• TABLE OF CONTENTS
HIDE
 Red imported fire ants (Hymenoptera:...
 Seasonal population dynamics of...
 Resistance to two classes of insecticides...
 Range expansion and local population...
 Development of trapping methods...
 Host specificity of the microsporidian...
 Two new species of Dryinus Latreille...
 Brachymyrmex patagonicus (Hymenoptera:...
 Descriptions of late instars of...
 Molecular diagnostics of economically...
 Seasonal distribution and evaluation...
 Red oak borer (Coleoptera: Cerambycidae)...
 Biology, thermal requirements,...
 Presentation of artificial diet:...
 Mating biology of Austromusotima...
 A checklist of the aphids of Honduras...
 Impact of Harmonia axyridis (Coleoptera:...
 Influence of radiation does on...
 Host acceptance trials of parasitoids...
 Three-dimensional sampling method...
 Open field host specificity tests...
 Status of the fire ant decapitating...
 New parish records of Coptotermes...
 First record of Acizzia jamatonica...
 Confirmation of Asian cockroach...
 Ethanol preservation of fire ants...
 Comparison of Mexican fruit fly...
 First state records for Merope...
 The paramagnetic force in plant...
 Book reviews
 Back Matter














Group Title: Florida Entomologist
Title: The Florida entomologist
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Permanent Link: http://ufdc.ufl.edu/UF00098813/00353
 Material Information
Title: The Florida entomologist
Uniform Title: Florida entomologist (Online)
Abbreviated Title: Fla. entomol. (Online)
Physical Description: Serial
Language: English
Creator: Florida Entomological Society
Florida Center for Library Automation
Publisher: Florida Entomological Society
Place of Publication: Gainesville Fla
Gainesville, Fla
Publication Date: September 2007
Frequency: quarterly
regular
 Subjects
Subject: Entomology -- Periodicals   ( lcsh )
Insects -- Periodicals -- Florida   ( lcsh )
Genre: review   ( marcgt )
periodical   ( marcgt )
Periodicals   ( lcsh )
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Additional Physical Form: Also issued in print.
System Details: Mode of access: World Wide Web.
Language: In English; summaries in Spanish.
Dates or Sequential Designation: Vol. 4, no. 1 (July 1920)-
Issuing Body: Official organ of the Florida Entomological Society; online publication a joint project of the Florida Entomological Society and the Florida Center for Library Automation.
General Note: Title from caption (JSTOR, viewed Sept. 13, 2006).
General Note: Place of publication varies.
General Note: Latest issue consulted: Vol. 87, no. 4 (Dec. 2004) (JSTOR, viewed Sept. 13, 2006).
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Volume ID: VID00353
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 Related Items
Preceded by: Florida buggist (Online)

Table of Contents
    Red imported fire ants (Hymenoptera: Formicidae) increase the abundance of aphids in tomato
        Page 419
        Page 420
        Page 421
        Page 422
        Page 423
        Page 424
        Page 425
    Seasonal population dynamics of wireworms (Coleoptera: Elateridae) in Florida sugarcane fields
        Page 426
        Page 427
        Page 428
        Page 429
        Page 430
    Resistance to two classes of insecticides in southern chinch bugs (Hemiptera: Lygaeidae)
        Page 431
        Page 432
        Page 433
        Page 434
    Range expansion and local population increase of the exotic ant, Pheidole obscurithorax, in the southeastern United States (Hymenoptera: Formicidae)
        Page 435
        Page 436
        Page 437
        Page 438
        Page 439
    Development of trapping methods with a synthetic sex pheromone of the pink hibiscus mealybug, Maconellicoccus hirsutus (Hemiptera: Pseudococcidae)
        Page 440
        Page 441
        Page 442
        Page 443
        Page 444
        Page 445
        Page 446
    Host specificity of the microsporidian pathogen Vairimorpha invictae at five field sites with infected Solenopsis invicta fire ant colonies in northern Argentina
        Page 447
        Page 448
        Page 449
        Page 450
        Page 451
        Page 452
    Two new species of Dryinus Latreille (Hymenoptera: Dryinidae) from China
        Page 453
        Page 454
        Page 455
        Page 456
    Brachymyrmex patagonicus (Hymenoptera: Formicidae), an emerging pest species in the southeastern United States
        Page 457
        Page 458
        Page 459
        Page 460
        Page 461
        Page 462
        Page 463
        Page 464
    Descriptions of late instars of three Littoral Cafius species (Coleoptera: Staphylinidae) by association of file stage with DNA sequences
        Page 465
        Page 466
        Page 467
        Page 468
        Page 469
        Page 470
        Page 471
        Page 472
        Page 473
        Page 474
    Molecular diagnostics of economically important clearwing moths (Lepidoptera: Sesiidae)
        Page 475
        Page 476
        Page 477
        Page 478
        Page 479
    Seasonal distribution and evaluation of two trap types for monitoring grape root borer Vitacea polistiformis (Lepidoptera: Sesiidae) in Florida vineyards
        Page 480
        Page 481
        Page 482
        Page 483
        Page 484
        Page 485
        Page 486
        Page 487
    Red oak borer (Coleoptera: Cerambycidae) flight trapping in the Ozark National Forest, Arkansas
        Page 488
        Page 489
        Page 490
        Page 491
        Page 492
        Page 493
        Page 494
    Biology, thermal requirements, and estimation of the number of generations
        Page 495
        Page 496
        Page 497
        Page 498
        Page 499
        Page 500
        Page 501
    Presentation of artificial diet: Effects of composition and size of prey and diet domes on egg production by Orius insidiosus (Heteroptera: Anthocoridae)
        Page 502
        Page 503
        Page 504
        Page 505
        Page 506
        Page 507
        Page 508
    Mating biology of Austromusotima camptozonale (Lepidoptera: Crambidae), a potential biological control agent of old world climbing fern, Lygodium microphyllum (Schizaeaceae)
        Page 509
        Page 510
        Page 511
        Page 512
        Page 513
        Page 514
        Page 515
        Page 516
        Page 517
    A checklist of the aphids of Honduras (Hemiptera: Aphididae)
        Page 518
        Page 519
        Page 520
        Page 521
        Page 522
        Page 523
    Impact of Harmonia axyridis (Coleoptera: Coccinellidae) on native arthropod predators in pecan and crape myrtle
        Page 524
        Page 525
        Page 526
        Page 527
        Page 528
        Page 529
        Page 530
        Page 531
        Page 532
        Page 533
        Page 534
        Page 535
        Page 536
    Influence of radiation does on the level of F1 sterility in the cactus moth, Cactoblastis cactorum (Lepidoptera: Pyralidae)
        Page 537
        Page 538
        Page 539
        Page 540
        Page 541
        Page 542
        Page 543
        Page 544
    Host acceptance trials of parasitoids from Indian Paratachardina lobata (Hemiptera: Kerriidae) on the invasive lobate lac scale in Florida
        Page 545
        Page 546
        Page 547
        Page 548
        Page 549
        Page 550
        Page 551
        Page 552
    Three-dimensional sampling method for characterizing ant mounds
        Page 553
        Page 554
        Page 555
        Page 556
        Page 557
        Page 558
    Open field host specificity tests in Brazil for risk assessment of Metriona elatior (Coleoptera: Chrysomelidae), a potential biological control agent of Solanum viarum (Solanaceae) in Florida
        Page 559
        Page 560
        Page 561
        Page 562
        Page 563
        Page 564
    Status of the fire ant decapitating fly Pseudacteon tricuspis (Diptera: Phoridae) in Louisiana
        Page 565
        Page 566
        Page 567
        Page 568
        Page 569
    New parish records of Coptotermes formosanus (Isoptera: Rhinotermitidae) in Louisiana
        Page 570
        Page 571
        Page 572
    First record of Acizzia jamatonica (Hemiptera: Psyllidae) in North America: friend or foe?
        Page 573
    Confirmation of Asian cockroach Blatella asahinai (Blattodea: Blattelidae) introduction to Texas based on genetics, morphology, and behavior
        Page 574
        Page 575
        Page 576
    Ethanol preservation of fire ants allows retrospective screening for Solenopsis invicta virus-1
        Page 577
        Page 578
    Comparison of Mexican fruit fly (Diptera: Tephritidae) capture between McPhail traps with torula and Multilure traps with biolures in south Texas
        Page 579
        Page 580
    First state records for Merope tuber (Mecoptera: Meropeidae) in Florida and biogeographical implications
        Page 581
        Page 582
        Page 583
        Page 584
    The paramagnetic force in plant growth and insect control
        Page 585
        Page 586
        Page 587
    Book reviews
        Page 588
        Page 589
        Page 590
    Back Matter
        Page 591
        Page 592
Full Text



Coppler et al.: Fire Ants Increase Aphid Abundance in Tomato



RED IMPORTED FIRE ANTS (HYMENOPTERA: FORMICIDAE)
INCREASE THE ABUNDANCE OF APHIDS IN TOMATO


LAURA B. COPPLER1, JOHN F. MURPHY AND MICKY D. EUBANKS2
Department of Entomology and Plant Pathology, Auburn University, Auburn AL

'Present Address: Virginia Tech University, Southern Piedmont AREC,
2375 Darvills Road, Blackstone, VA 23824-0448

2Corresponding author

ABSTRACT

Red imported fire ants, Solenopsis invicta (Buren) (Hymenoptera: Formicidae), are abun-
dant in many agroecosystems in the southern United States and can affect the abundance
of arthropods in these systems. We determined the effects of red imported fire ants on the
abundance of aphids, other herbivorous insects, and beneficial arthropods in Alabama to-
mato (Lycopersicon esculentum) by manipulating the density of red imported fire ants in
plots of tomato plants and by sampling fresh market tomato farms for two years (2003 and
2004). In both years of our study, aphid abundance was significantly greater in tomato plots
with high densities of fire ants than in plots where fire ant densities were suppressed. Fur-
ther, the abundance of fire ants was positively correlated with the abundance of aphids on
intensely managed tomato farms in both years. These aphids included many species that are
the primary vectors of economically-important plant viruses of tomato and other vegetable
crops. The positive effect of fire ants on aphid abundance was likely due to facultative fire
ant-aphid mutualisms. Other studies have demonstrated that fire ants protect honeydew-
producing insects from natural enemies, and we found that fire ants reduced the abundance
of beneficial arthropods in the second year of our field experiment. However, red imported
fire ants did not significantly reduce the abundance of non-aphid herbivores in either year
of our field experiment, suggesting that fire ants are not important biological control agents
of these insects in tomato. Fire ants may disrupt biological control of aphids in tomato fields
and suppression of fire ants on tomato farms may decrease the abundance of aphids.

Key Words: Solenopsis invicta, red imported fire ant, biological control, intraguild predation,
Lycopersicon esculentum, tomato, ant-aphid mutualism

RESUME

La hormiga de fuego roja importada, Solenopsis invicta Buren (Hymenoptera: Formicidae),
es abundante en various ecosistemas agricolas en el sur de los Estados Unidos y puede afectar
la abundancia de artr6podos en estos sistemas. Nosotros determinamos los efectos de la hor-
miga de fuego roja importada sobre la abundancia de afidos, otros insects herbivoros, y ar-
tr6podos ben6ficos en tomate (Lycopersicon esculentum) en el estado de Alabama por medio
del manejo de la densidad de la hormiga de fuego roja importada en parcelas de plants de
tomate y por el muestreo de fincas que produce tomate fresco para el mercado por 2 aios
(2003 y 2004). En ambos aios de nuestro studio, la abundancia de afidos fue significativa-
mente mayor en las parcelas de tomate con densidades altas de la hormiga de fuego que en
las parcelas donde la densidad de la hormiga de fuego fue suprimida. Ademas, la abundancia
de la hormiga de fuego fue correlacionado positivamente con la abundancia de afidos en fin-
cas de tomate bajo el manejo intensive para ambos aios. Estos afidos incluyeron muchas de
las principles species que son vectores de virus econ6micamente importantes en plants
de tomate y de otros cultivos de hortalizas. El efecto positive de la hormiga de fuego sobre la
abundancia de afidos probablemente fue debido al mutualismo facultativo entire la hormiga
de fuego y los afidos. Otros studios han demonstrado que la hormiga de fuego proteje insec-
tos que produce substancias azucaradas de sus enemigos naturales, y encontramos que la
hormiga de fuego redujo la abundancia de artr6podos ben6ficos en el segundo aio de nuestro
studio de campo. Sin embargo, la hormiga de fuego roja importada no redujo significativa-
mente la abundancia de herbivoros que no son afidos en ninguno de los aios de nuestro ex-
perimento, ello indica que las hormigas de fuego no son agents de control biol6gico
importantes de insects en tomate. La hormiga de fuego puede interrumpir el control biol6-
gico de afidos en campos de tomate y la supresi6n de la hormiga de fuego en estos campos de
tomate puede reducir la abundancia de afidos.







Florida Entomologist 90(3)


INTRODUCTION

Tomatoes (Lycopersicon esculentum) are the
second most important vegetable crop in the
United States with over 8 million tons produced
annually in the U.S. (Brunke et al. 2003). The eco-
nomic viability of tomatoes is threatened by nu-
merous insect pests, including lepidopteran lar-
vae, thrips (Thysanoptera: Thripidae), stinkbugs
(Hemiptera: Pentatomidae), whiteflies (Hemip-
tera: Aleyrodidae) and aphids (Hemiptera: Aphid-
idae) (Nault & Speese 2002; Kemble et al. 2004).
Aphids can be particularly serious pests of tomato
because aphid feeding reduces the yield and fruit
quality of tomatoes and, more importantly, aphids
are the primary vectors for plant viruses that can
devastate tomato production (Tomlinson 1987).
For example, epidemics of Cucumber mosaic virus
(CMV) have significantly reduced tomato produc-
tion in the southeastern U.S. and in China,
France, Italy, and Spain (Jorda et al. 1992; Kaper
et al. 1990; Sikora et al. 1998). Plant viruses like
CMV are often difficult to manage because they
may have extremely large host ranges (Edward-
son & Christie 1991) and can be transmitted by
dozens of species of aphid species in a nonpersis-
tent manner (Palukaitis et al. 1992). Viruses
transmitted by aphids in a nonpersistent manner
are acquired in seconds by the aphid while prob-
ing an infected epidermal cell and are retained on
the tip of the aphid's stylet. The virus can then be
immediately transmitted in the same general pro-
cess, i.e., a quick probe of a plant's epidermal cell
by the aphid. Because virus transmission of non-
persistent viruses like CMV by aphids is rapid,
chemical pesticides are ineffective because aphids
transmit the virus before they are intoxicated. Ad-
ditionally, some pesticides actually enhance the
spread of plant viruses by increasing aphid prob-
ing and movement among plants (Lowery & Boi-
teau 1988; Perring et al. 1999). Because aphids
are the primary vectors of most plant viruses, any
factor that increases aphid abundance is likely to
increase the spread of the virus and the potential
for a virus epidemic (Madden et al. 2000; Hull
2002; Jeger et al. 2004). For example, the abun-
dance of alate aphids is positively correlated with
the incidence of Beet mosaic virus in sugar beet,
Beta vulgaris (L.) (Dusi et al. 2000), with the inci-
dence of Barley yellow dwarf virus in cereals
(Power et al. 1991; Chapin et al. 2001; Fabre et al.
2003), and with the incidence of Potato virus Yand
Potato leafroll virus in potato, Solanum tubero-
sum (Basky 2002).
Our work in southeastern agricultural systems
has identified an important ecological interaction
that may alter the abundance of virus vectors: fire
ant-aphid mutualisms. Red imported fire ants,
Solenopsis invicta (Buren) are widespread, inva-
sive ants that are notoriously aggressive and vora-
cious (Vinson 1997; Holway et al. 2002). They were


originally introduced through the port of Mobile,
Alabama, in the early 1900s, spread across the
southern United States from North Carolina to
Arizona, and have recently invaded California
(MacKay & Fagerlund 1997; Vinson 1997). Fire
ants, like many ant species, readily form faculta-
tive mutualisms with honeydew-producing insects
such as aphids, scales, and whiteflies (Reilly &
Sterling 1983a, 1983b; Vinson 1997; Helms & Vin-
son 2002). Fire ants are attracted to plants in-
fested with aphids and are effective at protecting
aphids from predators. Fire ants, for example,
preferentially forage on aphid-infested cotton
plants compared with aphid-free cotton plants,
and fire ant and cotton aphid abundances are pos-
itively correlated in the field (Kaplan & Eubanks
2002, 2005). Fire ant predation of lady beetle lar-
vae is twice as high on aphid-infested plants as on
aphid-free plants (Kaplan & Eubanks 2002) and
predator larvae are more abundant in cotton fields
with suppressed densities of fire ants than in con-
trol fields with large fire ant populations (Kaplan
& Eubanks 2002, 2005; Diaz et al. 2004).
The positive effect of ants on aphid population
density may affect the movement of aphids. Un-
der low density conditions, developing aphids do
not produce wings when they molt and become
apterous or wingless adults. When crowded, how-
ever, nymphs develop wings as they molt and be-
come alate or winged adults (Dixon 1977; Muller
et al. 2001). Dispersing aphids typically search for
new host plants with no or few aphids (Dixon
1977; Hodgson 1991). If fire ant protection of
aphids results in a 3 to 5-fold increase in aphid
population density (i.e., Kaplan & Eubanks 2002,
2005), then it is likely that fire ant protection will
result in a 3 to 5-fold increase in the number of
alates. This could result in an increase in the
movement of aphids among different plant spe-
cies and ultimately increase virus spread and
incidence.
The effect of fire ants in agroecosystems is not
always negative. For example, fire ants have been
reported as predators of a wide variety of insect
pests including the velvetbean caterpillar, Antic-
arsia gemmatalis Hiibner (Lee et al. 1990), red-
necked peanutworm, Stegasta bosqueella (Cham-
bers) (Vogt et al. 2001), horn fly, Haematobia irri-
tans (L.), boll weevil, Anthonomus grandis (Ster-
ling 1978; Jones & Sterling 1979), cotton
bollworm, Helocoverpa zea (Diaz et al. 2004); beet
armyworm, Spodoptera exigua (Hubner) (Diaz et
al. 2004), and the sugarcane borer, Diatraea sac-
charalis (Zehntner) (Adams et al. 1981; Fuller &
Reagan 1988; Bessin & Reagan 1993). Thus, the
suppression of fire ants in tomato may result in
reduced aphid densities, but could increase the
abundance of other insect pests of tomato.
Our primary objective was to determine if fire
ants increase the abundance of aphids in tomato.
If fire ants do increase the abundance of aphids in


September 2007







Coppler et al.: Fire Ants Increase Aphid Abundance in Tomato


tomato fields, then the suppression of fire ants in
and around tomato fields may reduce the abun-
dance of aphids and potentially alter the spread of
aphid-vectored viruses. A secondary objective of
this study was to document the effect of fire ant
suppression on the abundance of other insect
pests in tomato so that any changes in pest abun-
dance could be included in future decisions re-
garding the suppression of fire ants in tomato.

