk tree (Albizia julibrissin) that oc-
curs widely in the eastern Palearctic Region:
China, Taiwan, and Japan (Udayagiri & Wadhi
1989; Morimoto 1990; Hua 2002). We list and map
locality records for 7 southeastern states (Ala-
bama, Florida, Georgia, Mississippi, North Caro-
lina, South Carolina, and Tennessee); give a diag-
nosis, description, and photographs of the adult to
facilitate its recognition among the U.S. bruchine
fauna; and provide observations on its seasonal
history and habits in the Southeast.

SYSTEMATICS
BRUCHIDIUS TERRENUS (SHARP)
(FIGS. 1-6)
Bruchus terrenus Sharp, 1886: 35.
Bruchidius terrenus: Chujo, 1937a: 194; 1937b: 61; Na-
kane, 1963: 319; Tan et al., 1980: 38; Morimoto, 1984:
266;1990:135.
Bruchidius notatus Chujo, 1937a: 196; 1937b: 64;Tan et
al., 1980: 39; Morimoto, 1990: 136.
Acanthoscelides terrenus: Zacher, 1952: 465; Udayagiri
and Wadhi, 1989: 66.
Diagnosis. Members of the Old World genus
Bruchidius are characterized by the presence of a
single minute acute tooth on the ventromesal mar-
gin of the metafemur at the apical 1/4; the absence
of marginal teeth or denticles on the pronotum;
pronotum conical or transverse, without lateral car-
ina; presence of a well-developed slender mucro at
the apex of the tibia ventrally; and the median lobe
of the male genitalia lacking "hinge sclerites" (Bot-
timer 1968; Borowiec 1987; Kingsolver 2004).
Bruchidius terrenus can be easily separated
from B. villosus and B. cisti by the characters in
the following description and revised key.
Description (Figs. 1-4). Male: Color.-Integu-
ment of head usually black, sometimes testa-
ceous; pronotum, elytra, and pygidium black,
sometimes testaceous (among specimens exam-
ined); ventral areas black except abdomen some-
times testaceous; antenna generally entirely


testaceous, but some specimens with apical seg-
ments black; fore and mid legs reddish orange;
metafemur usually with basal 1/2 black, apical
1/2 testaceous.
Vestiture.-Composed of fine silvery gray setae
evenly distributed over body but with dark brown
circular patches on either side of midline of pronotal
disk, each with conspicuous central gray spot
(Fig. 1); elytral interstices with dark brown spots
(Fig. 1); apices of elytra sometimes dark brown
(Fig. 3); pygidium (Fig. 3) usually immaculately sil-
very gray, sometimes with faint darker clouding.
Structure.-Head (Fig. 4) triangular, eyes pro-
tuberant, ocular sinus 3/5 length of eye, postocu-
lar lobe narrow; frons slightly convex, in some
specimens with brief frontal carina; pronotal disk
semicircular in outline, lateral margins gently
curvate; disk evenly convex with slight depres-
sions at caudal angles, densely, evenly punctate;
scutellum broad, bilobed; elytral striae shallow
but distinct, finely punctate; 3rd and 4th striae orig-
inating on marginal tubercle; interstices alternat-
ing in width, densely setose; pygidium shallowly
convex, uniformly, densely punctulate; fore and
mid legs not modified; metacoxae densely punctu-
late, lateral 1/2 of face densely setose; metatibia
with lateral carina complete and ending in a
short, acute tooth, ventral carina complete ending
in short mucro, mesal carina complete; basal ab-
dominal sternite with mesal pore; terminal stern-
ite emarginated to fit pygidial apex.
Male genitalia.-As in Fig. 5 and 6. Median
lobe (Fig. 5) 4x as long as its apical width; ventral
valve subtriangular with apex bluntly rounded;
internal sac densely lined with very fine spicules;
apical valve circular; lateral lobes (Fig. 6) sepa-
rated by deep cleft, apices spatulate and inwardly
curvate. The male genitalia were illustrated by
Morimoto (1990: p. 135).
Female.-Similar in all respects to male, ex-
cept basal sternite lacking medial pore; terminal
sternite evenly rounded, not emarginate.
Size.-Variable among specimens examined (n =
31); body length ranging from 2.46 to 4.07 mm; ma-
jority of specimens measured 3.69 to 3.82 mm.


REVISED KEY TO NORTH AMERICAN BRUCHIDIUS
(ADAPTED FROM BOTTIMER 1968 AND KINGSOLVER 2004)

1. Body and appendages entirely black (or only antennal segments 1-4 reddish brown); dorsal vestiture uniformly
white.................................................................... ........... 2
-. Body usually black, but appendages not entirely black; head, thorax, elytra, and ventral abdominal seg-
ments sometimes testaceous; antennae and legs testaceous to reddish orange; dorsal vestiture predom-
inantly silvery gray with brown mottling, especially on elytral surface (Figs. 1-2); southeastern United
States .............................................................. terrenus (Sharp)
2. All antennal segments black; 4th stria with prominent, subbasal denticle; mucro absent; western United States
and western Canada ............................................................... cisti (F.)
-. Basal 4 antennal segments reddish brown; striae lacking subbasal denticles; mucro one-eighth as long as basi-
tarsus; eastern United States and eastern Canada ................................... villosus (F.)






Florida Entomologist 92(3)


3 4 y 4

Figs. 1-4. Bruchidius terrenus. 1, Adult, dorsal aspect. 2, Adult, lateral aspect. 3, Pygidium, dorsal aspect. 4,
Head, frontal aspect. Scale line = 2 nunm.


HOST PLANT to China and Korea (Zheng et al. 2006). It was in-
troduced into the United States in 1785 (Spong-
Mimosa or silk tree (Albizia julibrissin) is na- berg 1990). With its attractive pink flowers and
tive to southern and eastern Asia, occurring east umbrella-like canopy, it has been widely culti-


September 2009







Hoebeke et al.: First North American Records of Bruchidius terrenus


Figs. 5-6. Bruchidius terrenus, male genitalia. 5, Me-
dian lobe. 6, Lateral lobes.


vated along roadsides because it is fast growing
and drought-tolerant; it is planted in gardens as a
highly prized ornamental (Spongberg 1990; Co-
thran 2004). Albizia julibrissin has become inva-
sive in the United States and has spread widely
from southern New England west to Missouri and
Illinois and south to Florida and Texas; it is culti-
vated in California and Oregon, where it has not
become invasive (Anonymous 2008).
Although B. terrenus appears to be a specialist
on seeds of A. julibrissin, it has been reported
from seeds of black locust, Robinia pseudoacacia
(L.), andAcacia confusa Merr. (Morimoto 1990). It
is one of 75 species of arthropods closely associ-
ated withAlbizia spp. in Asia and is considered an
important pest of A. julibrissin in Taiwan and
China (Zheng et al. 2006).

SEASONAL HISTORY AND HABITS

The seasonal history and habits ofB. terrenus
are based mainly on observations by the original
collector (T Barefoot, Elizabeth City, NC), D.L.S.
and A.G.W, supplemented by reference to the biol-
ogy ofB. villosus (Redmon et al. 2000).
Overwintered, sexually immature adults most
likely emerge in the Southeast in late spring and
disperse to mimosa where they probably attain
sexual maturity by feeding on pollen. In 2008 at
Clemson, South Carolina, A.G.W first found
adults on 10-VI (none were found during sam-
pling of the same trees on 2-VI) and observed
adults deep in flowers at Clemson and elsewhere
in the Southeast while surveying for the beetle.
The latest observation of an adult in the field (1


only, by A.G.W.) was on 15-IX-2007, in Emanuel
Co., GA (Swainsboro). Mimosa trees flower in
Georgia from May through Aug and the fruits
(pods) mature from Sep to Nov (Pardini and Ham-
rick 2008). In Telfair Co., GA (McRae), on 20-VI-
2007, A.G.W. noted the presence of ~5 inch pods.
Oviposition begins when green pods are forming,
probably in early Jul. Pods ripen from late Aug to
Nov and begin to disintegrate soon after but re-
main on the trees into winter. Females probably
lay eggs individually (in clusters) on the young
pods and cement them in place. Eggs probably
hatch in 1-2 weeks, the larvae emerging from the
underside of the egg and tunneling into the devel-
oping pod. A neonate larva burrows through the
pod wall into a soft green seed. In North Carolina,
early-instars infested most seeds of pods exam-
ined from early to mid-Jul in Elizabeth City (T.
Barefoot, in litt.) and the Raleigh area (D.L.S.,
personal observation). Unhatched eggs were still
observed (by D.L.S.) on the outside of pods in late
Jul 2004. By mid-Aug (11-VIII-2004), late instars
(~3-4 mm long) were found in seeds (T. Barefoot,
in litt.). During an examination of several trees in
Raleigh in Sep 2004, D.L.S. found seeds heavily
infested (>90% examined), whereas seeds of these
same trees in Sep 2007 were only slightly infested
(<5% examined). Although several larvae prob-
ably develop in each pod, D.L.S. noted most seeds
contained a single larva, occasionally 2, and
rarely 3 larvae; it is unknown if more than a sin-
gle larva can successfully develop in a seed. Pre-
sumably there are 4 instars. Pupation occurs
within a seed inside the closed pod. The pupation
period probably takes from 10 to 20 d. New-gener-
ation adults chew through the seed coat (Fig. 7)
and then chew through the pod coat to escape.
Adults emerged indoors in mid- to late Sep from
infested pods collected near Raleigh on 1-IX-2004
(D.L.S., personal observation). Adult emergence
holes were observed (by A.G.W.) on old seed pods
collected in early Sep at Clemson, SC (Fig. 8).
New adults, after emergence, probably feed on
pollen in the fall if flowers are still available.
Adults probably overwinter near host trees in
plant litter. Based on collecting (by A.G.W) in
2007 and 2008, adults are found on the host from
early to late Jun to mid-Sep. Bruchidius terrenus
appears to be univoltine.

NEW NORTH AMERICAN RECORDS

Distributional data for B. terrenus in North
America (see material examined below and Fig. 9)
are based primarily on collections from mimosa
(A. julibrissin) by A.G.W from Jun to Sep 2007,
unless noted otherwise. Parenthetical numbers
refer to adults collected. Voucher specimens are
deposited in the Cornell University Insect Collec-
tion (Ithaca, NY), the National Museum of Natu-
ral History, Smithsonian Institution (Washing-






Florida Entomologist 92(3)


I $ B I$I III k ~l1 11

________________ ~ iii1J~uI1IF )-. W2J7III~~~-ie~


Figs. 7-8. Reproductive structures ofAlbizia julibrissin infested by B. terrenus. 7, Seeds. 8, Seeds and pod coat.
Arrows denote exit holes by newly emerged adults. Scale line in millimeters.



ton, DC), North Carolina State University insect AL) and Adriean J. Mayor (Great Smoky Moun-
collection (Raleigh), the Florida State Collection tains National Park, Gatlinburg, TN).
of Arthropods (Gainesville, FL), and the personal Material examined: UNITED STATES: ALA-
collections of Robert H. Turnbow, Jr. (Enterprise, BAMA: Baldwin Co., Loxley, 30-VI-2007 (3). Bul-


September 2009







Hoebeke et al.: First North American Records of Bruchidius terrenus


Fig. 9. Known distribution of Bruchidius terrenus in
the southeastern United States (Alabama, Florida,
Georgia, Mississippi, North Carolina, South Carolina,
and Tennessee).


lock Co., Union Springs, 1-VII-2007 (2). Calhoun
Co., Oxford, 6-VII-2007 (3). Conecuh Co., SW of
Evergreen, 30-VI-2007 (1). Conecuh Co., Ever-
green, 30-VI-2007 (4). Covington Co., W of River
Falls, 30-VI-2007 (3). Crenshaw Co., S of Luverne,
1-VII-2007 (4). Jefferson Co., Hueytown, 6-VII-
2007 (6). Lee Co., Auburn, 1-VII-2007 (2). Macon
Co., Tuskegee, 1-VII-2007 (7). Pike Co., Troy, 1-
VII-2007 (5). St. Clair Co., Cook Springs, 6-VII-
2007 (3). Sumter Co., York, 6-VII-2007 (3). Tusca-
loosa Co., Tuscaloosa, 6-VII-2007 (3). FLORIDA:
Alachua Co., W of Hawthorne, 29-VI-2007 (2);
Gainesville, 24-IV-2007, M. C. Thomas, beating
flowers of Hydrangea quercifolia and Cornus foe-
mina (1, FSCA coll.). Gadsden Co., S of Quincy,
30-VI-2007 (1). Jackson Co., S of Cottondale, 30-
VI-2007 (8). Jefferson Co., S of Monticello, 29-VI-
2007 (9). Okaloosa Co., S of Crestview, 30-VI-2007
(4). Putnam Co., E of Palatka, 29-VI-2007 (8).
Santa Rosa Co., N of Avalon Beach, 30-VI-2007
(3). Walton Co., De Funiak Springs, 30-VI-2007
(4). GEORGIA: Coffee Co., Douglas, 28-VI-2007
(1); Pridgen, 28-VI-2007 (4). Emanuel Co.,
Swainsboro, 15-IX-2007 (1). Lanier Co., Stockton,
28-VI-2007 (62). Madison Co., jet. Hwys. 72 &
172, 1-VIII-2004, R. Turnbow (1, Turnbow coll.).
Mitchell Co., S of Camilla, 9-VIII-2008 (5). Oconee
Co., N of Bishop, 28-VI-2007 (1). Putnam Co., N of
Eatonton, 28-VI-2007 (5); Stephens Co., Toccoa
Falls College, 1-VIII-2004, R. Turnbow, on Sol-
idago (10, Turnbow coll.). Telfair Co., McRae, 28-


VI-2007 (8). Wilkinson Co., McIntyre, 28-VI-2007
(17). MISSISSIPPI: Kemper Co., De Kalb, 7-VII-
2007 (11). Lee Co., Tupelo, 7-VII-2007 (1). Noxu-
bee Co., E of Shuqualak, 7-VII-2007 (24).
Lowndes Co., W of Columbus, 7-VII-2007 (19).
Monroe Co., W of Amory, 7-VII-2007 (12). NORTH
CAROLINA: Carteret Co., Ft. Macon St. Pk., 10-
IV-2006, R. Newman (1, FSCA coll.). Polk Co., Co-
lumbus, 24-VI-2007 (3). Wake Co., 5 mi. NNW of
Raleigh, 1-IX-2004, D.L. Stephan (4). SOUTH
CAROLINA: Anderson Co., Powdersville, 24-VI-
2007 (1). Greenville Co., Greenville, 24-VI-2007
(1). Oconee Co., SE of Newry, 21-VI-2007 (2);
Westminster, 20-VI-2007 (4); Rt. 123, E of Chauga
River, 23-VI-2007 (4). Pickens Co., Lake Hartwell,
Clemson, 20 & 21-VI-2007 (5) & 10-VI-2008 (2);
Spartanburg Co., Inman, 24-VI-2007 (24). TEN-
NESSEE: Bradley Co., Cleveland, 8-VII-2007 (1).
Hamilton Co., E of Chattanooga, 8-VII-2007 (1).
Sevier Co., Old Gatlinburg Land Fill off Gnatty
Branch, 5-X-2004, M. Tomkasky (1, Adriean
Mayor coll.).

DISCUSSION

Two other Asian mimosa-associated insects
have been detected recently in North America.
The buprestid beetle Agrilus subrobustus Saun-
ders, whose native range includes China, Japan,
North and South Korea, and Taiwan, was discov-
ered in northern Georgia in 2006. In Japan, the
only known host of this little-studied species is
mimosa (Westcott 2007).Acizzia jamatonica (Ku-
wayama), a psyllid native to east Asia, was re-
ported from 2 localities in Georgia in 2006 (Hal-
bert 2007; Ulyshen & Miller 2007). Surveys of
this mimosa specialist in the southeastern United
States in 2007 and 2008 yielded records from 5
new states and additional counties in Georgia.
Acizzia jamatonica was detected in Europe in
2001, with establishment probably resulting from
shipments of mimosa from Asia as prized orna-
mentals (Wheeler & Hoebeke 2009).
Bruchidius terrenus might have been similarly
introduced to the southeastern states with mi-
mosa nursery stock. This seed predator might be-
come a pest of mimosa in landscape plantings and
could even be considered a beneficial addition to
our fauna by those who regard mimosa as an in-
vasive species and, therefore, an undesirable
plant.

ACKNOWLEDGMENTS
We thank Tony Barefoot (Elizabeth City, NC) for
bringing the discovery of an Asian seed beetle in mi-
mosa seeds to our attention initially through Extension
Entomologist Stephen Bambara (Department of Ento-
mology, North Carolina State University, Raleigh) in
August 2004. We are grateful to Paul Skelley (Florida
State Collection ofArthropods, Gainesville) for provid-
ing the photographic images of B. terrenus in Figs. 1-4,











to Kent Loeffler (Department of Plant Pathology, Cor-
nell University, Ithaca, NY) for photographing infested
seeds and pods of Albizia julibrissin, and to 2 anony-
mous reviewers for their helpful comments on a draft of
the manuscript.
This research was supported by the Cornell Univer-
sity Agricultural Experiment Station federal formula
funds, Project No. NYC-139404 to ERH, received from
Cooperative State Research, Education, and Extension
Service, U.S. Department of Agriculture.

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Florida Entomologist 92(3)







L6pez et al: Indicator of Quality in Mass-reared D. longicaudata


IS HOST SIZE AN INDICATOR OF QUALITY IN THE
MASS-REARED PARASITOID DIACHASMIMORPHA LONGICAUDATA
(HYMENOPTERA: BRACONIDAE)?

OLGA P. LOPEZ1, YANN HENAUT2, JORGE CANCINO1, MICHAEL LAMBIN3, LEOPOLDO CRUZ-LOPEZ2 AND JULIO C. ROJAS2
1Subdirecci6n de Desarrollo de M6todos, Programa Moscafrut SAGARPA-IICA, Central Poniente No. 14,
Col. Centro, 30700, Tapachula, Chiapas, M6xico

2Depto Entomologia Tropical, El Colegio de la Frontera Sur (ECOSUR), Apdo Postal 36, Tapachula Chiapas, M6xico

3Centre de Recherche sur la Cognition Animale, Universit6 Paul Sabatier, 118 Rte de Narbonne,
31062 Toulouse, France

ABSTRACT

Diachasmimorpha longicaudata (Ashmead) is an endoparasitoid of fruit flies reared for aug-
mentative biological control of several species ofAnastrepha in Mexico. During the production of
D. longicaudata different sizes of parasitoids are produced depending on the host size. Here, we
investigated whether host size influences the biological and behavioral parameters of the wasp
that emerged under laboratory conditions. Three different sizes of host (small, medium, and
large) were used for the experiments. Host size significantly affected the number of parasitoids
emerged as greater number of parasitoids emerged from medium size hosts compared to small
and large hosts. The ratio of females to males did not differ among the groups. Host body size in-
fluenced life expectancy of parasitoid females and males deprived of food, and parasitoids that
emerged from large hosts lived longer. In contrast, host body size did not significantly influence
the life expectancy of parasitoid females and males provided with food ad libitum. Host body size
also affected gross and net fecundity of emerged parasitoids; females that emerged from medium
and large host larvae were more fecund. Host size did not affect most of the behavioral parame-
ters evaluated, except the time of landing on source. Females that emerged from large host lar-
vae spent less time before landing on a host source than females from the smaller hosts. Host
body size did not affect the different trajectory indices evaluated, except the mean walking speed
ofD. longicaudata. Females developed in large hosts showed an increase walking speed com-
pared to females from medium and small hosts.

Key Words: mass rearing, host body size, biological control, longevity, fecundity, sex ratio,
host location behavior

RESUME

Diachasmimorpha longicaudata (Ashmead) es un endoparasitoide de las moscas de la fruta
usado como un agent de control de varias species de Anastrepha en M6xico. Durante la
producci6n masiva de D. longicaudata diferentes tamaios de individuos son producidos de-
pendiendo del tamaio del hospedero. Este trabajo investig6 si el tamaio del hospedero in-
fluye sobre los parametros biol6gicos y comportamentales de los parasitoides emergidos.
Tres diferentes tamaios de hospederos (pequeios, medianos y grandes) fueron usados para
los experiments. El tamaio del hospedero afect6 significativamente el numero de parasi-
toides emergidos debido a que un mayor numero de individuos emergi6 de las pupas de ta-
maio medio en comparaci6n con aquellos emergidos de las pupas de tamaio pequeio y
grande. La proporci6n de hembras/machos no fue significativamente diferente en los tres
grupos. El tamaio del hospedero influy6 en la esperanza de vida de los parasitoides hembras
y machos sin alimento, ya que los parasitoides grandes vivieron mis tiempo. En contrast,
el tamaio del hospedero no afect6 la esperanza de vida de los parasitoides a los cuales se les
proporcion6 alimento ad libitum. El tamaio del hospedero influy6 en la fecundidad de las
hembras emergidas, las hembras provenientes de hospederos medianos y grandes fueron
mis fecundas. El tamaio del hospedero no tuvo relaci6n con los parametros comportamen-
tales de los parasitoides emergidos, except el tiempo de aterrizaje sobre el hospedero. Las
hembras provenientes de hospederas grandes tardaron menos tiempo en aterrizar en com-
paraci6n con las hembras provenientes de hospederas pequeias. El tamaio del hospedero no
afect6 los diferentes indices de trayectoria evaluados, except la velocidad promedio de ca-
minar de D. longicaudata, las hembras provenientes de hospederos grandes mostraron un
incremento en su velocidad de caminar en comparaci6n con las hembras provenientes de
hospederos chicos y medianos.


Translation by the authors.







Florida Entomologist 92(3)


Diachasmimorpha longicaudata (Ashmead)
(Hymenoptera: Braconidae) is a koinobiont en-
doparasitoid native from the Indo-Australian re-
gion that attacks late instars of several fruit-in-
festing tephritid flies (Diptera: Tephritidae)
(Wharton & Gilstrap 1983). This parasitoid has
been employed in classical and augmentative bio-
logical control programs against several fruit
flies, including Bactrocera spp, Ceratitis capitata
(Wiedemann) and Anastrepha spp (Bess et al.
1961; Wharton & Gilstrap 1983; Eskafi 1990).
During the 1950s D. longicaudata was introduced
into Mexico (Jimenez 1963), and currently it is
mass-reared for augmentative biological control
of several species of Anastrepha (Sivinski et al.
1996; Montoya et al. 2000). One of the principal
reasons for its use as a biological control agent is
that its mass rearing has been developed success-
fully (Wong & Ramadan 1992). For instance, 50
million parasitoids are produced per week in the
mass-rearing program in Mexico (Cancino et al.
1996).
The quality of released individuals plays a fun-
damental role in a successful biological control
program (Van Lenteren 1991; Wong & Ramandan
1992). Although some techniques for quality eval-
uation of D. longicaudata have been developed
(Messing et al. 1993; Cancino & Yoc 1993; Purcell
et al. 1994), the search for new quality indicators
is still important because they may contribute in-
formation for quality improvement of released
parasitoids. The size of puparia of the parasitized
host may be a useful quality indicator of the indi-
viduals produced because it may have a direct re-
lationship with size of parasitoids. In several par-
asitoid species, body size is a reliable indicator of
parasitoid capacity as a biological control agent
(Godfray 1994; Van Lenteren 2002). In various
species of Trichogramma, large parasitoids are
more efficient than small ones (Greenber 1991;
Honda & Luck 2001).
The present study was undertaken to assess
the effect of the host body size on emergence, lon-
gevity, fecundity, and host location behavior of
mass reared D. longicaudata. We worked with 3
different sizes of a host ofD. longicaudata reared
in laboratory conditions. The results obtained
may be important in understanding the signifi-
cance of host size in relation to the quality control
ofD. longicaudata.

MATERIALS AND METHODS

Biological Material and Wasp Selection

Diachasmimorpha longicaudata and the host
Anastrepha ludens (Loew) were produced at the
"Moscafrut" mass rearing facilities located in
Metapa, Chiapas, Mexico according to procedures
described elsewhere (Cancino et al. 1996). The
parasitoids have been reared for more than 200


generations. Host larvae used for rearing the par-
asitoids were irradiated with 45 Gy to avoid emer-
gence of adults from unparasitized hosts. Mango
fruits (Mangifera indica L., var. Ataulfo), used as
an odor sources in host location experiments,
were selected from trees located in Tapachula,
Chiapas, Mexico. Fruits in trees were covered
with paper bags to avoid feral infestations. When
fruits were 3/4 ripe, 5 mangoes were placed into a
cage (1 x 1 x 1 m) and exposed for 15 min to 200
mated females ofA. ludens. Fruits with an aver-
age of 20 fruit fly third instars, about 15 d after
oviposition, were used in the following experi-
ments, which were conducted at 26 1C, 75 + 5
relative humidity, and a 12:12 h light: dark pe-
riod.
We selected different sizes ofA. ludens puparia
(14-d-old) to be parasitized by D. longicaudata in
order to obtain parasitoids from different size
hosts. Host puparia were selected from different
samples, measured, and put through the pupal
sizing and separating machine (FAO/IAEA/USDA
2003). The puparia were separated in 3 size
groups based on puparial diameter. Hereafter, we
called them large (diameter: 2.84 0.028 mm),
medium (2.43 0.032 mm), and small (2.02
0.031 mm) puparia. One lot of 30 female parasi-
toids that emerged from each size group was se-
lected and killed for measuring their hind tibia
length with an eyepiece micrometer.

Adult Emergence and Sex Ratio

Samples of 100 parasitized host puparia from
each size group were placed in plastic containers
(5 cm high x 7 cm diameter). The containers were
checked to determine adult parasitoid emergence
and sex-ratio. The experiment was performed 14
times.

Adult Parasitoid Longevity, Fecundity, and Flight Ability

Longevity of parasitoids was evaluated from
cohorts of 30 females and 15 males that emerged
from each size group; they were placed in Hawaii-
type cages (25 x 25 x 25 cm) (Wong & Ramadan
1992). Adult parasitoid longevity was evaluated
under 2 different conditions: those fed ad libitum
with honey and water or those deprived of food.
Longevity of individuals and their sex were re-
corded at 24-h intervals until the death of all
adults. The experiment was replicated 6 times.
Life expectative was estimated based on models
from Carey (1993).
Female fecundity was evaluated with cohorts
of 30 females and 15 males that emerged from
each size group, and were placed in Hawaii-type
cages, as above. The parasitoids were fed ad libi-
tum with honey and water. After the fifth day, 200
A. ludens last instars in artificial diet in Petri


September 2009







L6pez et al: Indicator of Quality in Mass-reared D. longicaudata


dishes (9.5 cm diameter x 0.5 cm depth) covered
were organdy cloth were placed into the cage and
exposed to the parasitoids for 2 h daily until the
last female of the parasitoid cohort died. After ex-
posure, the fruit fly larvae were washed from the
diet with water and placed in plastic containers (9
cm high x 5 cm diameter) with vermiculite for pu-
pation during the next 15 d. We recorded sex and
number of parasitoid adults that emerged. The
parasitoids that emerged were related with the
respective number of females alive each day. The
offspring production per female parasitoid was es-
timated in terms of gross fecundity (Xmx) and net
fecundity (Ro = 1lxmx) following the procedures
described by Carey (1993). The experiment was
performed 6 times.
Samples of 100 fruit fly puparia from each size
group after exposure to parasitoids were placed
inside PVC black cylinders (8 cm height x 10 cm
diameter) to evaluate the flight ability of emerged
wasps. The inside walls of the container were
coated with neutral powder to prevent parasitoids
from leaving the cylinders by walking. The cylin-
ders were placed in screened cages (60 x 60 x 60
cm) with a source of light (fluorescent tube, 75 w)
50 cm above the top of the cages. The cages were
checked daily and parasitoids that were able to fly
out of the cylinders were removed. After 10 d, the
number of parasitoid adults that remained within
cylinders (walkers) as well as the number of non-
emerged puparia were recorded. Flight ability
(percentage fliers) was estimated according to
Cancino et al. (2002). The experiment was repli-
cated 14 times.

Host Location Behavior

In the first experiment, we evaluated female
responses to long-range cues in a flight wind tun-
nel. The tunnel used was constructed of plexiglass
and measured 120 cm long, 30 cm high, and 30 cm
wide. A fan was used to pull air through the tun-
nel with a velocity of 0.4 m/s. Activated charcoal
filtered intake air. Illumination was provided by 2
fluorescent bulbs mounted 60 cm above the wind
tunnel that gave a light intensity of 230 lux. The
wasps were individually placed in a 5-cm high
plastic release container (4 cm i.d.) and allowed to
acclimatize to the wind tunnel room conditions for
at least 1 h before being observed. A mango fruit
infested withA. ludens larvae was used as an odor
source, placed in the center of the wind tunnel, 10
cm from the upwind end, and 8 cm above the wind
tunnel floor. Each observation began with placing
the release cylinder on a 14-cm high platform at
the downwind end of the tunnel and 1 parasitoid
was released. Tests lasted a maximum of 10 min
for each female. We recorded (frequency and time)
for taking flight, landing, and probing as defined
by Jang et al. (2000). Each female had only 1
flight opportunity and landing was only recorded


in case it was preceded by hovering. Treatments
were replicated 50 times for females from small
and medium size hosts, and 46 times for females
from large hosts. Tests were conducted in a ran-
dom order. In all experiments 5- to 8-d-old naive
female parasitoids were used.
In a second experiment, female response to
short-range cues was evaluated in an experimen-
tal arena composed of a white plastic box (20 cm
long x 15 cm wide x 5 cm high). A white filter pa-
per sheet (28 cm long x 22 cm wide, 127 g/m2, Per-
felter, Mexico city) was placed over the box
(Fig. 1). A rectangular area of 14 x 10 cm marked
in the center of the white filter paper was defined
as the observational zone. A video camera (SONY
model TRV 530, Tokyo) was focused on this zone
before the observations. A plastic container (3.7
cm diameter x 0.5 cm high) with 20 larvae ofA.
ludens (8-d-old) and 1 g of larval diet was at-
tached underneath the filter paper. This container
was placed at one edge of the observational zone.
A Petri dish (15 cm diameter x 0.5 cm high) con-
taining 40 mL of mango juice from infested fruits
was placed below the container with larvae.
Mango juice was important because without it,
wasps left the arena. The wasps of the 3 size
groups were individually placed in a plastic vial (5
x 1 cm) and they were allowed to acclimatize to
the arena room conditions for at least 30 min be-
fore being observed. Each observation began with
placing the vial containing the female in the ob-
servation zone at the extreme edge opposite
where host larvae were placed. Observation
stopped after 10 min or when the parasitoid at-
tempted to oviposit. The movements of the wasp
during the observation were recorded by the video
camera. The paper filter sheet and the container
with mango juice and larvae were changed after
each observation. All observations were carried




SGa Linhrl Lre



/-










Fig. 1. Schematic view of the arena used for evaluat-
ing the response of Diachasmimorpha longicaudata fe-
males to short-range cues.







Florida Entomologist 92(3)


out from 10 AM to 2 PM under 245 lux of light in-
tensity. Treatments (parasitoids from small, me-
dium, and large hosts) were replicated at least 10
times and were tested in a random order.
Video images were digitized on a personal com-
puter (1 frame every 40 ms) and the position of
the wasp in each frame was considered as point
coordinates on an X-Y grid. The image digitaliza-
tion program was developed by M. Lambin in the
Laboratory of Animal Behaviour Cognition of
Toulouse Sciences University, France. The digi-
tized search path was used to calculate total
length, total duration, duration of stops, mean of
stop duration, straightness, diffusion rate, mean
walking speed, and total walking speed (Henaut
et al. 2000, 2002).

Statistics

Data were analyzed with the Statistica Soft-
ware Package (version 6.1) (Stat Soft, Inc., 2003).
The relation between diameter and width of pu-
paria and adult size and hind tibia length of
emerged parasitoids was analyzed by correlation.
Data for adult emergence, longevity, fecundity,
and flight ability were analyzed by one-way anal-
ysis of variance (ANOVA). Bioassay data for long-
range cues (wind tunnel) were analyzed by G- test
and one-way ANOVA. Prior to statistical analysis,
data were checked for ANOVA assumptions and
transformed, if needed, to log (x + 1) or arcsine.
Significant differences were separated by Tukey
tests. Variances of sex-ratio data were still heter-
ogeneous after transformation, so randomization
one-way ANOVA (Manly 1991) was used to ana-
lyze those data. Bioassay data (trajectory data in-
dices) for short-range cues were analyzed by
Kruskall Wallis ANOVA by ranks test, and the
multiple comparisons between treatments were
made by Sprent (1993) procedure.

RESULTS

The diameter of fruit fly puparia was different
for the 3 groups used in this study (F = 705.8; df
= 2, 89; P < 0.001), with a positive relationship be-
tween diameter and width of puparia (R2 = 0.94, P
< 0.001).
There was a positive correlation between the
hind tibia length and the total body length of the
parasitoids that emerged from the 3 sizes of hosts
(R2 = 0.82; P < 0.001), with hind tibia measure-
ments as follows: (small = 1.45 + 0.02 mm, me-
dium = 1.86 + 0.02 mm, and large = 2.11 + 0.01
mm; F = 307.7; df = 2, 83; P < 0.001).