MATERIALS AND METHODS

Field experiments were conducted at the E.V.
Smith Research Center in Macon County, Ala-
bama, during 2003 and 2004. For both field sea-
sons, tomatoes (Lycopersicon esculentum variety:
Floralina) were grown from seeds in the green-
house for 1 month or until they reached 20 cm in
height. They were then placed outdoors for one
week of acclimatization, and then transplanted
into field plots. In 2003, twelve plots of tomato
plants, separated by at least 10 m, were estab-
lished on bare ground. Each plot consisted of 30
plants, spaced 45 cm apart and organized into
three rows separated by 75 cm each. In 2004, to-
matoes were transplanted into raised (15 cm)
beds of white plastic mulch, following fresh mar-
ket tomato production standards (Kemble et al.
2004). Twelve plots of tomato plants were estab-
lished during 2004 with the same plant and row
spacing as in 2003.
Plots were randomly divided into 2 treat-
ments: natural or high fire ant density (control) or
suppressed fire ant density. Suppression treat-
ments were established with Amdro, a commer-
cially available, ant-specific bait. Foraging fire
ant workers pick up the bait and return it to the
colony. The active ingredient of Amdro is hy-
dramethylnon which blocks the production of ATP
and kills ants by inhibiting energy production
(Valles & Koehler 1997). Amdro has very mini-
mal effects on non-target arthropods (Hu & Frank
1996; Eubanks et al. 2002), and has been success-
fully used to suppress fire ants in similar sized
and spaced plots in other studies (e.g., Harvey &
Eubanks 2004; 2005). Amdro was applied
weekly to plots assigned to the fire ant suppres-
sion treatment, beginning 1 week prior to sam-
pling, and fire ants were allowed to naturally col-
onize control (high fire ant) plots. Fire ant abun-
dance was quantified weekly within each plot
with traps consisted of a 2.5-cm piece of "hotdog"
placed in a 50-ml plastic tube. One trap was
placed in the center of each plot for = 45 minutes,
after which they were collected, sealed, returned
to the laboratory, and stored in a freezer until
ants were counted. All plants within each plot
were visually searched each week for aphids, fire
ants, and other arthropods. All abundance data
were log (n+l) transformed prior to analysis. Dif-
ferences in the abundance of fire ants, aphids,


non-aphid herbivores, and natural enemies were
compared between treatments with repeated
measures ANOVA (SAS Proc Mixed with repeated
statement; Khattree & Naik 1999). To avoid
pseudo-replication (i.e., counting each plant
within each plot as a replicate), data for each plot
were averaged to obtain mean plot values for
analysis.
In 2004, tomato plants in 4 fresh market to-
mato fields were sampled in Blount Co., Alabama
for the presence of fire ants and other arthropods.
Fields ranged from 5 to 7 acres and approxi-
mately 20 tomato plants per acre were visually
searched for aphids, fire ants, and other arthro-
pods weekly for 7 weeks. Additionally, "hotdog"
traps were used to quantify the relative abun-
dance of fire ants (1 trap per acre of tomato). In
2005 the same sampling procedures were re-
peated on 5 fresh market tomato fields in the
same area of Blount County. We found very few
non-aphid herbivores and natural enemies on to-
mato plants on the farms and, consequently, lim-
ited our analysis to a correlation of aphid abun-
dance and fire ant abundance in commercial to-
mato fields with seasonal averages.

RESULTS

In both 2003 and 2004 the abundance of fire
ants foraging on the ground in tomato plots was
significantly reduced by the application of Am-
dro, although fire ant suppression was better in
2003 (2003: 290.67 48.13 fire ants per trap ver-
sus 39.1 27.88 fire ants per trap, control versus
Amdro treated plots, respectively, F1,20 = 27.51;
P = <0.0001; 2004: 355.83 61.28 fire ants per
trap versus 200.33 31.41 fire ants per trap, con-
trol versus Amdro treated plots, respectively,
F129 = 4.63; P = 0.04).
We identified at least 6 different aphid species
on tomato plants in our plots, includingAphis gos-
sypii,A. fabae,Aulacorthum solani, Macrosiphum
euphorbae, Myzus persicae, and Uroleucon spp. In
2003, aphid abundance was significantly different
between treatments for apterous aphids (F1,3 =
9.82; P = 0.004) and for total aphids (apterous +
alate) (F1, = 8.57; P = 0.01) (Fig. 1A). The abun-
dance of alate aphids, however, was not statisti-
cally different between treatments (F130 = 1.76; P
= 0.19). In 2004, however, the abundance of alate
aphids (F,29 = 26.4; P < 0.0001) and total aphids
(F ,29 = 27.2; P < 0.0001) were significantly differ-
ent between the two treatments (Fig. 1B). In con-
trast to 2003, alate aphids made up over 95% of
total aphids in both control and suppression plots
in 2004. Likewise, during our survey of fresh mar-
ket tomato fields, the abundance of fire ants and
aphids was positively correlated in 2004 (one-
tailed test; r = 0.09; P = 0.04; n = 4) and during
2005 (one-tailed test; r = 0.91; P = 0.02; n = 5) and
greater than 95% of these aphids were alates.







Florida Entomologist 90(3)


2003


Iligh law
"irr A.n


2004


Fire Ants
Fig. 1. The number of aphids per tomato plant in
high fire ant plots and in low fire ant plots in 2003 (A)
and 2004 (B). Means one standard error plotted.


In 2003 and 2004, several non-aphid herbi-
vores were found feeding on tomato plants (Table
1), although their overall densities were quite low,
especially in 2004. Non-aphid herbivore abun-
dance, however, was not significantly different be-
tween treatments in both 2003 (2.25 0.2 per


plant versus 2.26 0.2 per plant, control versus
Amdro treated plots, respectively) (F,24 = 3.89;
P = 0.06) and 2004 (0.32 0.06 per plant versus
0.4 0.05 per plant, control versus Amdro
treated plots, respectively) (F,,,23 = 0.00; P = 0.96).
Several natural enemies also were present on
tomato plants in our experimental tomato plots in
2003 and 2004 (Table 2). In 2003, the most abun-
dant natural enemies were spiders and coccinel-
lid larvae (Coccinellidae). Natural enemy abun-
dance, however, was not significantly different be-
tween the 2 treatments (0.10 0.02 per plant ver-
sus 0.09 0.03 per plant, control versus Amdro
treated plots, respectively) (F1,4 = 0.01; P = 0.93).
In 2004, the most abundant natural enemies were
damsel bugs (Nabidae) followed by spiders and
minute pirate bugs (Anthocoridae). There was a
significant difference (F 29 = 4.31;P = 0.04) in nat-
ural enemy abundance between suppressed fire
ant plots and control plots with significantly more
natural enemies on tomato plants in fire ant sup-
pressed plots (0.13 0.03 per plant versus 0.20
0.03 per plant, control versus Amdro treated
plots, respectively).

DISCUSSION

Total aphid abundance in both 2003 and 2004
was significantly greater in tomato plots with
high densities of fire ants (control plots) than in
plots with suppressed fire ant densities. In 2004
alate aphids were more than twice as abundant in
high fire ant plots as in low fire ant plots and
there was a positive correlation between the
abundance of fire ants and alate aphids in com-
mercial tomato fields in 2004 and 2005. Taken to-
gether, these results suggest that fire ants in-
crease the abundance of important aphid pests of
tomato. These results are consistent with studies
conducted in other agricultural crops indicating
that fire ants often increase aphid abundance. For
example, fire ant 'tending' of aphids increases the


TABLE 1. MEAN NUMBER ( ONE STANDARD ERROR) OF NON-APHID HERBIVORES FOUND PER TOMATO PLANT IN CON-
TROL PLOTS (HIGH FIRE ANT PLOTS) AND AMDRO TREATED PLOTS (LOW FIRE ANT PLOTS) IN 2003 AND 2004.

2003 2004

Herbivore Taxa High fire ant SE Low fire ant SE High fire ant SE Low fire ant SE

Aleyrodidae 2.25 0.20 2.24 0.20 0.027 0.013 0.038 0.01
Miridae 0.004 0.003 0.002 0.002 0.007 0.007 0.002 0.002
Coreidae 0.002 0.002 0 0 0 0 0 0
Lepidoptera larvae 0 0 0.007 0.007 0.17 0.03 0.23 0.04
Chrysomelidae 0 0 0 0 0.09 0.02 0.1 0.01
Gryllidae 0 0 0 0 0 0 0.002 0.002
Acrididae 0 0 0 0 0.007 0.007 0.008 0.004
Cercopidae 0 0 0 0 0 0 0.002 0.002
Pentatomidae 0 0 0 0 0 0 0.002 0.002


September 2007







Coppler et al.: Fire Ants Increase Aphid Abundance in Tomato


TABLE 2. MEAN NUMBER ( ONE STANDARD ERROR) OF NATURAL ENEMIES FOUND PER TOMATO PLANT IN CONTROL
PLOTS (HIGH FIRE ANT PLOTS) AND AMDRO TREATED PLOTS (LOW FIRE ANT PLOTS) IN 2003 AND 2004.

2003 2004

Natural enemy Taxa High fire ant SE Low fire ant SE High fire ant SE Low fire ant SE

Spiders 0.09 0.004 0.09 0.03 0.05 0.03 0.09 0.01
Coccinellidae adults 0 0 0 0 0 0 0.005 0.005
Coccinellidae larvae 0.004 0.01 0 0 0 0 0 0
Reduviidae 0 0 0 0 0.01 0.009 0.01 0.005
Nabidae 0 0 0 0 0.06 0.02 0.03 0.01
Chrysopidae larvae 0 0 0 0 0.007 0.07 0.03 0.01
Anthocoridae 0 0 0 0 0.04 0.02 0.02 0.007
Hymenoptera 0 0 0 0 0.007 0.007 0.008 0.004
Lygaeidae 0 0 0 0 0.007 0.007 0.005 0.003


abundance of cotton aphids (Aphis gossypii) in
cotton (Kaplan & Eubanks 2002, 2005; Diaz et al.
2004) and brown citrus aphids (Toxoptera citri-
cida) in citrus (Michaud & Browning 1999). By es-
sentially doubling aphid abundance in tomato,
fire ants may significantly reduce tomato yield
and fruit quality, especially if these aphids are
carrying plant viruses that infect tomato. All of
the aphids that we identified in our field experi-
ment (A. gossypii, A. fabae, A. solani, M. euphor-
bae, M. persicae, and Uroleucon spp.) have been
identified as vectors of CMV and other plant vi-
ruses that can devastate tomato production
(Sikora et al. 1998; Palukaitis et al. 1992). Our re-
sults suggest that suppression of fire ants in and
around agricultural fields will significantly re-
duce the abundance of aphids and may reduce the
spread of aphid-vectored plant viruses. We plan to
test this hypothesis in the near future.
We found limited evidence that fire ants af-
fected the abundance of non-aphid herbivores in
tomato. This is surprising because many previous
studies conducted in other crops have found that
fire ants suppress herbivores (Eubanks, 2001;
Vogt et al. 2001; Diaz et al. 2004; Harvey & Eu-
banks 2004). For example, Eubanks (2001) found
that densities of S. invicta workers were nega-
tively associated with densities of several herbi-
vores in both cotton and soybean. Likewise, Diaz
et al. (2004) found that fire ants destroyed signifi-
cant numbers of bollworm (Helicoverpa zea) and
beat armyworm eggs (Spodoptera exigua (Hub-
ner)) in cotton. Additionally, Vogt et al. (2001)
found that fire ants forage on many herbivores in
peanut fields, suggesting that fire ants may have
negative effects on many herbivorous taxa. One
reason for the difference in our findings from other
studies could be the composition of herbivores
present or their relative abundance. The non-
aphid herbivores in tomatoes were dominated by
whiteflies and lepidopteran larvae (Table 1). Like
aphids, whiteflies produce honeydew and are
sometimes tended by fire ants (Queiroz & Oliveria


2001), so it is unlikely that fire ants are significant
predators of whiteflies in tomato. In 2004 the most
abundant herbivores were first and second instar
lepidopteran larvae, but they were present at rel-
atively low densities. Predation of caterpillars by
fire ants has been shown to be density-dependent
and to some extent size-dependent in other crops
(e.g., collards (Harvey & Eubanks 2004) and soy-
bean (Styrsky et al. 2006)) and their low densities
in tomato may have resulted in low fire ant preda-
tion during our plot experiments.
The number of natural enemies was not signif-
icantly different between treatments in 2003.
These results are inconsistent with results pub-
lished by Bugg & Dutcher (1989), Eubanks et al.
(2002), Kaplan & Eubanks (2002), and Vogt et al.
(2001). The conflicting results may be attributed
to the low density of natural enemies present and
the behavior of the natural enemies. In 2003, only
2 kinds of natural enemies, spiders and lady bee-
tle larvae (Coccinellidae), were found during vi-
sual searches and spiders were much more abun-
dant than lady beetle larvae. Some spiders can
avoid predation by foraging fire ants by rapidly
moving to other parts of the plant, dropping off of
the plant on silk lines, or stretching to reduce con-
tact with the plant surface (Eubanks et al. 2002).
In 2004, however, fire ants did negatively affect
the abundance of natural enemies. A greater
diversity of natural enemies was found during
visual searches in 2004 than in 2003. Other stud-
ies have shown that damsel bugs and minute pi-
rate bugs, the first and third most abundant nat-
ural enemies in our tomato plots in 2004, were
much more susceptible to fire ants than spiders
(Eubanks 2001; Eubanks et al. 2002). Thus, the
difference in natural enemies among the 2 years
of the study may explain the greater impact of fire
ants on beneficial insects in 2004.
In conclusion, fire ants had strong, positive ef-
fects on aphid abundance and insignificant effects
on non-aphid herbivores. Taken together, the re-
sults of this study suggest that fire ants may dis-











rupt biological control of aphids in this crop and
the suppression of fire ants will decrease aphid
populations in tomato.

ACKNOWLEDGEMENTS

We thank N. Bieser, M. Buckman, K. Rice, K. Rose, J.
Styrsky, and J. Tindle for field and laboratory assis-
tance. We also thank J. Burkett and his staff at E.V.
Smith Agricultural Experiment Station. This work was
supported by an Alabama Agricultural Experiment Sta-
tion Foundation grant to M. D. E and J. F. M. and by
funding from the Alabama Fire Ant Management Pro-
gram to M. D. E. We thank Henry Fadamiro and John
Styrsky for helpful comments on an earlier draft.

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Florida Entomologist 90(3)


September 2007


SEASONAL POPULATION DYNAMICS OF WIREWORMS
(COLEOPTERA: ELATERIDAE) IN FLORIDA SUGARCANE FIELDS

RON CHERRY
Everglades Research and Education Center, 3200 E. Palm Beach Road, Belle Glade, FL 33430

ABSTRACT

Wireworms are important insect pests of Florida sugarcane. The objective of this study was
to determine the seasonal population dynamics of wireworms in Florida sugarcane. In a pre-
liminary test, significantly more total numbers of wireworms were found under sugarcane
stools than between stools in a row or between rows. Hence, the sugarcane stool was used as
the sampling site for wireworms in the population dynamics study. A sugarcane field was
sampled for wireworms monthly from Jun 2004 to Jun 2006. The total number of wireworms
was significantly less in the summer than other seasons and these wireworms were also sig-
nificantly smaller at this time. Data in this study should be useful to southern Florida grow-
ers in understanding expected wireworm damage in different seasons.

Key Words: Elateridae, wireworms, sugarcane

RESUME

El gusano alambre (Coleoptera: Elateridae) es una plaga insectil important en la cana de azu-
car en el estado de la Florida. El objetivo de este studio fue determinar la dinamica estacional
de la poblaci6n del gusano alambre en la cana de azucar en el estado de la Florida. En un es-
tudio preliminary, el numero total de gusanos de alambre fue significativamente mayor en la
parte localizada debajo de plants de cana de azucar que entire plants en un mismo surco o en-
tre los surcos. Por lo tanto, la plant de cana fue usada como el sitio de muestreo para gusanos
alambres en el studio de la dinamica de poblaci6n. Un campo de cana de azucar fue mues-
treado para el gusano alambre mensualmente dejunio de 2004 hastajunio de 2006. El numero
total de gusanos de alambre fue significativamente menor en el verano que en las otras esta-
ciones y estos gusanos de alambre fueron significativamente mas pequeios en este tiempo. Los
datos de este studio deben ser tiles para los agricultores en el sur de la Florida para entender
los danos que pueden suceder por el gusano alambre en las diferentes estaciones.


Wireworms are important insect pests of Flor-
ida sugarcane. These insects attack the under-
ground portions of the plant by feeding on the
buds and root primordial during germination and
on shoots and roots after germination. Wire-
worms are primarily a pest in newly planted sug-
arcane although the insects are also found in ra-
toon sugarcane. Of the different wireworm spe-
cies found in Florida sugarcane, Melanotus com-
munis (Gyllenhal) is the most important pest.
Damage by this wireworm has been thoroughly
documented in studies by Hall (1985, 1990).
Cherry & Hall (1986) reported on the flight activ-
ity of M. communis in Florida sugarcane and
Cherry (1988) noted the distribution and abun-
dance of the species in Florida sugarcane.
Detailed studies of wireworm biology are rare
(Lefko et al. 1998). This paucity or information
has resulted largely because most wireworms are
difficult to collect in large numbers and have ex-
tremely prolonged life cycles, thereby making
them rather intractable for study (Keaster et al.
1975). However, understanding the seasonal pop-
ulation dynamics of wireworms in a crop may be
important in predicting expected wireworm dam-
age. For example, Bynum et al. (1949) reported


that wireworm damage was more severe to fall
planted sugarcane than summer planted sugar-
cane in Louisiana. Also, Burrage (1963) noted
that wireworm damage to potatoes increased
with rising soil temperatures in the spring to a
peak in Jun in Canada.
Currently, the seasonal population dynamics
of wireworms in Florida sugarcane are not
known. This information is important in under-
standing the basic biology of these important
pests. Moreover, besides sugarcane, wireworms
attack other crops such as corn and potatoes in
southern Florida. Hence these data may help pre-
dict wireworm seasonal damage in these crops
also. The objective of this research was to deter-
mine the seasonal population dynamics of wire-
worms in Florida sugarcane.

MATERIALS AND METHODS

Spatial Distribution

This first study determined where to sample for
wireworms within a sugarcane field since this in-
formation was necessary for later population dy-
namic study. Five sugarcane fields on muck soils in







Cherry: Wireworm Population Dynamics


southern Florida were sampled. These fields were
2 to 4 years old because since fewer wireworms are
found in newly planted sugarcane fields due to
pre-planting practices such as flooding, discing, or
insecticide application at planting. Samples were
obtained from post-harvest fields from Oct 2003 to
Feb 2004 because it is much easier to sample these
fields than pre-harvest standing sugarcane.
Six sugarcane plants within each field were
randomly selected for sampling. Three samples
were taken at each sugarcane plant (stool) that
included soil from under the stool, in the row be-
tween stools, and between rows of stools. Soil
sampling for soil insect pests by digging is labori-
ous and time-consuming (Toba & Turner 1983;
Robertson & Simpson 1989). Hence, each sample
consisted of a 40 x 40 x 30-cm deep volume of soil
which was dug-up and visually examined on a
table in the field by one person for 1 h. The 1-h
period was necessary to thoroughly examine the
large volume of soil to find wireworms, especially
smaller wireworms. If more than one person was
involved, observation time was divided by people
present. Wireworms were collected and stored in
alcohol. Based on the author's experience, it was
thought that this methodology and sample num-
ber would generate reasonable numbers of wire-
worms collected for analysis. Taxonomic identifi-
cation of wireworms was later conducted with mi-
croscopes in a laboratory. All samples were pooled
and the mean number of wireworms of different
species in each of the 3 sample areas (under stool,
etc.) was analyzed with a Least Significant Differ-
ence (LSD) test (SAS 2006).

Seasonal Population Dynamics

A commercial 2-year-old sugarcane field on
muck soil in southern Florida was sampled for
wireworms. The previous spatial distribution
study showed that most wireworms were found
under sugarcane stools. Hence, soil samples were
taken as previously described except that sam-
ples were taken only under stools. Five samples
were taken monthly from Jun 2004 to Jun 2006.
Based on the author's experience, it was thought
that this methodology and sample number would
generate reasonable numbers of wireworms col-
lected for analysis. Wireworms were collected,


immediately stored in alcohol, and identified as
described previously. Wireworm length also was
measured to determine possible seasonal changes
in wireworm size. Samples from the 2 years were
pooled for analysis. Also, in order to determine
possible seasonal differences in wireworm popu-
lations, samples from 3-month periods were
pooled. For the purposes of this paper, winter is
defined as Dec, Jan, and Feb, spring is Mar, Apr,
and May, summer is Jun, Jul, and Aug, and fall is
Sep, Oct, and Nov. These definitions correspond to
seasonal definitions for the North Temperate
Zone (Guralnik 1982). Mean differences in wire-
worm population abundance and wireworm size
between seasons was determined by the Least
Significant Difference (LSD) test (SAS 2006).

RESULTS AND DISCUSSION

The spatial distribution of wireworms around
sugarcane plants is shown in Table 1. Similar
numbers of Conoderus spp. were found under the
stool and between stools in the row with signifi-
cantly fewer between rows. Likewise, similar
numbers of CG,.i .... bimarginatus Schaeffer
were found under the stool and between stools in
the row with significantly fewer between rows. In
contrast, significantly more M. communis were
found under stools than between stools in the row
or between rows. Of the three habitats samples,
65% of M. communis were found directly under
sugarcane stools versus 41% for Conoderus spp.
and 44% for G. bimarginatus. These data show
that M. communis aggregates more at sugarcane
stools than Conoderus spp. or G. bimarginata for
reasons unknown.
Significantly more total numbers of wire-
worms were found under sugarcane stools than in
the other 2 habitats. These data show that the
sugarcane stool was an appropriate sample unit
for studying seasonal population dynamics in this
study. Similarly, Cherry (1984) reported that
third instar grubs of two scarab pests, Cycloceph-
ala parellela Casey and Ligyrus subtropicus
Blatchley were found in highest density under
stools in Florida sugarcane. These data are con-
sistent with Southwood's (1969) observation that
most soil dwelling pests of sugarcane become ag-
gregated around sugarcane plants.


TABLE 1. SPATIAL DISTRIBUTION OF WIREWORMS AROUND SUGARCANE PLANTS.

Under stool' Between stools in row' Between rows'

Conoderus spp. 1.1+ 1.1A 1.1+ 1.3A 0.5 0.7 B
G. bimarginatus 2.4 2.4 A 2.3 2.4 A 0.8 1.1 B
M. communis 3.4 2.5 A 1.0 1.4 B 0.8 0.8 B
Total 6.9 4.0 A 4.4 2.6 B 2.1 1.7 C

Means + SD. Means in a row followed by the same letter are not significantly different (alpha = 0.05) based on the Least Sig-
nificant Difference test (SAS 2006).







Florida Entomologist 90(3)


The seasonal population dynamics of wire-
worms in Florida sugarcane fields is shown in
Fig. 1 and statistical analysis is given in Table 2.
Combining all seasons shows that M. communis
was the most abundant wireworm found. Hall
(1988) reported that M. communis is an impor-
tant soil pest of Florida sugarcane and that insec-
ticides are routinely applied at planting time for
control. Conoderus spp. was second most abun-
dant. Hall (1988) reported 4 Conoderus species
commonly associated with Florida sugarcane.
However, Conoderus species were not determined
in this study. Conoderus have been reported to be
pests of sugarcane in Louisiana (Bynum et al.
1949) and Hawaii (Stone 1976). The third most
common wireworm was G. bimarginatus. Hall
(1988) reports G. bimarginatus to be small wire-
worms often present in Florida sugarcane. Unlike
M. communis, the pest status of Conoderus spp.
and G. bimarginatus has not been determined in
Florida sugarcane.
The greatest number of Conoderus spp. and
G. bimarginatus were found in the summer and
fall for reasons not fully understood. It is interest-
ing to compare Conoderus data in this study with
Conoderus data obtained in sweet potato fields in
the adjacent state of Georgia (Seal et al. 1992).
Analogous to this study, wireworm numbers were
high in summer months and decreased in the fall.


14
-4-Conoderus spp.
-O-G. bimarginatus
12 --M. communis


Melanotus communis were at their lowest num-
ber in the summer followed by a large upsurge in
numbers in the fall. This is explained by noting
that many M. communis pupate in the spring
with maximum flight activity during the summer
(Genung 1972; Cherry & Hall 1986) and maxi-
mum oviposition during Jun (Cherry & Hall
1986). Hence, few M. communis larvae are
present in the summer and because of summer
oviposition there is a large increase in larval pop-
ulations in the fall. The total number of wire-
worms was significantly less in the summer than
the other seasons primarily due to the large de-
crease ofM. communis in the summer
Seasonal size of wireworms in Florida sugar-
cane fields is shown in Table 3. In Conoderus spp.,
variation in size was not great between seasons
with no season being significantly different from
all other seasons. Likewise, in G. bimarginatus,
no season was significantly different from all
other seasons. In contrast, M. communis were sig-
nificantly smaller in the fall than in all other sea-
sons. These smaller larvae were probably new lar-
vae from the main period of M. communis oviposi-
tion which takes place May to Jul in southern
Florida sugarcane fields (Cherry & Hall 1986).
Unfortunately, ovipositional data for Conoderus
spp. and G. bimarginatus in Florida sugarcane
are not known. The average size of all wireworms


Summer 04 Fall 04 Winter 04 Spring 05 Summer 05 Fall 05 Winter 05 Spring 06
Season


Fig. 1. Seasonal population dynamics of wireworms in Florida sugarcane fields.


September 2007







Cherry: Wireworm Population Dynamics


TABLE 2. SEASONAL POPULATION DYNAMICS OF WIREWORMS IN FLORIDA SUGARCANE FIELDS.

Conoderus spp. G. bimarginatus' M. communis" Totala

Winter 0.8 1.4 B 0.5 1.0 BC 8.8 5.9 A 10.1 5.2 A
Spring 0.2 0.6 B 0.1 0.2 C 10.3 4.3 A 10.6 4.4 A
Summer 3.5 4.8 A 0.9 1.4 B 2.1 2.0 B 6.6 6.9 B
Fall 1.0 1.3 B 1.6 t 1.6 A 8.7 5.1 A 11.3 4.9 A

"Means + SD. Means in a column followed by the same letter are not significantly different (alpha= 0.05) based on the Least Sig-
nificant Difference test (SAS 2006).


TABLE 3. SEASONAL SIZE (MM LENGTH) OF WIREWORMS IN FLORIDA SUGARCANE FIELDS.

Conoderus spp. G. bimarginatus' M. communis" Totala

Winter 9.7 2.5 AB 9.2 1.7 A 17.3 4.4 A 16.3 4.9 AB
Spring 9.0 1.6AB 10.0 2.0 A 17.6 7.0 A 17.3 4.1A
Summer 8.9 3.2 B 7.8 2.7 A 16.7 7.0 A 11.1 6.0 D
Fall 10.7 4.1 A 7.8 2.0 A 14.5 4.4 B 13.1 4.8 C

"Means + SD. Means in a column followed by the same letter are not significantly different (alpha= 0.05) based on the Least Sig-
nificant Difference test (SAS 2006).


(total number) was significantly smaller in the
summer than in any other season.
Various temporal manipulations of crops have
been used to reduce insect damage. These include
providing trap crops at suitable times, crop rota-
tions, using fast growing varieties, and adjusting
planting times so that susceptible crop stages oc-
cur during periods of low pest incidence (New
2005). This study shows that the total wireworm
complex was least abundant (Table 2) and wire-
worms were smallest (Table 3) in the summer in
Florida sugarcane. Interestingly, Bynum et al.
(1949) also reported decreased wireworm damage
in Louisiana sugarcane planted in the summer as
compared to the fall. Both the Bynum et al. (1949)
study and this study are consistent in showing ex-
pected decreased wireworm feeding on sugarcane
during the summer. Data in this study should be
useful to southern Florida growers in under-
standing expected wireworm damage at different
times of the year.

REFERENCES CITED

BURRAGE, R. 1963. Seasonal feeding of larvae of Cten-
icera destructor and Hypolithus bicolor (Coleoptera:
Elateridae) on potatoes placed in the field at weekly
intervals. Ann. Entomol. Soc. Amer. 56: 306-313.
BYNUM, E., J. INGRAM, AND L. CHARPENTIER. 1949. Con-
trol of wireworms attacking sugarcane in Louisiana.
J. Econ. Entomol. 42: 556-557.
CHERRY, R. 1984. Spatial distribution of white grubs
(Coleoptera: Scarabaeidae) in Florida sugarcane. J.
Econ. Entomol. 77: 1341-1343.
CHERRY, R. 1988. Correlation of crop age with popula-
tions of soil insect pests in Florida sugarcane. J. Ag-
ric. Entomol. 5: 241-245.


CHERRY, R., AND D. HALL. 1986. Flight activity of Mel-
anotus communis (Coleoptera: Elateridae) in Florida
sugarcane fields. J. Econ. Entomol. 79: 626-628.
GURALNIK, D. 1982. Webster's New World Dictionary.
Simon and Schuster, New York.
GENUNG, W. 1972. Seasonal occurrence of click beetles
(Coleoptera: Elateridae) in the Everglades as mea-
sured by two types of traps. Florida Entomol. 55: 35-
41.
HALL, D. 1985. Damage by the corn wireworm, Melano-
tus communis (Gyll.) to plant cane during germina-
tion and early growth. J. Amer. Soc. Sugar Cane
Tech. 4: 13-17.
HALL, D. 1988. Insects and mites associated with sugar-
cane in Florida. Florida Entomol. 71: 130-150.
HALL, D. 1990. Stand and yield losses in sugarcane
caused by the wireworm Melanotus communis (Co-
leoptera: Elateridae) infesting plant cane in Florida.
Florida Entomol. 73: 298-302.
KEASTER, A., G. CHIPPENDALE, AND B. PILL. 1975. Feed-
ing behavior and growth of the wireworms Melano-
tus depressus and Limonius dubitans: effect of host
plants, temperature, photoperiod, and artificial di-
ets. Environ. Entomol. 4: 591-595.
LEFKO, S., L. PEDIGO, W. BATCHELOR, AND M. RICE.
1998. Spatial modeling of preferred wireworm (Co-
leoptera: Elateridae) habitat. Environ. Entomol. 27:
184-190.
NEW, T. 2005. Invertebrate Conservation and Agricul-
tural Ecosystems. Cambridge Univ. Press, New York.
354 pp.
ROBERTSON, L., AND G. SIMPSON. 1989. The use of ger-
minating-seed baits to detect soil insect pests before
crop sowing. Australian J. Exper. Agric. 29: 403-407.
SAS INSTITUTE. 2006. SAS Systems for Windows. SAS
Institute, Cary, NC.
SEAL, D., R. MCSORLEY, AND R. CHALFANT. 1992. Sea-
sonal abundance and spatial distribution on wire-
worms (Coleoptera: Elateridae) in Georgia sweet
potato fields. J. Econ. Entomol. 85: 1802-1808.







430 Florida Entc



SOUTHWOOD, T. 1969. Population studies of insects at-
tacking sugarcane, pp. 427-461 In J. R. Williams, J.
R. Metcalfe, R. W. Montgomery, and R. Mathes [eds.],
Pests of Sugarcane. Elsevier, New York. 690 pp.
STONE, M. 1976. Notes on the biology of the introduced
elaterid Conoderus exsul (Sharp) (Coleoptera: Elat-
eridae). Pan Pacific Entomol. 52: 304-310.


)m


ologist 90(3) September 2007



TOBA, H., AND J. TURNER 1983. Evaluation of baiting
techniques for sampling wireworms (Coleoptera:
Elateridae) infesting wheat in Washington. J. Econ.
Entomol. 76: 850-855.







Cherry & Nagata: Chinch Bug Resistance to Insecticides


RESISTANCE TO TWO CLASSES OF INSECTICIDES IN
SOUTHERN CHINCH BUGS (HEMIPTERA: LYGAEIDAE)

RON CHERRY AND RUSSELL NAGATA
Everglades Research and Education Center, University of Florida/IFAS,
3200 E. Palm Beach Road, Belle Glade, FL 33430

ABSTRACT

Southern chinch bugs were tested from 10 locations in Florida to determine possible resis-
tance to 4 insecticides. Resistance of varying degrees was found in all 4 insecticides:
bifenthrin, deltamethrin, imidacloprid, and lambda-cyhalothrin. This study is the first to
show southern chinch bug resistance to the latter 3 insecticides. Our data also show that
multiple locations are necessary for insecticidal testing for southern chinch bugs since re-
sults from 1 location can be very misleading.

Key Words: chinch bugs, insecticide resistance, St. Augustinegrass

RESUME
Los chinches vellosos en 10 sitios del estado de la Florida fueron evaluados para determinar
su possible resistencia a 4 insecticides. Se encontr6 diferentes grades de resistencia en los 4
insecticides estudiadas: bifenthrin, deltamethrin, imidacloprid y lambda-cyhalothrin. Este
es el primer studio que muestra la resistencia del chinche velloso en los 3 insecticides ilti-
mos. Nuestros datos tambi6n indican que se necesitan varias localidades para probar los in-
secticidas usados contra el chinche velloso dado que los results de un solo sitio pueden
darnos una falsa guia.


St. Augustinegrass, Stenotaphrum secunda-
tum (Walt.) Kuntze, lawns are used throughout
the southern United States for their climatic ad-
aptation and their ability to tolerate full sun to
moderate shade. The southern chinch bug, Blis-
sus insularis Barber, is the plant's most damaging
pest (Crocker 1993). The adaptability of this in-
sect is shown by developing resistance to insecti-
cides (Reinert & Portier 1983; Cherry & Nagata
2005) and overcoming host plant resistance
(Busey & Center 1987; Cherry & Nagata 1997).
Insecticide resistance in southern chinch bugs
(SCB) was first noted in 1953 in Miami where
Wolfenbarger (1953) showed poor control with
chlordane. By 1958, Kerr and Robinson (1958)
documented resistance to DDT at Sarasota, Flor-
ida. The chinch bugs had become resistant to par-
athion at Fort Lauderdale, Florida by 1960 (Kerr
1960). Chinch bug resistance to both chlorpyrifos
and diazinon was confirmed in 1977 at Pompano
Beach, Florida (Reinert & Niemczyk 1982). And,
in 1983, Reinert & Portier (1983) reported a 9.2-
fold level of resistance to the carbamate insecti-
cide, propoxur by SCB. Hence, by 1983, SCB had
shown resistance to chlorinated hydrocarbon, or-
ganophosphate, and carbamate insecticides.
In recent years, synthetic pyrethroid insecti-
cides have become increasingly used for SCB con-
trol in Florida. Bifenthrin is a synthetic pyre-
throid compound used as a contact and stomach
poison insecticide/acaricide (Thomson 1998).
Bifenthrin has been and still is being used for


SCB control in Florida. During 2003, reports of
difficulty in controlling SCB with bifenthrin in
Florida came to our attention. Cherry & Nagata
(2005) were the first to show SCB resistance to
bifenthrin. The objective of this study was to de-
termine if SCB has developed resistance to other
synthetic pyrethroids and/or imidacloprid, a neo-
nicotinoid insecticide in a different chemical class.