Adult Emergence and Sex Ratio

The size of host influenced the number of
wasps that emerged, and percent emergence of D.
longicaudata was greater in medium size hosts


compared to those that emerged from small and
large hosts (65.35 + 1.53, 46.25 + 2.15, and 49.59
+ 1.75, respectively, (F = 33.15; df = 2, 39; P <
0.001)). Host puparial size did not affect the ratio
of female to male wasps (F = 0.428; df= 2, 39; P =
0.134), although the ratio showed a trend toward
more females as size of host increased. Female to
male ratio averaged 1.82 0.27, 2.11 + 0.12 and
3.22 0.26 for wasps that emerged from small,
medium, and large hosts, respectively.

Adult Parasitoid Longevity, Fecundity, and Flight Ability

Host size affected life expectancy of unfed fe-
males (F = 11.21; df= 2, 15; P = 0.001), but not un-
fed males (F = 0.84; df= 2, 15; P = 0.44). Unfed fe-
males that emerged from medium and large hosts
lived longer than those from small ones (5.25
0.35, 5.46 + 0.29, and 3.63 0.25 d, respectively).
Unfed males that emerged from small, medium,
and large hosts lived 3.39 0.40, 4.78 + 0.34, 4.56
0.31 d, respectively. Host size did not influence
the life expectancy of females (F = 0.02, df= 2, 15,
P = 0.98) or males (F = 1.03, df = 2, 15, P = 0.38)
with food. Fed females emerging from small, me-
dium, and large hosts lived 19.23 + 0.79, 20.13
1.47, and 19.14 0.72 d, respectively. Fed males
emerging from small, medium, and large host
lived 13.81 + 1.40, 16.13 1.54, and 16.41 1.05
d, respectively.
The period of fecundity between different size
females was similar, 70% of the eggs were pro-
duced when females were 7-18-d-old (Fig. 2).
However, gross fecundity (F= 5.24; df = 2, 15; P =
0.01) and net fecundity rate (F = 14.57; df = 2, 15;
P = 0.0003) were affected by host size, being
higher in parasitoids that emerged from medium
and large hosts. The rate of gross fecundity was
53.6, 63.7, and 60.5 for wasps that emerged from
small, medium, and large hosts, respectively. The
rate of net fecundity was 37.4, 45.7, and 46.7 for
parasitoids that emerged from small, medium,
and large hosts, respectively. The offspring sex-ra-
tio was not affected by the host size (F = 1.67; df=
2, 15; P = 0.22).
Host size did not influence the flight capability
of parasitoids (F = 0.87; df= 2, 39; P = 0.42); flying
adults averaged 89.1 + 0.6, 89.78 0.8 and 90.5
0.9% for parasitoids emerging from small, me-
dium, and large hosts, respectively.

Host Location Behavior

The frequency and latency of the behavioral
activities performed by the 3 groups of parasitoids
are shown in the Table 1. None of the activities
evaluated were affected by host size (taking off: G
= 1.15, df = 2, P = 0.56; hovering: G = 2.27, df = 2,
P = 0.32; landing: 2.27, df = 2, P = 0.32; and prob-
ing: G = 3.56, df= 2, P = 0.17). Latency for taking
off (F = 0.29; df = 2, 89; P = 0.74) and probing (F =


September 2009







L6pez et al: Indicator of Quality in Mass-reared D. longicaudata


4-


3.5- a-o- small
--- medium

3 large


S2.5




2 15 -


1-


0.5-

0 I I 7 I I T T 1 I T
Q IV b, lb l) ,1Z N11 Nt ,ro N't, +
Age (days)

Fig. 2. Daily net fecundity of small, medium, and large Diachasmimorpha longicaudata females.


1.06; df = 2, 82; P = 0.35) was not influenced by
host size. However, latency for landing (time be-
tween take off and landing) was affected by host
size (F = 3.13; df= 2, 89; P = 0.048), with females
developing in large hosts spending less time to
land on source than those that emerged from
small hosts.
There were not clear differences between the
results of trajectory recorded (Table 2). The para-
sitoids emerging from different sizes of hosts had
similar response except in the mean walking
speed (H = 7.31;P = 0.02), with females emerging
from large hosts showing an increase in walking


speed compared to females from medium and
small hosts.

DISCUSSION

In the present study we found contrasting re-
sults about the influence of host size on the biolog-
ical and behavioral parameters of D. longicau-
data. The adult emergence ratio was affected by
host size and these results agree with those previ-
ously reported for this parasitoid species (Wong &
Ramadan 1992; Messing et al. 1993). We found
significantly more wasps that emerged from me-


TABLE 1. RESULTS OF SEARCHING BEHAVIOR PARAMETERS OF DIACHASMIMORPHA LONGICAUDATA FEMALES THAT
EMERGED FROM 3 DIFFERENT HOST SIZES.

Size Take off Landing Probing

Frequency (% of response)
Small 72 a 60 a 52 a
Medium 76 a 70 a 66 a
Large 76 a 74 a 70 a
Latency (sec)
Small 1.15 + 0.2 a 0.17 0.0 a 1.97 0.3 a
Medium 1.39 0.4 a 0.14 0.0 ab 1.27 + 0.1 a
Large 1.05 0.2 a 0.13 0.0 b 1.52 0.3 a

Values followed by the same letter are not significantly different for comparisons within columns.







Florida Entomologist 92(3)


TABLE 2. COMPARISON OF TRAJECTORY PARAMETERS OF DIACHASMIMORPHA LONGICAUDATA FEMALES THAT EMERGED
FROM 3 HOST SIZES.

Size

Parameter Small Medium Large P,

Total length (mm) 127.01 5.0 120.01 6.0 130.01 5.0 ns
Total duration (s) 89.01 9.0 76.01 13.0 67.01 5.0 ns
Duration of stops (s) 71.01 8.0 61.01 12.0 53.01 5.0 ns
Mean of stops duration (s) 10.72 1.2 9.71 0.8 9.11 0.5 ns
Straightness 0.81 0.0 0.81 0.0 0.81 0.0 ns
Diffusion 0.13 0.0 0.13 0.0 0.15 0.0 ns
Speed (mm.s-1) 1.71 0.2 2.00 0.2 2.32 0.2 ns
Walking speed (mm.s-1) 8.40 0.4 a 8.61 0.4 a 9.91 0.4 b

*Indicates the level of significance of Kruskal-Wallis ANOVA test.
ns, non-significant.
*significant at P = 0.05.


dium size hosts compared to those emerging from
small and large hosts. The low emergence rates
from small size hosts are in agreement with sev-
eral studies which have shown that small host
larvae are not preferred by D. longicaudata fe-
males and generally a greater proportion of males
emerged from such hosts (Messing et al. 1993;
Cancino et al. 2002). The low percentage of emer-
gence from large hosts contrasts with results ob-
tained by Messing et al. (1993), who found that
larger hosts commonly result in higher emer-
gence rates. The difference between our results
and those of Messing et al. (1993) may be due to
several factors including methodological and
rearing conditions. Host species used for rearing
D. longicaudata in the studies were not the same
and different hosts may provide qualitatively and
quantitatively different resources. In Telenomus
lobatus Johnson & Bin, the percentage eclosion
from the eggs of ('C., b ..., i., species was higher
than that from eggs of ('b.. i *....I species (Ruber-
son et al. 1989). The low emergence of parasitoids
from larger hosts in the present study may be due
to several factors, such as (1) larger hosts are less
abundant within the lot exposed to parasitization
and this may affect the probability to be encoun-
tered by ovipositing females, (2) larger larvae
may have a higher mobility than small and me-
dium size larvae enabling them to escape from
parasitization, and (3) larger hosts may be more
mature and not as suitable hosts for parasitoid
development. Lawrence et al. (1976) found a de-
cline in the suitability of Anastrepha suspense
(Loew) larvae for development ofD. longicaudata
when hosts were older than 5 d. Wong & Ra-
madan (1992) showed that a peak of overall emer-
gence of D. longicaudata is achieved sometime
during the middle third instar ofBactrocera dor-
salis (Hendel), after which emergence rates de-
cline again. Large host larvae that are close to pu-


pation may be related to low wasp emergence
(Lawrence et al. 1978; Wong & Ramadan 1992).
The sex ratio ofD. longicaudata did not signif-
icantly vary between the adults that emerged
from small, medium, and large hosts, which dis-
agree with previous reports on this species (Mess-
ing et al. 1993; Cancino et al. 2002). In contrast to
our results, several studies have shown that soli-
tary parasitoid wasps tend to lay male eggs in
small hosts and female eggs in large hosts (God-
fray 1994; Heinz 1998; Sagarra et al. 2001). Our
results may indicate that ovipositing females do
not discriminate between the different host lar-
vae quality, here based on host size. However, fac-
tors such as mortality, host species, host density,
mating, and competence with other foragers may
explain the results obtained (Godfray 1994). Ash-
ley & Chambers (1979) found that the sex ratio of
D. longicaudata did not remain constant and was
affected by the age and the density of ovipositing
females.
We found that the host body size only affected
the parasitoid longevity when they were deprived
of food; females from medium and large hosts
lived longer than those from small ones. Host
body size did not affect longevity ofD. longicau-
data females when food was provided ad libitum.
Hardy et al. (1992) found that larger individuals
of the bethylid parasitoid Goniozus nephantidis
Muesebeck lived longer than small individuals
when supplied with food, but the reverse was true
when parasitoids were starved. The fact that fe-
males from medium and large hosts lived longer
in absence of food may be due to these parasitoids
having more energy reserves than those from
small hosts. Several studies have reported a high
relationship between host size and parasitoid sur-
vival (Tillmon & Cate 1993; Jervis & Copland
1996; Sagarra et al. 2001). In parasitoids, as well
as other insects, large adult body size is often re-


September 2009







L6pez et al: Indicator of Quality in Mass-reared D. longicaudata


lated with increased resource carry-over from the
larval stage, and this is manifested as higher en-
ergy reserves (Riviero & West 2002; Ellers &
Jervis 2003).
The duration of the oviposition period of the
synovigenic D. longicaudata in the present study
ranged from 11.8 to 12.1 d and was similar to that
found previously for this species (Vargas et al.
2002). Interestingly, the oviposition period was
not affected by the body size of females, but body
size did affect the fecundity because medium and
large females were more fecund than the small
ones. In female parasitoids, fecundity is often cor-
related with body size, a measure of the resources
available to the developing larvae (King 1989). It
has been discussed that this relationship is par-
ticularly evident in proovigenic species, where the
maximal number of eggs an individual female can
lay is proportional to the amount of stored nutri-
ent reserves obtained during larval stage (Ueno,
1999). In synovigenic species, where egg produc-
tion should depend on availability of food for
adult females (Jervis & Kidd 1986; Heimpel &
Collier 1996), body size would not be always a
good indicator of female fecundity (Ueno 1999).
However, several studies have also shown that
there is a positive correlation between body size
and female fecundity in synovigenic parasitoids
(Godfray 1994).
This study showed that body size of D. longi-
caudata females did not affect most of the activi-
ties related to its host finding behavior, except the
latency for landing and the walking speed, which
are important parameters during host location.
Many other studies have shown that parasitoid
size influences important behavioral traits such
as host searching or host acceptance ability (Big-
ler 1989; Bourchier et al. 1993; Honda & Luck,
2001). Larger females of Trichogramma walk far-
ther per unity of time than their smaller counter-
parts (Bigler 1989; Honda & Luck 2001) and en-
counter more hosts (Kazmer & Luck 1995). In
contrast, Van Hezewijk et al. (2000) found that
the size of Trichogramma minutum Riley was not
a significant factor affecting either searching or
walking speed of wasps.
The lack of relationship between host body size
and some of the parameters evaluated in this
study may be due to several factors. Firstly, we
used females reared in the laboratory for more
than 200 generations and adaptation to the mass
rearing conditions is inevitable. Cancino et al.
(2002) compared the performance of mass reared
and wild strains of D. longicaudata in laboratory
conditions, and found that wild females were big-
ger, lived longer, and were more fecund than mass
reared females. The searching speed of a strain of
T minutum did not increase with age as com-
pared with another strain possibly as a result of
this trait being lost during more than 100 gener-
ations in culture (Van Hezewijk et al. 2000). Sec-


ondly, our study was performed in laboratory;
parasitoids may not behave in the same way in
the wild where abiotic and biotic factors fluctuate.
Thus, further studies are necessary to investigate
the performance of mass reared D. longicaudata
in field conditions.
In conclusion, this study showed that adult
emergence, longevity, fecundity, and walking
speed ofD. longicaudata females are affected by
host body size. A previous knowledge of these pa-
rameters is important in the adequate mainte-
nance of the mass-rearing and the field release of
this parasitoid. Thus, host puparia size could be
used as an indicator of the quality of emerged par-
asitoids.

ACKNOWLEDGMENTS

We are grateful with Javier Valle-Mora (ECOSUR)
for his statistical help, and Pablo Montoya (Programa
Moscafrut) for comments on a previous draft of this
manuscript.

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Florida Entomologist 92(3)


September 2009


RELEASES, DISTRIBUTION AND ABUNDANCE OF GRATIANA BOLIVIANA
(COLEOPTERA: CHRYSOMELIDAE), A BIOLOGICAL CONTROL AGENT OF
TROPICAL SODA APPLE (SOLANUM VIARUM, SOLANACEAE) IN FLORIDA

W. A. OVERHOLT1, R. DIAZ1, K. L. HIBBARD2, A. L. RODA3, D. AMALIN3, A. J. FOX4, S. D. HIGHT', J. C. MEDAL6,
P. A. STANSLY7, B. CARLISLE8, J. H. WALTER', P. J. HOGUEO1, L. A. GARY", L. F. WIGGINS12, C. L. KIRBY13
AND S. C. CRAWFORD12
1University of Florida, Fort Pierce, Florida

2Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Fort Pierce, Florida

3USDA/APHIS/PPQ, Miami, Florida

4Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville, Florida

'USDA/ARS/ CMAVE at Center for Biological Control, FAMU, Tallahassee, Florida

nUniversity of Florida, Gainesville, Florida

7University of Florida, Immokalee, Florida

'Cooperative Extension Service, Polk Co., Florida

'Cooperative Extension Service, Brevard Co., Florida

'oCooperative Extension Service, Okeechobee Co., Florida

"Cooperative Extension Service, Hardee Co., Florida

2Cooperative Extension Service, Hendry Co., Florida

1Cooperative Extension Service, Manatee Co., Florida

ABSTRACT

From 2003 to 2008, 176,643 Gratiana boliviana Spaeth (Coleoptera: Chrysomelidae) were
released in Florida as part of a biological control program targeting tropical soda apple
(TSA) Solanum uiarum Dunal (Solanaceae). The spatial distribution of releases was clus-
tered with more beetles released in south/central Florida than further north. A survey con-
ducted in the fall of 2008 found G. bolviana present at >70% of randomly selected locations
between 26 and 29 latitude, but no beetles were found at sites further north. The presence
of beetles and beetle damage were associated with smaller TSA plants and fewer fruits per
plant. The absence of beetles in northern Florida may be due to the fewer number released
in that area, but also could be influenced by land cover and climate.

Key Words: Biological control, tropical soda apple, Gratiana boliviana, establishment,
spread, invasive species

RESUME

Del 2003 hasta el 2008, se liberaron 176,643 individuos de Gratiana boliviana Spaeth (Co-
leoptera: Chrysomelidae) en la Florida como parte de un program de control biol6gico del
"tropical soda apple" (TSA) (Solanum viarum Dunal (Solanaceae)). La distribuci6n espacial
de las liberaciones fue agrupada con mas escarabajos liberados en el sur/central de la Flo-
rida que en areas mas al norte. Un sondeo realizado en otono de 2008 encontr6 G. bolviana
present en un >70% de los lugares seleccionados al azar entire la latitud de 26o y 29o, pero
ningun escarabajo fue encontrado en los sitios mas al norte. La presencia de los escarabajos
y el daho causado por ellos fueron asociados con plants de TSA mas pequenas y menos fru-
tos por plant. La ausencia de los escarabajos en el norte de la Florida puede ser debido al
numero menor de individuos liberados en esta area, pero tambi6n puede haber sido influen-
ciada por la cubertura de la tierra y el clima.







Overholt et al.: Distribution and Abundance of G. boliviana in Florida


Tropical soda apple (TSA), Solanum viarum
Dunal (Solanaceae), is a prickly, perennial weed
from South America that was first reported in
Florida in 1988 (Mullahey et al. 1993). The plant
quickly spread throughout Florida and into sev-
eral other states including Georgia, South Caro-
lina, North Carolina, Alabama, Georgia, Missis-
sippi, Tennessee and Texas (EDDMapS 2008,
The Plants Database 2008). TSA invades range-
lands, improved pastures, and natural areas
with an estimated 1 million acres infested in
Florida (Mullahey 1996). Although cattle do not
consume TSA leaf tissue, they readily consume
fruits, and in doing so vector seeds to new areas
in their digestive tracts (Brown et al. 1996). Cat-
tle ranchers in Florida spend an estimated $6.5
to $16 million annually to control TSA (Thomas
2007).
Exploration for classical biological control
agents of TSA was initiated in 1994 in South
America. One of the agents discovered in Ar-
gentina and Paraguay was Gratiana bolviana
Spaeth (Coleoptera: Chrysomelidae) (Medal et
al. 1996). Gratiana boliviana is a highly spe-
cialized herbivore (Gandolfo et al. 2007; Medal
et al. 2002); larvae and adults feed on foliage in
the upper third of the canopy of TSA plants, re-
sulting in a distinctive shotgun-hole feeding
pattern on leaves. Eggs are laid individually on
the upper or lower surfaces of leaves, and eclose
after about 5 d at 25oC. Larvae complete 5 in-
stars in 15-18 d before pupating on the under-
side of leaves (Diaz et al. 2008). The beetles en-
ter an adult reproductive diapause during win-
ter months from about Nov to Apr in central
Florida. During this period, beetles are found in
leaf litter under TSA plants (Overholt, unpub-
lished data). Gratiana boliviana was first re-
leased in Florida in 2003 in Polk Co. and has
since been released at many other locations in
the state.
During the summer of 2008, beetles were
found at a number of locations several kilome-
ters from where they had been released in pre-
vious years, and thus efforts were being ex-
pended travelling to locations to make new re-
leases, only to find that beetles were already
present. The primary objectives of the present
study were to document the numbers and loca-
tions of beetles released in Florida, and to esti-
mate the abundance and spatial distribution of
G. boliviana in Florida. This information will be
used to select future release sites. Additionally,
plant size, cover and number of fruits per plant
were measured at survey sites to examine pos-
sible impacts of G. boliviana on plant perfor-
mance. Finally, we investigated differences in
land cover and climate along a south-north gra-
dient which may influence the presence and
abundance of beetles at various latitudes in
Florida.


MATERIALS AND METHODS

Insect Rearing

Gratiana boliviana was reared by 3 institu-
tions at 5 locations in Florida: University of Flor-
ida (UF) at Gainesville, Fort Pierce and Immoka-
lee; Florida Department of Agriculture and Con-
sumer Services (FDACS) at Fort Pierce; and
USDA/APHIS in Miami. Beetles were reared on
whole live plants (30-60 cm in height) either in a
greenhouse, screenhouse or large outdoor screen
cages (2 m x 2 m x 2.5 m). Immature and adult
beetles were transferred to new plants every 2-4
weeks when plants began to exhibit signs of ex-
cessive feeding (approximately 50% of leaf tissue
consumed). Non-diapausing colonies were main-
tained over the winter at 2 locations (FDACS/Fort
Pierce and UF/Gainesville) by rearing insects un-
der a 14h:10h (L:D) photoperiod. Each spring,
field collected beetles were added to these colo-
nies, and to re-establish seasonal colonies at other
rearing facilities.

Releases

Property owners and land managers were in-
formed of the availability of G. bolviana as a TSA
biological control agent through Cooperative Ex-
tension Service agents, extension publications
(Medal et al. 2006, 2007), an article in a ranching
trade journal (Overholt et al. 2008), and by word
of mouth from neighbors. A database of releases
was maintained throughout the project and in-
cluded the date of release, geographic coordinates
of release sites, number of beetles released, TSA
density, and property owners' names. At the time
of release, property owners and land managers
were asked not to apply herbicides or mow release
areas to increase the likelihood of beetle estab-
lishment. In total, 176,643 G. boliviana were re-
leased in Florida between May 2003 and Oct
2008. Because of a few missing records, geo-coor-
dinates are available only for the release of
163,185 beetles on 374 occasions. Beetles were re-
leased at 340 different sites, some sites receiving
repeated releases (2-4). The vast majority of re-
leases were of adult beetles, although a few re-
leases were made by placing plants infested with
a mixture of life stages directly in the field. The
number of beetles released on each occasion
ranged from 25 to 2700 and averaged 439 20
beetles (mean SE) per release.

Statewide survey

From Sep 4 to Oct 30, 2008, an extensive sur-
vey was conducted to estimate the distribution
and abundance of G. boliviana throughout Flor-
ida. The survey was made in the fall because the
abundance of G. boliviana was typically highest







Florida Entomologist 92(3)


at this time of year (Overholt, unpublished
data). Because tropical soda apple is mainly a
weed of pastures and rangelands, the survey
was primarily conducted in counties that had
more than 10,000 head of cattle (USDA/NASS
2008). Three counties (Miami-Dade, Seminole,
and Union) with less than 10,000 cattle were in-
cluded in the survey. Random points (10) were
generated for each of the 40 counties included in
the survey with a random geospatial coordinate
generator included in the GIS software program
AWHERE Professional (AWHERE, Inc.,
Golden CO). Personnel conducting the survey
selected 3-5 random sites per county based on
land cover (pasture), presence of TSA, and ac-
cessibility. In a few counties, only 1 or 2 sites
were sampled due to difficulty in finding loca-
tions with TSA, and in 2 counties (Bradford and
Gilchrist), no TSA was found. In total, 113 sites
in 38 were surveyed (Fig. 1). At each site, 10
plants were sampled by walking a straight line
through the center of the infested area and se-
lecting a plant every 4-5 steps. The following pa-
rameters were measured or estimated for each
selected plant:
*Height from the ground to the highest
foliage.
*Diameter of the crown at the widest
point.
*Number of fruit.
*Plant cover (visual estimate; scale of 1-5
with 1 = 0-19% of surface area under the
plant canopy covered, 2 = 20-39% cover, 3
= 40-59% cover, 4 = 60-79% cover, and 5 =
>80% cover.


Legend
Density of release sites
0 0.004951487
S0.005 0010
S0.010 -0.015
0.015-0.020
0.020 0.025
- 0.025 0.030
S0030 0.035
S0.035 0.040
0.040 0.045


Fig. 1. The spatial distribution of Gratiana boliviana
releases in Florida from 2003-2008. Density estimates
are interpolated values in units of releases/m2.


*Damage due to G. boliviana (visual esti-
mate; scale of 0-5 with 0 = no damage, 1
= 1-19% of leaf tissue consumed, 2 = 20-
39% consumed, 3 = 40-59% consumed, 4
= 60-79% consumed, and 5 = >80% con-
sumed.
*Number of G. boliviana larvae, pupae,
and adults.

Data analyses

All spatial analyses were conducted with
ArcMap 9.2 (ESRI, Redlands CA). Nearest
neighbor analysis was conducted with the 'Spa-
tial Statistical Tools' to examine the spatial dis-
tribution of release points. This tool calculates a
Z statistic to test the hypothesis that points are
randomly distributed in space. The average dis-
tance between nearest release sites was calcu-
lated with the 'Calculate Distance from Neigh-
bors Utility'. The average distance between sur-
vey sites and the nearest release site was calcu-
lated by the 'Near' tool. The density of release
points was analyzed with the 'Point Density'
tool in 'Spatial Analyst Tools' with a neighbor-
hood setting of 20 km.
The effect of latitude and sampling date on the
number of G. boliviana/plant was examined with
simple linear regression. To determine whether
there was a latitudinal bias in the temporal pat-
tern of sampling, latitude was regressed on sam-
pling date. An interpolated surface ofG. boliviana
intensity across the surveyed areas in Florida
was generated by the Inverse Distance Weighted
(IDW) method in the Geostatistical Analyst, with
6 nearest neighbors and a power setting of 2. IDW
is a simple deterministic interpolator which esti-
mates values at un-sampled locations as
weighted averages of observed values within the
designated neighborhood, with weights decreas-
ing as distance increases. The influence of the in-
tensity of releases (number of beetles released in
an area) on the number of beetles found at survey
sites was examined by creating raster datasets
(20 km x 20 km) from the IDW interpolated sur-
face of G. boliviana intensity and a similarly in-
terpolated surface of numbers released. Grid cell
values of beetle intensity and release density
were extracted from the 2 raster datasets and a
multiple regression was performed to examine
the relationship between G. boliviana intensity
and 2 independent variables; density of releases
and latitude.
To visualize the possible influence of cold
temperatures on the distribution of G. bolivi-
ana, the IDW method with 15 neighbors and a
power setting of two was used to generate a sur-
face of the first occurrence of freezing tempera-
ture (<0C) in the winters between 2003, when
beetles were first released, and 2008. Weather
data from 120 climate stations in Florida were


September 2009







Overholt et al.: Distribution and Abundance of G. boliviana in Florida


extracted from the Applied Climate Informa-
tion System (CLIMOD 2008).
Pasture cover in 6 latitudinal zones of Florida
(250-260, 26o-27o, 27o-28o, 28o-29o, 290-300, and
300-310) was examined by constructing boundary
polygons for each zone. Vegetation cover for Flor-
ida was acquired from the Habitat and Landcover
raster dataset (30 m x 30 m resolution), which
classified ground cover from remote sensing data
into 43 categories (FFWCC 2004). A GIS layer in-
cluding 2 of the categories, unimproved pasture
and improved pasture, was extracted from the
dataset and converted from raster to polygon. The
intersection of each zone and pasture cover was
used to calculate the total amount of pasture in
each zone, and the mean, minimum and maxi-
mum patch size per zone. Mean patch size was
compared between zones with analysis of vari-
ance and means separated with LSD by the
PROC GLM procedure of SAS (SAS Institute
2001).
Multiple linear regressions with backward
elimination of non-significant (P > 0.05) indepen-
dent variables were performed to examine the
relationships between plant performance vari-
ables (plant height, diameter, cover, and number
of fruit) and the independent variables of G.
bolviana damage score, site latitude, and sam-
pling date (model 1), and the number ofG. boliv-
iana/plant, latitude, and sampling date (model
2). Latitude and sampling date were included in
the models to estimate the effects ofG. boliviana
on plant performance independent of these vari-
ables. All regressions were performed with
PROC REG procedure of SAS (SAS Institute
2001).

RESULTS

The average distance between nearest release
sites was 4.4 + 0.7 km, with a minimum of 2.3 km
and a maximum of 217 km. The distribution of re-
lease sites was clustered (Z = -26.1, P < 0.01) with
several patches of high intensity as indicated in
Fig. 1. More beetles were released between 270
and 290 than in areas further north or south
(Fig. 2). The number of beetles released per year
increased with time and peaked at 86,205 in 2008
(Fig. 3).
Gratiana boliviana was found at 48% (54/
113) of the surveyed sites, with intensity aver-
aging 3.2 0.6 beetles/plant (range 0-38.2 bee-
tles/plant). Damage due to G. boliviana was
found at 2 sites where no beetles were found.
Latitude and sampling date affected the pres-
ence of beetles with significantly fewer beetles
as latitude increased (F,,111= 10.6, P=0.0015) and
as the date of sampling increased (F,,, = 6.23, P
= 0.014). The regression between latitude and
sampling date was significant (F1,,, = 4.09, P =
0.046), with sampling occurring slightly later in


90,000
80,000
70,000
S60,0 W
0 s,0o0
j 40,000
30,000
20,000

25'-26 26&-27' 27'-28 28-29* 29'30- 3D-31
Lorudbul tne

Fig. 2. Number of Gratiana boliviana released in
Florida by latitudinal zone.





the season as latitude increased (0.16 d later
with each increase of 1 degree latitude). The av-
erage date of sampling at latitudes below 29
was Sep 27 ( 1.7 d), and at latitudes greater
than 29o was Oct 1 ( 2.7 d). The average dis-
tance between sites where beetles were present
and the nearest release site was 6.3 0.9 (SE)
km, and the furthest that beetles were found
from a release site was 32.4 km. On average,
there were 488 62.6 d between the date of re-
lease at the nearest point and the date of the
survey at sites where beetles were found. As-
suming that beetles arrived at the survey sites
from the nearest release site, the average dis-
tance beetles traveled per year since their re-
lease was about 4.7 km. The beetles found at
32.4 km from the nearest release site had moved
about 8.1 km/year since their release.
Beetles were present at 77% (10/13) of sites be-
tween 26 and 27 latitude, 79% (30/38) of sites be-
tween 27 and 28 and 54% (14/26) of sites between
28 and 29. No beetles were found at 32 sites sur-


100,000
90.000
so.oon
70,000
60,000
50,000
40,000
so.ooo
30,000
20,000
o,000
0


2003 2004 2005 2006 2007 2008
rear


Fig. 3. Number of Gratiana boliviana released per
year in Florida.







Florida Entomologist 92(3)


veyed north of 29 latitude, at 2 sites sampled in ex-
treme south Florida or in a small area of the west
coast near Sarasota. The northernmost occurrence
of G boliviana was at 28.77 in Seminole Co. near
the town of Sanford. The interpolated intensity sur-
face indicates highest densities along a band run-
ning from approximately Naples on the west coast
to Fort Pierce on the east coast, and northward on
the east coast in Volusia county (Fig. 4). It should be
noted that the spatial interpolation was based on
data from 38 counties, but projected into counties
not sampled. However, the beetle was not predicted
to occur in any counties that were not sampled, and
thus, the interpolation is likely a conservative esti-
mation of the actual distribution. The earliest occur-
rence of freezing temperatures in Florida during the
period of study (2003-2008) is shown in Fig. 5.
The number of beetles released in 20 km x 20 km
cells, and the latitude of the cells influenced beetle
intensity in those cells with the number of beetles
increasing as the number released increased (par-
tial regression coefficient = 0.69, t= 11.5, P < 0.0001)
and decreasing as latitude increased (partial regres-
sion coefficient = -0.0004, t = -13.8, P < 0.0001).
The amount of pasture cover in different zones
ranged from 2,205 ha between 25 and 26 lati-
tude to 500,674 ha between 27 and 28 latitude.
The mean patch size ranged from 2.91 ha between
28 and 29 to 9.34 ha between 27 and 28. Patch
size was smaller in the 280-29 zone than all other
zones except 250-26, where there was very little
pasture (F5,, 232= 28.5, P < 0.0001, LSD) (Table 2).
Sampling date was not significant (P > 0.05)
and therefore removed from multiple regression
models of plant parameters against G. boliviana
damage estimates and beetle numbers, but lati-


Gratiana bolivianalplant
I o



>10- 15
>15 20
>20- 25
>25- 30
>30


Fig. 4. Interpolated values of Gratiana boliviana in-
tensity based on a state-wide survey conducted in the
fall, 2008.


o G. baolvian abent
A G. bolvana present
Date o eaullet frost 2003-200

Oct 22 -Nov 6
Nov6 -Nov 19
S Nov20-Dec3
Dec4 -Dec 17
S Dec 1 -Dec 31
SJan I Jan 14
SJa.n 16..Jn 2


- 31<








- 27

- 26*

25-


Fig. 5. Date of earliest frost in Florida during the pe-
riod 2003-2008 and locations where Gratiana boliviana
was found to be present or absent during a state-wide
survey conducted in fall, 2008.


tude was significant and retained. Plant height, di-
ameter, and cover were all negatively affected by
the number of G. boliviana/plant, but the number
of fruit was not different. As the G. boliviana dam-
age score increased, plant height, diameter, cover,
and the number of fruit were reduced (Table 1).