MATERIALS AND METHODS

Chinch bugs were collected by vacuuming in
infested St. Augustinegrass lawns. Southern
chinch bugs have been shown to have localized re-
sponses to insecticides (Reinert & Portier 1983;
Cherry & Nagata 2005). Hence, all SCB were ob-
tained only from a specific lawn at each location.
After collection, the insects were stored at 18"C in
buckets with St. Augustinegrass until used for
testing. The insects were collected from 10 differ-
ent urban areas in 5 counties in central and
southern Florida. Chinch bugs were not collected
from northern Florida since fewer chinch bugs are
found there and control difficulties there were not
brought to our attention. Three of the populations
came from locations where there was difficulty in
controlling chinch bugs and insecticidal resis-
tance was suspected. For comparison, 7 popula-
tions were selected randomly as encountered in
other areas with no knowledge of the insecticidal
use history of the location or efficacy of insecti-
cides against the insects.







Florida Entomologist 90(3)


Methods for testing closely approximated the
methods Reinert & Porter (1983) and Cherry &
Nagata (2005) used earlier in toxicological tests
against SCB. The 4 insecticides selected for test-
ing were bifenthrin, deltamethrin, lambda-cyhal-
othrin, and imidacloprid. The first 3 insecticides
are synthetic pyrethroids labeled for SCB control
in Florida. Imidacloprid is a neonicotinoid insecti-
cide labeled for suppression of southern chinch
bugs in Florida lawns. In the laboratory, serial di-
lutions were made from commercial formulations
of the 4 insecticides. Freshly harvested St. Au-
gustinegrass stolons (ca 10 cm long) were dipped
into dilutions and allowed to air dry. Stolons were
placed individually into Petri dishes (15 cm
diam.) containing moist filter paper to maintain
high humidity.
Preliminary tests showed that very high doses
of deltamethrin and lambda-cyhalothrin were
necessary to kill SCB in 24 h. Hence, 20 adult
SCB were placed into each Petri dish and held 24
h for bifenthrin and imidacloprid and 72 h for del-
tamethrin and lambda-cyhalothrin at 28C and
14 D/10 L. For each test, 5 to 7 concentrations
with a control were tested. Robertson et al. (1984)
noted that a sample size of 120 appears to be the
minimum necessary for reliable LC,0 estimation.
Our sample sizes of adults tested ranged from 120
to 360 for each insecticide at each location to esti-
mate LC,, for that location. Different numbers of
adults tested per insecticide depended on avail-
ability of adults. Since our own objective was to
estimate LC,0 values, we selected doses expected
to give 25 to 75% mortality for best LC,0 estima-
tion as suggested by Robertson et al. (1984). Mor-
tality is defined as virtually no movement by an
adult during a 5 min observation period through a
5x large magnifying lens. The no movement crite-


rion was used to avoid ambiguities of comatose,
unable to stand, moribund, etc. In a separate test,
98% of adults we classified as dead after insecti-
cide exposure did not regain movement after 24 h
and the 2% showed only small twitches. Hence we
believe the no movement criterion was a good
measure of mortality since adults classified as
dead still appeared dead 24 h later.
Because of the longer 72 h testing period, all
results were corrected for control mortality by Ab-
bott's formula (Abbott 1925). Lethal median con-
centrations (LC,0) and slopes were calculated for
each population by using probit analysis on Log10
dose (SAS 2006). Resistance ratios were deter-
mined for each insecticide at each location to com-
pare ratios. The resistance ratio is defined as the
LC,0 of the insecticide at that location divided by
the lowest LC,0 of the insecticide at any location.

RESULTS AND DISCUSSION

Toxicological data for the 10 chinch bug loca-
tions are shown in Tables 1 to 4. Lowery & Smirle
(2003) note that non-overlapping 95% confidence
intervals show that LC,0 values are significantly
different (P < 0.05). A wide range of LC,0 values
were observed in the 10 locations with many of
the LC,0 values within insecticides being signifi-
cantly different.
In bifenthrin tests (Table 1), 6 of the 10 loca-
tions had LC0,values significantly higher than the
lowest LC,0. These data show SCB resistance to
bifenthrin as previously reported by Cherry &
Nagata (2005). Chinch bugs at all 3 of the loca-
tions with control difficulties showed some resis-
tance to bifenthrin. In contrast, only 3 of the 7
randomly chosen locations showed any level of
SCB resistance to bifenthrin. Also, the mean LC50


TABLE 1. LETHAL CONCENTRATIONS OF BIFENTHRIN TO SOUTHERN CHINCH BUGS AT DIFFERENT LOCATIONS IN FLORIDA.

Location LC,, CLb Slope SE nW

Control difficulty
Clermont 102d 40-218 0.8 0.2 240
Lady Lake 18d 5-35 1.1+ 0.2 120
Palm Coast 59d 24-111 1.0 0.2 120
Random
Belle Glade 7 2-17 0.8 0.2 240
Fort Pierce 19 0-54 1.2 0.4 120
Gainesville 8 0-45 1.0 0.4 120
Port St. Lucie 1 0-2 0.4 0.2 120
Royal Palm Beach 65d 18-147 0.8 0.2 120
Wellington 12' 5-20 1.8 0.5 120
West Palm Beach 6d 4-7 2.7 0.6 240

ppm ofAI.
'95% confidence limits.
'Number tested.
Significantly different (P < 0.05) from lowest LC, obtained for insecticide.


September 2007







Cherry & Nagata: Chinch Bug Resistance to Insecticides


TABLE 2. LETHAL CONCENTRATIONS OF DELTAMETHRIN TO SOUTHERN CHINCH BUGS AT DIFFERENT LOCATIONS IN FLORIDA.

Location LCs, CLb Slope SE nW

Control difficulty
Clermont 171' 95-297 1.2 0.2 120
Lady Lake 634d 240-7264 0.6 0.2 120
Palm Coast 40d 13-80 1.0 0.2 120
Random
Belle Glade 6 3-11 1.1+ 0.2 120
Fort Pierce 8 0-38 0.4 + 0.1 360
Gainesville 1 0-8 0.6 0.3 120
Port St. Lucie 3 0-10 0.8 0.3 360
Royal Palm Beach 46d 16-147 0.5 + 0.1 240
Wellington 3 0-23 0.3 0.1 240
West Palm Beach 859d 333-11275 2.7 0.2 120

appm ofAI.
b95% confidence limits.
'Number tested.
dSignificantly different (P < 0.05) from lowest LC, obtained for insecticide.


from the locations with control difficulties was
59.7 ppm versus 16.9 ppm from random locations.
These data are consistent with Cherry & Nagata
(2005) who reported that most SCB populations
in Florida are still susceptible to bifenthrin.
In deltamethrin tests (Table 2), 5 of the 10 lo-
cations had LCs values significantly higher than
the lowest LC50. These data show SCB resistance
to deltamethrin which has not been reported pre-
viously. Chinch bugs at all 3 of the locations with
control difficulties showed some resistance to del-
tamethrin. The Clermont, Lady Lake and West
Palm Beach locations had very high LC50 values
indicating high deltamethrin resistance at these
locations.


In imidacloprid tests (Table 3), 3 of 10 locations
had LC50 values significantly higher than the low-
est LC50. These data show SCB resistance to imi-
dacloprid and this is the first report of SCB resis-
tance to any neonicotinoid insecticide. However,
the highest resistance ratio in imidacloprid was
37.2 versus 102 for bifenthrin and 859 for delta-
methrin. These latter data show that there was
less variability to SCB response to imidacloprid
than the more widely fluctuating LC50 values seen
in bifenthrin and especially deltamethrin.
In lambda-cyhalothrin tests (Table 4), 1 of the
9 locations had a LC50 significantly higher than
the lowest LC50. These data show SCB resistance
to lambda-cyhalothrin, which had not been re-


TABLE 3. LETHAL CONCENTRATIONS OF IMIDACLOPRID TO SOUTHERN CHINCH BUGS AT DIFFERENT LOCATIONS IN FLORIDA.

Location LC5 CLb Slope SE nW

Control difficulty
Clermont 121 40-279 1.0 0.2 240
Lady Lake 44 1-126 0.8 + 0.2 240
Palm Coast 180 17-465 0.6 0.2 120
Random
Belle Glade 65 11-228 0.6 + 0.1 240
Fort Pierce 157 2-435 0.8 0.3 240
Gainesville 385 47-1856 0.5 0.2 120
Port St. Lucie 180 95-284 1.5 0.3 120
Royal Palm Beach 1197' 492-7505 0.7 0.2 120
Wellington 654d 415-1084 1.5 0.3 120
West Palm Beach 1637' 834-6576 1.0 0.3 120

ppm ofAI.
'95% confidence limits.
'Number tested.
dSignificantly different (P < 0.05) from lowest LC, obtained for insecticide.







Florida Entomologist 90(3)


September 2007


TABLE 4. LETHAL CONCENTRATIONS OF LAMBDA-CYHALOTHRIN TO SOUTHERN CHINCH BUGS AT DIFFERENT LOCATIONS
IN FLORIDA.

Location LC,, CLb Slope SE nW

Control difficulty
Clermont NA-
Lady Lake 81d 39-166 1.6 0.4 360
Palm Coast 27 2-73 0.7 0.2 360
Random
Belle Glade 6 0-20 1.0 0.4 240
Fort Pierce 18 3-48 0.8 0.2 240
Gainesville 4 0-16 0.8 0.2 120
Port St. Lucie 7 0-22 0.7 0.2 120
Royal Palm Beach 4 0-26 0.3 + 0.1 240
Wellington 23 5-64 0.7 0.2 240
West Palm Beach 27 11-51 0.6 + 0.1 240

ppm ofAI.
'95% confidence limits.
'Number tested.
Significantly different (P < 0.05) from lowest LC, obtained for insecticide.
'NA = Not available due to insufficient number of insects.


ported previously. This one example of insecticide
resistance did occur at Lady Lake, which was a lo-
cation with control difficulties. Of the 4 insecti-
cides, lambda-cyhalothrin had the lowest number
of locations at which resistance was detected.
Corroborating these data, it should be noted that
the highest resistance ratio for lambda-cyhalo-
thrin was 20.3, which was the lowest comparable
ratio of the 4 insecticides.
As a last note, high variation in SCB response
to insecticides at different locations has been
found by Reinert & Portier (1983), Cherry & Na-
gata (2005) and in this study. These data show
that multiple locations are necessary for insecti-
cidal testing for SCB since results from 1 location
can be very misleading.


REFERENCES CITED

ABBOTT, W. 1925. A method of computing the effective-
ness of an insecticide. J. Econ. Entomol. 18:265-267.
BUSEY, P., AND B. CENTER. 1987. Southern chinch bug
(Hemiptera: Heteroptera: Lygaeidae) overcomes re-
sistance in St. Augustinegrass. J. Econ. Entomol. 80:
608-611.
CHERRY, R., AND R. NAGATA. 1997. Ovipositional prefer-
ence and survival of southern chinch bugs (Blissus
insularis Barber) on different grasses. Int. Turfgrass
Soc. J. 8: 981-986.
CHERRY, R., AND R. NAGATA. 2005. Development of re-
sistance in southern chinch bugs (Hemiptera: Lyga-


eidae) to the insecticide bifenthrin. Florida Entomol.
88: 219-221.
CROCKER, R. 1993. Chemical control of southern chinch
bug in St. Augustinegrass. Int. Turfgrass Res. J. 7:
358-365.
KERR, S. 1960. Insect control. Proc. Univ. Florida Turf-
grass Manag. Conf. 8: 116-118.
KERR, S., AND F. ROBINSON. 1958. Chinch bug control
tests, 1956-57. Florida Entomol. 41: 97-101.
LOWERY, D., AND M. SMIRLE. 2003. Comparison ofbioas-
say techniques for determining baseline susceptibili-
ties of imidacloprid for green apple aphid (Homoptera:
Aphididae). J. Econ. Entomol. 96: 1864-1871.
REINERT, J., AND H. NIEMCZYK. 1982. Insecticide resis-
tance in epigeal insect pests of turfgrass: southern
chinch bug resistance to organophosphates in Flor-
ida, pp. 77-80. In H. Niemczyk and B. Joyner (eds.),
Advances in turfgrass entomology. Hammer Graph-
ics, Inc. Piqua, OH.
REINERT, J., AND K. PORTIER. 1983. Distribution and
characterization of organophosphate-resistant
southern chinch bugs (Heteroptera: Lygaeidae) in
Florida. J. Econ. Entomol. 76: 1187-1190.
ROBERTSON, J., K. SMITH, N. SAVIN, AND R. LAVIGNE.
1984. Effects of dose selection and sample size on the
precision of lethal dose estimates in dose-mortality
regression. J. Econ. Entomol. 77: 833-837.
SAS INSTITUTE. 2006. SAS Systems for Windows. SAS
Institute. Cary, NC.
THOMSON, W. 1998. Agricultural chemicals, Book one-
insecticides. Thomson Publ., Fresno, CA.
WOLFENBARGER, D. 1953. Insect and mite control prob-
lems on lawn and golf grasses. Florida Entomol. 36:
9-12.







King & Tschinkel: Pheidole obscurithorax Expands its Range and Density



RANGE EXPANSION AND LOCAL POPULATION INCREASE
OF THE EXOTIC ANT, PHEIDOLE OBSCURITHORAX, IN THE
SOUTHEASTERN UNITED STATES (HYMENOPTERA: FORMICIDAE)

JOSHUA R. KING* AND WALTER R. TSCHINKEL
Florida State University, Department of Biological Science, Unit 1, Tallahassee, FL 32306, USA

*Corresponding author

ABSTRACT

The exotic ant, Pheidole obscurithorax Naves, is currently expanding its range and increas-
ing in local density in the southeastern United States. We describe new county records from
5 states and local density increases along roadside transects in the Tallahassee, Florida area.
The patchy distribution suggests that this species is being transported to new localities by
people. Throughout its introduced range, this species is largely confined to highly disturbed
habitats, such as lawns and roadsides and frequently co-occurs with the introduced fire ant,
Solenopsis invicta Buren. Locally, although the maximum density of nests per unit area has
not changed since 2002, the total area occupied, and the total number of nests of P. obscu-
rithorax is much greater. Beyond these data, little is known of the biology of this species.

Key Words: abundance, Florida, invasive species, roadsides

RESUME
La hormiga ex6tica, Pheidole obscurithorax Naves, actualmente esta expandiendo su rango
y localmente aumentando su densidad en el sureste de los Estados Unidos. Nuevos registros
para condados en 5 estados del pais y el aumento de la densidad local a lo largo de los cami-
nos que cruzan el area de Tallahassee, Florida son descritos. Su distribuci6n irregular indica
que esta especie esta siendo transportada a nuevas localidades por medio de gente. A trav6z
de su rango de introduci6n, esta especie es usualmente limitada a areas muy disturbadas,
como los cespedes y lo largo de los caminos y se encuentra a menudo junta con la hormiga
de fuego introducida, Solenopsis invicta Buren. En cuanto de su distribuci6n local, aunque
la densidad maxima de los nidos por unidad de area no ha cambiado desde el aio 2002, la
area total ocupada y el nmmero total de nidos de P. obscurithorax es much mayor. Mas alla
de estos datos, poco es conocido sobre la biologia de este especie.


The southeastern United States, particularly
Florida, is a global hot spot for biological invasion.
Among the invaders, there is a very large and
growing number of exotic ant species (Deyrup
et al. 2000; Deyrup 2003). The most conspicuous
members of this exotic ant fauna were trans-
ported from the South American region that
spans northern Argentina, Paraguay, and south-
ern Brazil in the vicinity of the Paraguay, La
Plata, and Parana Rivers. The best-known mem-
bers of this group include the imported fire ants
Solenopsis invicta Buren and S. richteri Forel,
and the Argentine ant Linepithema humile
(Mayr), all of which have achieved pest status.
The notoriety of these species is a direct result of
their pest status and now global distribution, al-
though they may also pose a threat to some native
flora and fauna (Holway et al. 2002; Tschinkel
2006). There have been a number of non-pest ant
species from this same South American region
that were probably first introduced and estab-
lished in the Mobile, Alabama area which may be
following a similar spread trajectory as the pest


species. These species may have the potential to
become pests or negatively impact native species
if their populations continue to grow and spread.
Storz & Tschinkel (2004) first documented the
recent spread and ecological associations of one
such species, Pheidole obscurithorax Naves.
Pheidole obscurithorax was probably first intro-
duced in Mobile, Alabama around 1950 (Naves
1985) and has subsequently slowly spread across
the panhandle of Florida (Wilson 2003; Storz &
Tschinkel 2004), appearing in the Tallahassee,
Florida area in the late 1990s. This is an unusu-
ally large and active species of Pheidole that at-
tracts the attention of entomologists, so its spread
has not gone unnoticed. Field characters are large
size (about twice the size of the common native
species, Pheidole dentata), large nest mounds,
usually with a single entrance, and spectacularly
fast recruitment to bait such as cookie crumbs.
More recently, P. obscurithorax has expanded its
range and become much more abundant. Al-
though this species is not currently a pest species,
it is an abundant invasive exotic about which we







Florida Entomologist 90(3)


know very little. We can presently make few pre-
dictions about its current or long-term impact and
range limits. Because it overlaps in distribution
with fire ants and is closely associated with hu-
mans it warrants careful monitoring. Moreover,
we already know that one species of Pheidole,
P megacephala, has had deleterious effects on
certain natural ecosystems (Hoffmann et al.
1999). Here we update the distribution of P ob-
scurithorax in the southeastern U.S. and show
that it is greatly increasing in local abundance.

MATERIALS AND METHODS

We gathered new records from both taxono-
mists (Lloyd Davis, Mark Deyrup, Joe MacGown)
working in the southern United States and our
own collections to estimate the introduced range
of P obscurithorax. We used county records as es-
timates for our distribution map (Fig. 1) because
this species now covers such a large area and
there has not been a comprehensive effort to doc-
ument its full range.
For our local density study, we resampled the
same localities in Tallahassee, Florida that were
sampled by Storz & Tschinkel (2004). Storz &
Tschinkel (2004) counted nests along one 50 x 1-m
transect located along roadsides scattered
throughout Tallahassee. To increase the area of
observation, and thus get a more representative
sample, we sampled in the same localities as
Storz & Tschinkel (2004) but quadrupled (two 100
x 2-m transects per locality) the length of
transects. We then calculated the average number
of nests in a 50 x 1-m transect area (total number
of nests divided by 4) for comparison with Storz &
Tschinkel (2004). This average value was used to
create accumulation curves (Fig. 4) of nest density
per 50 x 1 m with the program Ecosim (Gotelli &
Entsminger 2001).


RESULTS

New records indicate that since 2002, P obscu-
rithorax has spread to 16 new counties in 5 states,
including a record from Texas (Fig. 1). This pat-
tern of spread suggests that, in addition to
spreading locally by natural dispersal of winged
females, long-distance dispersal is almost cer-
tainly facilitated by human transport. Generally,
throughout its range this species is closely associ-
ated with the most disturbed sites such as lawns
and roadsides, although there have been a few
records in natural areas such as hardwood forests
(Wilson 2003). We also have collected this species
in pastures. The most commonly co-occurring con-
spicuous species in the Southeast are Dory-
myrmex bureni (Trager) and S. invicta. We have
observed nests of all 3 species within a meter or
less of one another.
In 2006, nests of P obscurithorax were present
at all sites and all but one 100-m transect. The
number of nests per transect ranged from 0 to 22,
and averaged about 7 nests. The frequency distri-
bution (Fig. 2) was right-skewed, with maximum
values about 3 times the mean. This might be the
pattern expected for an invasion in progress, and
if that were the case, sites that have been occu-
pied longer would be expected to host more nests.
For 2006, sites that were first occupied in 1999
hosted an average of 4.4 colonies; those first occu-
pied in 2002 had 5.1 colonies and those in 2006,
2.2 colonies. These differences were not signifi-
cant (ANOVA: adjustedR2 = 0.101;P < 0.081), and
fail to support an effect of time-occupied on nest
density.
Although we sampled the same sites in 2006 as
did Storz & Tschinkel (2004) in 1999, 2000, and
2002, we sampled an average of almost 4 times as
much area. Thus, we cannot directly compare the
numbers of nests found in each transect and site.


Fig. 1. The increase in range, by county, of Pheidole obscurithorax in the southeastern United States, 2002 to
2006.


September 2007







King & Tschinkel: Pheidole obscurithorax Expands its Range and Density


0 2 I o0 14 1
No. of P. obscurithorax nest


73 2


Fig. 2. Frequency distribution of number of P. obscu-
rithorax nests on 100-m transects in 2006.


Converting these data to densities (nests/m2) al-
lows the comparison presented in Fig. 3. Although
the maximum densities for 2000 and later were
not very different, the number and proportion of
sites from which P. obscurithorax was absent
were different. In 1999, 71% of sites lacked P ob-
scurithorax. This dropped to 65% in 2000, 39% in
2002, and 0% in 2006. At the same time, the num-


ber of sites with moderate to high densities in-
creased. In our 2006 resurvey of Storz &
Tschinkel's (2004) sites, we usually encountered
P obscurithorax nests within the first few meters.
A second comparison between the 2002 and
2006 surveys confirmed these findings. By resam-
pling transects randomly from the data, we deter-
mined the relationship between the number of
transects sampled and the number of P. obscu-
rithorax nests encountered, the so-called rarefac-
tion curves (Fig. 4). To adjust samples to be of
equal size, we divided each of our 2006 transects
into 4, each with the mean expected value of
nests. For the same number of transects ran-
domly resampled, nests accumulated much more
rapidly in 2006 than in 2002, confirming their
greater abundance, independent of the number of
transects. Much of this effect was the result of the
higher number of vacant transects in the 2002
samples.
What accounted for the differences in densities
among the sites in 2006? We have already ruled
out a large role for years-of-occupation. On the
other hand, the colonies are not randomly distrib-
uted among the transects-a comparison of the
observed with the expected Poisson distribution
shows a surplus of low and high values and a def-
icit of mid-range values (Fig. 5). This suggests the
operation of some non-random, site-specific factor
affecting the frequency of nests. A possible clue is
that sites with high densities of P obscurithorax
also had high densities of the fire ant, S. invicta
(Fig. 6) (regression: F,43 = 20.4; adjusted R2 = 31%;


1= 2000 2001 2002 2003


2M04 2005 20


This study
















S' LStorz & Tschlnkel 2002


0 10 20 30


Fig. 3. Densities of P. obscurithorax (nests/100 m2) at
the survey sites, 1999-2006. Size of the symbol indicates
the relative frequency of its y-value. Data prior to 2006
were taken from Storz & Tschinkel (2004).


40 50 60


Transacts
Fig. 4. The rate at which the number of P. obscuritho-
rax nests accumulate upon random resampling of our
data and those of Storz & Tschinkel (2004). Our 2006
transects were divided into 4 to be equivalent to those of
Storz & Tschinkel (2004).


is -



10
,2






S10


ii
* 0







Florida Entomologist 90(3)


9-~
0>






p


14 16 It 2? 22 mrtnv


Fig. 5. The observed frequency distribution of P. ob-
scurithorax nests among transects compared to the ex-
pected Poisson distribution. There are more high and
low values and fewer mid-values than expected from a
random distribution. Data for 2006 only.


P < 0.00005). For every additional fire ant colony
per square meter, an additional 0.7 colonies of
P obscurithorax was present (note however, that
fire ant colonies have 1.5 to 2 orders of magnitude
more workers). Fire ants appeared in the Talla-
hassee area in the 1960s and their densities have
been more or less stable for decades. This sug-
gested that the density differences might be the
result of site quality differences-in other words,



22 2006
020



18
E 6
14



S2 p *



08
6

2 *
*
0 2 4 6 8 10 12 14 16
Number of S. invicta nest in transact

Fig. 6. The densities of fire ant nests in relationship to
the densities ofP. obscurithorax nests in the 2006 survey.


-0- eperlad


I
\
<.\

"v /"Il,"
%0 _


what is good for the fire ant is also good for P ob-
scurithorax. There is also some evidence that
P obscurithorax collects dead fire ants (and occa-
sionally live ones) for food (Storz & Tschinkel
2004), often piling the spent fire ant corpses
around their nests. There is also evidence that fire
ants do not competitively suppress P obscuritho-
rax as removal of fire ants does not affect
P obscurithorax densities in pastures (King &
Tschinkel 2006).

DISCUSSION

Much like fire ants, P obscurithorax has
clearly found the southeastern United States to
be quite congenial to its needs and is currently ex-
panding its range across the disturbed habitat
that comprises the bulk of the region. Its ongoing
range expansion and biogeographic history sug-
gest that P. obscurithorax may eventually occupy
a range similar in size to that ofS. invicta, at least
in the United States. Unlike fire ants, however,
P obscurithorax is not considered a pest species,
despite its local density. The obvious reason for
this is that Pheidole do not sting and, in the case
of P obscurithorax, have never been documented
to infest human dwellings or structures. Probably
as a result of this, little is known of its biology. We
know that it is a monogyne, dimorphic Pheidole
species in the fallax group (Wilson 2003), occurs
primarily in disturbed habitats, has large colonies
(-10,000 workers, WRT unpublished data), and
does not directly compete with S. invicta (King &
Tschinkel 2006). We do not know the flight range
of mated queens. We do not know how it is trans-
ported from place to place, although it seems most
likely that mated queens (not colony fragments)
are being transported in substrates such as pot-
ted plants. This species is the largest Pheidole in
the southeastern United States (King & Porter
2007) and we do not know how its range expan-
sion and population increase may impact co-oc-
curring species, if at all. Because this species is
currently expanding its range, we think that it
might make an excellent study system for inva-
sive ants, and invasive Pheidole in particular.

ACKNOWLEDGMENTS

We thank Lloyd Davis, Mark Deyrup (Archbold Bio-
logical Station), and Joe MacGown (Mississippi State
University) for their collection records. Comments from
2 anonymous reviewers improved an earlier version of
the manuscript.

LITERATURE CITED

DEYRUP, M. 2003. An updated list of Florida ants (Hy-
menoptera: Formicidae). Florida Entomol. 86:43-48.
DEYRUP, M., L. DAVIS, AND S. COVER 2000. Exotic ants
in Florida. Trans. American Entomol. Soc. 126: 293-
326.


o 2 4 6 8 .0 1I
Number of Nest


September 2007







King & Tschinkel: Pheidole obscurithorax Expands its Range and Density


GOTELLI, N. J., AND G. L. ENTSMINGER 2001. EcoSim:
null models software for ecology. Version 7.0. http://
homepages.together.net/~gentsmin/ecosim.htm. Ac-
quired Intelligence, Inc. & Kesey-Bear
HOFFMANN, B. D., A. N. ANDERSEN, AND G. J. E. HILL.
1999. Impact of an introduced ant on native rain for-
est invertebrates: Pheidole megacephala in mon-
soonal Australia. Oecologia 120: 595-604.
HOLWAY, D. A., L. LACH, A. V. SUAREZ, N. D. TSUTSUI,
AND T. J. CASE. 2002. The causes and consequences
of ant invasions. Annu. Rev. Ecol. Syst. 33: 181-233.
KING, J. R., AND S. D. PORTER 2007. Body size, colony
size, abundance, and ecological impact of exotic ants
in Florida's upland ecosystems. Evol. Ecol. Res. 9:
757-774.
KING, J. R., AND W. R. TSCHINKEL. 2006. Experimental
evidence that the introduced fire ant, Solenopsis in-


victa, does not competitively suppress co-occurring
ants in a disturbed habitat. J. Anim. Ecol. 75: 1370-
1378.
NAVES, M. A. 1985. A monograph of the genus Pheidole
in Florida (Hymenoptera: Formicidae). Ins. Mundi 1:
53-89.
STORZ, S. R., AND W. R. TSCHINKEL. 2004. Distribution,
spread, and ecological associations of the introduced
ant Pheidole obscurithorax in the southeastern
United States. J. Ins. Sci. 14: 12, Available online: in-
sectscience.org/4.12
TSCHINKEL, W. R. 2006. The Fire Ants. Harvard Univer-
sity Press, Cambridge, USA. 723 pp.
WILSON, E. 0. 2003. Pheidole in the New World: A Dom-
inant, Hyperdiverse Ant Genus. Harvard University
Press, Cambridge, USA. 794 pp.







Florida Entomologist 90(3)


September 2007


DEVELOPMENT OF TRAPPING METHODS WITH A SYNTHETIC
SEX PHEROMONE OF THE PINK HIBISCUS MEALYBUG,
MACONELLICOCCUS HIRSUTUS (HEMIPTERA: PSEUDOCOCCIDAE)

ANTONIO FRANCIS1, KENNETH A. BLOEM2, AMY L. RODA3, STEPHEN L. LAPOINTE4,
AIJUN ZHANG5 AND OGHENEKOME ONOKPISE6
'Florida A&M University, College of Engineering Sciences, Technology, and Agriculture,
Center for Biological Control, Tallahassee, FL

2USDA-APHIS-PPQ-Center for Plant Health Science and Technology, Raleigh, NC

3USDA-APHIS-PPQ-Center for Plant Health Science and Technology, Miami, FL

4USDA-ARS, U.S. Horticultural Research Laboratory, Ft. Pierce, FL

5USDA-ARS-BARC-PSI-Chemicals Affecting Insect Behavior Laboratory, Beltsville, MD

'Florida A&M University, College of Engineering Sciences, Technology, and Agriculture, Tallahassee, FL

ABSTRACT

The pink hibiscus ii.. :-. 1.. ....--, ........ hirsutus (Green), threatens numerous crops of
economic importance and could spread from populations in California and Florida to 33
other states. Field experiments conducted in Florida evaluated 3 commercially available
trap designs baited with synthetic female sex pheromone for efficiency in trapping adult
male M. hirsutus as well as ease in processing. Delta traps and double-sided sticky cards
captured more males than Jackson traps. The Delta and Jackson traps were more effective
at minimizing the capture of non-target insects. The effect of lure age on males captured was
also evaluated by pre-aging lures outdoors for 0 to 8 months before testing. Fewer males
were caught in Delta traps as the age of the lure increased, with significantly fewer caught
in traps that had been pre-aged for 2 months. Monitoring of male flight activity throughout
diel cycle with baited Delta traps indicated that males were most active around dusk. The
field experiments also showed that the pheromone traps often capture males in areas where
no visual indication of an infestation is evident. The pheromone trap may serve as a valuable
tool to detect new infestations of pink hibiscus mealybug.

Key Words: Maconellicoccus hirsutus, sex pheromone, monitoring, trap design, lure longev-
ity, male flight activity

RESUME

La cochinilla rosada del hibiscus, Maconellicoccus hirsutus (Green) es una amenaza para va-
rios cultivos de importancia econ6mica y puede esparcirse de poblaciones presents en Cali-
fornia y Florida a otros 33 estados. Experimentos de campo realizados en Florida evaluaron
3 disehos de trampas comerciales disponibles con cebos de una feromona sexual sint6tica de
hembras para atrapar machos adults de M. hirsutus con eficiencia y facilitar su process de
elaboraci6n. Las trampas de tipo "Delta" y las tarjetas con los dos lados pegajosos capturaron
mas machos que las trampas de tipo "Jackson". Las trampas Delta y Jackson fueron mas
efectivas en minimizar la cantidad capturada de insects que no se busca controlar. El efecto
de la edad del seAuelo sobre la cantidad de machos capturados tambi6n fue evaluado por se-
Auelos pre-afejados en el exterior por cero a 8 meses antes de la prueba. La cantidad de ma-
chos capturados en las trampas Delta fue menor con el aumento en la edad del seAuelo, con
significativamente menos capturados en trampas que han sidas pre-afejadas por 2 meses.
El monitoreo de la actividad de vuelo de los machos a trav6z del ciclo de "diel" [un ciclo bio-
16gico de 24 horas] con trampas Delta cebadas indic6 que los machos fueron mas activos por
el tiempo alrededor del atardecer. Los experiments de campo tambi6n muestrearon que las
trampas de feromonas a menudo capturaron machos en areas donde no hubo una indicaci6n
visual evidence de una infestaci6n. La trampa de feromona puede servir como una herra-
mienta valiosa para detectar nuevas infestaciones de la cochinilla rosada del hibiscus.


The pink hibiscus mealybug, Maconellicoccus 1989), attacking many agricultural crops, forest
hirsutus (Green), has a reported host range of >125 trees, and ornamental plants (Persad 1995; Kairo
plant species (Ghose 1972; Williams 1986; Mani et al. 2000). Heavy infestations of this mealybug







Francis et al.: Trapping Methods for Pink Hibiscus Mealybug


can eventually kill the host plant (Stibick 1997;
Kairo et al. 2000). In the absence of effective con-
trol measures, the economic risk to U.S. agriculture
has been estimated at $750 million per year (Mof-
fitt 1999). Therefore, early detection is important
for the timely application of control measures to
suppress new outbreaks ofM. hirsutus and prevent
significant economic and environmental losses.
Serrano et al. (2001) first reported attraction of
adult male M. hirsutus to virgin females, suggest-
ing the action of an unidentified sex pheromone.
The active components of this female sex phero-
mone were recently identified (Zhang et al.
2004a) and synthesized (Zhang et al. 2004b;
Zhang & Nie 2005). Its effectiveness in sticky
traps under field conditions as a male attractant
has been demonstrated (Zhang & Amalin 2005).
The synthesized pheromone was found to be
highly specific and lure activity remained for up
to 21 weeks in the field.
Prior to deploying pheromone-based trapping
systems, the factors that may influence trap cap-
ture need to be assessed and trapping protocols
standardized. We evaluated 3 commercially avail-
able trap designs for effectiveness in capturing
the target species and selectivity for non-target
captures, and more closely evaluated the longev-
ity of the sex pheromone lure in the field at differ-
ent sites. We also used pheromone-baited traps to
determine the diurnal pattern of flight by male
M. hirsutus. An effective monitoring program
with M. hirsutus sex pheromone should include
recommendations on frequency of lure replace-
ment and an effective trap design that would al-
low detection at low population levels.

MATERIALS AND METHODS

Lures
Lures for all of the field trials were prepared at
the USDA-ARS-BARC facility, Beltsville, MD,
with gray halo-butyl rubber septa (5 mm, West
Pharmaceutical Services, Kearney, NE) impreg-
nated with 1 pg of M. hirsutus synthetic sex pher-
omone (Zhang & Amalin 2005). They were stored
in air-tight plastic bags and shipped by express
carrier on the same day. Lures for all experiments
were from the same batch and were stored in a
deep freezer at -25 + 2C until needed. The blank
control septa used in these field trials were not
loaded with solvent of any type. Chemical and op-
tical purities of synthetic pheromone components
used in current study were the same as previously
reported (Zhang & Amalin 2005).

Trap Design Study
Field trials were conducted from April 13 to
May 18, 2005 at 2 sites on the USDA-ARS Sub-
tropical Horticulture Research Station, Miami,
FL. Prior sampling with lure-baited traps re-


vealed high numbers of adult male M. hirsutus in
the area despite the lack of visual signs of infesta-
tion (e.g., mealybug colonies, 'bunchy top', etc.) on
station property. The 2 sites were separated by
>300 m and were >15,000 m2 each. The first site
had a mixture of large non-fruit bearing trees
such as Ficus spp., while the second site had plots
of mature mango and avocado trees.
The traps tested were Delta traps (Scentry,
Inc., Buckeye, AZ), Jackson traps (Scentry Inc.,
Buckeye, AZ), and Pherocon V, scale-monitoring,
two-sided sticky cards (Tr4ce, Inc., Salinas, CA).
All traps were white and constructed from
weather-resistant paperboard. These traps are
commercially available, relatively inexpensive,
and widely used in other insect monitoring pro-
grams. The Delta trap has a triangular-shaped
design with perforated ends that can be folded in-
wards to create a narrower opening when assem-
bled and has a total sticky surface area of 575.25
cm2 on its 3 interior surfaces. The Jackson trap is
also triangular-shaped, but the 2 ends are com-
pletely open and only the removable bottom in-
sert has a sticky surface with an area of 120.97
cm2. The Pherocon V sticky card is a 2-dimen-
sional, rectangular trap with total sticky surface
area of 193.6 cm2 (96.8 cm2 on each side).
A randomized complete block design was used
with 2 replicates at each site. Each replicate con-
sisted of 6 treatments-the 3 trap designs baited
with pheromone lures or blank septa. Lures and
blank control septa were placed inside plastic bas-
kets and secured with wire holders to the top inte-
rior of the Delta and Jackson traps. For the sticky
cards, the baskets/lures were suspended from the
top of 1 of the 2 exposed sides. The traps were then
mounted to stakes buried in the ground through-
out both sites at a height of 0.45 m (Zhang & Ama-
lin 2005) and were arranged in a 3 x 4 grid with
the traps spaced 30 m apart. Traps were replaced
and re-randomized weekly for 4 weeks and the
lures were transferred to the new traps. Collected
traps were placed into separate transparent plas-
tic bags and brought to the laboratory to count
trapped M. hirsutus males and non-target insects.
Under >5x magnification, adult M. hirsutus males
generally range from 2.5-3 mm in length (exclud-
ing wings and caudal filaments). The 2 caudal fil-
aments are about as long as the body. However, be-
cause previous work had indicated that >99% of
male mealybugs captured in traps baited with
pink hibiscus mealybug pheromone were M. hir-
sutus (Zhang et al. 2004a), and initial identifica-
tions in these experiments provided by G. Hodges
(FL Department of Agriculture and Consumer
Services, Gainesville, FL) showed similar find-
ings, no efforts were made to determine if other
mealybug species were found on traps. At the end
of the trial, used lures were collected and stored in
individual plastic bags held in a freezer as previ-
ously described until analyses.