DISCUSSION

The survey demonstrated that G. boliviana is
firmly established in south/central Florida, and
has dispersed from the original release sites. TSA
plants were smaller and produced fewer fruit as
the number of beetles and damage increased,
clearly suggesting that beetles are having a nega-
tive impact on TSA plants. The absence of beetles
at the southernmost survey sites in Miami-Dade
and Broward counties, and in the west coast area
near Sarasota, is not surprising as no beetles
were released in these areas prior to the time of
the survey. However, the absence of beetles and
beetle damage in northern peninsular Florida
was unexpected, particularly considering evi-
dence that beetles are established at some loca-
tions in northern Florida (Medal et al. 2007), and
in southern Georgia and Alabama (Roda, unpub-
lished data). Moreover, cold tolerance studies sug-
gested that G. boliviana could establish as far
north as 32-33 (central Georgia) (Diaz et al.
2008). Thus, it appears that direct effects of cold
temperatures may not explain the absence of bee-
tles at surveyed locations in north Florida. At
least 4 non-mutually exclusive hypotheses could
explain the absence of G. boliviana at sites sur-
veyed in north-central Florida. First, fewer bee-
tles were released in this area compared to south-
ern locations, and therefore it may be simply a
matter of time until population densities increase
and beetles spread in northern areas. The signifi-


September 2009








Overholt et al.: Distribution and Abundance of G. boliviana in Florida


0



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a,

a,

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8
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P,










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cant relationship between the number of beetles
released in 20 km x 20 km grid cells and the num-
ber of beetles found at randomly selected sites in
those cells provides support for this hypothesis.
A second possible explanation is that the sur-
vey was conducted slightly later in the year at
more northern latitudes. Gratiana boliviana en-
ters an adult reproductive diapause in the fall,
during which time they are found in leaf litter un-
der TSA plants. Sampling at northern latitudes
may have occurred after beetles had entered dia-
pause, such that they were not found actively
feeding on TSA leaves. However, differences in
sampling dates between latitudes were small,
and therefore we do not believe that the presence
of beetles was greatly affected. Moreover, damage
characteristic of G. bolviana feeding was not
found at any sites north of 29 latitude, strongly
suggesting that beetles were absent from north-
ern survey sites for at least several weeks prior to
the survey.
Landscape features may provide an explana-
tion for the lack of beetles at northern Florida lat-
itudes. TSA is primarily an invader of pastures/
rangelands, and the spatial distribution of habi-
tats suitable for TSA may vary with latitude. Com-
parison of patch size of pasture between latitudi-
nal zones does not provide support for this hypoth-
esis, as patch size was lower in the 280-29 latitu-
dinal zone, where beetles were found, compared to
zones further north where they were absent. How-
ever, the presence of pasture is not equivalent to
the presence of TSA. Unfortunately, there is no in-
formation available on the spatial distribution of
TSA in Florida at a resolution that would allow an
examination of patch size, and connectivity be-
tween patches. Moreover, the spatial distribution
of TSA is likely dynamic. The plant can rapidly col-
onize new areas through transport in the digestive
tracks of livestock and wild mammals (Mullahey
et al. 1998), and may disappear from areas due to
active management by mowing and herbicide ap-
plication, or from natural events such as flooding
(Mullahey et al. 1998).
The most likely explanation for absence of G.
boliviana at northern survey sites may be asyn-
chrony in seasonal phenologies of TSA and G. bo-
liviana. Freezing temperatures do not usually kill
TSA plants, but all above-ground parts die back
(Mullahey et al. 1998); the only food source for the
beetles. Gratiana boliviana enters diapause in
the fall as day length decreases, although the crit-
ical day length for diapause induction is not yet
established. If a freeze arrives prior to beetles en-
tering diapause, immatures would certainly be
killed as they do not migrate from the plant on
which eggs that they hatched from were laid, and
food availability for adults would be greatly di-
minished. The earliest occurrence of freezing tem-
peratures during the period of the study (2003-
2008) coincides with the time of year when bee-


00 00
V V
vv




CO C d0
00 00 Cj ~







Florida Entomologist 92(3)


September 2009


TABLE 2. PASTURE COVER IN DIFFERENT LATITUDINAL ZONES OF FLORIDA.

Latitudinal Number Mean patch Minimum Maximum Total
zone of patches size SE (ha)* patch size (ha) patch size (ha) pasture (ha)

25 -26 505 4.37 1.lab 0.06 454 2,205
26 -27 22,806 5.66 0.5a 0.001 8377 128,997
27-28 30,306 9.34 0.7a 0.0006 22,782 500,674
28 -29 86,231 2.91 0.2b 0.0007 14,657 303,820
29 -30 34,435 4.64 0.3a 0.0001 5780 178,663
30 -31 18,501 7.34 0.3a 0.01 2129 135,869

*Means followed by the same lower case letter are not statistically different (LSD, P < 0.05).


tles enter diapause, and may be a factor that in-
fluences the distribution of G. boliviana. Simi-
larly, the increase of TSA in the spring, through
regrowth from root tissue and seed germination
occurs later in more northern areas of Florida
than in the south, where plants may even con-
tinue growing throughout the winter in some
years (Overholt, unpublished data). If G. bolivi-
ana diapause terminates before TSA increases in
abundance in the spring, food would be scarce and
population increase of beetles may be affected. A
lack of synchrony between diapause and climate
has been shown to negatively affect the distribu-
tion of Diorhabda elongata deserticola (Brulle)
(Coleoptera: Chrysomelidae), a biological control
agent released in the western US for control of
Tamarix spp. The insect established north of 38
latitude, but not further south due to day length
not being long enough to maintain a non-diapaus-
ing population for more than 1 generation (Bean
et al. 2007; Lewis et al. 2007). Bean et al. (2007)
also speculated that there was a lack of synchrony
between univoltine populations south of 38 and
their host plants that negatively affected sur-
vival.
The fall 2008 survey provides information on
the distribution and abundance of G. boliviana in
Florida that will be useful for selecting locations
for future releases. With the exception of the area
near Sarasota where no beetles were found, no
further releases appear to be warranted in cen-
tral/south Florida below 29 latitude, where bee-
tles were found at > 70% of release sites. However,
releases should be made in the spring/summer of
2009 at locations north of 29.


ACKNOWLEDGMENTS

We thank Larry Markle, Jackie Markle, Ben Anufo-
rum (University of Florida), Joe DeMarco, and Jose
Diaz (FDACS/DPI) for assistance in rearing, release,
and field evaluation of G. boliviana. For assistance with
field surveys, we express appreciation to John Maes,
Stuart Reitz, Chirs Albanese, Michael Getman (USDA-
ARS, Tallahassee, FL), Derek Barber (Cooperative Ex-
tension Service, Columbia Co., FL), Shep Eubanks (Co-
operative Extension Service, Holmes Co., FL), Daniel


Fenneman (Cooperative Extension Service, Madison
Co., FL), Larry Halsey (Cooperative Extension Service,
Jefferson Co., FL), Doug Mayo (Cooperative Extension
Service, Washington Co., FL), Elena Toro (Cooperative
Extension Service, Suwannee Co., FL), and Chris Vann
(Cooperative Extension Service, Lafayette, Co., FL). We
thank the Florida Department of Agriculture and Con-
sumer Services, Division of Plant Industry, and the
United States Department of Agriculture, Animal Plant
Health Inspection Service for financial support for the
tropical soda apple biological control program. Mention
of trade names or commercial products in this publica-
tion is solely for the purpose of providing specific infor-
mation and does not imply recommendation or
endorsement by the U. S. Department of Agriculture.


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Florida Entomologist 92(3)


September 2009


HOST SPECIFICITY OF ANTHONOMUS ELUTUS (COLEOPTERA:
CURCULIONIDAE), A POTENTIAL BIOLOGICAL CONTROL AGENT OF
WETLAND NIGHTSHADE (SOLANACEAE) IN FLORIDA

J. MEDAL', N. BUSTAMANTE', J. BARRERA2, O. AVILA', J. MONZON4 AND J. CUDA1
'University of Florida, Department of Entomology and Nematology, Gainesville, FL 32611

2Colegio de la Frontera Sur, Tapachulas, Chiapas, M6xico

3Ministerio de Agricultura, San Jos6, Costa Rica

4Museo de Entomologia, Guatemala, Guatemala

ABSTRACT

Multiple-choice and no-choice tests were conducted at the Department of Agriculture-Divi-
sion of Plant industry Quarantine facility in Gainesville to determine the specificity of the
Mexican/Central-American flower-bud weevil Anthonomus elutus Clark, a candidate for bi-
ological control of Solanum tampicense Dunal (wetland-nightshade) in Florida. Eighty-
seven plant species in 17 families were included in the feeding-oviposition multiple-choice
tests including the target weed and the 6 major cultivated Solanaceae Capsicum annuum L.
Capsicum frutescens L., Lycopersicon esculentum Mill., Nicotiana tabacum L., Solanum mel-
ongena L., and Solanum tuberosum L. Plant bouquets with flower-buds of 8 to 10 plant spe-
cies randomly selected, including always S. tampicense, were simultaneously exposed to 20-
26 A. elutus adults during approximately 2 weeks. Observation of oviposition and feeding
were made twice a week. No-choice host-specificity tests were conducted with A. elutus
adults on potted plants in cages made of clear-plastic cylinders. TenA. elutus adults were ex-
posed to 30 plant species individually tested during 2 weeks. Plant species in each test were
replicated 3-4 times. Results indicated thatA. tenebrosus fed and laid eggs only on the target
weed. No eggs were deposited on any of the other 86 plant species tested. The host-specificity
tests indicated that a host range expansion ofA.elutus to include any of the major cultivated
Solanaceae species is highly unlikely. A petition for field release in Florida was submitted to
the Technical Advisory Group for Biological Control Agents of Weeds (TAG) in Dec 2008.

Key Words: host-specificity tests, weed biological control, wetland-nightshade, Solanaceae

RESUME

Pruebas de ovoposici6n y alimentaci6n (con y sin elecci6n), se realizaron para evaluar la es-
pecificidad del picudo del bot6n floral, de origen Mexicano-Centroamericano, Anthonomus
elutus Clark, como agent potential para el control biol6gico de Solanum tampicense Dunal
en Florida, USA. Las pruebas se efectuaron en la cuarentena del Departamento de Agricul-
tura de la Florida-Divisi6n de Industria de Plantas en Gainesville. Ochenta y siete species
de plants, en 17 families, fueron incluidas en las pruebas de especificidad de elecci6n mul-
tiple, incluyendo la maleza objetivo y las seis plants cultivadas pertenecientes a la familiar
Solanaceae mas importantes: Capsicum annuum L. Capsicum frutescens L., Lycopersicon es-
culentum Mill., Nicotiana tabacum L., Solanum melongena L., y Solanum tuberosum L. En
cada prueba se utilizaron racimos florales de ocho a diez plants escogidas al azar inclu-
yendo siempre la plant objetivo las cuales fueron expuestas simultaneamente a 20-26 adul-
tos de A. elutus durante aproximadamente dos semanas. Registros de alimentaci6n y
ovoposici6n fueron realizados dos veces por semana. Pruebas de alimentaci6n/ovoposici6n
sin elecci6n fueron tambi6n realizadas usando plants creciendo en macetas yjaulas cilin-
dricas hechas de plastico claro transparent. Diez adults deA. elutus fueron expuestos a 30
species de plants en forma individual durante dos semanas. Cada prueba tuvo 3-4 repeti-
clones. Los resultados indicaron que A. elutus se aliment6 y coloc6 posturas unicamente en
la maleza objetivo wetland-nightshade. Ninguna postura fu6 depositada en las otras 86 es-
pecies de plants evaluadas. Las pruebas de especificidad indicaron que la posibilidad deA.
elutus de llegar a ser una plaga de las Solanaceae cultivadas es muy remota. La solicitud a
TAG para liberar el picudo en Florida fue hecha en Diciembre 2008.


Translation by the authors.







Medal et al.: Host Specificity ofAnthonomus elutus


Wetland-nightshade (also known as aquatic
soda apple), Solanum tampicence Dunal (Solan-
aceae) is a prickly perennial shrub that has the
potential to become a serious weed in Florida wet-
land habitats (Fox & Bryson 1998; Coile 1993). A
synonym that has been used in the past is
Solanum houstonii Dunal but the currently ac-
cepted name is Solanum tampicense. It was first
recorded in the Dry Tortugas (Monroe County),
Florida in 1974, and found 9 years later (1983) in
mainland southwest Florida (Charlotte County).
Two years later, it was reported in Highlands
County (at Fisheating Creek Wildlife Refuge),
and it was detected at Glades, De Soto, and Lee
counties in the early 1990s. How this plant ar-
rived in Florida in the 1970s is not known but it is
spreading rapidly, forming moderate to dense
stands replacing native vegetation mainly in wet-
land areas, open marsh, and in shaded woody ar-
eas (oak hammocks) at the edge of rivers. Wet-
land-nightshade infests a significant portion of
the Peace River system. Currently, this weed is
spreading into adjacent areas in at least 2 loca-
tions. The invaded wetland areas in Florida have
been estimated at 200-300 ha and along at least
100 km of river (Fox & Bryson 1998; Coile 1993).
This exotic weed was placed on the Florida Nox-
ious Weed List, and on the Federal Noxious Weed
List in 1998/99.
Wetland-nightshade is native to southern
Mexico, Guatemala, Belize (Gentry & Standley
1974), the Caribbean region (Sauget & Liogier
1957), Nicaragua, Costa Rica (Medal personal ob-
servation), and probably has also spread into
other regions including the northern part of
South America. This plant has enormous repro-
ductive potential through vegetative parts (sec-
tions of stems) as well as seed production. A single
plant growing in an open sunny area can produce
up to 8,620 seeds during an annual growing sea-
son that occurs from early spring to fall in south-
ern Florida (Fox & Wigginton 1996b; Langeland
& Burks 1998). Dispersion of the seeds may occur
through wildlife feeding on the fruits as is com-
mon with other Solanum species (Medal et al.
2002; Medal & Cuda 2000). Since the mid 1990s,
some initial research efforts involving state agen-
cies have focused mainly on chemical weed con-
trol along with a few studies on the biology and
ecology of the weed (Fox & Wigginton 1996a, b).
Currently, recommended management prac-
tices for wetland-nightshade in Florida wetlands
are based on herbicide applications (Langeland &
Stocker 1997). Herbicide treatment of this species
is labor intensive, expensive, and may result in
damage to non-target species. Many of the in-
fested areas are not accessible by boat or other
equipment, which makes herbicide applications
even more difficult. Biological control research ef-
forts with insects and nematodes against wet-
land-nightshade were initiated by University of


Florida researchers who included this plant in the
host-specificity tests against tropical soda apple,
Solanum viarum Dunal, a highly invasive terres-
trial congener of wetland-nightshade native to
the southern part of South America (Cuda et al.
1998, 2002a; Medal et al. 1999). Several leaf-feed-
ing insects were tested, including 3 chrysomelid
beetles (Metriona elatior Klug, Gratiana bolivi-
ana Spaeth, and Platyphora sp.) that were found
feeding on tropical soda apple in South America
(Medal et al. 1996, 2003, 2007). Quarantine-labo-
ratory screening tests with these insects indi-
cated only minor feeding damage and no larval
development on wetland-nightshade (Medal et al.
1999, 2002; Medal et al. unpublished data). Host-
feeding tests were also conducted from 1995 to
1997 with a foliar and stem-galling nematode Di-
tylenchus phyllobius (Thorne) Filipje, and two
leaf-beetles Leptinotarsa texana Schaeffer and
Leptinotarsa defecta Stal from Texas (Cuda et al.
1998, 2002b). Although the nematode and the 2
Leptinotarsa leaf-beetles caused significant dam-
age to silverleaf nightshade Solanum elaeagnifo-
lium Cav. (their preferred host plant), they were
unable to survive on wetland-nightshade in star-
vation tests. We initiated a classical biological
control project against this exotic weed in the fall
of 2005 because importation of specialized natu-
ral enemies that attack wetland-nightshade in its
native range, Mexico-Central America-Caribbean
region, may prevent or reduce the spread of this
weed. Field surveys of potential biocontrol agents
in the native range of wetland-nightshade, and
host-specificity tests with the selected agents
were conducted in southern Mexico and Central
America, and at the Gainesville quarantine facil-
ity during a 3-year period.
Wetland-nightshade can grow up to 5 m tall
sometimes forming dense stands mainly in
swamps/wetland areas or along river margins.
Stems and leaf veins are densely covered with
short (0.5 cm long) recurved or straight spines.
Leaves alternate in pairs or individually with a
petiole up to 3 cm long. Leaf blades are longer
(5.5-16 cm) than wide (2.2-5.5 cm) with lobed or
indented margins. The corolla is white with an-
thers up to 4 mm long. Fruits are relatively small
(up to 1 cm in diameter), and rounded, forming
small clusters. Fruits are initially green, turning
orange and finally red when ripe. Each fruit con-
tains 10-60 yellowish, flat round seeds 2-2.5 mm
long. Seeds can be viable for at least 12 months.
Wetland-nightshade can reproduce by forming
new stems from the stem base or regrowing from
crowns. It is shade tolerant but will grow in full
sun (Fox & Wigginton 1996b; Gentry & Standley
1974; Langeland & Burks 1998).
The flower-bud weevil, Anthonomus elutus
Clark (Coleoptera: Curculionidae) was tested as a
biological control agent of wetland nightshade in
Florida. This insect was first found and collected







Florida Entomologist 92(3)


on wetland nightshade in Monterrico, Departa-
mento Santa Rosa, Guatemala (N:1354' 50.7",
W:9032' 26.1") in Feb 2006. The identity ofA. elu-
tus was confirmed by Drs. Wayne Clark (Auburn
University, AL) and Germano Rosado Neto (Uni-
versidade Federal do Parand in Curitiba, Brazil).
Voucher specimens of A. elutus are deposited at
Auburn University, Alabama, at the Univer-
sidade Federal do Parand- Curitiba campus, Bra-
zil, at the Colegio de la Frontera Sur, Tapachula,
Chiapas, Mexico, at the Ministerio de Agricultura
in San Jose, Costa Rica, and at the Florida State
Collection of Arthropods, Division of Plant Indus-
try in Gainesville, Florida. This species does not
have a common name in Central America. The
only known host plant of A. elutus in Central
America is S. tampicense. The distribution of A.
elutus in southern Mexico and Central America is
not well known.
In this paper we report the results of the host-
specificity tests conducted at the Florida Depart-
ment of Agriculture-Division of Plant Industry
Quarantine facility in Gainesville with the flower-
bud weevil A. elutus as a potential biological con-
trol agent of the non-native weed wetland-night-
shade.

MATERIALS AND METHODS

Host-specificity tests with A. elutus adults
were conducted from Feb 2006 to Nov 2008 at the
quarantine facility in Gainesville, Florida. Adults
were first collected Feb 2006 on S. tampicense
plants in Alajuela Province, Costa Rica, and
Santa Rosa, Guatemala, and introduced into
quarantine in Florida where they were placed
with S. tampicense clusters of leaves/flower-buds
in screened plastic containers. These field-col-
lected adults and their offspring born in quaran-
tine were used for host-specificity testing.

Multiple-Choice Feeding and Oviposition Tests

Eighty-seven plant species in 17 families were
included in the feeding and oviposition preference
tests in Gainesville-quarantine (Table 1). The
plants tested included 62 species in the family of
the target weed (Solanaceae) of which 33 were
from the genus Solanum and 29 from 15 other
genera that include plants of agricultural and/or
ecological importance. Eight species representing
3 families (Boraginaceae, Convolvulaceae, Po-
lemoniaceae) related phyllogenetically very
closely to the Solanaceae, in the same order Po-
lemoniales (according to Heywood 1993), or in the
Solanales (based on the most recent classification
made by Chase 2003) were also included. Twenty-
five plant species representing 16 families, most
of them with an economic and/or environmental
value in North America, were tested. The major
target weed (S. tampicense) and other 9 plant spe-


cies in the Solanaceae were tested at least 3-
times. They include Solanum donianum Walpers
that is in the list of Florida threatened plants
(Coile 1998); 2 non-native invasive weeds
(Solanum viarum Dunal, Solanum torvum Sw.);
and the 6 major cultivated Solanaceae Capsicum
annuum L. (bell-pepper), Capsicum frutescens L.
(chile), Lycopersicon esculentum Mill. (tomato),
Nicotiana tabacum L. (tobacco), Solanum melon-
gena L. (eggplant), and Solanum tuberosum L.
(potato).
The plant species tested were obtained from lo-
cal nurseries, from fields were they grew natu-
rally, or were grown from seeds obtained from a
commercial nursery. All plants were grown before
testing in 1-gallon pots with a mixture of 2/3 parts
top soil and 1/3 part sand. The plants were main-
tained out-of-door in a screened area (50% shade),
provided with water as needed, and fertilized ev-
ery 2-3 months. Cut branches or bouquets (10-15
cm) with foliage/flower buds from the potted
plants were healthy, not fed upon by herbivorous,
and had approximately similar amounts of foliage
and numbers of flower buds (5-10) for each plant
species in a given test. The cut branches or bou-
quets of the test plants were placed in 30-mL
clear plastic cups filled with water. The top of the
cup was covered with a plastic lid that had a small
hole punched in the middle to insert the bouquet.
The water cups with bouquets were placed indi-
vidually on each cell of an egg-carton to keep the
plant bouquet erect. Eight to 10 plant species ran-
domly selected, including always the target weed,
were simultaneously exposed to 20-26 A. elutus
(approximately 50% male, 50% female) in clear
plastic round containers (26 cm diameter by 9 cm
height, with four 4-7 cm diameter vents drilled
along the sides of the container to allow for air cir-
culation). At the beginning of each test, the in-
sects were placed at the bottom center of each con-
tainer to observe their orientation to the tested
plants. Plant species in each test were replicated
3-4 times (1 replication of tested plants in each
separate container). Plants tested were exposed
toA. elutus adults over a period of 10 to 14 d. Ob-
servations of oviposition and feeding were made
twice a week. Plant bouquets containing dark-
ened or loosened flower-buds were replaced as
needed, and dissected to observe possible oviposi-
tion. The flower-bud and leaf area consumed was
visually estimated based upon a scale from 0 to 5
(0 = no feeding, 1 = probing or 5% of area con-
sumed, 2 = light feeding or 5-20% of the area, 3 =
moderate feeding or 21-40%, 4 = heavy feeding,
and 5 = intense feeding or >60 of the area con-
sumed).

No-Choice Adult Feeding Tests

No-choice host specificity tests were also con-
ducted with A. elutus adults at the Gainesville-


September 2009









TABLE 1. HOST RANGE, ADULT FEEDING, AND OVIPOSITION TEST WITH ANTHONOMUS ELUTUS IN FLORIDA.

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

Category 1. Genetic types of the target weed species found in North America


SOLANACEAE
Tribe Solaneae
Genus Solanum
Subgenus Leptostemonum
Section Micracantha
Solanum tampicense Dunal


Wetland nightshade


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


Tribe Solaneae
Genus Solanum
Subgenus Leptostemonum
Section Acantophora
Solanum capsicoides All.
Solanum mammosum L.
Solanum viarum Dunal

Section Lasiocarpum
Solanum quitoense Lam.
Solanum pseudolulo Heise
Solanum sessiliflorum Dunal

Section Micracantha
Solanum jamaicense Mill.

Section Melongena
Subsection Androceras
Solanum citrullifolium A. Braun
Solanum heterodoxum Dunal
Solanum rostratrum Dunal
Solanum sisymbriifolium Lam.


Red soda apple
Nipplefruit
Tropical soda apple



Naranjilla
Falso lulo
Nightshade



Jamaican nightshade



Watermelon nightshade
Melonleaf nightshade
Buffalobur nightshade
Sticky nightshade


Each test 3-4 replications with 20-26 adults (50% males, 50% females) per rep.
*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 =


Heavy (41-60%), 5 = Intense (>60% area).








TABLE 1. (CONTINUED) HOST RANGE, ADULT FEEDING, AND OVIPOSITION TEST WITH ANTHONOMUS ELUTUS IN FLORIDA.

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


Subsection Lathyrocarpum
Solanum carolinense L.
Solanum dimidiatum Raf.
Solanum elaeagnifolium Cav.

Subsection Melongena
Solanum bahamense
Solanum melongena L.
Solanum torvum Sw.
Solanum verbascifolium L.

Subgenus Solanum
Solanum americanum Mill.
Solanum diphyllum L.
Solanum erianthum Don.
Solanum jasminoides Paxt.
Solanum mauritianum Scop.
Solanum nigrescesns Mart. & Gal
Solanum nigrum L.
Solanum parishii heller
Solanum ptycanthum Dunal
Solanum pumillum Dunal
Solanum retroflexum Dunal
Solanum scabrum Mill.
Solanum seaforthianum Andr.
Solanum tuberosum L.


Horse nettle*
Western horsenettle*
Silverleaf nightshade


Bahama nightshade
Eggplant
Turkeyberry
Mullein nightshade*



American nightshade*
Two-leaf nightshade*
Potato tree"
White potato vine
Earleaf nightshade
Divine nightshade*
Black nightshade*
Parish nightshade*
Wonder berry
Rock outcrop Solanum
Wonderberry
Garden huckleberry
Brazilian nightshade
Potato


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


GenusAcnistus
Acnistus australe (Griseb.) Griseb.

Genus lochroma
lochroma sp.


Acnistus



Iochroma


Each test 3-4 replications with 20-26 adults (50% males, 50% females) per rep.
*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 =


Intense (>60% area).








TABLE 1. (CONTINUED) HOST RANGE, ADULT FEEDING, AND OVIPOSITION TEST WITH ANTHONOMUS ELUTUS IN FLORIDA.

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


Genus Physalis
Physalis angulata L.
Physalis arenicola Kearney
Physalis crassifolia Benth
Physalis gigantea L.
Physalis ixocarpa Brot.
Physalis pubescens L

Tribe Daturae
Genus Brugmansia
Brugmansia sanguinea (Ruiz & Pav.) Don

Genus Datura Datura iscolour Bernh
Datura metel L.
Datura meteloides D.
Datura stramonium L.

Tribe Lycieae
Genus Lycium
Lycium carolinianum Walt.
Lycium fremontii Gray.

Genus Lycopersicon
Lycopersicon esculentum Mill.

Tribe: Nicandreae
Genus: Nicandra
Nicandra physaloides (L.) Gaertn.

Tribe Nicotianae
Genus Nicotiana
Nicotiana tabacum L.
Nicotiana rustica L.
Nicotiana sylvestris Speg. & Comes


Cutleaf Ground-Cherry
Cypresshead
Ground-Cherry
Ground-cherry
Tomatillo
Strawberry tomato



Red floripontio

Angels'trumpet
Downy thorn apple
Datura
Jimson weed



Christmas berry
Lycium


Tomato



Apple of Peru



Tobacco
Wild tobacco
Tobacco


Each test 3-4 replications with 20-26 adults (50% males, 50% females) per rep.
*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 =


Heavy (41-60%), 5 = Intense (>60% area).








TABLE 1. (CONTINUED) HOST RANGE, ADULT FEEDING, AND OVIPOSITION TEST WITH ANTHONOMUS ELUTUS IN FLORIDA.

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

Genus Nierembergia
Nierembergia scoparia Sendtri Cupflower 2 120 0 0

Genus Petunia
Petunia x hybrida Garden-petunia 2 120 0 0

Tribe Salpiglossidae
Genus Salpiglossis
Salpiglossis sinuata Ruiz & Pav Painted tongue 2 120 0 0

Genus Schizanthus
Schizanthus spp. Butterfly flower 2 120 0 0

Tribe Solandeae
Genus Solandra
Solandra glandiflora Swartz Chalice vine 2 120 0 0

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

Section Torva
Solanum donianum Walpers Mullein nightshade" 5 400 0 0

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

BORAGINACEAE
Heliotrope sp. Heliotrope 1 60 0 0
Myosotis alpestris Schmidt Forget-Me-Not 1 60 0 0

CONVOLVULACEAE
Convolvulus purpurea L. Convolvulus 2 120 0 0
Ipomoea batata (L.) Lam. Sweet-potato 2 120 0 0
Evolvulus muttallianus Evolvulus 2 120 0 0

EHRETIACEAE
Cordia sebestena L. Geiger tree 1 60 0 0

Each test 3-4 replications with 20-26 adults (50% males, 50% females) per rep.
*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area).








TABLE 1. (CONTINUED) HOST RANGE, ADULT FEEDING, AND OVIPOSITION TEST WITH ANTHONOMUS ELUTUS IN FLORIDA.

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


NOLANACEAE
Nolana paradoxa Lindl.

POLEMONIACEAE
Cobaea scandens Cav.
Gilia tricolor Benth
Phlox panuculata L.


Chilean bellflower


Cobaea
Bird's-eyes
Phlox


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


ANACARDIACEAE
Anacardium occidental L.
Mangifera indica L.
Pistacia vera L.


APIACEAE
Daucus carota L.


ASTERACEAE
Helianthus annuus L.
Lactuca sativa L.


Cashew
Mango
Cultivated pistachio


Carrot


Annual sunflower
Lettuce


CAMPANULACEAE
Campanula persicifolia L

CARICACEAE
Carica papaya L.

CRUCIFERAE
Brassica oleracea L. var. Botrytis

MALVACEAE
Abelmoschus esculentus (L.) Moench


Each test 3-4 replications with 20-26 adults (50% males, 50% females) per rep.
*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 =


Heavy (41-60%), 5 = Intense (>60% area).


Bell flower


Papaya


Broccoli


Okra


















TABLE 1. (CONTINUED) HOST RANGE, ADULT FEEDING, AND OVIPOSITION TEST WITH ANTHONOMUS ELUTUS IN FLORIDA.

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

ROSACEAE
Fragaria x ananassa Duchesne Strawberry 1 60 0 0

RUTACEAE
Citrus sinensis (L.) Osbeck Sweet orange 1 60 0 0
Citrus paradise Mcfady Grapefruit 1 60 0 0

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

MALVACEAE
Gossypium hirsutum L. Cotton 3 200 0 0

SOLANACEAE
Genus Capsicum
Capsicum annuum L. Bell pepper 5 400 0 0
Capsicum frutescens L. Chile 5 380 0 0

Each test 3-4 replications with 20-26 adults (50% males, 50% females) per rep.
*0 = No feeding, 1 = Probing (<5% of flower bud/leaf area), 2 = Light (5-20%), 3 = Moderate (21-40%), 4 = Heavy (41-60%), 5 = Intense (>60% area).







Medal et al.: Host Specificity ofAnthonomus elutus


quarantine facility with potted plants (20-60 cm
height) in cages.Anthonomus elutus adults were ex-
posed to 30 plant species in 3 families including S.
donianum in the list of Florida threatened plants,
and all major cultivated Solanaceae (Table 2). Five
to 6 plant species were individually tested each time
due to limitation in cage numbers. Ten A. elutus
adults (5 males, 5 females) per replication (3 replica-
tions) were exposed to plants for 2 weeks. Cages
were made of clear-plastic cylinders (15 cm diame-
ter, 50-60 cm height), with a mesh screen at the top
and covering 6-circular holes (6 cm diameter) lo-
cated in pairs at the bottom, middle, and upper part
of the cylinder to allow for air circulation. Adults
tested originated from F2- F3 generations reared in
quarantine from adults collected on wetland-night-
shade plants in Costa Rica and Guatemala. The
adults tested were young (2-3-week old). Plants
were replaced as needed. At the end of the testing
periods, feeding and oviposition were recorded.


RESULTS AND DISCUSSION

Multiple-Choice Feeding and Oviposition Tests

In the Florida-quarantine multiple-choice
tests (Table 1), Anthonomus elutus adults fed
heavily to intensively (41-100% of the area of-
fered) on the target weed wetland-nightshade. A
minor or exploratory feeding (<5% of the area of-
fered) was observed on the non-natives Solanum
capsicoides, Solanum mammosum, Solanum si-
symbriifolium, and Solanum torvum. No feeding
was observed on any of the other 82 plant species
that were tested. Anthonomus elutus laid from 3
to 10 eggs per female inside the wetland-night-
shade flower-buds during the duration of the test
(Table 1). No eggs were deposited on any of the
other 86 plant species tested. Results indicated
that this potential biocontrol agent fed and laid
eggs only on the target weed wetland-nightshade.


TABLE 2. HOST RANGE OF ANTHONOMUS ELUTUS ADULTS IN NO-CHOICE FEEDING TESTS IN FLORIDA QUARANTINE.

Plant family / Species Common names Feeding Score* Eggs/Female


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


Bell pepper
Chile
Tomato
Tobacco
Cupflower
Ground-cherry
Strawberry tomato
American nightshade
Horse nettle
Watermelon nightshade
Western horsenettle
Two-leaf nightshade
Mullein nightshade
Silverleaf nightshade
Jamaican nightshade
White potato vine
Eggplant



Black nightshade
Rock-outcrop
Wonder berry
Sunberry
Garden huckleberry
Wetland nightshade
Turkeyberry
Potato
Tropical soda apple


Sweet-potato


Moderate feeding (21-40%), 4 = Heavy


Each test included 3-4 replications with 10 adults (5 females, 5 males) per replication.
*0 = No feeding, 1 = Probing (<5% ofleaf/flower bud area), 2 = Light feeding (5-20%), 3 :
feeding (41-60%), 5 = Intense feeding (>60% of the area).







Florida Entomologist 92(3)


No-Choice Adult Feeding Tests

The no-choice host specificity tests withA. elu-
tus adults exposed to individual potted plants (30
species in 3 families) in cages at the Gainesville-
quarantine facility (Table 2) indicated that the in-
sects fed and laid eggs (range: 3-6, average: 5 eggs
per female) only on wetland-nightshade. Feeding
on wetland-nightshade was moderate (21-40% of
the area offered) compared to a probing or explor-
atory feeding (<5%) observed on S. elaeagnifolium
and on S. torvum. No eggs were laid on any of the
29 non-target plant species tested including the 3
eggplant cultivars (Black Beauty, Market, Asian
Long Purple).
The high specificity showed by this weevil in
the host-range tests feeding and development
only on the target weed in the Micrantha section,
indicated no adverse impacts would be expected
on the 6 solanaceous species that were not tested
and are listed as threatened or endangered in Ha-
waii and Puerto Rico. Indirect beneficial effects on
wildlife populations associated with release and
establishment ofA. elutus may be expected due to
recolonization by native plants that have been
displaced by the rapidly growing and highly com-
petitive wetland-nightshade plants.
The host-specificity tests at the Gainesville
quarantine indicated that A. elutus is safe to re-
lease. Occasional temporary feeding might occur
on some close related Solanum species. These spe-
cies are the non-native weeds S. torvum (in the
Federal Noxious Weed list, introduced from Cen-
tral/South America (Kissman & Groth 1995), S.
capsicoides, S. sisymbriifolium, and S. jamai-
cense. Noticeable damage to economic/native
Solanaceae plants is unlikely to occur based on
our host tests. The lack of records as a crop pest in
the native range of the weevils support our find-
ings on the specificity and safety ofA. elutus as a
biocontrol agent of wetland-nightshade in Flor-
ida.
One of the strongest arguments in favor of re-
leasing A. elutus against wetland-nightshade in
Florida was the inability of this weevil to feed,
oviposit, or develop on eggplant, tomato, potato,
bell-pepper, chile, native Solanaceae plants, and
other economic crops in the no-choice and choice
experiments. These findings suggest that a host
range expansion ofA. elutus to include any of the
tested non-target plants is highly unlikely. The
quarantine feeding-oviposition tests exposing A.
elutus to 87 plant species in 17 families indicated
that this weevil is nearly a monophagous herbi-
vore which feeds on a few Solanum species, in-
cluding S. torvum which is an exotic weed of in-
creasing concern in Florida due to its potential to
invade wildlife areas and displace native vegeta-
tion. The primary goal of this biological control
program is to bring down the wetland-nightshade
population density below a threshold that does


not cause economic or ecological damage. We do
not consider eradication of an invasive plant that
has already been established to be a realistic goal.
However, if local eradication were observed (e.g.,
as a consequence of herbicide/intense mowing ap-
plications), and occasional feeding on a non-target
plant occurred, the evidence accumulated indi-
cates that the possibility ofA. elutus becoming a
problem on a non-target plant is remote.
Based on the specificity ofA. elutus feeding and
developing only on the target weed in the Micran-
tha section, we consider this weevil safe for field
release against wetland-nightshade. Therefore, a
petition to release the Mexican-Central-American
flower-bud weevil A. elutus for the control of wet-
land- nightshade in Florida was submitted to the
USDA-APHIS-PPQ Technical Advisory Group
(TAG) members on Dec 2008.