Florida Entomologist 90(3)


Lure Longevity Study

Fresh to 8 MonthsAged Lures: Field trials were
conducted from Jun 2 to Jun 30, 2005 at 3 sites on
the USDA-ARS research station. The 2 sites from
the trap design study were again used, as well as
a third site that had similar characteristics to the
site with avocado and mango trees and was sepa-
rated from the other 2 sites by approximately 300
m. As previously described, male M. hirsutus
were captured in monitoring traps prior to run-
ning the experiment at each of these sites, al-
though no visible signs of infestation or colonies
could be found. Lures were aged outdoors in Delta
traps (Ecogen, Inc., Billings, MT) that were sus-
pended in tree canopies for periods ranging from
1-8 months. This 'aging' process commenced on
Oct 1, 2004 and ended on Jun 1, 2005. Aged lures
of 1, 2, 4, 6, and 8 months, a 'fresh' (not aged) lure
and a blank control septum, were then tested in
white Delta traps to determine the influence of
age on their attractiveness to male M. hirsutus.
The Delta trap was chosen based on the results
from the trap design study.
Procedures for preparation and field place-
ment of the 7 treatments were the same as those
described for the trap design study. Traps were
arranged in a randomized complete block design
with 2 replicates at each of the 3 sites. All traps
and the lures of the'fresh lure' treatment were re-
placed weekly for 4 weeks. The lures in the 6
aged-lure treatments were transferred to new
traps each week for the duration of the study. Col-
lected traps were put into separate transparent
plastic bags and brought to the laboratory to
count only trapped males. Used lures were col-
lected at the end of the trial and stored in a
freezer as previously described. These were later
sent to the USDA-ARS-BARC for analysis to de-
termine the amount of sex pheromone remaining
in each septum.
To determine pheromone residue, 3 lures from
each of the 6 different time periods were placed
individually into 3 mL hexane in a 4-mL vial and
soaked for 8 h. Extracts (20 pL each) were diluted
with hexane to an approximate volume (ca.10 ng
per pL) for gas chromatography-mass spectrome-
try (GC-MS) analyses. Electronic impact GC-MS
analyses of pheromone lures were conducted on a
Hewlett-Packard 6890 GC coupled to a HP 5973
Mass Selective Detector with a DB-WAXETR cap-
illary column (J&W Scientific, Inc., Folsom, CA,
60 m x 0.25-mm ID, 0.25-pm film-thickness, 50C
for 2 min, then programmed to 230C at 15C per
min and held for 15 min) with helium as carrier
gas. A 70 eV electron beam was employed for sam-
ple ionization. The ions, m/z 93, 121, and 136
were selected as the monitor ions and remaining
pheromone concentrations were obtained by com-
parison with synthetic RS pheromone standards
at the same conditions.


Fresh to 7 Weeks Aged Lures: To further verify
that lures <2 months old were as effective as fresh
lures at attracting male M. hirsutus, a second
field experiment was conducted from Oct 12 to
Oct 19, 2006 at one site on the USDA-ARS re-
search station. As previously described, lures
were aged outdoors in Delta traps that were
placed in tree canopies for periods ranging from 1
week to 7 weeks. Aged lures of 1, 2, 4, and 7 weeks
and a 'fresh' (not aged) lure were tested in white
Delta traps to determine the influence of short
term ageing on their attractiveness to male
M. hirsutus. Traps were arranged in a random-
ized complete block design with 5 replicates of
each lure age. After 1 week, traps were processed
and all male M. hirsutus were counted as previ-
ously described.

Male Flight Activity Study

Field studies of male flight activity over a 24-h
period were conducted from Jun 2 to Jun 5, 2005
at 2 residential properties, Miami, FL. The 2 sites
had hibiscus hedgerows of >50 m infested with
low to moderate levels ofM. hirsutus. Sample pe-
riods were defined as 1 sample covering 0800 to
1600, hourly samples taken from 1600 to 2400,
and a single sample covering 0000 to 0800. Three
white Delta traps baited with sex pheromone
lures and mounted to 0.45 m high stakes were
used at each site, spaced 15 m apart and 1 m from
the hedge. Traps were initially placed at 0800 on
the first day and replaced at the start of each sam-
ple period with lures being transferred to the new
traps. Traps and lures were processed as previ-
ously described.

Statistical Analyses

Data from all males captured during the field
studies were analyzed by analysis of variance
(ANOVA) by Standard Least Squares (JMP Sta-
tistical Discovery 6.0.2, SAS Institute 2006). Fac-
tors of variation in the statistical models were
trap design, baiting of traps, site, and week for
the trap design study; and lure age, site, and week
for the lure longevity study. The numbers of male
M. hirsutus and non-target insects caught on
traps were the dependent variables. When the
models indicated significant treatment effects
and/or significant interactions, differences among
means were separated by Tukey's honestly signif-
icant difference (HSD) test at a = 0.05 for multi-
ple comparisons.
Residual amounts of pheromone recovered
from lures were analyzed by regression analyses
(SPSS 10.0 for Windows, George & Mallery 2002)
with time as the factor of variation in the statisti-
cal model and amount of pheromone residue as
the dependent variable. The numbers of male
M. hirsutus caught by traps baited with the differ-


September 2007







Francis et al.: Trapping Methods for Pink Hibiscus Mealybug


ent lure-treatments also were analyzed by regres-
sion analyses (SPSS 13.0 for Windows Student
Version, SPSS, Inc. 2005) with time as the factor
of variation in the statistical model and numbers
of males caught as the dependent variable.
Sample periods (time intervals in hours) and
site were the factors of variation for the male
flight activity study. The number of male M. hir-
sutus caught on traps was the dependent vari-
able. When the models indicated significant treat-
ment effects and/or significant interactions, dif-
ferences among means were again separated by
Tukey's HSD test.

RESULTS

Trap Design

Analysis of the data for males captured with
the different trap designs showed that the model
was significant (df = 9,84; F = 20.86; P < 0.0001)
and that there were significant differences in
male trap catch among the different baited traps
(df = 2,84; F = 5.06; P = 0.008). The only signifi-
cant interaction was between trap baiting status
(baited versus unbaited) and site (df = 1, 84; F =
26.42; P < 0.0001). Unbaited traps caught a total
of only 3 male M. hirsutus during the course of the
experiment. Males captured in baited traps were
higher at site 1 than they were at site 2. Delta
traps generally caught about 1.5x as many males
as did sticky cards, although the difference was
not significant, and they caught significantly (ap-
proximately 2.5x) more males than Jackson traps
(Fig. 1). Captures with Jackson traps and sticky
cards were not significantly different (Fig. 1).
Analysis of data for non-target insects cap-
tured by the different trap designs showed that
the model was significant (df= 8,93; F = 12.61 P <
0.001) and that there were no significant interac-
tions. There was no significant difference in the
number of non-target insects caught between
baited and non-baited traps (df = 1,85; F = 0.36;
P = 0.55); however, there were significant differ-
ences among the 3 trap designs (df = 2,85; F =
37.27; P < 0.0001). Sticky cards caught signifi-
cantly more (>2x) non-target insects than either
Jackson traps or Delta traps (Fig. 1). Captures
with Jackson traps and Delta traps were not sig-
nificantly different (Fig. 1). Non-target catches for
both baited and unbaited traps were primarily
hymenopteran and dipteran species, but notice-
ably not the mealybug parasitoids Anagyrus ka-
mali Moursi and Gyranusoidea indica Shafee,
Alam and Agarwal.

Lure Longevity

Fresh to 8 Months Aged Lures: The amount of
pheromone in the septa decreased as the lures
aged (Fig. 2). The greatest decrease in residual


A ---- Dlta
* Jackson
0 Sticky card


Male N. hirsutus


Non-target


Fig. 1. Mean number of male M. hirsutus and non-
target insects captured per week in different types of
pheromone baited traps located at the USDA ARS Sub-
tropical Horticulture Research Station, Miami, FL. Sig-
nificant differences between means of male M. hirsutus
and non-target insects captured in the different traps
are indicated by different letters (a = 0.05).


pheromone occurred during the first 2 months,
dropping from an initial load of 1.00 pg/septum
(0.01 SE) to 0.30 pg (+0.03 SE). Thereafter, the
decrease was more gradual, dropping from 0.30
pg to 0.07 pg after 9 months. This decrease of
pheromone over time was best described by the
equation In y = -0.2794x-0.4477; r2 = 0.85. Based
on this equation, the half-life of the lure was ap-
proximately 0.9 months (25 d).
Analysis of data for male M. hirsutus captured
in traps with fresh and aged lures by site by week
showed that the model was significant (df= 83,84;
F = 8.18; P < 0.0001), and that all primary and
secondary interactions also were significant. This
indicates that there were factors other than the
different lure ages at each site and from week-to-
week that were influencing trap captures. To al-
low for a simpler interpretation of the treatment
effects, males captured for site 1, week 1 were an-
alyzed separately. Site 1 was selected because this
site had the highest overall number of males
trapped. Week 1 was selected because any influ-
ence of the aging of lures over the 4-week dura-
tion of the experiment was excluded. This analy-
sis indicated that lure age did significantly affect
males captured (df = 7, 6; F = 13.07; P = 0.003).
Based on Tukey's HSD separation of means (a =
0.05), traps baited with fresh lures did not cap-
ture significantly greater numbers of males than
traps baited with lures aged 1 month; however,
they did capture significantly more males than
traps baited with lures aged 2 months or longer
(Fig. 2). Traps baited with lures aged 2, 4, 6, and







Florida Entomologist 90(3)


.----- b
0 1 2 3 4 5 6 7 8 9
Lure ace (months)


tween 1700 h and 2000 h, with 1.5 (0.8 SE), 1.6
( +0.6 SE), and 3.4 (+1.1 SE) males captured/trap
During the sample periods 1700 to 1800, 1800 to
1900, and 1900 to 2000 h, respectively. No males
o. 75 were captured after 2000 h.


DISCUSSION


o
0
.so
0.50 f

m
0.25 a


+I


Fig. 2. Relationships of lure age to number of male
M. hirsutus caught in pheromone-baited traps during a
week-long field trial at one site on the USDA-ARS Sub-
tropical Horticulture Research station, Miami, FL; as
well as the relationship of lure age to amount of M. hir-
sutus pheromone residue in the lures. Septa were
loaded with 1 pg of pheromone and aged for 1, 2, 4, 6,
and 8 months prior to use. Significant differences be-
tween means of male M. hirsutus and pheromone resi-
due are indicated by different letters (a = 0.05).


8 months in the field prior to the experiment did
not capture significantly greater numbers of
males than traps baited with blank septa. Regres-
sion analysis of the full model data for the num-
ber of males caught by different aged lures was
best described by the equation y = 16.346e-1745 ; r2 =
0.94. Based on this equation, the half-life of the
fresh lure in terms of males captured was about
3.5 months.
Fresh to 7 Weeks Aged Lures: No differences in
male M. hirsutus captures were found among
traps baited with fresh lures and lures aged for 1,
2, 3, 4 and 7 weeks (df = 2,24; F = 0.39; P = 0.85).
The average number of males trapped (SE) was
found to be 16 (4.9), 31.8 (14.1), 17.4 (3.2), 20.6
(8.8), 25.4 (9.8), and 21. 4 (10.3) for fresh, 1, 2, 3,
4, and 7 weeks aged lures, respectively. The pher-
omone residue remaining in the lures used for
this experiment was not determined.

Timing of Male Flight Activity

Significantly different numbers of male M. hir-
sutus were captured at different times of the day
(df= 9,179; F = 5.91; P < 0.0001), with the highest
male captures of 6.5/trap (2.0 SE) occurring from
1600 h to 2400 h. No males were captured during
the sample period 0000 h to 0800 h, and only 1.0
male/trap (+0.5 SE) was captured from 0800 h to
1600 h. During the sample period 1600 hours to
2400 hours, hourly changes of traps were made.
This revealed that male activity was greatest be-


The sex pheromone developed by Zhang et al.
(2004a) was an effective attractant for adult male
M. hirsutus as shown previously by Zhang &
Amalin (2005). As suggested by its unique chem-
istry (Zhang et al. 2004a), the pheromone appears
to be specific to this mealybug based on observa-
tional data from males captured in traps, al-
though this was not specifically tested and sur-
veys to determine what other mealybug species
were in the area were not conducted. Differences
in trap design affected total trap capture of both
target and non-target insects, with Delta traps
being the most selective to male M. hirsutus.
There appeared to be a correlation between the
number of males captured and the area of sticky
trapping surface; i.e., male catch decreased as a
function of decreasing area of sticky surface
among trap designs.
There was no significant difference in the cap-
ture of non-target insects between baited and un-
baited traps, suggesting that they were the result
of insects randomly flying into the sticky surfaces.
As such, the degree of exposure of the sticky sur-
face influenced the number of non-target insects
captured to a greater extent than the area of
sticky surface. Sticky cards with fully exposed
sticky surfaces caught significantly greater num-
bers of non-target insects than either Delta or
Jackson traps. Delta traps, which had the largest
sticky surface area but the smallest trap opening,
caught the fewest non-target insects. Sticky cards
also had the most debris (e.g., leaves, sticks, sand)
stuck to them (author, unpublished data). Because
adult male M. hirsutus are small, large amounts
of debris and non-target insects greatly increase
the time to process a trap and the probability of
missing individual males. This is a particularly
important consideration for a monitoring program
if early detection of a new infestation is the goal.
Our findings support previous work on trap de-
signs for other mealybugs. Millar et al. (2002) re-
ported that Delta traps were more effective at
trapping male Planococcus ficus Signoret than
sticky cards. Although Zada et al. (2004) reported
that plate traps caught more male P. citri (Risso)
than Delta traps, they did find that larger traps
(900 cm2 of trapping surface) caught more males
than smaller traps (225 cm2 of trapping surface).
Vincent & Simard (1986) similarly found that
sticky traps accumulated large amounts of non-
target insects and debris. Adams et al. (1989) re-
ported that this was particularly problematic
when processing traps for very small insects such


60
lj


September 2007







Francis et al.: Trapping Methods for Pink Hibiscus Mealybug


as mealybug males, because non-target species
increased the time required to service traps. An
area of concern for pest monitoring programs is
attraction of natural enemies to baited traps;
however, Zhang & Amalin (2005) reported that
there was no statistical difference between sticky
traps baited with lures of the M. hirsutus sex
pheromone and blank control traps for mealybug
parasitoids caught. No parasitoids (A. kamali and
G. indica) were found in pheromone-baited traps
in this study. These findings suggest that large-
scale monitoring programs would not adversely
affect biological control efforts.
Characteristics of the lure dispenser can affect
the longevity of the pheromone release, the uni-
formity of the release rate over time, pheromone
stability, and the pheromone release rate (Sand-
ers 1989). Release rates also can be affected by en-
vironmental conditions such as temperature, rel-
ative humidity, and wind speed (Bierl-Leonhardt
et al. 1979; Walton et al. 2004; Zhang & Amalin
2005). Therefore, the effectiveness of a lure over
time needs to be determined in order to maintain
maximum trap capture, especially when monitor-
ing in areas with very low pest numbers.
Preliminary field experiments conducted in
2004 suggested that males captured were not sig-
nificantly different when traps were baited with
lures that were 0-4 weeks of age (author, unpub-
lished data). In the 2005 study, males captured
also were not significantly different between Delta
traps baited with fresh lures changed weekly and
traps with lures aged for one month. Similarly, the
Oct 2006 field experiments showed that the effec-
tiveness of fresh lures was not different from lures
aged 1, 2, 3, 4, and 7 weeks. However, males cap-
tured were significantly lower when traps were
baited with lures aged for 2 months; effectiveness
continued to decline numerically thereafter with
increasing lure age. Traps baited with lures aged
for 2 months caught about 32% of the number of
males that were captured in traps baited with
fresh lures; traps baited with lures aged for 8
months only captured about 4% of what was
caught in traps baited with fresh lures. In fact,
traps containing lures that were >2 months old did
not catch significantly more insects than traps
with blank septa, i.e., they often caught no insects
even though traps with fresh and 1-month old
lures indicated that males were present. Based on
these findings, we recommend that the lures be re-
placed every 1-2 months when using these traps
for early detection of new (low level) infestations.
Lures used in our field longevity studies ap-
peared to maintain their attractiveness to male M.
hirsutus for a shorter period of time relative to
those of equal loading dose used by Zhang & Ama-
lin (2005). They found the lures attracted males
for up to 21 weeks. A possible explanation for the
difference might be that these authors used loca-
tions in south Florida with high infestation levels


of M. hirsutus and the baited traps were placed
close to infested hibiscus plants. Typically 100-800
males were captured per trap per week. In con-
trast, in our experiments the baited traps were
placed in fields with no visible signs of infestation
in the immediate vicinity and only 10-80 males
were captured per trap per week. Thus, although
the residual pheromone in used lures in both stud-
ies was similar (=0.18 pg after 21 weeks in Zhang
& Amalin (2005) and =0.17 pg after 20 weeks in
our studies), it is not surprising they caught sig-
nificant numbers of males for a longer time period.
Also, in our lure longevity studies trap captures of
males with 2 and 4 month-old lures were signifi-
cantly different from zero at a = 0.1).
Studies have shown a similar duration of lure
activity in the field for other mealybug species.
For example, lures with 100 pg of the sex phero-
mone for P ficus remained attractive for >12
weeks in vineyards in California (Millar et al.
2002). Walton et al. (2004) also reported that lures
of the same loading dose as those in California
continued to attract P. ficus males for up to 10
weeks in South African vineyards. Zada et al.
(2004) reported that lures with 200 pg of P citri
sex pheromone could remain attractive for up to
16 weeks in Israel.
The greatest decrease in pheromone residue oc-
curred during the first 2 months with a more grad-
ual decrease after that period. A lower pheromone
release rate may explain why traps baited with
older lures caught fewer male M. hirsutus. Septa
loaded with a higher initial amount of pheromone
might prolong the period of high release rate and
increase the length of time that the lures are max-
imally attractive (i.e., as attractive as fresh lures
with 1 pg of pheromone). However, Zhang & Ama-
lin (2005) reported that high levels of pheromone
could have an inhibitory effect on males and sug-
gested using high release rates of the pheromone
for mating disruption. Additional studies to evalu-
ate the impact of pheromone release rate on males
captured and applications of the pheromone to dis-
rupt mating seem warranted.
Many mealybug species fly mainly around sun-
set while others are early-morning flyers (Aldrich
1996). Male M. hirsutus were active in the late af-
ternoon to early evening hours with almost no
flight activity at other times. This study was con-
ducted over 3 consecutive days with heavy show-
ers in the late afternoons to early evenings. Con-
sequently, male activity may have been disrupted
to some degree. However, because the crepuscular
diel activity pattern emerged despite the inclem-
ent weather at this time of day, we are confident of
the findings. Our own unpublished data suggest
that oriented flight by male mealybugs to phero-
mone plumes occurs at <100 m. For small insects
like M. hirsutus males, environmental factors like
rain and wind above, landscape features, and
landscape management such as mowing probably











have significant impacts on dispersal and there-
fore their capture in baited traps. These factors
may help explain the interactions we found
among treatments, sites, and sampling dates.
Further work is needed to better understand the
dynamics of male dispersal and the influence of
environmental factors and cultural practices. Un-
derstanding male flight activity could provide in-
formation critical to locating an infestation once
males have been detected in a trap.