ACKNOWLEDGMENTS

We thank Howard Frank (University of Florida),
and Julieta Brambila (United States Department of Ag-
riculture, Animal and Plant Health Inspection Service)
for reviewing the manuscript. We thank Wayne Clark
(Auburn University) for identification of Anthonomus
elutus. This research was funded by Florida Fish and
Wildlife.

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Florida Entomologist 92(3)


September 2009


A NEW GENUS AND NEW SPECIES OF THE SUBTRIBE CICADINA
(HEMIPTERA: CICADIDAE: CICADINI)

YOUNG JUNE LEE
University of Connecticut, Department of Ecology and Evolutionary Biology, 75 North Eagleville Road,
Storrs, CT 06269, USA

ABSTRACT

A new genus, Qurana gen. nov. and a new species, Qurana ggoma sp. nov. of the subtribe
Cicadina (Hemiptera: Cicadidae: Cicadini) are described from Cambodia. This new genus is
allied to the genera Purana Distant, Calcagninus Distant, and Gudaba Distant but is dis-
tinguished by a long rostrum, a short male abdomen, a non-dentate pronotal collar, and a
widely truncate uncus.

Key Words: taxonomy, Qurana, Qurana ggoma, Purana, Calcagninus, Gudaba, Cambodia

RESUME

Se describe un nuevo g6nero, Qurana gen. nov., y una nueva especie, Qurana ggoma sp.
nov., del subtribu Cicadina (Hemiptera: Cicadidae: Cicadini) de Cambodia. Este nuevo g6-
nero es aliada con los g6neros Purana Distant, Calcagninus Distant y Gudaba Distant pero
puede ser distinguida por su largo rostro, el abdomen corto del macho, el collar del pronoto
sin dientes y el uncus ancho y truncado.


A new species is described from Cambodia. It
was found among undetermined material in the
collections of the Institut royal des Sciences na-
turelles de Belgique, Brussels (IRSNB). A new ge-
nus is described here as well in order to include
this unique new species. It is clear that this new
species belongs to the subtribe Cicadina of the
tribe Cicadini in the subfamily Cicadinae, follow-
ing the classification of Lee (2008), because of its
small body size, scale-like male operculum, and
the structure of the male genitalia. The species is
allied to the species of the genera Purana Distant,
1905, Calcagninus Distant, 1892, and Gudaba
Distant, 1906. However, it cannot be placed in
these or any other existing genera of Cicadina be-
cause of its unique morphology as discussed be-
low. Morphological measurements were made
with a MitutoyoTM vernier caliper in mm.

Taxonomy
Genus Qurana gen. nov.
Type Species. Qurana ggoma sp. nov. (Cambodia)

Distinguishing Features. This genus is closely al-
lied to the genus Purana in having a small and slen-
der body and 2 pairs of tubercles on the abdominal
sternite. Within Purana, Qurana looks more allied
to the Purana carmente species group proposed by
Schouten & Duffels (2002) in having male tymbal
covers each with a striking black mark and the
forewing with the bases of apical cells 2 and 3 not
being infuscated (Schouten & Duffels 2002; Lee
2009). Qurana is distinguished by the following
characters: body much smaller (male body length
approximately 15.6 mm, but longer than 19 mm in


Purana); rostrum very long, passing center of stern-
ite III (Fig. 1F); forewing very long (Fig. 1A, B); male
abdomen short, about as long as or slightly shorter
than distance from head to cruciform elevation (Fig.
1A); male operculum short and ellipsoidal (Fig. 1B,
F); anterolateral pronotal collar not dentate; and
uncus with a widely truncate apex and a rounded
medial indent in ventral view (Fig. 1C).
Qurana is also similar to the genera Gudaba
and Calcagninus in having a very small and slen-
der body, 2 pairs of tubercles on the abdominal
sternite, and short and transverse male opercula,
but it is distinguished by the following characters:
postclypeus not prominent; rostrum very long,
passing center of sternite III (about reaching pos-
terior coxae in Gudaba and just passing posterior
coxae in Calcagninus); anterolateral pronotal col-
lar not dentate; hind wing with 6 apical cells (6 in
Calcagninus but 5 in Gudaba); male abdomen
short (longer than distance from head to cruci-
form elevation in Calcagninus and Gudaba); and
tymbal cover nearly complete (very short in Cal-
cagninus and Gudaba).
Description. Body small (male body length ap-
proximately 15.6 mm). Postclypeus moderately
swollen. Postclypeus with 2 longitudinal fasciae
connecting medial ends of transverse fasciae. Ros-
trum long, usually passing center of sternite III and
sometimes reaching posterior margin of sternite III.
Pronotum long, longer than twice the length of
head. Inner area barely marked between central
fasciae and lateral fissures. Anterolateral pronotal
collar not dentate. Wings hyaline, not infuscated.
Forewing long, approximately 1.3 times the length
of body. Costal vein considerably bent and narrowed







Lee: New Genus and New Species of Cicadina


A B


CD


Fig. 1. Qurana ggoma sp. nov. male, Siem Reap, Cambodia, 28 May 2005 (IRSNB). A. holotype in dorsal view.
B. holotype in ventral view. C. pygofer in ventral view. D. pygofer in lateral view. E. body in laterodorsal view. F. ab-
domen in lateroventral view.


after node. Anterior longitudinal vein of apical cell 5
about as long as anterior longitudinal vein of apical
cell 7. Male operculum ellipsoidal, not or slightly
passing posterior margin of sternite II. Male abdo-
men about as long as or slightly shorter than dis-
tance from head to cruciform elevation. Male tym-
bal cover with a distinct, large black mark laterally.
Male abdominal sternites III and IV each with a
pair of tubercles on lateral surfaces. Uncus not bi-
furcate, with a widely truncate apex and a rounded
medial indent in ventral view. Basal lobe of pygofer
spine-shaped with a narrow apex.
Er ... .,..,. The generic name is an arbitrary
combination of letters, suggesting that this is a
genus next to Purana.

Qurana ggoma sp. nov.
(Fig. 1)

Type Material. Holotype: male (Fig. 1A, B),
"Coll. I.R.Sc.N.B. // CAMBODIA // (Siem Reap


prov) // Kbal Spean, Light Trap // 28 V 2005 // Leg
Var&Grootaert" (printed yellow label) (IRSNB).
Paratypes: 5 males and 2 females, same data as
holotype (IRSNB).
ErLt, ...... ... The specific name is a noun derived
from the Korean noun ggoma, meaning "a small
person", referring to the small sized body of this
species, which is considerably smaller than the
species of its allied genera.
Measurements of Types (6 males, 2 females;
mean (range)). Length of body: male 15.6 (15.4-
15.6), female 15.1 (14.5-15.7); width of head in-
cluding eyes: male 4.9 (4.8-5.0), female 4.9 (4.9-
4.9); wing span: male 44.9 (43.8-46.4), female 45.4
(44.7-46.1).
Description of Male (Fig. 1A, B, E, F). Head
ochraceous with the following black to fuscous
marks: a median large mark enclosing ocelli,
with its anterior end nearly reaching frontocly-
peal suture; a pair of upside-down "L"-shaped
marks on the sides of the median mark, nearly


-~--
c~ .----'







Florida Entomologist 92(3)


connecting with the median mark; a fascia along
posterior margin of head; and a pair of short lon-
gitudinal fasciae along inner margin of com-
pound eyes. Distance between lateral ocelli and
compound eyes about as wide as twice the dis-
tance between lateral ocelli. Postclypeus moder-
ately swollen. Antennae brown to dark brown.
Ventral part of head ochraceous with black to
fuscous marks. Postclypeus with transverse fas-
ciae along transverse grooves, with posterior
ones being very short, and 2 longitudinal lines
connecting medial ends of transverse fasciae.
Anteclypeus with 2 pairs of longitudinally ar-
ranged spots, with posterior ones reaching poste-
rolateral margins of anteclypeus. Rostrum fus-
cous to black apically; usually passing center of
sternite III and sometimes reaching posterior
margin of sternite III. Lorum with a longitudinal
fascia along inner margin of lorum, except ante-
rior part. Gena with a transverse fascia between
postclypeus and compound eye.
Pronotum (Fig. 1A, E) ochraceous. Inner area
of pronotum with the following fuscous marks: a
pair of central longitudinal fasciae slightly broad-
ened at both anterior and posterior ends; a pair of
longitudinal indistinct marks between median
parts ofparamedian fissures and posterior ends of
lateral fissures; a pair of fasciae along lateral fis-
sures; and a pair of curved fasciae along lateral
margins of inner area. Pronotal collar with (some-
times without) a narrow transverse fascia along
posterior margin and with a pair of narrow fas-
ciae along lateral margins, and a pair of small
(sometimes indistinct) spots at lateral inner cor-
ner. Anterolateral pronotal collar slightly devel-
oped, but not dentate.
Mesonotum (Fig. 1A, E) ochraceous, but
partly darker or brighter, with the following
black to fuscous marks: a median longitudinal
fascia slightly broadened posteriorly to reach to
posterior margin of cruciform elevation; a pair of
small roundish spots enclosing scutal depres-
sions; a pair of inwardly curved fasciae along
parapsidal sutures; and a pair of longitudinal
fasciae on lateral sigilla, with their posterior
ends hooked outwardly. Cruciform elevation
ochraceous with a median longitudinal fascia
continued from mesonotum and a pair of fasciae
along lateral margins, fuscous. Ventral part of
thorax ochraceous.
Legs ochraceous. Fore-femur with a small
subapical spine as well as primary and second-
ary spines; with a fuscous fascia along midline of
ventral side. Fore- and mid-tarsi castaneous api-
cally. Pretarsal claws dark brown to fuscous.
Wings (Fig. 1A, B) hyaline. Forewing without
infuscation; long, approximately 1.3 times the
length of body. Coastal vein ochraceous; consider-
ably bent and narrowed after node. Basal cell
slightly tinged with ochraceous. Basal membrane
and hind wing jugum gray.


Operculum (Fig. 1F) bright ochraceous without
marks; short, obliquely ellipsoidal, and not or
slightly passing posterior margin of sternite II.
Two opercula widely separated, with gap of about
half to two-thirds as wide as operculum.
Abdomen (Fig. 1E, F) about as long as or
slightly shorter than distance from head to cru-
ciform elevation. Abdomen mostly ochraceous,
but tergite 8 mostly dark brown to fuscous.
Tergite 2 sometimes with a longitudinal median
fuscous fascia. Posterior margin of tergite 3
about as wide as anterior margin of mesono-
tum. Tymbal cover ochraceous but fuscous on
about lateral one-third to half; quarter round or
semicircular, slightly wider than long, with lat-
eral margin nearly linear. Ventral part of abdo-
men ochraceous except posterior margin of ster-
nite II, posterior area of sternite VII, and most
area of sternite VIII, fuscous. Sternites III and
IV each with a pair of tubercles on lateral sur-
faces, bright ochraceous and protruding poste-
riad.
Male genitalia (Fig. 1C, D): Pygofer oval in
ventral view. Uncus with a widely truncate apex
and a rounded medial indent in ventral view;
curved downward in lateral view. Distal shoulder
of pygofer smoothly curved without extension.
Dorsal beak very short. Basal lobe of pygofer
spine-shaped with a narrow apex.
Description of Female. Operculum ochraceous
without distinct marks; very small, not touching
posterior margin of sternite II. Ventral part of ab-
domen ochraceous to brown. In one specimen, ster-
nite VII mostly black. Abdominal segment 9 fus-
cous dorsally but bright ochraceous ventrally. Ovi-
positor sheath fuscous, protruding far beyond anal
styles. Dorsal beak about as long as anal styles.

ACKNOWLEDGMENTS

I am indebted to Dr. Jerome Constant (Institut royal
des Sciences naturelles de Belgique, Brussels, Belgium)
for the loan of specimens. I am grateful to anonymous
reviewers for suggestions that improved the manu-
script. This work benefited from support from the Uni-
versity of Connecticut and the National Science
Foundation under Grant Numbers DEB 05-29679 and
DEB 07-20664. Any opinions, findings, and conclusions
or recommendations expressed in this material are
those of the author and do not necessarily reflect the
views of the NSF.

REFERENCES CITED

DISTANT, W. L. 1892. A monograph of oriental Cica-
didae, parts 5-7. Indian Museum, Calcutta, pp. i-xiv/
97-158, pls. 10-15.
DISTANT, W. L. 1905. Rhynchotal notes-XXIX. Annals
and Magazine of Natural History (7)15: 58-70.
DISTANT, W. L. 1906. Rhynchota Vol. III (Heteroptera-
Homoptera). The Fauna of British India, including
Ceylon and Burma. Taylor and Francis, London,
xiv+503 pp.


September 2009







Lee: New Genus and New Species of Cicadina


LEE, Y. J. 2008. A checklist of Cicadidae (Insecta: Hemi-
ptera) from Vietnam, with some taxonomic remarks.
Zootaxa 1787: 1-27.
LEE, Y. J. 2009. Descriptions of two new species of the
Purana abdominalis species group (Hemiptera: Ci-
cadidae: Cicadini) from the Philippines, with a key


to the species groups of Purana. J. Natural History
43: 1487-1504.
SCHOUTEN, M. A., AND DUFFELS, J. P. 2002. A revision of
the cicadas of the Purana carmente group (Ho-
moptera, Cicadidae) from the Oriental region. Tijd-
schrift voor Entomologie 145: 29-46.







Florida Entomologist 92(3)


September 2009


ANT TENDING OF MIAMI BLUE BUTTERFLY LARVAE
(LEPIDOPTERA: LYCAENIDAE): PARTNER DIVERSITY
AND EFFECTS ON LARVAL PERFORMANCE

MATTHEW D. TRAGER1' AND JARET C. DANIELS''
'Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611

2McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History,
University of Florida, Gainesville, FL 32611

ABSTRACT

The larvae of many lycaenid butterflies (Lepidoptera: Lycaenidae) are tended by ants that
protect them from natural enemies in return for sugar-rich secretions that the larvae pro-
duce to attract and retain their ant guards. We investigated the relationship between larvae
of the endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri Comstock & Hun-
tington) and potentially mutualistic ants. We observed 10 ant species interacting with Mi-
ami blue larvae in the field, and raised larvae successfully in captivity with 3 of these and 6
other ant species that are not known to tend larvae in the wild. In an experimental assess-
ment of ant effects on larval performance, we found no differences in age at pupation, pupal
mass, length of pupation, total time as an immature or ratio of time as a larva to time as a
pupa among larvae raised with Camponotus floridanus Buckle. L '- .. i. - -.r humile Mayr,
or in a no-ant control. Larvae raised with C. floridanus were significantly more likely to pu-
pate in the ant harborage than larvae in the other treatments. We did not observe ants be-
having antagonistically toward Miami blue larvae in field, laboratory, or experimental
conditions; even ant species previously identified as potential predators tended larvae. Our
results demonstrate that Miami blue larvae can elicit typical tending behaviors across di-
verse ant taxa and that ant tending does not substantially alter larval development, findings
that may have implications for conservation and population restoration of the Miami blue
butterfly.

Key Words: Camponotus floridanus, Cyclargus thomasi bethunebakeri, Linepithema humile,
lycaenid, mutualism, myrmecophily

RESUME

Las orugas de las mariposas en la familiar Lycaenidae a menudo son atendidas por hormigas
que las protegen de enemigos naturales a cambio de secreciones ricas en azucares produci-
das por las orugas para atraer y retener sus hormigas guardias. Investigamos la relaci6n en-
tre larvas de la mariposa Miami blue (Cyclargus thomasi bethunebakeri Comstock &
Huntington), un especie en peligro de extinci6n, con las hormigas posiblemente mutualistas.
Observamos 10 species de hormigas interactuando con las orugas de la Miami blue en el
campo, y criamos satisfactoriamente las orugas en cautiverio con 3 de 6sas y con 6 otras es-
pecies de hormigas no observadas al tender orugas en el habitat natural. Hicimos un en-
sayo experimental para observer los efectos de las hormigas para el desempeio de las larvas,
en que medimos los variables de respuesta siguientes: edad a la pupaci6n, masa de la pupa,
duraci6n del estadio pupal, plazo total como inmadura, y relaci6n de los plazos larva:pupa.
No encontramos distinciones en ninguno del los variables entire las orugas criadas con Cam-
ponotus floridanus (Buckley) o con Linepithema humile (Mayr) o en un ensayo control sin
hormigas. Sin embargo, hallamos que las larvas criadas con C. floridanus lograron significa-
tivamente mas probable hacerse en pupa con las hormigas que en los otros tratamientos. No
observamos comportamiento antagonista por las hormigas hacia las orugas, ni ain por es-
pecies previamente identificadas como posibles predadores de estas larvas, que de hecho
atiendieron las orugas. Nuestros resultados demuestran que las orugas de la Miami blue
provocan comportamientos tipicos de atenci6n por diversas species de hormigas y aunque
el cuidado de las hormigas no altera significativamente el desenvolvimiento larval, hallazgo
que puede tener implicaciones para la conservaci6n y la rehabilitaci6n de populaciones de la
mariposa Miami blue.


Translation provided by the authors.







Trager & Daniels: Ant Tending of Miami Blue Butterfly Larvae


The larvae of many lycaenid butterflies (Lepi-
doptera: Lycaenidae) interact with ants in rela-
tionships ranging from brood parasitism in ant
colonies to mutualism in which both species ben-
efit. Approximately 60% of the ant-associated ly-
caenid species are facultative myrmecophiles that
are tended by several different ant species, usu-
ally on the larval host plant (Pierce et al. 2002).
Lycaenid larvae in such relationships generally
emit semiochemicals from a pair of tentacular or-
gans and secrete a sugar-rich solution from a dor-
sal nectary organ to attract and retain their ant
guards (Axen et al. 1996; Axen 2000; Daniels et
al. 2005), which then protect the larvae from
predators and parasitoids (Pierce & Mead 1981;
Fiedler et al. 1996). Additionally, larvae of some
lycaenid species pupate inside ant nests, where
the ants presumably protect them until adult
eclosion (Wagner 1995; Dejean & Beugnon 1996).
Thus, these associations entail an exchange of
goods and services that usually results in net ben-
efits for both partners (Pierce et al. 1987; Cush-
man et al. 1994). However, there is often substan-
tial variation in quality among potential partners
in facultative, diffuse mutualistic systems that
may result in negative or neutral consequences
for the survival and performance of the interact-
ing species (Bronstein 2001; Miller 2007).
Evaluating partner quality in purportedly mu-
tualistic relationships is necessary to understand
the ecological conditions and evolutionary pro-
cesses that influence the outcome of the relation-
ship (Bshary & Grutter 2002; Ness et al. 2006). A
central question in research on lycaenid-ant mu-
tualisms is how interactions with different ant
species affect larval performance (Axen 2000;
Fraser et al. 2001; Weeks 2003). The physiological
cost of provisioning ants with a sugar-rich exo-
crine secretion leads to reduced growth of ant-
tended larvae in some systems (Pierce et al. 1987;
Baylis & Pierce 1992), but growth of ant-tended
larvae in other systems is similar to or greater
than that of untended larvae (Fiedler & H11-
dobler 1992; Cushman et al. 1994; Wagner & Del
Rio 1997; Fraser et al. 2001). Because pupal mass
is often correlated with adult reproductive output
in Lepidoptera (Gotthard 2008), the growth con-
sequences for larvae of ant tending could have
subsequent effects on individual fitness (Elgar &
Pierce 1988). Furthermore, because the relative
importance of adult size and timing of adult emer-
gence may differ between conspecific males and
females (Fagerstrom & Wiklund 1982; Gotthard
et al. 2000), in some lycaenid species there are sex
differences in the effects of ant tending (Fiedler &
Hlldobler 1992). Assessing the effects of ant
tending on lycaenid larvae and identifying the
factors that explain variation in the interaction
among and within species are central issues for
furthering our understanding of these complex
relationships.


Larvae of the imperiled Miami blue butterfly,
Cyclargus thomasi bethunebakeri (Comstock &
Huntington) (Lycaenidae: Polyommatinae), asso-
ciate with several species of ants (Minno & Em-
mel 1993; Smith et al. 1994; Saarinen & Daniels
2006). However, we know very little about the
costs and benefits of this relationship (Florida
Fish and Wildlife Conservation Commission
2003; Saarinen & Daniels 2006). In addition to re-
porting new observations of ant tending in the
wild and under laboratory conditions, our study is
the first to quantify experimentally the effects of
ant tending on performance of Miami blue larvae.
Specifically, we observed the behavioral compo-
nents of interactions between Miami blue larvae
and different ant species in the field and in the
laboratory and raised larvae with several ant spe-
cies to assess the nature of these interactions. We
then quantified the effects of ant tending and but-
terfly sex on larval survival, time until pupation,
mass at pupation, and length of pupation with an
experiment in which we raised larvae with 2 ant
species and a no-ant control. We also noted the lo-
cation of pupation for larvae in this experiment.
The implications of our work for the conservation
of wild Miami blue butterfly populations and fu-
ture reintroduction efforts are discussed.

MATERIALS AND METHODS

Study Organism

The Miami blue butterfly is a small, sexually
dimorphic lycaenid formerly found in much of
coastal peninsular Florida and outlying barrier
islands (Smith et al. 1994; Calhoun et al. 2002;
Carroll & Loye 2006). Miami blue larvae have an
onisciform or sluglike shape typical of ant-tended
lycaenids and have prominent ant-associated or-
gans for communicating with and provisioning
ant guards beginning in the third instar
(Saarinen & Daniels 2006). The larvae elicit tend-
ing behavior from ants through chemical commu-
nication originating from a pair of eversible tenta-
cles and, perhaps, other specialized epidermal
glands (Pierce et al. 2002). Ants typically respond
by rapidly antennating around the head, tentacu-
lar organs, and dorsal nectary organ of the larvae
and accepting sugar-rich secretions from the lat-
ter (Saarinen & Daniels 2006). Although ants
may protect Miami blue larvae against parasi-
toids and predators in the wild, we have not re-
corded parasitism or predation of any larvae de-
spite extensive field observations.
The Miami blue butterfly is currently listed by
the state of Florida as an endangered species
(Florida Fish and Wildlife Conservation Commis-
sion 2003). More information on the history and
current status of this species can be found in Cal-
houn et al. (2002), Carroll & Loye (2006), and
Saarinen & Daniels (2006).







Florida Entomologist 92(3)


Field and Laboratory Observations of Ant Tending

Over 3 growing seasons (2006-2008), we con-
ducted field observations of ant tending at Bahia
Honda State Park (BHSP), where there is a wild
population of Miami blue butterflies, as well as at
2 other sites where we introduced captive-bred
larvae, Elliott Key in Biscayne National Park
(BNP) and Dagny Johnson Key Largo Hammock
Botanical State Park (DJSP). The introduced lar-
vae were from a colony maintained at the Univer-
sity of Florida as part of the Miami blue butter-
fly's state-mandated management and recovery
plan (Florida Fish and Wildlife Conservation
Commission 2003). These larvae, and all others
used in the studies we report here, originated
from stock sourced from a wild population at
BHSP and were raised on Caesalpinia bonduc L.
(Roxb.) as the host plant. Observations at all 3
sites were opportunistic because larvae were of-
ten difficult to locate once on the host plant, were
not always tended, and ants did not always imme-
diately find recently introduced larvae. We were
particularly interested in the behaviors of both
ants and Miami blue larvae upon first encounter
and then subsequent interactions that would in-
dicate whether ants ignored, tended or depre-
dated larvae.
We observed interactions between Miami blue
butterfly larvae and several ant species in the lab-
oratory. We raised dozens of larvae with the 2 fo-
cal ant species in this study, Camponotus florida-
nus Buckley and Linepithema humile Mayr, prior
to the experimental assessment described below.
For the other ant species, we raised from 2 to 4
Miami blue butterfly larvae simultaneously with
a single, queenright ant colony that had no previ-
ous interactions with Miami blue butterflies. We
introduced larvae to the ant colonies at the second
or early third instar, observed the initial interac-
tion to assure that the ants did not attack the lar-
vae and replaced the host plant daily until the
larvae pupated. The only 2 exceptions to this pro-
tocol were our discovery of Pheidole moerens
Wheeler tending larvae after entering the labora-
tory without our knowledge and when we found
Camponotus floridanus workers tending larvae in
an outdoor flight cage.

Experimental Design and Analysis

We quantified the effects of ant tending on Mi-
ami blue larvae with an experiment in which we
raised larvae from the second instar to pupation
with 2 ant species, Camponotus floridanus and
Linepithema humile, or in a no ant control. Cam-
ponotus floridanus is a large formicine that is the
most common ant found tending Miami blue lar-
vae in the wild, thereby making it relevant spe-
cies for our study. Linepithema humile is a small,
non-native dolichoderine that readily tends hon-


eydew-producing Hemiptera and other lycaenid
larvae (Agrawal & Fordyce 2000; Tillberg et al.
2007). Although we had no evidence that L. hu-
mile interacts with Miami blue larvae in the wild,
this species is amenable to experimentation and
in our study served as a surrogate for smaller, ac-
tive ant species that we have frequently observed
tending Miami blue larvae in the field (e.g.,
Paratrechina longicornis, P. bourbonica, Tapi-
noma melanocephalum).
We conducted 2 trials in which we randomly
assigned 3 larvae to each of 4 replicate shoebox-
style trays in each of the 3 ant treatments (total of
72 larvae). We placed new groups of ants in each
tray between trials. Larvae were from eggs laid by
multiple females in the captive colony and were
all the same age in each of the 2 trials. We re-
placed the host plant daily and manually trans-
ferred larvae from the old to the new cuttings.
Each tray contained an ant harborage comprising
a 90-mm diameter Petri dish containing a layer of
dental plaster that we wetted occasionally to
maintain humidity and covered with dark paper.
For the C. floridanus treatment, we placed 50
workers in each tray, and for the L. humile treat-
ment we placed 100 workers and 1 queen in each
tray. These ants had been maintained in captivity
for approximately 6 months prior to the experi-
ment and were allowed a week to acclimate to the
tray before we introduced the Miami blue larvae.
To assure that malnourishment would not affect
the ants' interactions with the Miami blue larvae,
we provisioned ants with water, 10% sucrose solu-
tion, and cut mealworms (larvae of Tenebrio sp.);
these were also provided to the control trays. We
coated the sides of the trays with a fluoropolymer
resin slippery barrier (Insect-a-Slip, BioQuip
Products, Rancho Dominguez, CA) to contain the
ants but, even with this preventative measure, a
small number of Miami blue larvae escaped dur-
ing our experiments.
We monitored the presence of all larvae each
day. Upon pupation, we recorded the location of
the pupae, measured their mass with a digital an-
alytic balance accurate to 0.01mg (Denver Instru-
ments SI-215D), and kept them in individual vi-
als in the laboratory under fairly constant tem-
perature and humidity until adult emergence (24-
28 C with 40-50% relative humidity). We mea-
sured the wing chord length (mm) and recorded
the sex of each adult. Thus, our response vari-
ables for statistical analyses were age at pupa-
tion, location of pupation, pupal mass, length of
pupation, and adult wing chord. From these data
we calculated 2 additional response variables-
the proportion of time spent in the pupal and lar-
val stadia (length of pupation divided by age at
pupation) and total time as an immature (age at
pupation plus length of pupation)-to increase
our understanding of the potential effects of ants
on larval development of Miami blue butterflies.


September 2009







Trager & Daniels: Ant Tending of Miami Blue Butterfly Larvae


We conducted correlation analysis to test for re-
lationships between the measures of larval perfor-
mance to elucidate patterns in developmental strat-
egies of Miami blue larvae. We analyzed differences
among ant treatments in the location of pupation
with Fisher's exact test. To assess the influence of
ant treatment and sex on larval performance, we
conducted linear mixed-effects analyses in which
we tested the effects of ant treatment and sex (both
fixed effects) on age at pupation, pupal mass, length
of pupation, and the 2 compound variables de-
scribed above. All models contained a nested ran-
dom effect of tray within trial to account for vari-
ance due to differences between the 2 experimental
trials or among groups of the same ant species. All
analyses were conducted in the R language and en-
vironment for statistical computing (R Develop-
ment Core Team 2009) and followed protocols for
model specification and interpretation described by
Pinheiro & Bates (2002) and Faraway (2006).

RESULTS

Field and Laboratory Observations of Ant Tending

We observed 8 ant species tending Miami blue
larvae in the field, including observations at the
natural population in Bahia Honda State Park
and the reintroduction sites in Biscayne National
Park and Dagny Johnson Key Largo Hammock
Botanical State Park (Tables 1 and 2). Saarinen &
Daniels (2006) reported 1 additional species, Fore-
lius pruinosus, tending Miami blue larvae but we
have not since observed this interaction. We most


frequently observed 2 Camponotus species, C.
floridanus and C. planatus, tending both wild and
recently introduced larvae. Additionally, at both
reintroduction sites, Pseudomyrmex gracilis was
common and tended recently released larvae.

Experimental Assessment of Ant Effects

We collected data on age at pupation, location
of pupation, pupal mass, length of pupation, and
wing chord from 64 Miami blues. Of the original
72 larvae, 5 escaped from the ant trays and were
excluded from further study and 3 individuals did
not successfully eclose from the pupal stage so
could only be included in some analyses. Pupal
mass and wing chord were highly positively corre-
lated (r = 0.69, t = 7.31, df = 59, P < 0.0001), and
so we used pupal mass as a measure of size for our
analyses. There was no correlation between age at
pupation and pupal mass (r = -0.14, t = -1.13, df=
62, P = 0.26), but pupal mass was positively corre-
lated with length of pupation (r = 0.31, t = 2.54, df
= 62, P = 0.014) and age at pupation was nega-
tively correlated with length of pupation (r = -
0.49, t = -4.42, df= 62, P < 0.0001).
Most larvae (45 of 64) pupated under the paper
that shaded the Petri dish harborages regardless
of ant presence or identity, but the frequency of
pupal location was non-random among the 3 ant
treatments (Fisher's exact test P = 0.029). Five of
22 larvae raised with C. floridanus pupated inside
the Petri dish with the ants, whereas no larvae
from either the L. humile or no ant treatments
pupated in that location


TABLE 1. ANT SPECIES OBSERVED TENDING MIAMI BLUE LARVAE IN THE FIELD AND THE INTERACTION STATUS SUG-
GESTED BY SAARINEN & DANIELS (2006), IF AVAILABLE. THE TENDING BEHAVIOR WE OBSERVED FOR ALL
ANTS LISTED HERE WAS CONSISTENT WITH A MUTUALISTIC INTERACTION.

Ant taxa Interaction status Sites and observers

Subfamily Pseudomyrmicinae
Pseudomrymex gracilis P DJSP2, BNP2
Subfamily Myrmicinae
Crematogaster cf. ashmeadi S BHSP1'2
Monomorium floricola u BHSP1'2
Subfamily Dolichoderinae
Forelius pruinosus S ENP1, BHSP1
Tapinoma melanocephalum S BHSP'2
Subfamily Formicinae
Camponotus floridanus S BHSP1'2, BNP2, ENP1
Camponotus inaequalis KWNWR3
Camponotus planatus S DJSP2, BNP2, BHSP1
Paratrechina bourbonica pS BHSP2
Paratrechina longicornis pS BHSP2, KWNWR3
Interaction status suggested by Saarinen & Daniels (2006): P = potential predator, pS = potential symbiont (i.e., potential mu-
tualist), S = symbiont (i.e., mutualist), u = unknown. 1 = observations reported by Saarinen & Daniels (2006); 2 = new observations
by the authors; 3 = new observations by P. Cannon (pers. comm.).











TABLE 2. ANT SPECIES OBSERVED TENDING MIAMI BLUE
LARVAE UNDER EXPERIMENTAL LABORATORY
CONDITIONS AND THE INTERACTION STATUS
SUGGESTED BY SAARINEN & DANIELS (2006),
IF AVAILABLE. WE SUCCESSFULLY RAISED AT
LEAST 2 LARVAE FROM APPROXIMATELY THIRD
INSTAR TO PUPATION WITH ALL OF THE ANT
SPECIES LISTED HERE UNDER CONDITIONS DE-
SCRIBED IN THE MATERIALS AND METHODS EX-
CEPT PHEIDOLE MOERENS, WHICH WE
DISCOVERED TENDING LARVAE IN THE LABORA-
TORY.

Ant taxa Interaction status

Subfamily Pseudomynnicinae
Pseudomyrmex ejectus
Pseudomyrmex gracilis P
Subfamily Myrmicinae
Pheidole moerens
Solenopsis invicta P
Subfamily Dolichoderinae
Dorymyrmex bureni
Linepithema humile
Tapinoma melanocephalum S
Subfamily Formicinae
Brachymyrmex patagonicus
Camponotus floridanus S

Interaction status suggested by Saarinen & Daniels (2006):
P= potential predator, pS = potential symbiont (i.e., mutualist),
S =symbiont (i.e., mutualist), u = unknown.


We present summarized values of the perfor-
mance parameters measured in Table 3 and the
results of all statistical analyses of the effects of
larval sex and ant treatment on larval perfor-
mance in Table 4. There was no significant effect
of ant treatment on any of the measures of larval
performance, although the analysis suggested
that larvae raised with C. floridanus may have a


September 2009


shorter length of pupation relative to the time
spent in the larval stadium. Male larvae pupated
at a significantly smaller mass than females but
females tended to complete pupation faster.
Although we did not quantify the frequency of
tending behaviors in this study, both C. floridanus
and L. humile regularly tended Miami blue but-
terfly larvae throughout the course of the experi-
ment. We rarely found untended larvae, particu-
larly in the later instars, with either ant species.
The interactions conformed to the common behav-
ioral pattern of the larvae everting their tentacu-
lar organs and secreting from the dorsal nectary
organs, followed by the ants antennating the lar-
vae and consuming the nectar. However, upon dis-
turbance associated with maintaining the experi-
ment, L. humile usually abandoned the larvae
whereas C. floridanus showed typical defensive
behaviors such as running around larvae, tapping
on the host plant or other surfaces with their
mandibles, and antennating the air with open
mandibles.

DISCUSSION

Relationships between lycaenid butterfly lar-
vae and ants range dramatically in the effects on
the interacting species. Previous studies have
suggested that some ant species may depredate
Miami blue butterfly larvae or may opportunisti-
cally tend larvae without providing protection
against predators or other benefits (Saarinen &
Daniels 2006). However, through both field obser-
vations and laboratory trials, we recorded a uni-
versal tending response among ants consistent
with a mutualistic interaction. Indeed, including
the observations we report here, a total of 17 ant
species have been observed tending Miami blue
larvae either in wild populations, in reintroduc-
tion sites following releases of captive raised lar-
vae or in the laboratory (Saarinen & Daniels
2006; Carroll & Loye 2006). We have not observed


TABLE 3. SUMMARY OF DEVELOPMENTAL PARAMETERS (MEANS SE) FOR MIAMI BLUE BUTTERFLY LARVAE, SEPA-
RATED BY ANT SPECIES TREATMENT, AND SEX. FOR EASE OF INTERPRETATION, MEANS AND SE HERE ARE
POOLED ACROSS TRIALS AND TRAYS; TEST STATISTICS FROM THE MIXED-EFFECTS MODEL ANALYSIS ON THE
EFFECTS OF ANT SPECIES AND SEX ON DEVELOPMENTAL VARIABLES ARE PRESENTED IN TABLE 4.

Age Pupal Length of Time Length
at pupation (d) mass (mg) pupation (d) as immature (d) pup./Age at pup.

Ant treatment
C. floridanus (n = 22) 19.09 0.31 60.52 1.56 8.73 0.13 27.82 0.27 0.460 0.013
L. humile (n = 21) 18.81 0.44 58.11 2.23 8.76 0.19 27.57 0.39 0.471 0.019
No ants (n = 21) 18.57 0.44 62.06 2.23 9.0 0.19 27.57 0.39 0.489 + 0.019
Sex
Male (n = 39) 18.51 0.36 58.92 1.86 9.10 0.15 27.51 0.32 0.490+ 0.015
Female (n = 25) 19.32 0.28 62.27 1.45 8.56 0.12 27.88 0.25 0.446+ 0.012
All (n = 64) 18.83+ 0.18 60.23 0.92 8.82 0.079 27.66 0.16 0.473 0.008


Florida Entomologist 92(3)







Trager & Daniels: Ant Tending of Miami Blue Butterfly Larvae


TABLE 4. RESULTS OF MIXED-EFFECTS MODEL ANALYSES TESTING THE EFFECTS OF ANT TREATMENT, SEX, AND THEIR
INTERACTION ON MEASURES OF LARVAL PERFORMANCE. TRAY IN TRIAL WAS A NESTED RANDOM EFFECT FOR
ALL ANALYSES AND VARIANCE COMPONENTS ARE PRESENTED AS STANDARD DEVIATIONS FOR BOTH TERMS
FOR EACH MODEL. ALTHOUGH PRESENTED TOGETHER IN THIS TABLE, WE TESTED EACH RESPONSE VARIABLE
INDEPENDENTLY.

Response variables Predictor variables F-value (df) P-value

Age at pupation Ant treatment 2.15 (2, 51) 0.13
Sex 0.41 (1,51) 0.53
Ant treatment Sex 0.23 (2, 51) 0.80
Trial o = 1.16; Tray in Trial o = 0.10
Pupal mass Ant treatment 1.68 (2, 51) 0.20
Sex 8.91 (1,51) 0.0043
Ant treatment Sex 0.18 (2, 51) 0.84
Trial o = 0.0021; Tray in Trial o = 3.57 107
Length of pupation Ant treatment 1.40 (2, 51) 0.26
Sex 3.60 (1, 51) 0.063
Ant treatment Sex 0.76 (2, 51) 0.47
Trial o = 0.27; Tray in Trial ( = 4.91 105
Time as immature Ant treatment 0.57 (2, 51) 0.57
Sex 0.17 (1, 51) 0.68
Ant treatment Sex 0.20 0.82
Trial o = 0.87; Tray in Trial ( = 0.089
Length of pupation/ Ant treatment 2.90 (2, 51) 0.064
Age at pupation Sex 3.14 (1, 51) 0.082
Ant treatment Sex 0.81 (2, 51) 0.45
Trial o = 0.045; Tray in Trial o = 1.79 106


any depredation of Miami blue larvae by ants in
the field, and laboratory trials suggest that even
ant species identified by Saarinen & Daniels
(2006) as potential predators in fact tend larvae
in ways consistent with a mutualistic interaction.
These results are notable for the large number of
potential ant partners, the consistency of behav-
iors toward larvae among distantly related ant
taxa and the nearly complete lack of obviously an-
tagonistic interactions.
Despite the high diversity of potential ant
partners, our observations suggest that only a
small subset of ant species accounts for the vast
majority of interactions with Miami blue butterfly
larvae in the field. Previous studies have reported
that ant species in the genus Camponotus most
commonly tended larvae (Minno & Emmel 1993;
Carroll & Loye 2006; Saarinen & Daniels 2006),
and we most commonly found C. floridanus and C.
planatus associated with wild and recently re-
leased larvae. Notably, this pattern was consis-
tent across 3 sites (BHSP, BNP, and DJSP) that
likely differ dramatically in ant community com-
position (Deyrup et al. 1988). There are at least 4
likely explanations for the apparent reciprocal af-
finity between Miami blue larvae and Campono-
tus. First, Camponotus commonly forage on nick-
erbean for plant exudates, honeydew-producing
Hemiptera, and insect prey even in sites where


Miami blues do not occur, so frequent tending
could be opportunistic association resulting from
high rate of encounters compared to other poten-
tial ant partners. Second, after discovering Miami
blue larvae, even those tended by other ant spe-
cies, Camponotus may competitively exclude
other ants from interacting with them through
constant tending and defense. Third, following
initial contact, the Miami blue larvae may allo-
cate more resources to retaining Camponotus
compared to other ant species because Campono-
tus are higher quality defenders. Finally, Cam-
ponotus are quite large and active and therefore
researchers may be more likely to find larvae
tended by these ants.
Larvae of some facultatively ant-tended lycae-
nid species pupate inside ant nests (e.g., Wagner
1995), but this has not been previously reported
for the Miami blue butterfly. We found that a
higher than expected number of larvae pupated
in the ant harborages in the laboratory when
tended by C. floridanus and no larvae pupated in
that location when kept with L. humile or raised
without ants, suggesting that this aspect of the
relationship may have been overlooked. Clearly,
future studies are required to elucidate the pre-
cise mechanisms that account for the persistent
and geographically widespread association be-
tween Camponotus species, particularly C. flori-







Florida Entomologist 92(3)


danus and C. planatus (and possibly C. inequae-
lis), and Miami blue larvae.
In most facultative ant-lycaenid mutualisms,
the primary direct cost for the lycaenid larvae is
metabolic expense required to produce sugary se-
cretions for ant defenders (Pierce et al. 1987;
Daniels et al. 2005). Because the secretions are
induced by ant tending (Agrawal & Fordyce
2000), we might expect to find reduced larval
growth rate or pupal mass in ant-tended larvae.
In our experimental assessment of ant effects on
performance of Miami blue butterfly larvae, both
C. floridanus and L. humile almost constantly
tended larvae and we frequently observed both
species consuming secretions from the dorsal
nectary organ. Carroll & Loye (2006) found that
Camponotus sp. raised with Miami blue larvae
lived longer than ants raised without any food
source, demonstrating that larval secretions are
nutritionally valuable for tending ants. However,
contrary to theoretical predictions and the results
of studies in similar ant-lycaenid systems, we
found no effect of ant presence on any of our mea-
surements of larval performance. It is possible
that these secretions are less metabolically ex-
pensive than some studies have suggested or that
ants tend larvae even when they receive only
small nutritional rewards (Fiedler & Saam 1995).
Alternatively, larvae may compensate for meta-
bolic expenses of provisioning ants by feeding
more efficiently when tended (Fiedler & H6ll-
dobler 1992; Wagner & del Rio 1997). If the latter
is true for Miami blue butterfly larvae, we may
have found no effect of ant association on larval
performance because increased growth of tended
larvae offset the costs of the sugary secretions.
Regardless of the specific mechanism, it appears
that the net costs of ant tending for Miami blue
butterflies do not substantially affect their larval
development compared to untended conspecific
larvae.
The primary benefit of ant association for ly-
caenid larvae is defense against natural enemies
(Atsatt 1981; Pierce & Mead 1981; Weeks 2003).
We did not test this important aspect of the rela-
tionship for Miami blue butterfly larvae in this
study, nor have we observed ants actively protect-
ing larvae against attack by predators or parasi-
toids in the wild. However, in the laboratory C.
floridanus displayed strong defensive behaviors
(e.g., rapidly circling larvae, recruiting nearby
workers and lunging at forceps) when disturbed,
particularly when we moved Miami blue butterfly
larvae to new host plant. The large size of this ant
species and nearly constant tending may serve as
a visual deterrent to potential attackers. Al-
though L. humile workers assiduously tended Mi-
ami blue larvae in our experiment, they are sub-
stantially smaller than C. floridanus and did not
show such a strong defensive response; instead,
they usually ran away from the larvae and source


of disturbance. Despite these qualitative observa-
tions, it is important to emphasize that we have
no definitive evidence from this study that C.
floridanus are more effective defenders of Miami
blue butterfly larvae than small-bodied ant spe-
cies. Given the apparently small physiological
costs for Miami blue larvae of associating with
ants, any predator or parasitoid deterrence pro-
vided by ant defenders could substantially in-
crease their probability of survival to the adult
stage.
Male and female Lepidoptera larvae may have
divergent developmental strategies, and may
therefore exhibit variation in behaviors, such as
foraging or prolonging the larval period that rep-
resent trade-offs between growth and mortality
(Gotthard 2008). In lycaenid larvae, such differ-
ences could result in sex-specific interactions with
ants, particularly in species for which timing of
adult emergence and adult size have different ef-
fects on the relative reproductive success of males
and females (Elgar & Pierce 1988). Indeed,
Fiedler & Hlldobler (1992) found that Polyom-
matus icarus males benefited from ant associa-
tion through increased pupal mass, but ant tend-
ing appeared to be energetically costly for fe-
males. This species has a strongly protandrous
mating system with frequent male-male competi-
tion for access to females (Lundgren 1977), so it is
reasonable that male larvae would interact with
ants to optimize adult size as long as the costs to
development time were not too great. Similarly,
Baylis & Pierce (1992) found that male Jalmenus
evagoras lost less mass from secretions for ants
during the nonfeeding, prepupal instar. By con-
trast, studies on other lycaenid species have
found no sex-related differences in ant associa-
tion (e.g., Fraser et al. 2001). It is worth noting
that most experiments testing lycaenid larval
performance with and without ants have not rec-
ognized sex as a potentially important predictor
of the interaction and its effects. We found that
male and female Miami blue larvae differed in
some performance parameters, but the sex x ant
treatment term did not explain a significant
amount of variation in any of the analyses. As
such, our results suggest that Miami blue larvae
did not have sex-specific costs or benefits of inter-
acting with ants, at least under our experimental
conditions.
The state-mandated management plan for the
Miami blue butterfly identified examining the re-
lationship between larvae and ants as one of the
research goals to inform conservation efforts
(Florida Fish and Wildlife Conservation Commis-
sion 2003). Saarinen & Daniels (2006) generated
further interest in the implications of this rela-
tionship for protecting the Miami blue by suggest-
ing that some ant species, particularly the non-
native Solenopsis invicta and Pseudomyrmex gra-
cilis, could be predators of Miami blue larvae. In-


September 2009







Trager & Daniels: Ant Tending of Miami Blue Butterfly Larvae


deed, non-native ants can disrupt coevolved mu-
tualistic interactions through competition with
native ants or failure to provide the benefits to
partner species (Ness & Bronstein 2004). The ant
fauna of the Florida Keys contains numerous non-
native species, including some that are extremely
invasive and have been implicated as major pred-
ators of arthropods (Deyrup et al. 1988; Deyrup et
al. 2000). However, we found a universal tending
response toward Miami blue larvae and very little
antagonism across a wide range of ant species, in-
cluding those previously identified as potential
predators. This is a similar result to studies on
honeydew-producing Hemiptera that have gener-
ally mutualistic relationships with ants, includ-
ing non-native species (Helms & Vinson 2003;
Mondor & Addicott 2007). Different ant species
may not have equivalent effects on Miami blue
larvae but, based on our observations, we doubt
that many ant species regularly depredate larvae.
We suggest that ants may be important for pro-
tecting larvae from natural enemies, but the iden-
tity of the ant attendants may not be particularly
important for other measures of larval perfor-
mance. Furthermore, because the 2 ant species
that most commonly tend larvae in the wild and
show a strong defensive response, C. floridanus
and C. planatus, are common throughout south-
ern Florida, it is likely that at least 1 of these spe-
cies would be present at any potential reintroduc-
tion site. Our results suggest that the facultative
and diffuse interactions between ants and Miami
blue larvae are unlikely to be the most important
determinant of conservation success for the im-
periled butterfly.

ACKNOWLEDGMENTS

We thank E. V. Saarinen and 2 anonymous reviewers
for helpful comments on a previous version of this pa-
per. Research for this study was conducted under Flor-
ida Fish and Wildlife Conservation Commission permit
WX02525f. Funding for this work was provided by the
Florida Fish and Wildlife Conservation Commission, E.
O. Dunn Foundation, U.S. Fish and Wildlife Service and
the National Fish and Wildlife Foundation; additionally,
M. D. Trager was funded by a National Science Founda-
tion Graduate Research Fellowship. The opinions ex-
pressed herein are solely those of the authors and do not
represent the position of any of these funding sources.

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Florida Entomologist 92(3)







Aldrich et al: Invasion of the Brown Marmorated Stink Bug


SEMIOCHEMICALLY BASED MONITORING OF THE INVASION
OF THE BROWN MARMORATED STINK BUG AND UNEXPECTED
ATTRACTION OF THE NATIVE GREEN STINK BUG
(HETEROPTERA: PENTATOMIDAE) IN MARYLAND

J. R. ALDRICH1,*, A. KHRIMIAN1, X. CHEN3** AND M. J. CAMP2
'Invasive Insect Biocontrol & Behavior Laboratory, Agricultural Research Service,
United States Department of Agriculture, B-007, rm 313, Agricultural Research Center-West, Beltsville, MD 20705

2Biometrical Consulting Service, Agricultural Research Service, United States Department of Agriculture,
B-005, Agricultural Research Center-West, Beltsville, MD 20705

3Department of Entomology, University of California, Riverside CA 92521

To whom correspondence should be addressed. E-mail: Jeffrey.Aldrich@ars.usda.gov

"Present address: Small Molecule Synthesis Facility, Chemical Biology Program, Department of Chemistry,
Duke University, Durham, NC 27708-0354

ABSTRACT

The brown marmorated stink bug, Halyomorpha halys (Stal) (Pentatomidae), is a newly in-
vasive species in the eastern U. S. that is rapidly expanding its range from the original point
of establishment in Allentown, Pennsylvania. Although an attractant pheromone has yet to
be identified for H. halys, in its native Asian range the insect is cross-attracted to the pher-
omone of another pentatomid Plautia stali Scott whose males produce methyl (E,E,Z)-2,4,6-
decatrienoate. Previous tests of methyl 2,4,6-decatrienoate isomers in the U. S. verified that
H. halys is highly attracted to methyl (E,E,Z)-2,4,6-decatrienoate, and that the native green
stink bug, Acrosternum hilare (Say), also is attracted to this compound. Using traps baited
with methyl 2,4,6-decatrienoates and the reported pheromone of A. hilare (trans- and cis-
(Z)-a-bisabolene epoxides), we monitored populations of the brown marmorated and green
stink bugs at the Agricultural Research Center, Beltsville, Maryland, for the 2004-2008
growing seasons. Over this time period, the H. halys population rose from being undetect-
able in 2004 to becoming much more abundantly trapped than the native A. hilare. Further-
more, A. hilare was significantly more attracted to methyl (E,E,Z)-2,4,6-decatrienoate than
the blend of bisabolene epoxides reported as its pheromone. Supplemental material online
at http://www.fcla.edu/FlaEnt/fe923.htm#InfoLink1

Key WordsAcrosternum, Halyomorpha, aggregation, kairomone, pheromone, Hemiptera, ta-
chinid

RESUME

La chiche marr6n marmol, Halyomorpha halys (Stal) (Pentatomidae), es una nueva especie
invasora en el este de Estados Unidos, la cual esta expandiendo su rango de distribuci6n ra-
pidamente desde su punto original de entrada en Allentown, Pennsylvania. Aunque su fero-
mona ain no se ha identificada, este insecto es atraido en su habitat native (Asia) por la
feromona de otro pentat6mido, Plautia stali Scott, cuyos machos produce (E,E,Z)-2,4,6-de-
catrienoato de metilo. Experimentos previous con distintos is6meros de 2,4,6-decatrienoato de
metilo en Estados Unidos demostraron que H. halys es fuertemente atraida por (E,E,Z)-
2,4,6-decatrienoato de metilo, y que la chiche verde nativaAcrosternum hilare (Say) es tam-
bi6n atraida por este compuesto. Usando trampas cebadas con 2,4,6-decatrienoatos y con la
feromona reportada para A. hilare (ep6xidos de (E)- y (Z)-a-bisaboleno), realizamos un se-
guimiento poblacional de ambas species en la Agricultural Research Center, Beltsville, Ma-
ryland, durante las temporadas de crecimiento 2004-2008. En este period, las poblaciones
de H. halys aumentaron desde niveles no detectables en 2004, a valores de captures sustan-
cialmente mayores que los correspondientes a la especie native. Asimismo, A. hilare fue sig-
nificativamente mas atraida al (E,E,Z)-2,4,6-decatrienoato de metilo que a la mezcla de
ep6xidos de a-bisabolenos, reportados como la feromona de esta especie.


Translation provided by Dr. Andr6s Gonzalez Ritzel.







Florida Entomologist 92(3)


The brown marmorated stink bug, Halyomor-
pha halys (Stal) (Heteroptera: Pentatomidae), is
an exotic species (Hoebeke & Carter 2003) that is
rapidly expanding its range from its original
point of establishment in Allentown, Pennsylva-
nia (Hamilton 2009). Collection records for H.
halys date from 1996, although it was not realized
that the insect was foreign until 2001 after the
population had exploded. Halyomorpha halys is
native to northeast Asia and, in Japan, has be-
come a serious fruit pest since post-world war II
reforestation of old-growth deciduous forest with
Japanese cedar, Crytomeria japonica, and false
cypress, C'i/... ......... .. obtusa (Kiritani 2007).
According to Kiritani (2007), H. halys and 2 other
fruit-feeding stink bugs are dependent on cones to
complete their life cycle. The bug also has an ex-
traordinary propensity to form aggregations
(Toyama et al. 2006), often in houses and other
buildings where they are a nuisance similar to
boxelder bugs (Tinker 1952). In fact, it has been
suggested that an aggregation ofH. halys entered
the U.S. in a cargo container (Hoebeke & Carter
2003) and, subsequently, an aggregation was ap-
parently transported via some type of vehicle to
Oregon where H. halys also is established (Anon-
ymous 2007). More recently, H. halys has been
found established in the Ziirich region of Switzer-
land (Wermelinger et al. 2008).
Pheromone-baited traps offer a convenient and
potentially powerful means to monitor insect pop-
ulations, and some stink bug pheromones are
known (Millar 2005), but no pheromone is known
for H. halys. However, in Japan H. halys is cross-
attracted (Tada et al. 2001a,2001b; Funayama
2008) to the pheromone of another pentatomid,
Plautia stall Scott, whose males produce methyl
(E,E,Z)-2,4,6-decatrienoate (Sugie et al. 1996). In
tests begun in 2003 at Allentown (Khrimian 2005;
Khrimian et al. 2008), we verified that H. halys
adults and nymphs are, indeed, attracted to me-
thyl (E,E,Z)-2,4,6-decatrienoate and related geo-
metric isomers. Surprisingly, methyl 2,4,6-dec-
atrienoates also attracted significant numbers of
the native green stink bug, Acrosternum hilare
(Say) (Aldrich et al. 2007), even though methyl
2,4,6-decatrienoates have not been found fromA.
hilare and the reported pheromone components of
this bug (trans-/cis-(Z)-a-bisabolene epoxides)
(Aldrich et al. 1989; Aldrich et al. 1993; McBrien
et al. 2001) are chemically unlike methyl 2,4,6-de-
catrienoates.
Here we report the results of our efforts to
monitor H. halys and A. hilare at the Beltsville
Agricultural Research Center from the 2004
through 2008 growing seasons using traps baited
with methyl 2,4,6-decatrienoates and trans-/cis-
(Z)-a-bisabolene epoxides, the reported phero-
mones of Plautia stall (Sugie et al. 1996) and A.
hilare (Aldrich et al. 1989; McBrien et al. 2001),
respectively. Over this time period, the population


ofH. halys in Maryland increased from being un-
detectable in 2004 to becoming much more abun-
dantly trapped than A. hilare in 2007 and 2008.
Previously reported data for 2004 and 2005 (in-
cluded herein, Aldrich et al. 2007) showed thatA.
hilare is particularly attracted to methyl (E,E,Z)-
2,4,6-decatrienoate.

MATERIALS AND METHODS

Chemical Standards and Treatments

Methyl (E,Z,Z)-, (Z,E,Z)- and (E,E,Z)-2,4,6-dec-
atrienoates were synthesized as previously de-
scribed (Khrimian 2005), as were (4S)-cis- and (4S)-
trans-(Z)-a-bisabolene epoxides (Z)-(1R,2S,4S)-4-
(1',5'-dimethyl 1',4'-hexadienyl)-1,2-epoxy-l-meth-
ylcyclohexane and (Z)-(1S,2R,4S)-4-(l',5'-dimethyl
1',4'-hexadienyl)-1,2-epoxy-l-methylcyclohexane),
respectively, (Chen et al. 2000).
Lures for methyl 2,4,6-decatrienoate treat-
ments were prepared by impregnating gray rub-
ber septa (West Pharmaceutical Services, Kear-
ney, NE) with single synthetic isomers (Khrimian
2005) as previously described (Aldrich et al. 2007;
Khrimian et al. 2008). For treatments with the re-
ported pheromone ofA. hilare, septa were impreg-
nated similarly with a 5:95 ratio of trans-/cis-(Z)-
a-bisabolene epoxides. Lures containing methyl
2,4,6-decatrienoates were loaded with 4.0 or 2.5
mg of each active ingredient per septum, and bis-
abolene epoxide lures were loaded with a total of
2.5 mg of the epoxide mixture per septum. In tests
combining the bisabolene epoxides with methyl
(E,E,Z)-2,4,6-decatrienoate, 2 septa were placed
together in traps, 1 with the methyl ester and a
second with the epoxides. Lures were rebaited
weekly or biweekly as specified in figure legends.

Field Trapping

Field experiments were carried out at BARC,
Prince George's County, Maryland, from 2004
through 2008 beginning in late spring or early
summer through mid-Oct. A summary of the field
experiments is presented in Table 1.
From 2004 through 2007, four replicates per
treatment were tested with traps hung in the
same location each year about 1.8 m above ground
from tree branches (>20 m apart) in patches of
mixed deciduous forest bordering a field consist-
ing of alternating strips of corn and soybean (see
supplemental material). Replicates were grouped
into sets of traps, with (>100 m between sets), and
traps were rotated one position within a set every
2-3 weeks. In 2004 and 2005, container traps (30
cm tall transparent plastic, 10 cm removable bot-
tom, and two 9.5 inwardly projecting mesh fun-
nels see supplemental material) were fabricated
by Sterling International, Inc. (Spokane, WA). In
2006, a new kind of baffle trap (see supplemental


September 2009







Aldrich et al: Invasion of the Brown Marmorated Stink Bug


TABLE 1. FIELD EXPERIMENTS CONDUCTED AT BETLSVILLE, MD, 2004-2008.

Year' Duration Site Description2 Treatment3 Trap4 N

2004 1 Jul-26 Oct Deciduous/soybean-corn EZZ C 4
ZEZ C 4
2005 11 Aug-19 Oct Deciduous/soybean-corn EZZ C 4
ZEZ C 4
2006 6 Jun-26 Oct Deciduous/soybean-corn EEZ C 4
EEZ B 4
15 Aug-26 Oct Deciduous/soybean-corn BX C 4
BX B 4
EEZ+BX C 4
EEZ+BX B 4
2007 11 May-18 Oct Deciduous/soybean-corn EEZ B 4
BX B 4
5 xBX B 4
9 Aug-18 Oct Deciduous/soybean-corn live male HH C-L 4
18 Sep-18 Oct Deciduous/soybean-corn dead male HH C-L 4
2008 18 Apr-31 Oct Deciduous/soybean-corn EEZ B 2
18 Apr-31 Oct Coniferous/corn EEZ B 2

'Data for 2004 and 2005 from Aldrich et al. (2007).
Aerial view of field sites in supplemental online material.
*EZZ = methyl (E,Z,Z)-2,4,6-decatrienoate; ZEZ = methyl (Z,E,Z)-2,4,6-decatrienoate; EEZ = methyl (E,E,Z)-2,4,6-decatrienoate;
BX = 5:95 ratio of trans-/cis-(Z)-a-bisabolene epoxides. Note: EZZ readily rearranges in sunlight to EEZ and other isomers (Khrim-
ian et al. 2008).
'Photographs of traps shown in online supplemental material; C = container-type trap; B = baffle-type trap; C-L = container trap
with bottom insert for laboratory-reared H. halys (HH) males.


material) was tested side-by-side with the con-
tainer traps deployed for the first 2 years (4 repli-
cates/treatment/trap type) (Table 1). Baffle traps
were constructed from 0.25 inch corrugated plas-
tic (Coroplast, Dallas, TX) after the design of Mi-
zell (1996) (61 cm tall and 30.5 cm at the base), ex-
cept that the collector consisted of the top of a re-
usable yellowjacket trap (Sterling International,
Inc.) held in place with wire pins. In 2007 and
2008, the baffle trap was used exclusively except
when traps were baited with H. halys males as
described below (Table 1).
From 9 Aug through 18 Oct 2007, 4 traps were
added in which 2-3 live, laboratory-reared H.
halys males were used to bait traps. Halyomorpha
halys were reared as previously for other stink
bugs (Aldrich et al. 1991) but with organically
grown green beans (Phaseolus vulgaris) (My Or-
ganic Market, College Park, MD) and buckwheat
(Fagopyrum esculentum) seeds in addition to sun-
flower seeds (Helianthus annuus). Adult males at
least 2 weeks old were placed with a bean in
transparent plastic half pint cups having several
3-4- mm diameter holes punched in the bottom
halves of the cups, then the cups were inverted
and inserted into the bottom of the container
traps instead of the normal trap bottom such that
volatiles from the male bugs would be released in-
side the trap. Another set of traps was added on
18 Sep prepared as described above except that


instead of live H. halys males, specimens of meth-
ylene chloride-extracted males were pinned in-
side the plastic cups to test for possible visual re-
sponse to chemically neutral H. halys males.
From 18 Apr through 31 Oct 2008, four baffle
traps baited with methyl (E,E,Z)-2,4,6-decatri-
enoate and 4 unbaited control traps were deployed
as described above except that 2 sets of traps were
hung from deciduous trees around the perimeter of
the agricultural field in the same location as in pre-
vious years, and 2 sets of traps were deployed in 2
patches of mature coniferous trees located approx-
imately 500 m northeast of the deciduous/soybean-
corn site (see supplemental material). One conifer
patch consisted exclusively of loblolly pine (Pi-
naceae: Pinus taeda L.); the other patch consisted
mainly of eastern white pine (Pinus strobus L.),
plus some Austrian (Pinus nigra Arnold) and Vir-
ginia pines (Pinus virginiana).
Among phytophagous stink bug species in the
subfamily Pentatominae (Heteroptera: Pentato-
midae), sexual communication is a bimodal pro-
cess whereby long-distance attraction is mediated
by sex pheromones, and shorter-range mate loca-
tion is mediated by substrate-borne vibrational
songs (Bagwell et al. 2008). Thus, pentatomines
attracted chemically from long-range may fail to
enter traps in the absence of vibrational signals;
therefore, bugs in and within 10 cm of traps were
counted as being attracted.







Florida Entomologist 92(3)


Feeding Choice Test

Seeds of 2 Asian conifer species that are fed
upon by H. halys in Japan (Kiritani 2007) were
obtained from the U.S. National Arboretum,
Washington, D.C., for feeding choice experiments:
Japanese cedar, Cryptomeria japonica, and false
cypress, C'/ .. ........". i obtusa. 2 treatments (sun-
flower seeds versus Cr. japonica, and sunflower
seeds versus Ch. obtusa), each with 8 replicates,
were tested. A Petri dish with a filter paper in the
bottom served as an arena for each replicate. One
shelled sunflower seed, and either clumps of 8 Cr.
japonica or 6 Ch. obtusa seeds were glued to the
filter paper with wallpaper paste (Metylan stan-
dard, Roman Adhesives, Inc., Calumet City, IL).
The numbers of conifer seeds were chosen to ap-
proximate the weight of a sunflower seed. Addi-
tionally, a cotton-plugged, glass shell vial of water
was provided. Each Petri dish received 5 second
instar H. halys, and treatments were placed on
the same shelf in an environmental chamber at
25C, 72% relative humidity, and 16:8 h (L:D)
photoperiod. The number of nymphs found on
each seed treatment was recorded at 1-2-h inter-
vals each day for 5 consecutive days, and dead
nymphs were removed and replaced as they were
found.

Statistical Analysis

Fisher's exact chi-square, exact binomial and
exact multinomial tests were performed for the
analyses by StatXact (Mehta & Patel 2005). When
applicable the numbers of insects were analyzed
as an R x C table with Fisher's chi-square test to
determine if the distribution of the insects dif-
fered for the various factors. If the overall test
was significant, then Fisher's Chi-square tests for
2 x 2 tables, binomial or multinomial tests were
performed to determine where the distributions
differed. In some instances there was no table,
and only binomial or multinomial tests could be
used to test for differences.

RESULTS

Halyomorpha halys populations increased
from being undetectable in 2004 to being abun-
dantly trapped in 2007 and 2008 (Fig. 1). Adults
of both sexes and nymphs (second through fifth
instars) of each species were caught in roughly
equal numbers (data for A. hilare not shown).
Captures of A. hilare adults remained constant
from 2006 through 2008, while captures of H.
halys adults went from being less than those ofA.
hilare in 2006 to being about 4 times greater than
A. hilare for 2007 and 2008 (Fig. 2). In tests con-
ducted in 2008 deploying traps from deciduous or
coniferous trees bordering agricultural fields (see
online supplemental material), all A. hilare indi-


2004 2006 2006 2007 2008
Fig. 1. The total number of Halyomorpha halys adults
and nymphs caught inside or within 10 cm of 2 traps
baited with isomers of methyl 2,4,6-decatrienoate and de-
ployed at the same location in the field at Beltsville, Mary-
land, from 2004 through 2008 (2004: treatment = methyl
(E,Z,Z)-2,4,6-decatrienoate*, 4 mg active ingredient/rub-
ber septum in container trap, rebaited biweekly); 2005:
methyl (E,Z,Z)-2,4,6-decatrienoate, 4 mg a.i./rubber sep-
tum in container trap, rebaited biweekly; 2006-2008: me-
thyl (E,E,Z)-2,4,6-decatrienoate, 2.5 mg a.irubber
septum in baffle trap, rebaited weekly). [*Note: Methyl
(E,Z,Z)-2,4,6-decatrienoate readily isomerizes to methyl
(E,E,Z)-2,4,6-decatrienoate and other isomers when ex-
posed to light (Khrimian et al. 2008).]


viduals and all but 1 adult male H. halys were
captured at the deciduous/soybean-corn site (P <
0.0001). Significant numbers of bugs were never
caught in control traps (usually none were
caught); therefore, data for control traps are omit-
ted from Figs. 1-5.
The 2006 data for the 2 different types of traps
showed that baffle traps worked as well as or bet-







.--5







2006 2007 2008
Fig. 2. The total number of Halyomorpha halys and
Acrosternum hilare adults caught inside or within 10 cm of
2 traps baited with methyl (E,E,Z)-2,4,6-decatrienoate
and deployed at the same location in the field at Beltsville,
Maryland, from 2006 through 2008 (2.5 mg active ingredi-
ent/rubber septum in baffle trap, rebaited weekly). Aster-
isks over bars indicate significant differences between
captures for each species within a year (P < 0.0001).