ACKNOWLEDGMENTS

We gratefully acknowledge the following individuals
of the University of Florida/USDA-APHIS at USDA-
ARS SHRS, Miami, FL for field assistance: Divina Ama-
lin, Lizandra Nieves, Jason Bravo, Roger Coe, Luis
Bradshaw, Shawron Weingarten, and Juang-Horng
Chong. We thank Kathy Moulton and Carol Wyatt-
Evens at the USHRL, Fort Pierce, FL for field assis-
tance. Dr. Greg Hodges of FL-DPI, Gainesville helped
with initial mealybug identifications and commented on
pheromone specificity. Edward Jones, USDA-APHIS,
and Gilbert Queeley, Florida A&M University, provided
statistical assistance. USDA-APHIS and Florida A&M
University funded this research and we acknowledge
both institutions. Mention of trade names or commer-
cial products in this publication is solely for the purpose
of providing specific information and does not imply rec-
ommendation or endorsement by the U.S. Department
of Agriculture.

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Florida Entomologist 90(3)







Porter et al.: Host Specificity of Fire Ant Pathogen


HOST SPECIFICITY OF THE MICROSPORIDIAN PATHOGEN
VAIRIMORPHA INVICTAE AT FIVE FIELD SITES WITH INFECTED
SOLENOPSIS INVICTA FIRE ANT COLONIES IN NORTHERN ARGENTINA

SANFORD D. PORTER1, STEVEN M. VALLEYS TIMOTHY S. DAVIS2, JUAN A. BRIANO3,
LUIS A. CALCATERRA', DAVID H. OI1 AND R. AUSTIN JENKINS2
'USDA-ARS, Center for Medical, Agricultural and Veterinary Entomology,
1600 SW 23rd Drive, Gainesville, FL, USA

2Clemson University and Sandhill Research and Education Center, Columbia, SC, USA

3USDA-ARS, South American Biological Control Laboratory,
Bolivar 1559 (1686) Hurlingham, Buenos Aires, Argentina

ABSTRACT

The microsporidian pathogen Vairimorpha invictae is being evaluated for release in the
United States as a potential self-sustaining biological control agent for imported fire ants.
We examined the host range of this pathogen at 5 sites in northern Argentina where Sole-
nopsis invicta Buren fire ant colonies had high levels of infection (28-83%). At 3 sites near
the city of Corrientes, we examined 509 non-Solenopsis ants from 61 collections, 12 genera,
and 19 species with a polymerase chain reaction-based screening procedure. No V invictae
infections were detected in any of the samples. At 2 sites near San Javier in Santa Fe Prov-
ince, 350 km to the south, we screened another 438 non-Solenopsis ants from 44 baits, 4 gen-
era, and 4 species, again with no infections. At the Corrientes sites, we also examined 235
non-ant arthropods from 10 orders, 43 families, and more than 80 species. None were in-
fected with V invictae. The results of this study indicate that, in its native South American
range, V invictae is specific to Solenopsis fire ants.

Key Words: biocontrol, host range, Formicidae, Microsporidia

RESUME

El microsporidio pat6geno Vairimorpha invictae esta siendo evaluado en los Estados Unidos
como agent potential de control biol6gico clasico para las hormigas de fuego (hormigas bra-
vas). Examinamos el rango de hospederos de este pat6geno en cinco sitios del norte Argentino
en donde colonies de la hormiga brava Solenopsis invicta tenian altos niveles de infecci6n (28-
83%). En tres lugares cerca de Corrientes, examinamos 509 hormigas en 12 g6neros (excluido
Solenopsis), 19 species y 61 cebos usando la t6cnica de acci6n en cadena de la polimerasa
(PCR), pero ninguna estuvo infectada con V invictae. En dos sitios cercanos a San Javier, Pro-
vincia de Santa Fe, 350 km hacia el sur, examinamos otras 438 hormigas no Solenopsis en 4
g6neros, 4 species y 44 cebos, otra vez sin infecci6n. En los sitios de Corrientes, tambi6n exa-
minamos otros 235 artr6podos en 10 6rdenes, 43 families y mas de 80 species. Otra vez, nin-
guno fue infectado con V invictae. Los resultados de este studio indican que, en su area
native de Am6rica del Sur, V invictae es especifico de hormigas del g6nero Solenopsis.


Translation provided by the authors.


Solenopsis fire ants are 5-10 times more abun-
dant where they occur in the United States than
they are in their South American homelands (Por-
ter et al. 1992; Porter et al. 1997). Escape from
numerous natural enemies left behind in South
America (Williams et al. 2003) is a likely explana-
tion for this difference. Natural enemies include
almost 2 dozen species of phorid flies (Porter &
Pesquero 2001; Brown et al. 2003), a parasitic ant
(Calcaterra et al. 1999), several nematodes (Will-
iams et al. 2003), a virus (Valles et al. 2004b;
Valles et al. 2007), and two species of microsporid-
ian pathogens: Thelohania solenopsae Knell,


Allen & Hazard and Vairimorpha invictae Jouve-
naz & Ellis.
The first microsporidian, T solenopsae, was de-
scribed by Knell et al. (1977) from Brazil. Briano
et al. (1995a, 1995b, 1995c, 1996) published ex-
tensively on its distribution, abundance, and im-
pacts at sites in Argentina. This pathogen was
first detected in the United States by Williams et
al. (1998) who found it widespread and appar-
ently specific to imported fire ants.
The second microsporidian, V invictae, was de-
scribed by Jouvenaz & Ellis (1986). This pathogen
occurs in both monogyne and polygyne fire ant







Florida Entomologist 90(3)


colonies (Valles & Briano 2004) and appears to be
more common in the red fire ant S. invicta than in
the black fire ant S. richteri (Briano & Williams
2002). Its occurrence in the field is associated
with smaller colonies and declining fire ant popu-
lations (Briano 2005). Recent laboratory studies
have shown that this pathogen can be transmit-
ted among fire ant colonies by the transfer of lar-
vae, pupae, or even dead workers and that in-
fected colonies grew 85% slower than uninfected
control colonies (Oi et al. 2005).
Terrestrial microsporidian pathogens, as a
group, are normally limited to one host or a group
of related hosts (Solter & Maddox 1998; Briano et
al. 2002; Keeling & Slamovits 2004). Narrow host
ranges also apparently apply to Vairimorpha
(Solter et al. 2000); however, some species like
Vairimorpha necatrix appear to have relatively
broad physiological host ranges in the laboratory
when lepidopteran larvae challenged with high
spore doses. Nevertheless, actual ecological host
ranges in the field are likely less broad (Maddox
et al. 1981).
Vairimorpha invictae is a promising self-sus-
taining or classical biocontrol agent for fire ants
because of(1) documented impacts on fire ant col-
onies, (2) its prevalence on red fire ants, and (3) its
absence from the United States. Furthermore, V.
invictae appears to be specific to fire ants. Briano
et al. (2002) reported that none of the 9 genera of
non-Solenopsis fire ants collected at 167 baits
from >25 sites or from 50 colonies at >20 sites
were infected with V invictae. This absence of ob-
served infections occurred despite the fact that V.
invictae was found in 61 of 535 fire ant colonies
collected at 18 of 90 sites.
The objective of the current study was to fur-
ther examine the host range of V invictae by in-
vestigating its presence in arthropods and other
species of ants that co-occur in close proximity
with V invictae infected fire ant colonies in South
America.

MATERIALS AND METHODS

We found 3 populations of mostly polygyne
S. invicta infected with V invictae in Apr 2004, 5-20
km east of Corrientes city, Argentina. The first site
was a campground and boat launch area along the
Parana River (S 2722.601', W 5840.891'). The sec-
ond site was by a bridge over a small stream near a
family farm (S 27024.283', W 5841.127'). The third
site was along Route 12 at km marker 1034 just
outside the Corrientes Airport (S 2727.414', W
5845.743'). Two additional sites with V invictae
infected colonies were found near San Javier, Santa
Fe Province about 350 km south of Corrientes. The
first site was near Estancia Liriolay (S 3030.951W
6001.052). The second site was near an intersec-
tion with a clay road marked by white tires on the
roadside (S 3031.615, W 6000.730). The propor-


tion of polygyne colonies at these 2 sites was 33%,
based on PCR tests done for a recent study (Briano
et al. 2006).
In order to examine the host range of V invic-
tae at the five study sites, we used small vials (12
by 85 mm) baited with pieces of hotdog to collect
other species of co-occurring ants. Sugar water
baits were also used at the San Javier sites. At
each site, 20-50 baits were set out at 5-m intervals
in several transects. Some hand collecting was
also used for ants not attracted to baits. At the
Corrientes sites, we also used sweep nets to col-
lect co-occurring arthropods. A supplemental
sample of 79 non-ant Hymenoptera collected in
Jan 2005. Ants and other arthropods were stored
submerged in 95% ethanol to preserve the DNA
(King & Porter 2004).
Infected fire ant colonies were detected by
macerating about 30 workers with an electric tis-
sue grinder. A drop of aqueous extract was then
examined under a phase-contrast microscope for
the presence of spores (Briano 2005). At baits, we
macerated 15-20 fire ant workers for microscopic
examination if that many were available. These
sampling rates should have been sufficient on av-
erage to detect V invictae in samples with worker
infection rates as low as 3-7%.
PCR was used to detect V invictae in non-Sole-
nopsis ants and other kinds of arthropods (Valles
et al. 2004a). Briefly, PCR was carried out with
primer pairs specific for the 16S rRNA gene of
V invictae (p90, 5'CACGAAGGAGGATAACCAC-
GGT and p93, CGCAATCAGTCTGTGAATCTCT-
TCA) by using the hot start method in a PTC 100
thermal cycler (MJ Research, Waltham, MA) un-
der the following optimized temperature regime: 1
cycle at 94C for 2 min, then 35 cycles at 94C for
15 s, 55C for 15 s, and 68C for 45 s, followed by a
final elongation step of 5 min at 68C. The reaction
was conducted in a 25 pL volume containing 2 mM
MgCl2, 200 pM dNTP mix, 1 unit of Platinum Taq
DNA polymerase (Invitrogen, Carlsbad, CA), 0.4
pM of each primer, and 0.5 pL of the genomic DNA
preparation (10 to 50 ng). PCR products were sep-
arated on 1.2% agarose gel and visualized by
ethidium bromide staining. For all experiments,
positive and negative controls were run alongside
treatments. In situations in which verification of V
invictae positive non-Solenopsis insects was re-
quired, the amplicon was cloned and sequenced.
Amplicons were gel-purified, ligated into the pCR-
4 vector and transformed into TOP10 competent
cells (Invitrogen, Carlesbad, CA). Insert-positive
clones were identified and DNA sequences of in-
serts were elucidated by the University of Florida,
Interdisciplinary Center for Biotechnology Re-
search (Gainesville, FL).
At the Corrientes sites, samples of ants from
several baits were pooled, generally by genus and
site, into 18 samples. Most pooled samples con-
sisted of ants from 2-5 baits and contained 20-50 in-


September 2007







Porter et al.: Host Specificity of Fire Ant Pathogen


dividuals depending on their size and availability.
The arthropod samples were similarly pooled,
mostly by order or family, into 33 samples. At the
San Javier sites, ant samples from non-fire ant gen-
era were similarly pooled into 16 samples with each
sample containing 20-40 workers from 2-3 baits.
PCR was also conducted to verify that the test
could detect the V invictae target gene in other ant
genera (Linepithema, Dorymyrmex, Crematogaster,
Paratrechina). This experiment was done by spik-
ing 30-70 non-Solenopsis workers with 5 Solenopsis
workers from a V invictae-positive colony prior to
maceration for DNA extraction. Similar tests were
not done with non-ant arthropods, because DNA
extraction techniques are quite reliable, sample
sizes were limited, and it would have considerably
increased the number of samples needing testing.
We also used PCR to test 3 samples of infected fire
ant workers from San Javier that were initially de-
tected by microscopic inspection. Based on the ex-
perience from a previous study conducted in Argen-
tina (Valles & Briano 2004), V invictae is always de-
tected with PCR tests if it was previously detected
by microscopic examination.

RESULTS

As expected (Valles & Briano 2004), the PCR
technique identified V invictae in fire ant workers
in which spores had been detected by light micro-
scopy (n = 3). Similarly the PCR technique de-
tected V invictae in samples of Paratrechina, Cre-
matogaster, and Linepithema ants spiked with 5
infected fire ants, thereby showing that ants in
these genera do not appear to have chemistry that
would mask detection of V invictae DNA. Vairi-
morpha invictae was not detected in the first sam-
ple with Dorymyrmex ants, perhaps because none
of the 5 fire ant workers used to spike the sample


were infected (Briano & Williams 2002) or per-
haps because of a faulty extraction. A retest with
these ants, however, produced a positive result.
Infected fire ants were detected in both fire ant
mounds and among foraging workers collected at
baits (Table 1). At the Corrientes sites, the percent-
age of positive baits was lower than the percentage
of positive mounds when we included baits with <4
workers. However, when we only compared baits
with >5 workers, the percentages were similar.
This comparison is more appropriate because only
10-60% of workers in a mound are infected (Briano
& Williams 2002) and mound samples always con-
tained many workers (15-30) while bait samples,
with only a few workers, may have produced false
negatives relative to their colony of origin. How-
ever, at the clay road site near San Javier (Table 1),
the percent of infected baits was still about half
that of the infected mounds.
All of the non-ant arthropod samples from the
Corrientes area (Table 2) were negative for V in-
victae (Table 1). One sample from the camp-
ground area containing 2 solitary bees and 2
sphecid wasps produced a faint positive band, but
sequencing confirmed that the amplicon was not
from the V invictae 16S rRNA gene.
All of at least 19 species of ants from 12 non-So-
lenopsis genera including 947 individuals from 107
samples collected near Corrientes and San Javier
(Table 3) were also negative for V invictae (Table 1).
When we resampled ant colonies at the 3 Cor-
rientes sites 20 months after the initial sampling
(Dec 2006) and tested them for V invictae infec-
tions, we found that V invictae had disappeared
from the bridge site (0/8) and only one weakly pos-
itive sample was found each at the campground
and airport sites (1/12; 1/19), a considerable de-
cline from what we found in 2004 (Table 1). This
drop in frequency of V invictae infections was not


TABLE 1. PERCENT OF FIRE ANTS, OTHER KINDS OF ANTS, AND OTHER INSECTS INFECTED WITH THE MICROSPORIDIAN
PATHOGEN VAIRIMORPHA INVICTAE AT 5 SITES IN ARGENTINA. NUMBERS IN PARENTHESES SHOW POSITIVES
DIVIDED BY TOTAL SAMPLE.

Sites

Taxon Campground' Airport' Bridge' Liriolay2 Clay Rd.2

Fire Ants
Mounds 69% (9/13) 28% (4/14) 83% (10/12) 43% (15/35) 62% (16/26)
Baits (all) 45% (5/11) 17% (2/12) 50% (7/14) -33% (6/18)
(>5 ants) 56% (5/9) 29% (2/7) 64% (7/11) -33% (6/18)
Other Ants
Baits3 0% (0/31) 0% (0/23) 0% (0/9) 0% (0/29) 0% (0/15)
Other Arthropods
Individuals 0% (0/184) 0% (0/39) 0% (0/12) -

'Near Corrientes, Argentina.
Near San Javier, Santa Fe Province, Argentina.
Seven Acromyrmex samples were from nests rather than baits.







Florida Entomologist 90(3)


September 2007


TABLE 2. NON-ANT ARTHROPODS COLLECTED AT CORRIENTES SITES, APR 2004. NUMBER OF MORPHOSPECIES, FAMI-
LIES, AND INDIVIDUALS GROUPED BY ORDER AND SITE WHICH WERE TESTED BY PCR FOR THE PRESENCE OF
THE MICROSPORIDIAN PATHOGEN VAIRIMORPHA INVICTAE.