September 2009








Aldrich et al: Invasion of the Brown Marmorated Stink Bug


UContainrtypeTrap
W B"Iateype Trap








A. Nmaret In A. han I on 4hys In H. Ilys/ on
Species / Iocation

Fig. 3. Comparison of the efficiency of container-type
and baffle-type traps for the total number of adults of
Halyomorpha halys and Acrosternum hilare during
2006 ("in" = inside the trap; "on" = standing on the trap
or within 10 cm of the trap; treatment = methyl (E,E,Z)-
2,4,6-decatrienoate, 4 baffle traps, 2.5 mg active ingre-
dient/rubber septum, rebaited weekly). Asterisks over
bars indicate significant differences between captures
for the trap types (P < 0.0001).



ter than container traps in capturing H. halys and
A. hilare adults (Fig. 3, and supplemental online
material). The distribution of insects and location
(i.e., inside versus on traps) were significantly dif-
ferent for container- and baffle-type traps (x2 =
10.5, P < 0.0147, df = 3). Halyomorpha halys
adults and nymphs were more apt to enter baffle
traps than container traps (P < 0.0001); therefore,
in 2007 baffle traps were used for all treatments
except when container traps were baited with H.
halys males.
The combined captures (bugs inside and
within approximately 10 cm of traps) of H. halys
andA. hilare for 2006 and 2007 by date are shown
in Figs. 4 and 5, respectively. For H. halys,


4Q S'l W7 O7


e7 ( A s2002 12007)14
Date (2006 & 2007)


Fig. 5. The total number Acrosternum hilare adults
and nymphs caught inside or within 10 cm of traps
baited with methyl (E,E,Z)-2,4,6-decatrienoate by date
during 2006 and 2007 (4 baffle traps, 2.5 mg active in-
gredient/rubber septum, rebaited weekly).


nymphs were the first individuals to be caught in
mid-summer, followed by the appearance of
adults from late Aug to early Oct. For A. hilare, a
few adults were caught in May and Jun, and then
nymphs appeared in mid-summer, followed by
adults from Aug to early Oct. Some of the adults
for both H. halys andA. hilare that were caught in
the latter part of the season had soft, incom-
pletely sclerotized cuticle, indicating that they
were newly molted adults.
The attraction H. halys and A. hilare adults in
2006 to traps baited with methyl (E,E,Z)-2,4,6-de-
catrienoate (EEZ), a 5:95 ratio of trans-/cis-(Z)-a-
bisabolene epoxides (BX), and the combination
EEZ+BX, versus unbaited control traps is shown
in Fig. 6. The distribution ofH. halys andA. hilare
was significantly different for these treatments


s stB 2T ea57 7/1 1015
Date (2006 & 2007)


Fig. 4. The total number Halyomorpha halys adults
and nymphs caught inside or within 10 cm of traps
baited with methyl (E,E,Z)-2,4,6-decatrienoate by date
during 2006 and 2007 (4 baffle traps, 2.5 mg active in-
gredient/rubber septum, rebaited weekly).


cOntM EE EE*BX BX
Treatment

Fig. 6. The total number of Halyomorpha halys and
Acrosternum hilare adults caught inside or within 10
cm of traps baited in 2006 with methyl (E,E,Z)-2,4,6-de-
catrienoate (EEZ), a 5:95 ratio of trans-/cis-(Z)-a-bisab-
olene epoxides (BX), or EEZ+BX, versus unbaited
control traps (4 baffle traps/treatment, 2.5 mg/lure, re-
baited weekly). Bars followed by different letters are
significantly different (P < 0.0001).


S1A hfe nyph a



0 0
a M DC r .
w on *rmor*


*rharysadulR
1 0 it hafys nymphs 0



D0 *


aooc .o o -


I-i 11/1







Florida Entomologist 92(3)


for each species (x2 = 93.82 and 83.07, respec-
tively, P < 0.0001, df = 3). H. halys and A. hilare
adults of both sexes were attracted to traps baited
with methyl (E,E,Z)-2,4,6-decatrienoate, but nei-
ther species was attracted to traps baited with the
bisabolene epoxide blend. Combining methyl
(E,E,Z)-2,4,6-decatrienoate with the bisabolene
epoxide blend did not significantly increase cap-
tures of either bug (P = 0.1548 and P = 0.063 forA.
hilare and H. halys, respectively). The 2007 test
results involving EEZ and BX treatments corrob-
orated the 2006 results showing attraction of both
species to EEZ-baited but not to BX-baited traps,
and baiting traps with a 5 times greater dose of
the bisabolene epoxide blend did not result in in-
creased attraction of either species (Fig. 7). In ad-
dition, the following tachinid fly parasitoids were
caught in 2006 and 2007 in or near traps baited
with methyl (E,E,Z)-2,4,6-decatrienoate: Gymno-
soma par (Walker) (11 females), Euthera tentatrix
Loew (4 females), Euclytia flava (Townsend) (6 fe-
males and 3 males); or traps baited with the blend
of bisabolene epoxides: Trichopoda pennipes (F.)
(9 females and 3 males), E. flava (3 females and 2
males).
In 2007, baiting traps with live, laboratory
reared H. halys males significantly attracted con-
specific adults of both sexes and nymphs (4 fe-
males, 11 males and 6 nymphs; 29 Aug to 1 Oct,
2007, P < 0.0001). Traps containing solvent ex-
tracted, dead adult H. halys pinned males did not
attract any conspecific individuals from 18 Sep
through 16 Oct, during which time traps baited
with live males caught 5 male and 2 female H.
halys adults.
Finally, in feeding choice experiments between
sunflower seeds and seeds of Japanese cedar
(Cryptomeria japonica) or false cypress (Chame-




4b 0



B



A a A AJ
-7 a -


contrl


EM ex 6. OX
Trealmemt


Fig. 7. The total number of Halyomorpha halys and
Acrosternum hilare adults caught inside or within 10
cm of traps baited in 2007 with methyl (E,E,Z)-2,4,6-de-
catrienoate (EEZ), a 5:95 ratio of trans-/cis-(Z)-a-bisab-
olene epoxides (BX), or 5 septa loaded with BX, versus
unbaited control traps (4 baffle traps/treatment, 2.5 mg/
lure, rebaited weekly). Bars followed by different letters
are significantly different (P < 0.0001).


cyparis obtusa), second instar H. halys showed an
overwhelming preference for sunflower seeds. For
the sunflower/Japanese cedar test, 78 nymphs
were observed aggregated on sunflower seeds,
and none were observed on Japanese cedar seeds.
For the sunflower/ false cypress test, 78 nymphs
were observed aggregated on sunflower seeds,
and 2 nymphs were observed on false cypress
seeds.

DISCUSSION

Previous trapping experiments at Allentown,
Pennsylvania, showed that methyl (E,E,Z)-2,4,6-
decatrienoate is apparently essential for attrac-
tion ofH. halys, but methyl (E,Z,Z)- and (Z,E,Z)-
2,4,6-decatrienoates can be used to attract H.
halys because these isomers substantially isomer-
ize to methyl (E,E,Z)-2,4,6-decatrienoate when
exposed to sunlight (Khrimian et al. 2008). There-
fore, although our 2004 and 2005 tests used me-
thyl (E,Z,Z)- and (Z,E,Z)-2,4,6-decatrienoates, we
are confident that the trap counts recorded at
Beltsville, Maryland, from 2004 through 2008 are
a true reflection of the range expansion of H.
halys into central Maryland. From a practical
standpoint it is fortunate that methyl (E,E,Z)-
2,4,6-decatrienoate is most active because this is
the most convenient of the methyl 2,4,6-decatri-
enoate isomers to synthesize (Khrimian 2005),
and isomerization of methyl (E,E,Z)-2,4,6-decatri-
enoate in sunlight may even increase attractive-
ness to H. halys (Khrimian et al. 2008).
Our data confirm that H. halys is truly an in-
vasive species (Richardson et al. 2000; Colautti &
MacIsaac 2004) because in central Maryland the
population has risen from being undetectable in
2004 to being more abundantly trapped than the
indigenous A. hilare. Halyomorpha halys ex-
panded its range some 280 k to reach Beltsville,
Maryland, where it was first detected in traps in
2005 (1 adult and 3 nymphs) (Aldrich et al. 2007).
Thus, the lag time (Mooney & Cleland 2001) from
establishment to population explosion is rela-
tively short for H. halys. In North America, the
brown marmorated stink bug now also has been
collected in Ohio, Virginia and Massachusetts,
and populations in Pennsylvania and New Jersey
have reached damaging levels in some apple or-
chards (Hamilton 2009). The extent to which H.
halys becomes an agricultural pest, in addition to
a nuisance when overwintering in dwellings, re-
mains to be seen. However, the insect is continu-
ing to spread in North America, and its northern
range may be gradually extended as a result of
global warming (Kiritani 2006, 2007). The discov-
ery of H. halys in Switzerland (Wermelinger et al.
2008) portends a similar situation in Europe.
Our finding that traps baited with live, labora-
tory-reared H. halys males attracted conspecific fe-
males, males and nymphs suggests that H. halys


September 2009







Aldrich et al: Invasion of the Brown Marmorated Stink Bug


males do produce a pheromone despite our inability
thus far to isolate any male-specific compounds
from them. Alternatively, the captured conspecifics
could have been attracted by substrate-borne vibra-
tions from caged males. If attraction was due to the
presence of a pheromone, the apparent low level of
pheromone production by laboratory-reared H.
halys males may be caused by an inadequate diet
(see Morishima et al. 2005). In this regard it could
be significant that we must feed the insects organi-
cally grown green beans for them to survive yet the
colony never flourishes (J. R. A., personal observa-
tion). Nevertheless, in our feeding tests of Japanese
cedar and false cypress seeds versus sunflower
seeds, second instars showed no propensity to feed
on the conifer seeds notwithstanding Kiritani's
(2007) claim that H. halys must feed on cones of
these conifers to complete their life cycle. Funayama
(2005) presented evidence that the mature seeds of
Japanese cedar are suitable for the development of
H. halys nymphs, but that indehiscent cones are
not. He concluded that most H. halys in his region of
study (Akita Prefecture, Japan) cannot develop into
adults or lay eggs by feeding on Japanese cedar
cones because cones do not dehisce until Oct. On the
other hand, H. halys adults laid eggs on Japanese
bird cherry trees, Prunus grayana (Rosaceae), in
early Jun, and nymphs from these eggs completed a
generation by mid-Sep feeding on bird cherry fruit
(Funayama 2007). Funayama believes that H. halys
females in his region develop their ovaries while
feeding on early maturing wild cherries (P apatala,
P verecunda, and P sargentii), and then immigrate
into the later maturing bird cherry (P grayana) to
lay eggs (personal communication; Fruit-tree Ex-
periment Station, Akita Prefectural Agriculture,
Forestry and Fisheries Research Center, Daigo,
Hiraka, Yokote, Akita, 013-0102, Japan; funaya-
mak@pref.akita.lg.jp). The observation of
Funayama (2007) that Prunus spp. are excellent
hosts for H. halys in Japan, raises the possibility
that North American Prunus spp., such as black
cherry, Prunus serotina, are early season hosts ofH.
halys in the U.S.
The near total absence of H. halys in the conif-
erous forest site at Beltsville is further evidence
that the establishment ofH. halys in North Amer-
ica is not associated with coniferous forests or or-
namentals. Our results suggest that H. halys was
primarily associated with soybean at our study
site, and sweep-net sampling of the soybean strips
at the Beltsville deciduous/soybean-corn site con-
firmed the presence ofH. halys nymphs and adults
(J. R. A., unpublished data). Indeed, in Oct 2008 we
observed a spectacular invasion of a home near
Hagerstown, Maryland, by thousands of H. halys
adults that were apparently originating from a
soybean field about 50 m downhill from the resi-
dence (J. R. A. and A. K., personal observation).
The abundance of overwintering adults in dwell-
ings where the H. halys population has exploded


suggests that the failure to catch adults early in
the season in traps baited with methyl (E,E,Z)-
2,4,6-decatrienoate reflects a seasonal difference
in the responsiveness of H. halys to this compound.
Perhaps the main reason why the population
ofH. halys is rapidly increasing is that native sce-
lionid wasp (Arakawa et al. 2004) and tachinid fly
parasitoids (Aldrich et al. 2006) have yet to fully
exploit H. halys as a new host. The only tachinid
to emerge from H. halys adults collected in Allen-
town (2 flies from 834 H. halys adults) was Tri-
chopoda pennipes (F.) (Aldrich et al. 2006), which
is the sole known tachinid parasitoid ofA. hilare
in North America (Arnaud, Jr. 1978). Tachinid
flies use pheromones of heteropterans to home-in
on potential hosts (Aldrich 1995), so parasitism of
H. halys by T pennipes may be a clue that the
pheromone of H. halys is chemically similar to
(Z)-a-bisabolene epoxide. Furthermore, we found
that T pennipes is attracted to the reported "pher-
omone" ofA. hilare (Aldrich et al. 1989; Aldrich et
al. 1993; McBrien et al. 2001) even thoughA. hi-
lare itself is not attracted to the 5:95 blend of cis-
and trans-(Z)-a-bisabolene epoxides. Combining
(Z)-a-bisabolene epoxides with methyl (E,E,Z)-
2,4,6-dectrienoate did not increase attraction ofA.
hilare, nor did increasing the dosage of (Z)-a-bis-
abolene epoxides affect A. hilare attraction. Thy-
anta spp. are the only New World bugs known to
use a methyl 2,4,6-decatrienoate (the (E,Z,Z)-iso-
mer) as a pheromone component (Millar 1997;
McBrien et al. 2002; Moraes et al. 2005); there-
fore, A. hilare may be eavesdropping on phero-
mone calling Thyanta males. Interestingly, fe-
males of the solitary wasp, Astata occidentalis
Cresson (Sphecidae) use methyl (E,Z,Z)-2,4,6-de-
catrienoate as a kairomone to find Thyanta adults
with which to provision their nests (Millar et al.
2001; Aldrich et al. 2007). Evans (1957) also lists
Hymenarcys nervosa (Say) and Banasa calva
(Say) (Pentatomidae) as preferred prey forA. occi-
dentalis, suggesting that these stink bugs may
employ methyl 2,4,6-decatrienoates in their pher-
omones.
Recently, Funayama (2008) has provided the
most direct evidence that H. halys in Japan is at-
tracted to the pheromone of P stall to find food.
By calculating a nutritional level index [= live
weight (mg)/pronotum width (mm)3] for adults
collected in traps baited with methyl (E,E,Z)-
2,4,6-dectrienoate versus adults hand-collected
on food plants, he found that the semiochemically
collected insects were nutritionally inferior to
those hand-collected from host plants. Our cur-
rent and past data (Aldrich et al. 2007) indicate
that the cross-attraction propensity of H. halys
for the P stall pheromone has been maintained in
the population of H. halys invading North Amer-
ica. The fact that pentatomid nymphs, even young
nymphs, are often attracted to synthetic phero-
mones of conspecific males (even when no adult







Florida Entomologist 92(3)


conspecifics are present in traps to potentially
produce substrate vibrations) (Aldrich 1988,
1995) also supports the idea that pheromones
may be associated with food because nymphs are
obviously not seeking a mate. Funayama (per-
sonal communication) suspects that H. halys
adults and nymphs may need to feed on a series of
host plants for optimal development. Similarly,A.
hilare has a distinct preference for certain native
hosts, mainly wild bushes and trees, and a succes-
sion of hosts appears to be necessary to sustain a
population (Schoene 1933).
Under field conditions in Georgia, Tillman et al.
(2009) verified our finding that traps baited with a
5:95 trans- to cis-(Z)-a-bisabolene epoxide blend
failed to attract A. hilare, whereas traps baited
with methyl (E,E,Z)-2,4,6-dectrienoate signifi-
cantly attracted A. hilare. Additionally, however, it
was demonstrated for the first time in the field that
the southern green stink bug, Nezara viridula (L.),
can be trapped using its reported pheromone, a 3:1
trans- to cis-(Z)-a-bisabolene epoxide blend (Ald-
rich et al. 1987; Baker et al. 1987), but that N. ir-
idula is not attracted to methyl (E,E,Z)-2,4,6-dec-
trienoate. Males of Acrosternum and Nezara spp.
produce distinctive ratios of the same trans/cis-(Z)-
a-bisabolene epoxides, butA. hilare produced only
about 0.3 gg/male/day versus 9 pg/male/day for N.
viridula and about 20 pg/male/day for A. pennsyl-
vanicum (Aldrich et al. 1989). Although we now
have some indication that H. halys males do pro-
duce an attractant pheromone, our difficulty in iso-
lating the pheromone suggests that the amount of
pheromone produced is naturally low, as is the case
as well for A. hilare.
In conclusion, H. halys seems to have suppressed
pheromonal communication while relying more on
pheromone cross-attraction to find the array of host
plants required for optimal development. The semi-
ochemical situation for A. hilare may be similar
since production of male-specific compounds is low,
and the insect apparently requires a series of host
plants to complete development as for H. halys. The
semiochemical parallels between H. halys andA. hi-
lare would be validated if and when native North
American species of bugs are found that produce
methyl (E,E,Z)-2,4,6-dectrienoate as a part of their
aggregation pheromones.

ACKNOWLEDGMENTS

We thank Dr. David Rider, North Dakota State Uni-
versity, for identification ofHalyomorpha halys, and Drs.
Joseph Kirkbride and Richard Olsen, USDA-ARS Floral
& Nursery Plants Research Unit, Beltsville, Maryland,
for conifer seeds and identifications, respectively. We are
also grateful to Dr. Jocelyn Millar, Department of Ento-
mology, University of California at Riverside, for the gift
of bisabolene epoxides used in early years of this study.
Mr. Robert Bennett helped maintain the colony of H.
halys and Mr. Edward Clark helped with field trapping,
for which we are grateful. This article reports the results


of research only. Mention of a proprietary product does
not constitute an endorsement or a recommendation by
the USDA for its use. The authors thank Dr. Andr6s
Gonzalez Ritzel, Universidad de la Republica Gral., Mon-
tevideo, Uruguay, for the Spanish Resumen.

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Bull. Soc. Entomol. Suisse 81: 1-8.










Supplemental information for the paper by Jeffrey Aldrich et al.


Online Figure 1: The two types of traps used in field tests:
a) Container trap and, b) Baffle trap.


Online Figure 2: Aerial view of BARC-West 2008 field sites: a) deciduous trees
bordering strips of soybean and corn and, b) mature stands of coniferous trees


I I If.
U q'







Online Figure 3: The brown marmorated stink bug, Halyomorpha halys:
a) Adult female and, b) adult male (top) and fifth-instar nymph
(approximately 2.5 x life size).
a3 b
LAo

!!1







Florida Entomologist 92(3)


September 2009


DEVELOPMENT OF SPALANGIA CAMERONI AND MUSCIDIFURAX RAPTOR
(HYMENOPTERA: PTEROMALIDAE) ON LIVE AND FREEZE-KILLED
HOUSE FLY (DIPTERA: MUSCIDAE) PUPAE

PHILLIP E. KAUFMAN' AND CHRISTOPHER J. GEDEN2
'Entomology and Nematology Department, University of Florida, Box 110620, Gainesville, FL 32611-0620

2USDA-ARS, Center for Medical, Agricultural and Veterinary Entomology, 1600 SW 23d Dr., Gainesville, FL 32608

ABSTRACT

The use of pteromalid parasitoids for muscoid fly control is becoming increasingly common.
Two species that are often recommended for releases are Spalangia cameroni Perkins and
Muscidifurax raptor Girault and Sanders. This study was conducted to determine if the re-
duced suitability of freeze-killed pupae for Pteromalidae, particularly S. cameroni, is due to
freezing, freezing duration, or the type of freezer used. Processing pupae through freezing
had a considerably greater negative effect on rearing of S. cameroni than on M. raptor. Al-
though freezing pupae did reduce M. raptor progeny production, there was no effect of length
of storage or type of freezer used. Freezing pupae for 5 months resulted in significantly fewer
S. cameroni progeny than freezing pupae for 2 weeks. Although not significant, pupae held
in a frostfree freezer produced more progeny than those in a non-frostfree freezer. Measure-
ments of weights of parasitized pupae over time indicated that freezing did not result in ac-
celerated desiccation of hosts. These results document that the use of prolonged freezing and
type of freezer minimally impacts M. raptor, but that commercial insectaries and research-
ers should be cautious in the use of frozen pupae when rearing or surveying for S. cameroni.

Key Words: Muscidifurax raptor, Spalangia cameroni, Pteromalidae, house fly, Musca do-
mestica, biological control

RESUME

El uso de parasitoides de la familiar Pteromalidae para el control de moscas del tipo muscoi-
dea es cada dia mas comun. Dos species que se recomiendan a menudo para liberar son
Spalangia cameroni Perkins y Muscidifurax raptor Girault y Sanders. Este studio fue rea-
lizado para determinar si la reducida susceptibilidad de las pupas (de las moscas) matadas
por congelaci6n para criar las species de Pteromalidae, particularmente en S. cameroni, si
es debido a la congelaci6n, la duraci6n de la congelaci6n o a la clase de congelador que se usa.
Al procesar las pupas por la congelaci6n se present un efecto negative notablemente mayor
sobre la crianza de S. cameroni en comparici6n con lo de M. raptor. Aunque la congelaci6n de
las pupas redujo la producci6n de las progenies de M. raptor, no hubo un efecto causado por
la duraci6n del almacenamiento o de la clase de congelador que se uso. La congelaci6n de las
pupas por 5 meses result en un numero signativamente menor de progenies de S. cameroni
en comparici6n a las pupas congeladas por 2 semanas. Aunque no es significativo, las pupas
mantenidas en el congelador de clase "frostfree" (que no crea escarcha) producieron mas pro-
genies que las pupas mantenidas en congeladores que no son de clase "frostfree". Las medi-
das del peso de las pupas parasitizadas sobre tiempo indicaron que la congelaci6n no result
en la desecaci6n acelerada de los hospederos. Estos resultados documentan que el uso de
congelaci6n prolongada y la clase de congelador tiene un impact minimo sobre M. raptor,
pero los insectarios y los investigadores deben ser precavidos con el uso de pupas congeladas
cuando se cria o monitorea la especie S. cameroni.


House flies are a primary pest with confined
livestock, particularly dairy and poultry. With the
continued development of insecticide resistance
and the general movement toward more sustain-
able management systems, including IPM, in-
creasing numbers of producers are using biologi-
cal control. On livestock and poultry facilities,
augmentation of naturally-occurring pteromalid
parasitoids can be helpful in managing fly popu-
lations and has become more common (McKay &
Galloway 1995; Kaufman et al. 2001; Geden &


Hogsette 2006). Evaluation of the effectiveness of
parasitoid releases continues to rely on the use of
either live sentinel muscoid pupae or collection of
wild pupae as hosts of the parasitoids. There are
merits to both approaches, and Kaufman et al.
(2001) summarized their advantages and disad-
vantages. A compromise system that would allow
for longer field exposures of sentinel pupae is de-
sirable because this would combine the opera-
tional advantages of the sentinel pupae approach
(repeatability, rapid location, high recovery rate)







Kaufman & Geden: Fly Parasitoid Development on Freeze-killed Pupae


with the increased species diversity characterized
by collections of wild pupae.
Manipulation of muscoid pupae to increase
field longevity of sentinel hosts over live pupae
has received considerable attention (McKay &
Galloway 1995; Floate & Spooner 2001; Gibson
& Floate 2004; Geden & Hogsette 2006). In ad-
dition to surveillance, the use of killed pupae
has 2 advantages to the biocontrol community.
First, use of killed and stored pupae would al-
low commercial insectaries to stockpile hosts in
anticipation of increased demands for products
(Floate 2002). Second, placement of masses of
killed pupal hosts in livestock facilities has
been demonstrated to enhance parasitism of
natively occurring and augmented parasitoids
by providing an opportunity for in situ amplifi-
cation of local populations (Pickens & Miller
1978; Petersen 1986).
The impacts of killed pupae on pteromalid
development and parasitization have been in-
vestigated by several authors (Morgan et al.
1986; Petersen & Mathews 1994; Geden &
Kaufman 2007). Recently, we documented mul-
tiple methods of pupal preservation with heat
shock being the most readily adoptable proce-
dure (Geden & Kaufman 2007). In that study
we also found that pupae that were killed by
brief exposure to freezing followed by refriger-
ated storage conditions resulted in diminished
suitability for parasitism by Spalangia cam-
eroni Perkins.
Many researchers have reported that the use
of freeze-killed pupae with S. cameroni results
in significantly reduced productivity or utiliza-
tion, suggesting that this species may be averse
to killed hosts (Roth et al. 1991; Floate 2002;
Geden & Kaufman 2007). However, in our pre-
vious publication, we reported that S. cameroni
production remained at 73-78% of that with live
pupae when freeze-killed pupae were stored for
up to 8 weeks at 4C. Additionally, with many of
the early publications on freeze-killed pupae,
pupae were held in frost-free freezers. These
freezers remain free of frost by systematically
raising the temperature in the freezer to liber-
ate frozen water particles into a gaseous state,
wherein the water is removed when the com-
pressor removes the water vapor, resulting in
lost moisture from the material in the freezer.
We see this effect as freezer burn, and its effect
on the quality of stored fly pupae is unknown.
The primary objective of this study was to
determine whether the type of freezing condi-
tions (frost-free versus non-frost-free) affects
the suitability of freeze-killed pupae for para-
sitism by pteromalids, particularly S. cameroni.
A second objective was to determine whether
the length of time that pupae are held in either
freezer type impacts host suitability for the par-
asitoids.


MATERIALS AND METHODS

House flies were obtained from an established
colony maintained at the University of Florida.
Flies were held under laboratory conditions that
included 27C, 75% RH and a photoperiod of 16:8
(L:D) h. Larvae were reared on a diet of 2:3:15:8
ratio of calf protein supplement, wood chips, bran
and tap water, respectively, and adults were
maintained on water, nonfat dry milk, and su-
crose. Following pupariation, extracted fly pupae
were weighed to obtain individual pupal weights.
The daily collection was then divided in half and
placed into double-layered zippered plastic
freezer bags. Air was expelled from the bags by
hand to the greatest extent that was possible
without crushing pupae. One double-bag each
was placed into either a vertical frost-free or non-
frost-free freezer (both set at -20C) and held fro-
zen until experiment initiation. Pupae were al-
lowed to thaw on the benchtop for 4 h prior to use.
Live pupae were collected from rearing bins the
day of parasitoid introduction. Freezer treat-
ments consisted of 2 freezer types (frost-free and
non-frost-free) and 2 freezing durations (2 weeks
or 5 months), resulting in 5 treatments. Muscidi-
furax raptor Girault and Sanders and S. cameroni
were Florida strains and reared as previously de-
scribed (Geden 2002).
Evaluation methodology was similar to that
described by Geden & Kaufman (2007). Briefly, at
experiment initiation, pupae were counted into
groups of 100 with 5 sets of pupae per each pupal-
treatment and parasitoid species. Parasitoid ex-
posures consisted of 5 female M. raptor or 10 fe-
male S. cameroni and all containers were held at
27C. Parasitoids were removed from pupae fol-
lowing 24 h exposure and pupae were held in a
75% RH, 27C rearing chamber for fly and parasi-
toid emergence. Following house fly emergence, S.
cameroni-exposed pupae were placed in gelatin
capsules to aid in enumeration. The experiment
was conducted on 3 separate occasions with dif-
ferent batches of pupae and parasitoids.
An additional test was conducted to measure
water loss in live, freeze-killed, and irradiated pu-
pae parasitized by the 2 parasitoid species. The
pupae were all obtained from a single cohort of
10,000 fly pupae (2 d old) that was divided into 3
equal portions. One portion (live) was held at 8C
for 3 d before the test. The second portion (freeze-
killed) was placed in a frost-free freezer as de-
scribed in the previous section for 3 d. The third
portion (irradiated) was irradiated as before and
held for 3 d in a refrigerator set at 4C. On the day
of the test, pupae of each treatment were exposed
to oviposition by M. raptor and S. cameroni at
host:parasitoid ratios of 10:1 and 5:1, respectively,
for 24 h. After the parasitoids were removed, the
pupae were transferred to screen-topped plastic
holding containers and placed in a growth cham-







Florida Entomologist 92(3)


ber maintained at 25C, 70% RH, and constant
light. Aliquots of 50 pupae were removed from the
chamber and weighed individually for 2 (M. rap-
tor) or 3 weeks (S. cameroni) after oviposition.
Each pupa was dissected immediately after being
weighed, and weight data were analyzed only for
those pupae that contained live immature parasi-
toids. Numbers of observations ranged from 20
(live, day 7, M. raptor) to 49 (irradiated, day 21, S.
cameroni). Initial live weights of the pupae on the
day of collection averaged 16.55 + 0.35 mg per
pupa.
Data on progeny production were analyzed
separately for each species with pupal treatment
and replication as main effects in the General
Linear Models (GLM) Procedure of the Statistical
Analysis System (SAS Institute 1995). Treatment
effects were further evaluated by 3 single degree
of freedom orthogonal contrasts with the Con-
trast statement in the GLM Procedure: (1) live
versus frozen pupae; (2) pupae kept frozen for 2
weeks versus 5 months; and (3) pupae stored in a
frost-free versus non-frost-freezer. Data were nor-
malized by square-root transformation prior to
ANOVA. Treatment effects on weights of parasit-
ized pupae were evaluated separately for each
species and time interval by one-way ANOVA in
the GLM Procedure of SAS.

RESULTS AND DISCUSSION

Processing pupae through freezing had a con-
siderably greater negative effect on S. cameroni
than on M. raptor (Table 1). The overall treatment
effect for the ANOVA was significant for M. raptor
(F4,66 = 2.62;P < 0.05). Live pupae produced nearly
33 M. raptor progeny, while previously frozen


progeny production ranged from 21 to 27 (F166 =
6.67; P < 0.05). With M. raptor, no differences
were observed between length of freezer exposure
or type of freezer (frost-free or non-frost-free).
Our results are similar to those reported by
Floate & Spooner (2002) who froze pupae for 1-4
wks at -20C without significant reductions in M.
raptor parasitism. In their study, overall parasit-
ism was higher than in our study; however, para-
sitoids in their study were allowed to attack the
killed pupae for 4 d. Additionally, the type of
freezer used was not mentioned. In our study, we
held pupae in the freezer in airtight, double-lay-
ered zipper-style bags, while Floate & Spooner
(2002) held pupae in non-airtight containers.
Geden & Kaufman (2007) also reported that M.
raptor progeny production was not impacted by
several pupal killing treatments that included
freezing and subsequent refrigeration.
The overall treatment effect with S. cameroni
was highly significant (F4,66 = 34.13; P < 0.01),
demonstrating again that S. cameroni survival is
highest when live puparia are used (Table 1). Live
house fly pupae exposed to S. cameroni produced
42 progeny, which was significantly greater (F, 66 =
91.32;P < 0.01) than any of the frozen treatments,
where a range of from 8 to 27 progeny were recov-
ered. With S. cameroni, the length of time that pu-
pae were in the freezer was significant (F,66 =
35.84; P < 0.01) as was the type of freezer (F16 =
4.71;P < 0.05).
Several researchers have studied the suitabil-
ity of killed pupae for use with Spalangia spp.
with varied results. Rueda & Axtell (1987) deter-
mined that freezing for short periods of time did
not reduce S. cameroni production in comparison
to live pupae. Morgan et al. (1986) reported that


TABLE 1. PROGENY PRODUCTION OF MUSCIDIFURAX RAPTOR AND SPALANGIA CAMERONI ON LIVE HOUSE FLY PUPAE
AND PUPAE KILLED BY PLACING IN FROSTFREE AND NON-FROSTFREE -20C FREEZERS FOR EITHER 2 WEEKS
OR 5 MONTHS.

Mean (SE) no. adult parasitoid progeny produced:

Treatment M. raptor S. cameroni

Live 32.7 (3.3) 41.9 (3.1)
2-wk, Frostfree 27.3 (3.9) 26.9 (4.5)
2-wk, Non-frostfree 23.3 (4.8) 15.9 (3.0)
5-mo., Frostfree 25.3 (3.6) 7.9(1.1)
5-mo., Non-frostfree 21.1(4.7) 8.1(1.2)
ANOVA F
Treatment overall 2.62* 34.13**
Live vs frozen 6.67* 91.32**
2 wk vs 5 mo storage 0.57ns 35.84**
Frostfree vs Non 2.87ns 4.71*

n = 3 replicates of 5 sets of 100 pupae per treatment exposed to either 5 (M. raptor) or 10 (S. cameroni) female parasitoids for 24 h.
**, P < 0.01.
*, P < 0.05; ns, P > 0.05; df = 4,66 (overall treatment effect) or 1,66 orthogonall contrasts).


September 2009







Kaufman & Geden: Fly Parasitoid Development on Freeze-killed Pupae


gamma-irradiation did not impact the parasi-
toidism of Spalangia endius Walker. However,
these results seem to be in the minority, particu-
larly with respect to the use of frozen pupae.
Considerably more research suggests that killed
fly pupae, particularly frozen fly pupae are less
suitable for Spalangia than are live pupae (Roth
et al. 1991; Floate 2002; Geden & Kaufman
2007). Floate (2002) determined that refrigera-
tion at 15C reduced suitability as hosts for S.
cameroni. Geden & Kaufman (2007) found that
S. cameroni production from pupae killed by
heat shock or gamma radiation was not different
on the day the pupae were killed, but freezing (-
80C for 10 min) reduced progeny production by
16%. Furthermore, they report that production
from freeze-killed and refrigerated was reduced
to 73-78% during the first 8 weeks of storage and
was 28% of live pupal production by 4 months of
storage.
Petersen & Matthews (1984) using Muscidi-
furax zaraptor Kogan and Legner theorized
that reduced suitability of previously frozen
house fly pupae as hosts (over 96 h thawed) was
due to desiccation rather than putrefaction of
the host. Our theory held that the suitability of
pupae held in the freezers for extended time pe-
riods would decrease more quickly with frost-
free freezers as pupae moisture levels dropped.
Our bioassay results show that the effects of
freezing were more profound for S. cameroni
than for M. raptor. Our data do not support the
desiccation theory and suggest that the length
of freezing had a more profound impact on pu-
pal suitability than did the type of freezer. Data
on weights of parasitized pupae over time pro-
vide further evidence that differential desicca-
tion rates do not account for the unsuitability of
freeze-killed hosts for S. cameroni (Table 2). Al-
though parasitized pupae in all treatments lost
substantial amounts of weight over time, there
was no evidence for accelerated weight loss as a
result of freezing.
After eliminating desiccation as a hypothe-
sis, the reason for the poor performance by


S. cameroni on frozen pupae remains uncertain.
Because we found in our previous study that
this species readily attacked and developed on
pupae that were killed by other means (heat
and irradiation), the poor suitability of freeze-
killed pupae is not simply due to the fact that
they are not alive (Geden & Kaufman 2007).
While dissecting puparia for this experiment,
we found that the freeze-killed hosts were rap-
idly putrefying compared with live hosts. From
these observations as well as from examination
of the condition of hosts killed by heat shock
and irradiation, we suggest that freezing re-
sults in damage to the puparium that makes
the killed host within more vulnerable to colo-
nization by decomposing and saprophytic mi-
croorganisms. We suggest that hosts killed by
freezing simply deteriorate and decay faster
than hosts that were initially live or that were
killed by irradiation and minimal heat. If this is
the case, the reason for the relatively high sur-
vival of M. raptor on freeze-killed hosts may be
that its shorter development time allows this
species to complete larval development before
host quality deteriorates below a critical
threshold.
The application of the results of this study con-
firms that the use of pupae that are killed by and
stored under freezer conditions will substantially
impact progeny production with S. cameroni re-
gardless of freezer type. Although such pupae
could be useful for emergency provisioning of
hosts in a rearing program, their use for routine
production and in surveillance programs is not
recommended.


ACKNOWLEDGMENTS

We thank L. Wood, A. Campbell, M. Geden, J. Matta,
C. Scipioni, and J. Pecora for assistance with this
project. Support was provided in part by the University
of Florida Agricultural Experiment Station Federal For-
mula Funds, project FLA-ENY-04598 (Cooperative
State Research, Education and Extension Service, U.S.
Department of Agriculture).


TABLE 2. WEIGHTS OF LIVE, FREEZE-KILLED, OR IRRADIATION-KILLED HOUSE FLY PUPAE AFTER PARASITIZATION BY
MUSCIDIFURAX RAPTOR AND SPALANGIA CAMERONI.

Mean (SE) weight (mg) of parasitized pupae

Species Days since oviposition Live Irradiated Frozen ANOVA F


13.37 (0.26) 13.43 (0.47) 13.35 (0.45)
11.39 (0.34) 10.82 (0.22) 10.72 (0.31)


13.48 (0.31)
10.37 (0.26)
9.50 (0.17)


13.04 (0.32)
10.08 (0.30)
9.11(0.25)


13.63 (0.34)
11.00 (0.28)
9.04 (0.23)


0.01 ns
1.44 ns

0.85 ns
1.97 ns
1.25 ns


M. raptor
M. raptor


S. cameroni
S. cameroni
S. cameroni











REFERENCES CITED

FLOATE, K. D. 2002. Production of filth fly parasitoids
(Hymenoptera: Pteromalidae) on fresh and freeze
killed and stored house fly pupae. Biocontrol Sci.
Technol. 12: 595-603.
FLOATE, K. D., AND SPOONER, R. W. 2002. Parasitization
by pteromalid wasps (Hymenoptera) of freeze-killed
house fly (Diptera: Muscidae) puparia at varying
depths in media. J. Econ. Entomol. 95: 908-911.
FLOATE, K. D., COGHLIN, P., AND GIBSON, G. A. P. 2000.
Dispersal of the filth fly parasitoid Muscidifurax
raptorellus (Hymenoptera: Pteromalidae) following
mass releases in cattle confinements. Biol. Cont. 18:
172-178.
GEDEN, C. J. 2002. Effect of habitat depth on host loca-
tion by five species of parasitoids (Hymenoptera:
Pteromalidae, Chalcididae) of house flies (Diptera:
Muscidae) in three types of substrates. Environ. En-
tomol. 31: 411-417.
GEDEN, C. J., AND HOGSETTE, J. A. 2006. Suppression of
house flies (Diptera: Muscidae) in Florida poultry
houses by sustained releases of Muscidifurax rap-
torellus and Spalangia cameroni (Hymenoptera:
Pteromalidae). Environ. Entomol. 35: 75-82.
GEDEN, C. J., AND KAUFMAN, P. E. 2007. Development of
Spalangia cameroni and Muscidifurax raptor (Hy-
menoptera: Pteromalidae) on live house fly (Diptera:
Muscidae) pupae and pupae killed by heat shock, ir-
radiation, and cold. Environ. Entomol. 36: 34-39.
GIBSON, G. A. P., AND FLOATE, K. D. 2004. Filth fly par-
asitoids on dairy farms in Ontario and Quebec, Can-
ada. Canadian Entomol. 136: 407-417.
KAUFMAN, P. E., LONG, S. J., AND RUTZ, D. A. 2001. Im-
pact of exposure length and pupal source on Mus-
cidifurax raptorellus and Nasonia vitripennis (Hy-


September 2009


menoptera: Pteromalidae) parasitism in a New
York poultry facility. J. Econ. Entomol. 94: 998-
1003.
McKAY, T., AND GALLOWAY, T. D. 1995. Survey and re-
lease of parasitoids (Hymenoptera) attacking house
and stable flies (Diptera: Muscidae) in dairy opera-
tions. Canadian Entomol. 131: 743-756.
MORGAN, P. B., SMITTLE, B. J., AND PATTERSON, R. S.
1986. Use of irradiated pupae to mass culture the
microhymenopterous pupal parasitoid Spalangia
endius Walker (Hymenoptera: Pteromalidae). I.
Musca domestic L. (Diptera: Muscidae). J. Entomol.
Sci. 21: 222-227.
PETERSEN, J. J. 1986. Augmentation of early season re-
leases of filth fly (Diptera: Muscidae) parasites (Hy-
menoptera: Pteromalidae) with freeze-killed hosts.
Environ. Entomol. 15: 590-593.
PETERSEN, J. J., AND MATTHEWS, J. R. 1984. Effects of
freezing of host pupae on the production of progeny
by the filth fly parasite Muscidifurax zaraptor (Hy-
menoptera: Pteromalidae). J. Kansas Entomol. Soc.
57: 387-393.
PICKENS, L. G., AND MILLER, R. W. 1978. Using frozen
host pupae to increase the efficiency of a parasite-re-
lease program. Florida Entomol. 61: 153-158.
ROTH, J. P., FINCHER, G. T., AND SUMMERLIN, J. W.
1991. Suitability of irradiated or freeze-killed horn
fly (Diptera: Muscidae) pupae as hosts for hy-
menopterous parasitoids. J. Econ. Entomol. 84: 94-
98.
RUEDA, L. M., AND AXTELL, R. C. 1987. Reproduction of
Pteromalidae (Hymenoptera) parasitic on fresh and
frozen house fly (Musca domestic Linn.) pupae.
Philippines J. Sci. 116: 313-326.
SAS INSTITUTE. 1995. SAS users guide: statistics. SAS
Institute, Cary, NC.


Florida Entomologist 92(3)







Scientific Notes


PARASITISM OF THE BROWN CITRUS APHID IN DOMINICA
BY LYSIPHLEBUS TESTACEIPES AND LIPOLEXIS OREGMAE
(HYMENOPTERA: APHIDIINAE)

ARTURO COCCO1, AYYAMPERUMAL JEYAPRAKASH AND MARJORIE A. HOY
Department of Entomology and Nematology, University of Florida, Bldg. 970, Natural Area Drive,
Gainesville, FL, 32611-0629, USA

'Presently at Dipartimento di Protezione delle Piante, Sezione Entomologia Agraria, Universita di Sassari,
Via De Nicola 1, 07100 Sassari, Italy


The brown citrus aphid Toxoptera citricidus
(= citricida, Nieto Nafria et al. 2005) (Kirkaldy)
(Hemiptera: Aphididae) is an efficient vector of
the citrus tristeza virus (CTV), and an economi-
cally important pest in areas where citrus spe-
cies are grafted on rootstocks susceptible to CTV
(i.e., sour orange) (Rocha-Pena et al. 1995). Tox-
optera citricidus originated in Asia and invaded
Florida and the Caribbean basin during the
1990s, causing serious economic losses (Hoy et
al. 2007). The parasitoid Lysiphlebus testaceipes
Cresson (Hymenoptera: Aphidiinae) has been re-
corded parasitizing the brown citrus aphid in
Florida, Jamaica, and Puerto Rico (Yokomi &
Tang 1996; Persad et al. 2004; Hoy et al. 2007),
but control of the brown citrus aphid was poor.
As a part of a classical biological control program
directed against T citricidus, the endoparasitoid
Lipolexis oregmae Gahan (Hymenoptera: Aphi-
diinae) was evaluated for introduction into Do-
minica because it established in Florida and was
detected in Jamaica in 2004, where it was fortu-
itously introduced (Hoy et al. 2007; Persad et al.
2007).
Before importation and release ofL. oregmae
in Dominica, a survey was conducted during
Feb and Apr 2007 at 6 parishes to evaluate the
distribution and parasitism rates of brown cit-
rus aphids. Brown citrus aphids and aphids of
unknown species were collected in 95% ethyl al-
cohol and DNA was analyzed for parasitoid
DNA with the High-fidelity PCR protocol devel-
oped by Persad et al. (2004). Genomic DNA from
pooled brown citrus aphids was extracted with
Puregene reagents according to the method
suggested by the manufacturer (Gentra Sys-
tems, Minneapolis, MN) and resuspended in 10
pL of sterile water. The nuclear rRNA ITS2 se-
quences of L. oregmae were amplified with the
specific forward primer LO-ITSF 5'-GGCCAGT-
TGTCGAGTCC-3' in combination with the 28 S-
R reverse primer (5'-ATGCTTAAATT-
TAGGGGGTA-3'), while the rRNA ITS2 partial
sequences of L. testaceipes were amplified with
the forward primer LT-ITSF 5'-CTAGC-
GATAAATGAATGTTC-3' in combination with
the 28 S-R reverse primer (Persad et al. 2004).
PCR products were separated by electrophore-
sis on 2% agarose gel, stained with ethidium


bromide, and photographed. The L. oregmae-
specific primers produced a 270-bp PCR prod-
uct, while the ITS2 sequences amplified from L.
testaceipes produced a 520-bp PCR product.
Although L. oregmae was not purposefully re-
leased in Dominica, the survey indicated that
both parasitoids are present throughout the is-
land (Table 1), with L. oregmae found in all 6
parishes at 76% of the locations sampled, and L.
testaceipes found in 5 parishes at 53% of the
sampling sites. Both L. oregmae and L. testa-
ceipes were detected from aphids of unknown
species on weeds within citrus groves in St.
George parish at 3 locations. This indicated that
both parasitoids parasitize alternative aphid
hosts in the presence of the brown citrus aphid.
When and by which mechanisms L. oregmae was
introduced to Dominica are unknown. The fortu-
itous introduction of parasitoids of citrus pests
into different Caribbean islands indicates that,
as in Florida, it is difficult to prevent invasive
insect introductions.
Although evaluation of the effectiveness of
parasitoids of the brown citrus aphid was beyond
the goal of this study, the rate of parasitism on a
single date was assessed by selecting randomly 5
aphids from each of 4 parishes and testing them
individually with both L. testaceipes- and L. oreg-
mae-specific primers following the protocol de-
scribed above (Table 2). The percentage of parasit-
ized aphids was remarkably high, ranging from
80 to 100%. None of the brown citrus aphids
tested was positive for L. oregmae only, while 20,
20, and 40% of the samples from St. Mark, St.
David, and St. Peter, respectively, were positive
for L. testaceipes only. Most of samples were posi-
tive for both L. oregmae and L. testaceipes, indi-
cating that both parasitoids had parasitized the
brown citrus aphid. The oviposition sequence, age
of larvae, and larval development time are key
factors that affect the development of parasitoids
within parasitized brown citrus aphids, so it is
impossible to resolve which species would emerge
from the brown citrus aphid in these cases (Per-
sad & Hoy 2003). However, the parasitism level
estimated for L. oregmae and L. testaceipes
ranged from 80 to 100% overall in this limited
sample, suggesting that they are common parasi-
toids of the brown citrus aphid in Dominica. De-







Florida Entomologist 92(3)


TABLE 1. SURVEY ON DISTRIBUTION AND ABUNDANCE OF BROWN CITRUS APHID PARASITOIDS IN CITRUS-GROWING AR-
EAS OF DOMINICA DURING 2007 WITH A HIGH-FIDELITY PCR PROTOCOL AND SPECIES-SPECIFIC PRIMERS.


Sample date Parish


Location


Host


No. of pooled
aphids tested


L. oregmae' L. testaceipes'


Syndicate
Syndicate
Syndicate
Grand Bay
Sundalae
Melville Hall
Wesley
Soufriere
Marigot
Hatton Garden
Woodford Hill
Woodford Hill
Castle Bruce
Giraudel
More Prosper
Roseau
Giraudel


Citrus
Citrus
Citrus
Citrus
Citrus
Citrus
Sweet pepper
Citrus
Citrus
Citrus
Citrus
Citrus
Citrus
Composite weed
Composite weed
Citrus
Colocasia sp.


'+ = positive detection; = negative detection.
Adult wasps collected from a citrus grove.
'Aphids of unknown species collected from citrus or composite weeds within a citrus grove.


TABLE 2. PARASITISM ASSESSMENT OF 5 INDIVIDUAL BROWN CITRUS APHIDS AT EACH SITE BY L. OREGMAE AND L.
TESTACEIPES IN DOMINICA DURING 2007 WITH SPECIES-SPECIFIC PRIMERS AND A HIGH-FIDELITY PCR PRO-
TOCOL.

Percentage positive by PCR

Sample L. oregmae L. testaceipes
date Parish Location Host only only L. oregmae and L. testaceipes

19 Apr St. Peter Syndicate Citrus 0 40 60
20 Apr St. Andrew Marigot Citrus 0 0 100
23 Apr St. Mark Soufriere Citrus 0 20 60
20 Apr St. David Castle Bruce Citrus 0 20 80


spite this apparent abundance, natural enemies
cannot prevent transmission of diseases such as
CTV by aphids, so replanting with citrus on CTV-
resistant rootstocks should be considered.


SUMMARY

The brown citrus aphid parasitoids Lipolexis
oregmae and Lysiphlebus testaceipes are
present and widely distributed in Dominica. Li-
polexis oregmae was not purposefully released
and it is not clear when and by which pathway
the parasitoid was introduced to Dominica. The
brown citrus aphid samples tested were para-
sitized by both parasitoids (80-100%), suggest-
ing that both L. oregmae and L. testaceipes
might be effective parasitoids of T citricidus in
Dominica.


ACKNOWLEDGMENTS

The authors thank Peter Hill, Naomi Commodore,
Ryan Anselm, and Bernitta Serrant of the Ministry of
Agriculture, Fisheries and the Environment, Common-
wealth of Dominica, for assistance in collecting parasit-
ized aphids. This research was funded by the
Autonomous Region of Sardinia (A.C.) and the Davies,
Fischer and Eckes Endowment in Biological Control
(M.A.H.).


REFERENCES CITED

HOY, M. A., JEYAPRAKASH, A., CLARKE-HARRIS, D., AND
RHODES, L. 2007. Molecular and field analyses of the
fortuitous establishment of Lipolexis oregmae (Hy-
menoptera: Aphidiidae) in Jamaica as a natural en-
emy of the brown citrus aphid. Biocontrol Sci. Tech-
nol. 17: 473-482.


7 Feb
7 Feb
7 Feb
2 Feb
6 Feb
16 Feb
15 Feb
20 Apr
20 Apr
20 Apr
20 Apr
20 Apr
20 Apr
23 Apr
23 Apr
20 Apr
23 Apr


St. Peter
St. Peter
St. Peter
St. Patrick
Unknown
St. Andrew
St. Andrew
St. Mark
St. Andrew
St. Andrew
St. Andrew
St. Andrew
St. David
St. George
St. George
St. George
St. George


September 2009







Scientific Notes


NIETO NAFRiA, J. M., ALONSO-ZARAZAGA, M. A., AND
PEREZ HIDALGO, N. 2005. Toxoptera citricida or Tox-
optera citricidus? The validity of a specific name
(Hemiptera, Aphididae, Aphidini). Graellsia 61: 141-
142.
PERSAD, A. B., AND HOY, M. A. 2003. Intra- and inter-
specific interactions between Lysiphlebus testaceipes
and Lipolexis scutellaris on Toxoptera citricida. J.
Econ. Entomol. 96: 564-569.
PERSAD, A. B., JEYAPRAKASH, A., AND HOY, M. A. 2004.
High-fidelity PCR assay discriminates between im-
mature Lipolexis oregmae and Lysiphlebus testa-
ceipes (Hymenoptera: Aphidiidae) within their aphid
hosts. Florida Entomol. 87: 18-24.


PERSAD, A. B., HOY, M. A., AND NGUYEN, R. 2007. Es-
tablishment of Lipolexis oregmae (Hymenoptera:
Aphidiidae) in a classical biological control program
directed against the brown citrus aphid (Homoptera:
Aphididae) in Florida. Florida Entomol. 90: 204-213.
ROCHA-PENA, M. A., LEE, R. F., LASTRA, R., NIBLETT, C.
L., OCHOA-CORONA, F. M., GARNSEY, S. M., AND
YOKOMI, R. K. 1995. Citrus tristeza virus and its
aphid vector Toxoptera citricida. Threats to citrus
production in the Caribbean and Central and North
America. Plant Disease 79: 437-445.
YOKOMI, R. K., AND TANG, Y. Q. 1996. A survey of para-
sitoids of brown citrus aphid (Homoptera: Aphid-
idae) in Puerto Rico. Biol. Control 6: 222-225.







Florida Entomologist 92(3)


THE WHIP SCORPION, MASTIGOPROCTUS GIGANTEUS
(UROPYGI: THELYPHONIDAE), PREYS ON THE CHEMICALLY
DEFENDED FLORIDA SCRUB MILLIPEDE, FLORIDOBOLUS PENNERI
(SPIROBOLIDA: FLORIDOBOLIDAE)

JAMES E. CARREL'AND ERIC J. BRITT2'
1University of Missouri, Division of Biological Sciences, 209 Tucker Hall, Columbia, MO 65211-7400 USA

2University of South Florida, Division of Integrative Biology, 4242 East Fowler Avenue, SCA 110,
Tampa, FL 33620 USA

2'3Current address: Archbold Biological Station, 123 Main Drive, Venus, FL 33960


The rare Florida scrub millipede, Floridobolus
penneri Causey, is confined to xeric, sandy scrub
habitats in the southern part of the narrow Lake
Wales Ridge in Polk and Highlands Counties,
Florida (Deyrup 1994). Although large in size
(adult body length of about 90 mm and width of
about 11.5 mm), little is known about this cylin-
drical animal because it is restricted in distribu-
tion and is nocturnally active aboveground only in
mid-summer; it spends most of its secretive life
buried in sand (Deyrup 1994). Floridobolus is the
only genus and species in an entire family of
spirobolid millipedes, the Floridobolidae (Shelley
2000).
Floridobolus, like most millipedes, is well pro-
tected from predators. When disturbed, it quickly
coils into a tight spiral, shielding its head and all
of its appendages with its smooth, hard integu-
ment and may remain coiled for a long time, for
an average of 4.6 0.7 min (n = 10, + SEM) in lab-
oratory tests. In response to mild to strong provo-
cation, such as squeezing or biting, Floridobolus
discharges large amounts of a liquid defensive se-
cretion from paired glands that open along the
sides of its body (Attygalle et al. 1993; Eisner et
al. 1998). The volatile exudate, a blend of six 1,4-
benzoquinones, appears to act as a conventional
repellent to invertebrate and vertebrate enemies
and the quinone vapors are potent irritants of the
eyes of mice, birds, and humans (Eisner et al.
1978). Prior to this publication, the only known
predator of Floridobolus is the larva of a phengo-
did beetle, Phengodes laticollis meridiana Witt-
mer. The larva instantly paralyzes the millipede
by biting its neck with sharp mandibles and the
defensive quinones remain sealed in the glands
while the larva eats the uncontaminated internal
tissues, starting at the neck and moving toward
the anus, leaving behind empty rings of armored
body segments (Eisner et al. 1998; Eisner et al.
2005).
We now report that the giant whip scorpion,
Mastigoproctus giganteus (Lucas) (Arachnida:
Uropygi) consumes Floridobolus, even if the
quinone-based secretion is released upon attack.
In Aug-Sep 2008 we collected millipedes at 3-day


intervals in 96 pitfall traps arranged in sets of 12
each at 8 randomly chosen sites in scrubby flat-
woods near the southern end of the Archbold Bio-
logical Station, Highlands County, Florida (rang-
ing from 27008" 20" N, 81'21' 18" W to 27007' 19"
N, 81'21' 54" W, elevation 40-43 m). Each trap
consisted of a plastic bucket (17.5 cm diameter x
19 cm depth, 3.8 liter capacity) placed in the
ground so that the rim was flush with the sandy
soil and filled with 3-5 cm of sandy soil. During
the first 2 weeks of Aug, while checking all of the
buckets on 4 different days, we found 17 M. gigan-
teus individually in the traps. In 6 instances there
were partially consumed Floridobolus with them,
6 traps contained both M. giganteus and 1 or more
living Floridobolus, and 5 had M. giganteus and
no millipedes. An additional 35 traps each con-
tained 1-4 living but zero dead Floridobolus on
the days they were checked. In addition, at this
time we never found dead or injured Floridobolus
in the approximately 100 traps containing large
carnivorous carabid beetles, Pasimachus strenuus
LeConte, or in about 30 traps containing very
large lycosid spiders, Hogna osceola (Gertsch and
Wallace). Our field data suggested that millipede
mortality was highly correlated with the presence
of giant whip scorpions: dead, partially consumed
millipedes were found only in buckets where M.
giganteus were present. This conclusion subse-
quently was reinforced when on 3 occasions in the
field we discovered a giant whip scorpion holding
a millipede in its powerful pedipalps while it bit
the prey middorsally at an intersegmental mem-
brane (Fig. 1).
Fig. 1 shows sand grains adhering to the cuti-
cle of the millipede, a sign that the prey may have
discharged its defensive secretion in response to
squeezing or biting inflicted by the giant whip
scorpion. Secretory discharge was confirmed
when several, but not all millipedes, were quickly
attacked as they slowly approached M. giganteus
during laboratory trials staged in sand-filled are-
nas. In some instances the predator's attack did
not evoke release of secretion by the millipede
(Fig. 2). Microscopic examination of the interior of
the segmental rings a day after the millipedes


September 2009







Scientific Notes


Figs. 1-4. Giant whip scorpions, Mastigoproctus gigantea, preying on Florida scrub millipedes, Floridobolus pen-
neri. 1. Attack observed in field. Sand adhering to prey suggests millipede discharged after attack commenced. 2.
Attack staged in laboratory arena. When grasped and bitten, prey uncoiled and, while it was consumed, it did not
discharge defensive secretion. Scale bar = 1 cm. 3. Other staged attacks involved massive discharge of defensive se-
cretion, as visible (arrow) by dozen or so lateral droplets of quinones and adherent sand on other segments. 4. Sev-
eral hours post-attack, all millipedes were reduced to segmental rings and liquified internal tissues wrought by the
arachnid predator's digestive enzymes.











were eaten, with the technique of Eisner et al.
1998, confirmed the presence of replete defensive
glands.
However, in some instances a giant whip scor-
pion's powerful pedipalpal grasp and subsequent
middorsal bite caused the millipede immediately
to discharge quinones from many glands (Fig. 3),
but the predator was not deterred. The odor of the
secretion was very noticeable to us at this time. In
all instances, within 15-30 min after an attack
commenced, the whip scorpion had inflicted a fa-
tal incision between 2 segments of its prey and
liquefied tissues were ingested (Fig. 4).
Although we showed that Floridobolus is highly
vulnerable to attack by M. giganteus, Florida scrub
millipedes spend most of their lives underground
and giant whip scorpions are not common preda-
tors in scrub, so we suspect the rate of predation
might be low in the field. Our results extend the di-
etary diversity of M. giganteus beyond insects,
arachnids, and amphibians (Cloudsley-Thompson
1958, Punzo 2000) to the diplopods.
We thank Jan Weaver, Nicola Ihasz, Alan Riv-
ero, and Whitney Hummel for assistance in the
field, M. Deyrup and H. M. Swain for advice and
encouragement, Archbold Biological Station for
research facilities and a variety of services, and
the helpful comments of 2 anonymous reviewers.
This study was supported by the Archbold Biolog-
ical Station, by the Development Fund of Mis-
souri University, and by Rinker Materials and
Walt Disney World Hospitality and Recreation
Corporation awarded to H. R. Mushinsky and E.
D. McCoy at the University of South Florida.

SUMMARY

On several occasions in Aug-Sep 2008 we de-
tected giant whip scorpions, Mastigoproctus gi-


September 2009


ganteus, feeding on rare Florida scrub millipedes,
Floridobolus penneri, in bucket-style pitfall traps
in the field. Subsequently in laboratory feeding
trials we determined that M. giganteus will
readily attack, kill, and consume F penneri even
if the prey discharges its irritating defensive se-
cretion.

REFERENCES CITED

ATTYGALLE, A. B., Yu, S.-C., MEINWALD, J., AND Eis-
NER, T. 1993. Defensive secretion of the millipede
Floridobolus penneri. J. Nat. Prod. 56: 1700-1706.
CLOUDSLEY-THOMPSON, J. L. 1958. Spiders, Scorpions,
Centipedes and Mites. Pergammon Press, London.
118 pp.
DEYRUP, M. 1994. Florida scrub millipede Floridobolus
penneri Causey, pp. 254-256 In M. Deyrup and R.
Franz [eds.], Rare and Endangered Biota of Florida,
Volume IV. Invertebrates. University Press of Flori-
da, Gainesville, Fl. 798 pp.
EISNER, T., ALSO, D., HICKS, K., AND MEINWALD, J.
1978. Defensive secretions of millipeds, pp. 41-72 In
S. Bettini [ed.], Arthropod Venoms. Springer-Verlag,
Berlin. 977 pp.
EISNER T., EISNER, M., ATTYGALLE, A. B., DEYRUP, M.,
AND MEINWALD, J. 1998. Rendering the inedible edi-
ble: circumvention of a millipede's chemical defense
by a predaceous beetle larva. Proc. Natl. Acad. Sci.
USA. 95: 1108-1113.
EISNER, T., EISNER, M., AND SIEGLER, M. 2005. Secret
Weapons: Defenses of Insects, Spiders, Scorpions,
and Other Many-legged Creatures. Belknap Press of
Harvard University Press, Cambridge, MA. 372 pp.
PUNZO, F. 2000. Diel activity patterns and diet of the gi-
ant whipscorpion Mastigoproctus giganteus (Lucas)
(Arachnida, Uropygi) in Big Bend National Park
(Chihuahuan Desert). Bull. British Aracnol. Soc. 11:
385-387.
SHELLEY, R. M. 2000. Annotated checklist of the milli-
peds of Florida (Arthropoda: Diplopoda). Insecta
Mundi 14: 241-251.


Florida Entomologist 92(3)







Scientific Notes


A NEW PEST OF RICE IN MISSOURI: RANGE EXPANSION OF TRIOPS
LONGICAUDATUS (CRUSTACEA: NOTOSTRACA: TRIOPSIDAE) INTO THE
NORTHERN MISSISSIPPI RIVER ALLUVIAL PLAINS

K. V. TINDALL', K. FOTHERGILL2, W. MINSON3 AND B. OTTIS4
'University of Missouri, Division of Plant Sciences, Delta Research Center, P.O. Box 160, Portageville, MO 63873

2Conservation Seeding and Restoration, Inc. 506 Center Street West, Kimberly, ID 83341 U.S.A

3Bootheel Crop Consultants, 16000 County Road 624, Dexter, MO, 63841

4RiceTec, Inc., P.O. Box 17396, Jonesboro, AR 72403


Tadpole shrimp, Triops longicaudatus (Le-
Conte) (Notostraca: Triopsidae), are pests in Cal-
ifornia rice production systems. Tadpole shrimp
are an obligate species of ephemeral freshwater
aquatic habitats and in North America were con-
sidered primarily a species of the western United
State for many years. Taylor et al. (1987) reported
an eastward range expansion into Oklahoma.
Tadpole shrimp were not known to be in Missouri
until 1979 when a report was filed with the Mis-
souri Department of Conservation. There were 2
more records filed in 1983 and 2007. Early reports
were along the Missouri River with the 1979 and
1983 reports being from Jackass Bend (Jackson
County) and the 2007 record from Darst Bottoms
(St. Charles County) (Dorothy Butler, personal
communication). Additional populations of tad-
pole shrimp have been found in 2009 on the Ar-
kansas/Missouri state line north of Gosnell, AR in
Missouri (Dunklin County) and near Luxora, AR
(Mississippi County).
How tadpole shrimp came to be in Missouri is
unknown, but dispersal occurs via floodwaters
(Taylor et al. 1987), wind (Caceres & Soluk 2002;
Nathan et al. 2005; Graham & Wirth 2008), birds
(Green & Figuerola 2005), and via the pet trade
(Halliday 2008).
On June 8, 2007, a single specimen of an un-
known invertebrate was brought to the Delta Re-
search Center in Portageville, Missouri (Pemiscot
County) for identification. The specimen was col-
lected from a drill-seeded rice field in Pemiscot
County (near Bakerville). The specimen was de-
termined to be a tadpole shrimp but the species
was not determined. Growers were alerted of its
presence in the state at winter meetings.
On May 20, 2008, a phone call was received
about a 16-hectare field in Stoddard County (lo-
cated north and west of Catron), of water-seeded
hybrid rice that had not emerged. The water was
drained from the field and thousands of tadpole
shrimp were congregated in the remaining pud-
dles. No viable seeds were present and the field
was replanted (Ottis, personal observation). On
June 2, 2008, another call was received about
multiple fields in New Madrid County (near Lil-
bourn) that were infested. At least 1600 hectares


had tadpole shrimp present and of those infested,
nearly 800 hectares were economically impacted
and approximately 40 hectares were replanted
(Minson, personal observation).
Specimens collected from both locations in
2008 were yellow-brown in color and <5 cm in
length. The carapace covered slightly less than
the anterior half of the animal. Numerous ap-
pendages were present on the thorax and abdo-
men. Two close-together, sessile, compound eyes
with a simple eye in between were located on the
head. There were >35 body segments. Two tails
extended from the telson. The taxonomic treat-
ment of Longhurst (1955) recognizes T longicau-
datus as the only North American Triops species
and on the basis of this treatment, specimens
were identified as T longicaudatus. However, this
taxonomy may not be supported as new tech-
niques reveal genetic differences due to reproduc-
tive isolation (Sassaman et al. 1997).
Tadpole shrimp females lay an average of 81
eggs in 24 h; however, 1 individual laid 198 eggs
(594 eggs/ 3d) (Scott 1972). Eggs are laid on either
decaying or living plant material, algae, or in the
soil. Egg hatch is affected by pH (Scott 1972; Ha-
masaki & Ohbayashi 2000), soil type, age of egg
(Su & Mulla 2002), temperature (Scott 1972), sa-
linity (Horne 1967; Scott 1972) and depth of
burial in the soil (Scott 1972). Eggs require a des-
iccation period prior to hatching (Fry & Mulla
1992). When a larva ecloses, it feeds on diatoms
and protozoa in the mud (Longhurst 1955) during
early instars. Then it acquires feeding behaviors
similar to that of the adult, which consumes veg-
etative material and aquatic invertebrates (Wal-
ton et al. 1991) and is cannibalistic (Scott 1972).
The foraging behavior (i.e., movement in the mud)
of nearly mature and adult tadpole shrimp up-
roots small seedlings and muddies the water. Lar-
val development is influenced by temperature; al-
though individuals reared at 30C were smaller
than those reared at lower temperature, they
reached sexual maturity at an earlier age (Fry-
O'Brien & Mulla 1996).
Tadpole shrimp are problematic in California
water-seeded rice production systems when lar-
vae eclose after fields are flooded. Sexually ma-











ture tadpole shrimp are found as early as 9-12
days after floods are established (Scott 1972);
therefore, rice plants have <9 days to break the
surface of the flood (i.e., the time at which rice is
no longer vulnerable), before tadpole shrimp are
large enough to uproot seedling rice (Godfrey
2005). Rice planted by drill-seeded or dry-seeded
methods has an adequate root system when fields
are flooded, and tadpole shrimp are not pests in
these systems. Once rice is no longer vulnerable
to tadpole shrimp damage, tadpole shrimp may
serve as a biological control agent for mosquitoes
(Fry et al. 1994) and/or weeds (Takahashi 1977;
Yonekura 1979).
Hybrid rice varieties are planted at a lower
seeding rate (33-45 kg/ha) than conventional va-
rieties (100-120 kg/ha), making them more sus-
ceptible to tadpole shrimp damage than higher
seeding rates. For example, losing 10% of a stand
planted at 33 kg/ha is more detrimental than los-
ing 10% of a stand planted at 110 kg/ha.
Southeastern Missouri is part of the Missis-
sippi Alluvial Plain (USGS 2003). Historically, the
region was covered with swamp lands and heavy
timber (Nolen 1912), but much of which is now
croplands. These croplands include rice fields that
mimic ephemeral ponds inhabited by tadpole
shrimp. In 2008, <10% of the 80,000 hectares of
rice production in Missouri was water seeded.
Therefore, tadpole shrimp will impact only a
small percentage of hectares in Missouri. How-
ever, rice production also occurs on almost
757,000 ha in the Mississippi Alluvial Plain
states of Arkansas and Louisiana (NASS 2008),
and the percentage of water-seeded rice varies
each year, with many hectares of water-seeded
rice in those states that could be impacted if there
is further dispersal southward.

SUMMARY

In North America, tadpole shrimp, Triops lon-
gicaudatus, are pests of water-seeded rice produc-
tion in California. In 2008, tadpole shrimp were
documented to be a pest of rice of water-seeded
rice in Missouri for the first time. This occurrence
represents a range expansion into a new physio-
graphic region (Mississippi Alluvial Plain) and
the Southern U.S. rice producing region. A brief
review of the biology and implications of this pest
are described.

REFERENCES CITED

CACERES, C. E., AND SOLUK, D. A. 2002. Blowing in the
wind: a field test of overland dispersal and coloniza-
tion by aquatic invertebrates. Oecologia 131:402-
408.
FRY, L. L., AND MULLA, M. S. 1992. Effect of drying and
soil moisture on egg hatch of the tadpole shrimp (No-
tostraca: Triopsidae). J. Econ. Entomol. 85: 65-69.


September 2009


FRY, L. L., MULLA, M. S., AND ADAMS, C. W. 1994. Field
introductions and establishment of the tadpole
shrimp, Triops longicaudatus (Notostraca, Triop-
sidae), a biological-control agent of mosquitoes. Biol.
Control 4: 113-124.
FRY-O'BRIEN, L. L, AND MULLA, M. S. 1996. Optimal
conditions for rearing the tadpole shrimp, Triops
longicaudatus (Notostraca: Triopsidae), a biological
control agent against mosquitoes. J. American Mos-
quito Contr. 12: 446-453.
GODFREY, L. D. 2005. Rice Tadpole Shrimp. University
of California IPM Pest Management Guidelines:
Rice. UC ANR Publication 3465 Invertebrates. http:/
/www.ipm.ucdavis.edu/PMG/r682500111.ht-
ml#REFERENCE. Last accessed: 18 December
2008.
GRAHAM, T. B., AND WIRTH, D. 2008. Dispersal of large
branchiopod cysts: potential movement by wind
from potholes on the Colorado Plateau. Hydrobiolo-
gia 600: 17-27.
GREEN, A. J., AND FIGUEROLA, J. 2005. Recent advances
in the study of long-distance dispersal of aquatic in-
vertebrates via birds. Diversity and Distributions
11.2: 149-156.
HALLIDAY, S. 2008. MyTriops. Available at http://mytri-
ops.com. Last accessed: 18 December 2008.
HAMASAKI, K., AND OHBAYASHI, N. 2000. Effect of water
pH on the survival rate of larvae of the American
tadpole shrimp, Triops longicaudatus (LeConte) (No-
tostraca: Triopsidae). Appl. Entomol. Zool. 35: 225-
230.
HORNE, F. R. 1967. Effects of physio-chemical factors on
the distribution and occurrence of some southeast-
ern Wyoming phyllopods. Ecology 48: 472-477.
LONGHURST, A. R. 1955. A review of the Notostraca.
Bull. British Nat. Hist. 3: 1-55.
NATHAN, R., SAPIR, N., TRAKHTENBROT, A., KATUL, G.
G., BOHRER, G., OTTE, M., AVISSAR, R., SOONS, M. B.,
HORN, H. S., WIKELSKI, M., AND LEVIN, S. A. 2005.
Long-distance biological transport processes
through the air: can nature's complexity be unfolded
in silicon? Divers. Distrib. 11: 131-137.
NATIONAL AGRICULTURE STATISTICS SERVICE (NASS).
2008. Rice. http://www.nass.usda.gov. Last accessed:
18 December 2008.
NOLEN, J. H. 1912. Missouri's Swamp and Overflowed
Lands and Their Reclamation. The Hugh Stephens
Printing Company: Jefferson City, MO. 141 p.
SASSAMAN C., SIMOVITCH, M. A., AND FUGATE, M. 1997.
Reproductive isolation and genetic differentiation in
North American species of Triops (Crustacea: Bran-
chiopoda: Notostraca). Hydrobiologia 359: 125-147.
SCOTT, S. R. 1972. Laboratory and field studies of Triops
longicaudatus (LeConte) in California. M.S. Thesis,
University of California, Davis.
SU, T. Y., AND MULLA, M. S. 2002. Spatial occurrence
and hatch of field eggs of the tadpole shrimp Triops
newberryi (Notostraca: Triopsidae), a potential bio-
logical control agent of immature mosquitoes. J. Vec-
tor Ecol. 27: 128-137.
TAKAHASHI, F. 1977. Triops spp. (Notostraca: Triop-
sidae) for the biological control agents of weeds in
rice paddies in Japan. Entomophaga 22: 351-357.
TAYLOR, C. M., BRYANT, R. M., JR., AND HARTMAN, R. E.
1987. Eastward range extension of the tadpole
shrimp, Triops longicaudatus (Leconte), in Oklaho-
ma. Proc. Oklahoma Acad. Sci. 67:75-76


Florida Entomologist 92(3)







Scientific Notes


UNITED STATES GEOLOGICAL SURVEY (USGS). 2003. A
Tapestry of Time and Terrain, U.S. Dept. of the Inte-
rior. http://tapestry.usgs.gov/physiogr/physio.html.
Last accessed: November 12, 2008.
WALTON, W. E., TIETZE, N. S., AND MULLA, M. S. 1991.
Consequences of tadpole shrimp predation on may-


flies in some Californian ponds. Freshwater Biol. 24:
143-154.
YONEKURA, M. 1979. Biological control of weeds by tad-
pole shrimps in paddy field weed efficacy of tadpole
shrimps in transplanted rice fields. Weed Res. Japan
24: 64-68.







Scientific Notes


LENGTH OF MULTIPLE-FUNNEL TRAPS AFFECTS
CATCHES OF SOME BARK AND WOOD BORING BEETLES
IN A SLASH PINE STAND IN NORTHERN FLORIDA

DANIEL R. MILLER AND CHRISTOPHER M. CROWE
Southern Research Station, USDA Forest Service, Athens, GA, USA 30602


The multiple-funnel trap has gained broad ac-
ceptance for catching bark and ambrosia beetles
since the trap was developed more than 25 years
ago (Coleoptera: Scolytidae) (Lindgren 1983). The
trap consists of black plastic funnels aligned ver-
tically over each other, allowing for intercepted
beetles to fall through the funnels into a wet or
dry collection cup located on the bottom funnel.
Currently, there are 2 national programs in the
USA that use baited multiple-funnel traps for de-
tecting exotic species: the Cooperative Agricul-
tural Pest Survey (CAPS) and the Early Detection
and Rapid Response program (EDRR) (USDA
APHIS 2007; Rabaglia et al. 2008). Multiple-fun-
nel traps are available in several sizes or lengths,
expressed by the number of funnels (4-, 8-, 12- or
16-unit) (Contech Inc., Delta, BC; Synergy Semi-
ochemicals Corp., Burnaby, BC). The general ex-
pectation is that longer multiple-funnel traps
catch more beetles. In support of that position,
Hoover et al. (2000) found that catches of the
striped ambrosia beetle, Trypodendron lineatum
(Olivier) (Coleoptera: Scolytidae), in traps baited
with the pheromone lineatin, increased as the
length of traps were increased from 4 to 16 units.
Haack & Lawrence (1997) found that catches of
Tomicus piniperda (L.) were higher in 12- and 16-
unit traps than in 8-unit ones.
The objective of our study was to verify that
long multiple-funnel traps (16-unit) catch more
bark and wood boring beetles than short traps (8-
unit) in a slash pine (Pinus elliottii Engelm.)


stand in northern Florida. We focused our study
on common southern species attracted to the bi-
nary combination of ethanol and (-)-a-pinene
used in the national programs (Miller 2006;
Miller & Rabaglia 2009). We conducted 1 trapping
experiment in a mature slash pine stand on the
Osceola National Forest near Olustee, FL for 9
weeks in 2001 (29 Aug-8 Nov). PheroTech Inc.
(now Contech) supplied separate lures for releas-
ing ethanol and (-)-a-pinene at rates of approxi-
mately 0.6 and 2 g/d, respectively, as well as 8-
unit and 16-unit multiple-funnel traps. Traps
were set in 6 blocks of 2 traps per block with all
traps set 10-15 m apart. There were 2 treatments:
(1) 8-unit; and (2) 16-unit multiple-funnel traps.
One trap of each treatment type was randomly as-
signed to a position within each block. All traps
were baited with ethanol and (-)-a-pinene. Each
trap was suspended between trees by rope such
that the bottom of each was 0.2-0.5 m above
ground level. No trap was within 2 m of any tree.
Collection cups contained approximately 150 mL
of pink propylene glycol solution (Peak RV and
Marine Antifreeze, Old World Industries Inc.,
Northbrook, IL). Using SYSTAT ver. 11.00.01
(SYSTAT Inc., Point Richmond, CA), we con-
ducted two-sided t tests on data transformed by
ln(y + 1) to remove heteroscedasticity (Pepper et
al. 1997).
Catches of Arhopalus rusticus nubilus (Le-
Conte) (Cerambycidae) in 16-unit traps were
143% greater than those in 8-unit traps (Table 1).


TABLE 1. CATCHES OF SOME BARK AND WOOD BORING BEETLES IN 8-UNIT AND 16-UNIT MULTIPLE-FUNNEL TRAPS,
BAITED WITH ETHANOL AND (-)-A-PINENE, IN THE OSCEOLA NATIONAL FOREST, FL (N = 6).

Mean (+SE) beetle catches

8-Unit Trap 16-Unit Trap P value (t test)

Cerambycidae
Arhopalus r. nubilus 27.0 3.3 65.7 12.4 0.008
Xylotrechus s. sagittatus 23.3 6.4 34.2 12.7 0.790
Scolytidae
Dendroctonus terebrans 3.5 1.3 5.5 1.6 0.245
Ips grandicollis 3.7 1.7 4.7 1.0 0.329
Xyleborinus saxesenii 63.7 10.0 65.7 15.2 0.919
Xyleborus spp. 17.5 2.8 34.0 2.7 0.002
Curculionidae
Hylobius pales 59.2 11.8 27.2 5.6 0.015
Pachylobius picivorus 3.2 0.9 3.7 1.0 0.667







Florida Entomologist 92(3)


There was no effect of trap length on catches of
Xylotrechus sagitattus sagittatus (Germar).
Catches ofXyleborus Eichhoff spp (Scolytidae) in
16-unit traps were 94% greater than those in 8-
unit traps, whereas catches of Xyleborinus saxes-
enii (Ratzeburg) were unaffected by trap length.
Catches of 2 common bark beetles, Dendroctonus
terebrans (Olivier) and Ips grandicollis (Eichhoff),
were unaffected by trap length. The lack of signif-
icant differences for X. s. sagittatus, D. terebrans
and I. grandicollis may be a consequence of low
power due to low overall trap catches. Trap catch
differences of A. r. nubilus and Xyleborus spp be-
tween 8-unit and 16-unit traps could be related to
differences in trap surface area for interception of
beetles or to preferences for taller vertical silhou-
ettes (Hoover et al. 2000). In contrast, catches of
the reproduction weevil Hylobius pales Herbst
(Curculionidae) in 16-unit traps were 54% less
than those in 8-unit traps (Table 1), possibly rep-
resenting a preference for stumps over standing
trees. Pachylobius picivorus LeConte was unaf-
fected by trap length although trap catches were
low.
Trap selection is a compromise between cost,
ease of deployment, and trap performance. Cur-
rently, 8- and 16-unit traps with wet cups cost
US$ 39.24 and US$ 57.02, respectively (Contech,
Delta BC). The longer 16-unit traps can be un-
wieldy, particularly in areas with substantial un-
dergrowth. Our results suggest that managers
should consider the likelihood of increased trap
performance based on trap length for target spe-
cies in choosing trap type. For some species such
asX. saxesenii and Hylobius pales, longer traps do
not necessarily result in greater numbers of cap-
tures. Moreover, it is unclear if higher trap
catches relate directly to a higher probability of
capture for rare individuals such as newly-intro-
duced exotic species.
We thank J. L. Hanula and R. A. Rabaglia for
reviews of the manuscript and staff on the Os-
ceola National Forest for permission to conduct
the study. Funding for this research was provided
by the USDA Forest Service. The use of trade
names and identification of firms or corporations
does not constitute an official endorsement or ap-
proval by the US Government of any product or
service to the exclusion of others that may be suit-
able.


SUMMARY

In 2001, we compared catches of some common
species of bark and wood beetles in 8- and 16-unit
multiple-funnel traps baited with ethanol and (-)-
a-pinene in a slash pine stand on the Osceola Na-
tional Forest in northern Florida with bottoms of
both trap types at the same height above ground
level. MoreArhopalus rusticus nubilus (Ceramby-
cidae) andXyleborus spp (Scolytidae) were caught
in 16-unit traps than in 8-unit ones whereas
catches of Hylobius pales (Curculionidae) were
lower in 16-unit traps than in 8-unit ones. Trap
length had no effect on catches ofXylotrechus sag-
ittatus sagittatus (Cerambycidae), Dendroctonus
terebrans, Ips grandicollis, Xyleborinus saxesenii
(Scolytidae) and Pachylobius picivorus (Curcu-
lionidae).

REFERENCES CITED

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HOOVER, S. E. R., LINDGREN, B. S., KEELING, C. I., AND
SLESSOR, K. N. 2000. Enantiomer preference of Try-
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RABAGLIA, R., DUERR, D., ACCIAVATTI, R., AND RAGEN-
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[www.aphis.usda.gov/plant_health/plant_pest_info/
pest_detection]


September 2009







Florida Entomologist 92(3)


September 2009


EGG PARASITOIDS OF DALBULUS MAIDIS
(HEMIPTERA: CICADELLIDAE) IN JALISCO STATE, MEXICO

EDUARDO G. VIRLA1, ERICA LUFT ALBARRACIN'AND GUSTAVO MOYA-RAYGOZA2
'PROIMI-Biotecnologia, Div. Control Biol6gico, Av. Belgrano y Pje. Caseros (T4001 MVB), San Miguel de Tucuman.
Tucuman, Argentina

2Departamento de Botanica y Zoologia, CUCBA, Universidad de Guadalajara, km 15.5 carretera Guadalajara-
Nogales, Zapopan, C.P. 45110, Jalisco, Mexico
E-mail: gmoya@cucba.udg.mx


The corn leafhopper, Dalbulus maidis (Delong
& Wolcott) (Hemiptera: Cicadellidae) is broadly
distributed throughout the American continent,
from southeastern and southwestern USA to Ar-
gentina. It is the most important leafhopper pest
of maize, Zea mays L., in Latin America (Nault
1990), and causes great losses to corn crops be-
cause of its capacity to transmit efficiently corn
stunt spiroplasma (CSS), maize bushy stunt phy-
toplasma (MBSP), and maize rayado fino virus
(MRFV) (Nault & Ammar 1989).
Egg parasitoids are the most important natu-
ral enemies of leafhoppers (Freytag 1985). The
known egg parasitoids of D. maidis comprise 4
taxa of Mymaridae, 4 of Trichogrammatidae, and
1 Eulophidae (Table 1), but none of these are
known in Mexico, putative corn leafhopper origin
center (Nault 1990).
In the laboratory, 6-10 females of D. maidis,
which were 2 weeks old and obtained from Zapo-
pan site, were placed in polyethylen-terephta-
lathe (PET) cylindrical cages (35 cm high x 18 cm
diam.) on maize leaves in order to obtain senti-


nel eggs. Potted maize plants (pot of ca. 10 dm3)
in the vegetative stage (3 to 5 leaves) were
checked daily for eggs. Twice, on 17 Aug and 23
Aug, 10 plants containing less than 24-h-old eggs
were exposed in each site during 72-96 h. Potted
plants containing sentinel eggs were placed in-
side the cornfield at no more than 3 m from the
edge of the field. Sentinel eggs ofD. maidis were
exposed to parasitization in 2 cornfields in
Jalisco State from Aug to Sep, 2008 at Zapopan
site (2044'40.2"N, 10330'48.3"W, elevation
1,662 m), and El Grullo site (19047'50.4"N,
10412'43"W, elevation 869 m).
After 8 to 10 d, the leaves containing exposed
eggs were cut from the plant in the laboratory and
transferred to a petri dish with the bottom con-
taining wet tissue paper and covered with clear
plastic food wrapping to avoid desiccation, and to
keep wasps from escaping. Parasitized eggs were
checked daily to ensure leaf quality until the
emergence of the adult wasps. The parasitization
rates were not measured due to rotting or desicca-
tion of some leaves containing exposed eggs.


TABLE 1. SUMMARIZED INFORMATION ABOUT THE KNOWN EGG PARASITOIDS OF DALBULUS MAIDIS (HEMIPTERA:
CICADELLIDAE).

Family Parasitoid species Country reference

Mymaridae
Anagrus breuiphragma Soyka Argentina, Brasil (Triapitsyn 1997; Oliveira
& Spotti Lopez 2000; Virla,
2001)
Anagrus flaveolus Waterhouse Argentina, Peru (Marin 1987; Triapitsyn
1997)
Anagrus nigriventris Girault Argentina Luft Albarracin et al. (2006)
Anagrus sp. Nicaragua Gladstone et al. (1994)

Eulophidae
Aprostocetus (0.) infulatus De Santis Argentina Luft Albarracin &
Triapitsyn (2007)

Trichogrammatidae
Oligosita sp. Argentina, Brasil (Oliveira & Spotti Lopez
2000; Luft Albarracin et al.
2005)
Paracentrobia sp. Argentina, Nicaragua (Gladstone et al. 1994; Luft
Albarracin et al. 2005)
Paracentrobia subflava (Girault) Argentina Virla (1999)
Zagella sp. Argentina Luft Albarracin et al. (2005)







Scientific Notes


TABLE 2. NUMBER OF SPECIMENS AND PERCENT OF TOTAL EGG PARASITOIDS OF DALBULUS MAIDIS, OBTAINED FROM
SENTINEL EGGS DURING SUMMER 2008 IN 2 SITES OF JALISCO, MEXICO.

Site Site
Parasitoid species El Grullo Zapopan

Anagrus breuiphragma 40 (9.5%) 495 (98.2%)
Polynema sp. 2 (0.5%) 0
Paracentrobia nr subflava 360 (85.9%) 9 (1.8%)
Aphelinoidea sp. 9 (2.2%) 0
Pseudoligosita sp. 8 (1.9%) 0
TOTAL 419 504


From the approximately 1600 exposed eggs,
923 wasps emerged. The specimens belonged to 5
species: 2 Mymaridae (Anagrus breviphragma
Soyka and Polynema sp.), and 3 Trichogramma-
tidae (Paracentrobia nr subflava, Aphelinoidea
sp., and Pseudoligosita sp.). Anagrus brevi-
phragma and P. nr subflava were the most abun-
dant taxa in the Zapopan site and El Grullo site,
respectively, (Table 2).
Anagrus breviphragma belongs to the incarna-
tus species group, subgenus Anagrus s. str. It has
a very broad distribution that includes Japan,
England, France, Italy, Germany, Austria,
Greece, Guadeloupe, Guyana, Colombia, Brazil,
and Argentina (Chiappini 1989; Triapitsyn 1997).
The known hosts for A. breviphragma are Ci-
cadella viridis (L.), Dalbulus maidis (Cicadel-
lidae), Conomelus anceps (Germar), Delphacodes
kuscheli Fennah, Dicranotropis hamata (Bohe-
man), Muellerianella fairmairei (Perris), Peregri-
nus maidis (Ashmead) (Delphacidae), and Ortho-
tylus virescens (Douglas & Scott) (Miridae) (Tri-
apitsyn 1997; Virla 2001).
The species of Paracentrobia is very close to P.
subflava (Girault), but it has dense discal cilation
in the forewings, between the areas delimited by
the rows of the microtrichias, whereas in P sub-
flava, these areas are mostly bare, as reported by
Girault in the original description, and in voucher
specimens deposited in the entomological collec-
tion of La Plata Museum, Buenos Aires, Argen-
tina (MLPA). We cannot yet determine the species
of Polynema, Aphelinoidea, and Pseudoligosita
because of the lack of specific keys to these gen-
era.
Taking into account the importance of the corn
leafhopper in Mexico and the lack of information
about the egg parasitoid complex, we point out
the need for a proper evaluation of this parasitoid
guild and its influence on this leafhopper pest.
Slide-mounted and dried card-mounted
voucher specimens resulting from this study were
deposited in the collection of the Fundaci6n e In-
stituto Miguel Lillo at San Miguel de Tucuman,
Argentina (IMLA).
The research was carried out under the scien-
tific and technological cooperation (n' 0710) be-


tween Mexico (CONACYT) and Argentina (MIN-
CyT). Erica Luft Albarracin is a CONICET fellow-
ship holder.

SUMMARY

A survey of eggs parasitoids of the corn leaf-
hopper, Dalbulus maidis (DeLong & Wolcott) was
conducted in Jalisco State, Mexico. Samples were
collected during the summer of 2008 with sentinel
eggs. Five taxa, Anagrus breviphragma Soyka
and Polynema sp. (Mymaridae), Paracentrobia nr
subflava,Aphelinoidea sp., and Pseudoligosita sp.
(Trichogrammatidae) were reared. This is the
first reference to an egg parasitoid complex of the
corn leafhopper in Mexico, and A. breviphragma
is recorded for the first time occurring in Mexico.

REFERENCES CITED
CHIAPPINI, E. 1989. Review of the European species of
the Anagrus Haliday (Hymenoptera Chalcidoidea).
Boll. Zool. Agr. e Bachic., II, 21: 85-119.
FREYTAG, P. H. 1985. The insect parasites of leafhop-
pers, and related groups, pp. 423-467 In L. R. Nault
and J. Rodriguez [eds.], The leafhoppers and Plan-
thoppers. John Wiley & Sons, New York, 500 pp.
GLADSTONE, S., DE LA LLANA, A., RIOS, R., AND LOPEZ,
L. 1994. Egg parasitoids of the corn leafhopper Dal-
bulus maidis (De Long & Wolcott) (Homoptera: Ci-
cadellidae) in Nicaraguan maize. Proc. Entomol. Soc.
Washington 96: 143-146.
LUFT ALBARRACIN, E., VIRLA E., AND TRIAPITSYN, S.
2005. Diversidad e incidencia de los parasitoides 06-
filos del vector del Achaparramiento (CSS), Dalbulus
maidis (Hemiptera, Cicadellidae), en Tucuman, Ar-
gentina, VIII Congreso Nacional de Maiz, Rosario,
Argentina. p. 258-261.
LUFT ALBARRACIN, E., VIRLA E. G., AND TRIAPITSYN, S.
V. 2006. A new host record for the egg parasitoid
Anagrus nigriventris (Hymenoptera: Mymaridae) of
the corn leafhopper, Dalbulus maidis (Hemiptera:
Cicadellidae). Florida Entomol. 89: 284-285.
LUFT ALBARRACIN, E., AND TRIAPITSYN, S. V. 2007.
Aprostocetus (Ootetrastichus) infulatus (Hy-
menoptera: Eulophidae): description of the male,
new distribution and first host records. Zootaxa
1438: 65-68.
MARIN, R.1987. Biologia y comportamiento de Dalbulus
maidis (Homoptera: Cicadellidae). Rev. Peruana En-
tomol. 30: 113-117.







510 Florida Ento



NAULT, L. R. 1990. Evolution of an insect pest: maize
and the corn leafhopper, a case study. Maydica 35:
165-175.
NAULT, L. R., AND AMMAR, D. 1989. Leafhopper and
planthopper transmission of plant viruses. Annu.
Rev. Entomol. 34: 503-529.
OLIVEIRA, C., AND SPOTTI LOPEZ, J. 2000. Parasitoides
de ovos da cigarrinha-do-milho, Dalbulus maidis
(DeLong & Wolcott) (Hemiptera: Cicadellidae), em
Piracicaba. Rev. Agric. 75: 263-270.
TRIAPITSYN, S. V. 1997. The genus Anagrus (Hy-
menoptera: Mymaridae) in America south of the
United States: a review. CEIBA 38: 1-12.


1m


ologist 92(3) September 2009



VIRLA, E. G. 1999. Aportes acerca de la bionomia de
Paracentrobia subflava (Hymenoptera: Trichogram-
matidae), parasitoide de Hemipteros Cicadeloideos
argentinos. Rev. Soc. Entomol. Argentina 58: 17-22.
VIRLA, E. G. 2001. Notes on the biology of Anagrus
breuiphragma (Hymenoptera: Mymaridae), natural
enemy of the corn leafhopper Dalbulus maidis
(Hemiptera: Cicadellidae) and other plant diseases
vectors in South America. Bol. Sanidad Veg. "Plagas"
27: 239-247.







Scientific Notes


EFFECTS OF DIET ON DEVELOPMENT AND SURVIVORSHIP OF
NARNIA FEMORATA NYMPHS (HEMIPTERA: COREIDAE)

FAE NAGEON DE LESTANG AND CHRISTINE W. MILLER
Entomology and Nematology Department, University of Florida, Gainesville, FL 32605 USA


Little is known about the biology and natural
history of Narnia femorata, a cactus-feeding bug
(Fig. 1) (Stal 1862; Brailovsky 1975; Brailovsky et
al. 1994). This coreid is native to the southwest-
ern United States, Mexico, and parts of Central
America, and has recently been introduced to cen-
tral Florida (Baranowski & Slater 1986). Narnia
femorata feeds mainly on the flowers and fruit of
Opuntia species (prickly pears and cholla) (Bara-
nowski & Slater 1986; Miller et al. 2006), espe-
cially Opuntia imbricata, a cholla cactus native to
the southwest United States (Kinraide 1978; Ben-
son 1982).
In nature, deer, gophers, coyotes, birds, and ro-
dents frequently remove ripe and unripe Opuntia
cactus fruits (Gonzalez-Espinosa & Quintana-As-
cencio 1986; Janzen 1986; Hellgren 1994). Thus,
these structures are not always accessible to N.
femorata adults and nymphs. While adult N. fem-
orata can readily fly to a new host plant with
fruits if one is available, wingless nymphs have
limited mobility and can be stranded without ac-
cessible fruit for part or all of their growth and de-
velopment. In this study, we examined how fruit
availability affects the development and survivor-
ship ofN. femorata nymphs.
In Alachua County, Florida, Narnia femorata
feeds on Opuntia humifusa cactus, the only spe-
cies of Opuntia native to the area. Both N. femo-
rata and 0. humifusa were collected at Ordway-
Swisher Biological Station, University of Florida
(82W, 29041'N) from Oct through Nov 2007. We
collected 10 male and 10 female N. femorata from
across 40 hectares. To maximize genetic diversity
of our laboratory population, we did not collect


any insects from the same cactus patch. Collected
insects were paired and mated in a greenhouse
with a photoperiod of 12:12 (L:D). We collectedN.
femorata eggs from adult containers and placed
them into containers separate from adults.
After hatching, first instars were individually
transferred to discrete containers with either
Opuntia cladodes (cactus pads), or cladodes and
fruit. These containers included single 0. humi-
fusa cladodes planted in approximately 6.4 cm of
topsoil and potting soil mix. The lids of these con-
tainers were fitted with screening for ventilation.
Containers were kept in a greenhouse with a tem-
perature between 4 and 32C. Cacti were watered
weekly. Narnia femorata nymphs complete 5 in-
stars before closing as adults, and we tracked
survivorship and development instarr) of all
nymphs weakly for 10 weeks.
We used a total of 150 insects in the experi-
ment, with 75 juveniles placed in each treatment
cladodess with fruits or cladodes without fruit).
We employed an ordinal regression analysis to
compare developmental stage of insects at 10
weeks. Survivorship was compared with 2 differ-
ent statistical tools; we conducted a logistical re-
gression to analyze resulting survivorship after
10 weeks and a Kaplan-Meier Survival Analysis
with Tarone-Ware test to estimate survival curves
over the 10-week time period. All analyses were
run with SPSS 16.0.
At 10 weeks post-hatching, juveniles reared
with fruits were significantly further along in
their development than those reared without
fruits (x2 = 29.745, df = 1, P < 0.001) (Fig. 2). In
fact, 35 of the 37 surviving insects from the cla-


SA it
0401
03M1


Fig. 1. Narnia femorata
credit: C. W. Miller.


at the fifth instar. Photo


Cladode & fruit Cladode only
Nymphal treatment

Fig. 2. Developmental stages of N. femorata at the
tenth week post-hatching







Florida Entomologist 92(3)


dodes with fruits treatment had reached adult-
hood, compared to a mere 2 of the 30 surviving in-
sects from the cladodes without fruit treatment
(Fig. 2).
Resulting survivorship at 10 weeks for insects
from both treatments was not significantly differ-
ent (2 =1.161, df = 1, P = 0. 281), with 49% sur-
vival in cladodes with fruit and 41% survival in
the cladodes without fruit. The survivorship
curves of the 2 groups did not differ over the 10
weeks (2 = .020, df = 1, P > 0.50).
Our results demonstrate that N. femorata can
achieve growth and development without cactus
fruits, and thus may have adapted to some degree
to ephemeral nature of this resource. However, we
found that nymphs reared on cladodes without
cactus fruits have slower development, which
may have survival costs in natural situations. The
presence of predators in nature such as spiders,
assassin bugs, and lizards could result in a lower
survivorship for animals with longer develop-
ment time (Calef 1973; Pastorok 1981; Caswell
1983; Doughty & Roberts 2003). In a separate
study we found that N. femorata individuals
reared without fruits eclose as smaller adults
with reduced mating success (Nageon de Lestang,
unpublished data; Miller & Nageon de Lestang
unpublished data). Thus, the developmental envi-
ronment of nymphs likely has numerous conse-
quences for survival and reproduction in this spe-
cies and deserves further investigation.
We thank O. Liburd, S. Gillespie, E. Martinez,
C. Maxwell, L. Nguyen, D. Sasson, M. Tudor, and
R. Zaiser for comments on a previous version of
this manuscript. A special thanks to C. Barfield
for his support.

SUMMARY

We examined the influence of 2 natural nutri-
tional environments on the development and sur-
vivorship ofNarnia femorata in a greenhouse set-
ting. Narnia femorata raised on cactus with fruits
developed faster than those raised on cactus with-
out fruits, yet no significant difference in survi-
vorship was found between the 2 treatments.


REFERENCES CITED

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ville, FL.
BENSON, L. 1982. The Cacti of the United States and
Canada, Stanford University Press. Stanford, CA.
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Narnia Stal (Coreidae-Coreinae-Anisoscelini) y de-
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del Valle de Tehuacan, Puebla (Hemiptera-Het-
eroptera) I. Chelinidea staffilesi, C. tabulata y Nar-
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CALEF, G. W. 1973. Natural mortality of tadpoles in a
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CASWELL, H. 1983. Phenotypic plasticity in life-history
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DOUGHTY, P., AND ROBERTS, J. D. 2003. Plasticity in age
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poles: responses to variation in food levels and dete-
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F. 1986. Seed predation and dispersal in a dominant
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Tasks for Vegetation Science. 15: 273-284.
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Council. Kingsville, TX.
JANZEN, D. H. 1986. Chihuahuan desert nopaleras: de-
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KINRAIDE, T. B. 1978. The ecological distribution of chol-
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MILLER, T. E. X., TYRE, A. J., AND LOUDA, S. M. 2006.
Plant reproductive allocation predicts herbivore dy-
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PASTOROK, R. A. 1981. Prey vulnerability and size selec-
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September 2009




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