Species-Families (individuals) by site

Order Campground Airport Bridge

Araneae 8-? (8) 4-? (4) 4-? (4)
Odonata' 1-1(1) 1-1(1)
Orthoptera2 5-3 (17) 1-1(3) 2-2 (2)
Homoptera3 9-3 (10) 3-1(12)
Hemiptera4 8-5 (18) 6-3 (7) 2-2 (2)
Psocoptera 1-1 (1) -
Coleoptera5 12-5(16) 5-3 (6)
Diptera6 15->7 (23) 3-3 (3) 2-2 (2)
Lepidoptera7 5-4 (5) 2-2 (2)
Hymenoptera8 7-4 (7) 1-1 (1) 1-1 (1)
supplemental sample9 58-15 (79)

Estimated Totals 121-44 (184) 26-16 (39) 12-9 (12)

Identified Families: 'Libellulidae; 'Acrididae, Tettigoniidae, Gryllidae, Tetrigidae; 'Cicadellidae, Fulgoridae; 'Myridae, Lyga-
eidae, Coreidae, Pentatomidae, Berytidae; 5Curculionidae, Buprestidae, Chrysomelidae, Scolytidae, Dermestidae; 6Tephritidae,
Bombyliidae, Culicidae, Dolichopodidae, Tipulidae, Bibionidae, Rhagionidae, Sarcophagidae; 7Lycaenidae, Nymphalidae, Pieridae,
Sphingidae, Geometridae; 8Chrysididae, Halictidae, Sphecidae, Cynipidae, Apidae, Ichneumondidae, Braconidae, Diprionidae, Be-
thylidae, Vespidae, Anthophoridae; 'Collected 26 Jan 2005.


unexpected because similar drops in infection junction with lower nest densities (Briano 2005;
rates have been reported previously, often in con- Briano et al. 2006).


TABLE 3. THE NUMBER OF ANT SPECIES, BAITS, AND INDIVIDUALS (EXCLUDING FIRE ANTS) GROUPED BY GENUS AND SITE
WHICH WERE TESTED BY PCR FOR THE PRESENCE OF VAIRIMORPHA INVICTAE.

Species-Baits (individuals) by site
Subfamily
Genus Campground" Airporta Bridgea Liriolayb Clay Rd.b

Ponerinae
Ectatomma 1-2 (2)
Pachycondyla 1-1 (1) -
Myrmicinae
Acromyrmex -1-3 (15)c 1-4 (48)c
Crematogaster 4-4 (21) 1-8 (90) 1-2 (22)
Pheidole 4-5 (32)
Wasmannia 1-4 (42)
Zacryptocerus 1-1 (1)
Dolichoderinae
Dorymyrmex 1-3 (31) 1-4 (24) 1-5 (44)
Linepithema 1-11 (101)
Formicinae
Camponotus 1-1 (3) 1-2 (7) -1-1 (7)
Brachymyrmex 1-1 (30) 1-1 (20) -
Paratrechina 1-1 (1) 1-9 (66) -1-19 (191) 1-15 (148)

Total 16-31(234) 5-23 (214) 3-9 (61) 4-29 (290) 1-15 (148)

"Near Corrientes, Argentina.
bNear San Javier, Santa Fe Province, Argentina.
'Collected from nests rather than baits.







Porter et al.: Host Specificity of Fire Ant Pathogen


DISCUSSION

Overall, these data indicate that V invictae is
specific to Solenopsis fire ants because this patho-
gen was not found in any of the sympatric ants or
co-occurring arthropods. Briano et al. (2002)
failed to find V invictae in 217 collections of non-
Solenopsis ants from 9 genera in Argentina; al-
though, it appears that only 20% of the sites may
have had V invictae present in co-occurring fire
ants. While V invictae has not been found in other
ant genera, it has been reported from 3 species of
Solenopsis fire ants (S. invicta, S. richteri, S. mac-
donaghi; Briano et al. 2002), all in the saevissima
complex of South American fire ants (Trager
1991). It likely occurs in other South American
Solenopsis species as well. The known geographic
range of this pathogen extends from Mato Grosso,
Brazil (Jouvenaz & Ellis 1986) south through Par-
aguay and part of Bolivia (Briano et al. 2006) to
Buenos Aires Province, Argentina (Briano et al.
2002). Extensive searches in the United States for
T solenopsae and other pathogens (Jouvenaz et
al. 1977; Williams et al. 1998) have failed to detect
V invictae infections.
We currently do not know whether V invictae
has the ability to infect native Solenopsis fire ants
in the United States (e.g.; S. geminata, S. xyloni)
which belong to a different complex of fire ants
(geminata) or the more distantly related group of
very small Solenopsis thief ants. Laboratory tests
in quarantine are currently being conducted by
D. H. Oi in an effort to answer the question of
whether our native North American fire ants and
other species of ants are susceptible to V invictae
infections.
Low levels of infection in native fire ants would
probably be tolerable (Porter 2000) because the
native fire ants can be pests in their own right, es-
pecially the tropical fire ant S. geminata which
has become a world-wide exotic pest. However, in-
fections in other ant genera or in other families of
insects would be a complicating factor which
would likely require further tests and impact
evaluations across a range of ant genera or ar-
thropod families.

ACKNOWLEDGMENTS

Chuck Strong is thanked for conducting the PCR
tests and Euripides Mena is thanked for doing the visual
microscope scans. Laura Varone and Alicia Delgado
(SABCL) helped with field collections in Corrientes and
Juan P. Livore, a former SABCL intern, helped with field
collections at the San Javier sites. Roberto Pereira and
Yoshifumi Hashimoto are thanked for helpful comments.

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Florida Entomologist 90(3)







Xu et al.: New Species of Dryinus from China


TWO NEW SPECIES OF DRYINUS LATREILLE
(HYMENOPTERA: DRYINIDAE) FROM CHINA

ZAIFU XU', MASSIMO OLMI2 AND JUNHUA HE3
'College of Natural Resources and Environment, South China Agricultural University,
Guangzhou, Guangdong 510642, P.R. China

2Department of Plant Protection, University of Tuscia, 1-01100 Viterbo, Italy

'Institute of Insect Science, Zhejiang University, Hangzhou, Zhejiang 310029, P.R. China

ABSTRACT

Two new species, Dryinus chenae and Dryinus expolitus, are described from Jianfengling
National Nature Reserve (Hainan Province, China). Dryinus chenae can be recognized from
the related Oriental species D. lini Olmi and D. lianus Xu, Olmi & He by the different OPL/
TL ratio (OPL as long as TL in D. chenae, much shorter than TL in D. lini, much longer than
TL in D. lianus). Dryinus expolitus can be recognized from the related Oriental species D.
choui Olmi by the different sculpture (scutum granulated, with a few small areolae near the
posterior margin in D. expolitus; almost completely reticulate rugose, with only the anterior
surface of the median region granulated in D. choui) and color (pronotum black in D. expol-
itus, broadly testaceous in D. choui). Keys are provided for the determinations of the above
species with illustrations of female chelae.

Key Words: Taxonomy, Hymenoptera, Dryinidae, Dryinus, new species, China

RESUME

Se described las nuevas species Dryinus chenae y Dryinus expolitus de la Reserva Natural
y Nacional Jianfengling (Provincia de Hainan, China). Dryinus chenae puede ser diferen-
ciada de las species orientales relacionadas, D. lini Olmi y D. lianus Xu, Olmi & He, por di-
ferencias en la relaci6n OPL/TL (OPL tan largo como TL en D. chenae, mas corto que TL en
D. lini, y mas largo que TL en D. lianus). Dryinus expolitus puede ser diferenciado de D.
choui Olmi, que es la especie oriental mas relacionada, por diferencias en la escultura (en D.
expolitus escudo granulado, con pocas y pequeias areolas cercanas al margen posterior,
mientras que en D. choui el escudo es casi completamente reticulado rugoso, solo con la su-
perficie anterior de la region media granulada), y la coloraci6n (pronoto negro en D. expoli-
tus, y mayormente testaceo en D. choui). Se proven claves para la determinaci6n de las
species y ilustraciones de las quelas de sus hembras.


Translation provided by the authors.


Dryinidae (Hymenoptera: Chrysidoidea) are
parasitoids of Hemiptera Auchenorrhyncha (Gug-
lielmino & Olmi 1997, 2006). Dryinus Latreille,
1804, is a genus present in all zoogeographical
regions. About 242 species have been described
from all continents (Olmi 1999) and the genus
was revised by Olmi (1984, 1993).
The species of Dryinus inhabiting China were
studied in the last 20 years mainly by Xu & He
(1994a, 1994b, 1998), Yang (1994), Yang & Ma
(1994) and Xu et al. (2006). A recent book on Chi-
nese Dryinids was published by He & Xu (2002).
In 2006 we have examined additional specimens
of Dryinus from P.R. China, Hainan Province, and
have found 2 new species described herein.

MATERIALS AND METHODS

The descriptions follow the terminology used
by He & Xu (2002) and Olmi (1984, 1994, 1999).


The measurements reported are relative, except
for the total length (head to abdominal tip, with-
out the antennae), which is expressed in millime-
tres. In the descriptions, POL is the distance be-
tween the inner edges of the two lateral ocelli; OL
is the distance between the inner edges of a lat-
eral ocellus and the median ocellus; OOL is the
distance from the outer edge of a lateral ocellus to
the compound eye; OPL is the distance from the
posterior edge of a lateral ocellus to the occipital
carina; TL is the distance from the posterior edge
of an eye to the occipital carina.
All material studied in this paper is deposited
in the Hymenoptera collection of Zhejiang Uni-
versity (ZJUC), Hangzhou, Zhejiang, PR. China.

Dryinus chenae sp. nov. (Fig. 1)

Description. Holotype female. Fully winged.
Length 5.12 mm. Head black, with mandibles and







Florida Entomologist 90(3)


clypeus ferruginous; genae partly ferruginous.
Antennae with segments 1-8 testaceous; segments
9-10 missing in the holotype. Mesosoma black,
with posterior collar of pronotum testaceous.
Gaster brown. Legs testaceous, with coxae black.
Antennae clavate; antennal segments 1-8 in the
following proportions: 11:7:33:18:14:12:10:8 (last
2 segments missing in the holotype). Head dull,
hairless, slightly convex, with dorsal surface re-
ticulate rugose; ventral surface of the head with
medial area smooth and lateral areas sculptured
by many longitudinal keels; frontal line complete;
occipital carina complete, laterally not reaching
the eyes; POL = 8; OL = 2.5; OOL = 8; OPL = 2; TL
= 2; greatest diameter of posterior ocelli: 3. Prono-
tum shiny, smooth, without sculpture, except
many longitudinal striae around and on the sides
of the disc; pronotum with disc humped and pos-
terior collar short; pronotal tubercles not reach-
ing the tegulae. Scutum, scutellum and metano-
tum dull, reticulate rugose. Notauli almost com-
plete, almost reaching the posterior margin of the
scutum (but slightly visible near the posterior
margin of the scutum among the areolae). Me-
sopleura dull, reticulate rugose. Metapleura dull,
strongly transversely striate. Propodeum hair-


less, with dorsal surface reticulate rugose; poste-
rior surface reticulate rugose, with 2 complete
longitudinal keels; dorsal surface of propodeum
about as long as posterior surface. Forewing with
2 dark transverse bands; distal part of stigmal
vein about twice as long as proximal part (19:9).
Fore tarsal segments in the following proportions:
24:2.5:5.5:17:25. Enlarged claw spatulate (Fig. 1)
with a strong subdistal tooth and 1 row of 15
lamellae. Segment 5 of front tarsus (Fig. 1) with 3
rows of 6 (longer) + approximately 48 lamellae,
without interruption to the distal apex. Tibial
spurs 1, 1, 2.
Male. Unknown.
Holotype: Female, PR. CHINA, Hainan Prov.,
Jianfengling National Nature Reserve, 12-
15.vii.2006, T. F. Chen, No. 200700783 (ZJUC).
Etymology: The species is named after the col-
lector Miss T. F. Chen.
Remarks. Dryinus chenae is similar to D. lini
Olmi, 1993 (known from Taiwan and Guangxi),
and D. lianus Xu et al. (2006) (known from Yun-
nan). The above species can be recognized by the
different OPL /TL ratio (OPL as long as TL in
D. chenae, much shorter than TL in D. lini, much
longer than TL in D. lianus), as follows:


1 Head with OPL as long as TL .


. chenae sp. nov.


1' Head with OPL much shorter or much longer than TL ........................................... 2
2 Head with OPL much shorter than TL; dorsal surface of the propodeum reticulate rugose,
without parallel longitudinal keels ................... .............................. lini Olmi


2' Head with OPL much longer than TL; dorsal surface of the propodeum sculptured
by numerous parallel longitudinal keels ................................


Dryinus expolitus sp. nov. (Fig. 2)

Description. Holotype female. Fully winged.
Length 6.25 mm. Head black, with mandibles
testaceous; clypeus black, with anterior margin
whitish. Antennae brown, with segments 5-10
testaceous and ventral side of segment 1 whitish.
Mesosoma black. Gaster brown. Legs black, with
articulations, fore trochanters and distal third of
fore coxae testaceous. Antennae clavate; antenna
segments in the following proportions:
13:7:48:18:19:10:10:8:6:9. Head dull, hairless, ex-
cavated, with dorsal and ventral surface granu-
lated; frontal line incomplete, not present in the
anterior third of the frons; occipital carina com-
plete, laterally not reaching the eyes; ocellar tri-
angle with a few irregular keels joining the ocelli;
POL = 3; OL = 4; OOL = 11; OPL = 1; TL = 2;
greatest diameter of posterior ocelli: 3. Pronotum
shiny, smooth, with disc granulated; sides of
pronotum without sculpture and with many lon-
gitudinal striae around the disc; pronotum with
disc humped and posterior collar short; pronotal
tubercles not reaching the tegulae. Scutum dull,
smooth, granulated, with a few areolae near the


. lianus Xu, Olmi & He


posterior margin. Notauli complete, posteriorly
separated; minimum distance between the no-
tauli longer than POL (6:3). Scutellum dull, gran-
ulated. Metanotum with anterior half reticulate
rugose and posterior half smooth. Mesopleura
dull, reticulate rugose. Metapleura dull, strongly
transversely striate. Propodeum hairless, with
dorsal surface reticulate rugose; posterior surface
reticulate rugose, without longitudinal keels; dor-
sal surface of propodeum slightly longer than pos-
terior surface (13:8). Forewing with 3 dark trans-
verse bands; distal part of stigmal vein much
longer than proximal part (21:9). Fore tarsal seg-
ments in the following proportions: 24:4:9:28:43.
Enlarged claw not spatulate (Fig. 2) with a strong
subdistal tooth and 1 row of 14 lamellae. Segment
5 of front tarsus (Fig. 2) with 2 rows of approxi-
mately 36 lamellae; distal apex with a group of at
least 24 lamellae. Tibial spurs 1, 1, 2.
Male. Unknown.
Holotype: Female, P. R. CHINA, Hainan Prov.,
Jianfengling National Nature Reserve, 12-
15.vii.2006, T F. Chen, No. 200700723 (ZJUC).
Etymology: The specific name derives from the
Latin adjective expolitus (smooth).


September 2007







Xu et al.: New Species of Dryinus from China


Figs. 1-2. Chelae of holotypes. 1. Dryinus chenae (scale bar = 0.10 mm). 2. Dryinus expolitus (scale bar = 0.25 mm).


Remarks. Dryinus expolitus is similar to
D. choui Olmi, 1993 (known from Taiwan).The
above species can be recognized by the different
sculpture (scutum granulated, with a few small
areolae near the posterior margin inD. expolitus;


almost completely reticulate rugose, with only
the anterior surface of the median region granu-
lated in D. choui) and color (pronotum black in D.
expolitus; broadly testaceous in D. choui), as fol-
lows:


1 Pronotum broadly testaceous, with brown areas; scutum almost completely reticulate rugose,
with only the anterior surface of the median region granulated ..........................choui Olmi
1' Pronotum black; scutum granulated, with a few small areolae near the posterior
m argin .................................................................. expolitus sp. nov.


ACKNOWLEDGMENTS

We are grateful to Prof. Ducheng Cai (from South
China Tropical Agricultural University), Miss Tianfei
Chen, Miss Liqiong Weng, Mr. Jingxian Liu, Mr. Jiem-
ing Liu and Mr. Wenyong Zhang (from South China Ag-
ricultural University) for valuable assistance with the
collections of specimens.


REFERENCES CITED

GUGLIELMINO, A., AND M. OLMI. 1997. A host-parasite
catalog of world Dryinidae (Hymenoptera: Chrysi-
doidea). Contrib. Entomol. Internat. 2: 165-298.
GUGLIELMINO, A., AND M. OLMI. 2006. A host-parasite
catalog of world Dryinidae (Hymenoptera: Chrysi-
doidea): first supplement. Zootaxa 1139: 35-62.







456 Florida Entc



HE, J., AND Z. Xu. 2002. Hymenoptera Dryinidae.
Fauna Sinica 29. Science Press, Beijing: xii + 464 pp.
OLMI, M. 1984. A Revision of the Dryinidae (Hymenop-
tera). Mem. American Ent. Inst. 37: xii + 1913 pp.
OLMI, M. 1993. A new generic classification for Thauma-
todryininae, Dryininae and Gonatopodinae, with de-
scriptions of new species (Hymenoptera Dryinidae).
Boll. Zool. Agr. Bachic. (Ser. ii) 25: 57-89.
OLMI, M. 1994. The Dryinidae and Embolemidae (Hy-
menoptera: Chrysidoidea) of Fennoscandia and Den-
mark. Fauna Entomologica Scandinavica 30. Brill,
Leiden. 100 pp.
OLMI, M. 1999. Hymenoptera Dryinidae-Embolemi-
dae. Fauna d'Italia 37. Edizioni Calderini, Bologna:
xvi + 425 pp.
XU, Z., AND J. HE. 1994a. Two new species of Dryinidae
from Mt. Gutianshan, Zhejiang (Hymenoptera:
Chrysidoidea). Wuyi Sci. J. 11: 132-135.
XU, Z., AND J. HE. 1994b. Hymenoptera: Dryinidae, pp.
266-269 In T Zhu [ed.], Insects and Macrofungi of


Im


ologist 90(3) September 2007



Gutianshan, Zhejiang. Zhejiang Sci. & Tech. Pub-
lishing House, Hangzhou. 327 pp.
XU, Z., AND J. HE. 1998. The descriptions of two new
species of the genus Dryinus from China (Hy-
menoptera: Dryinidae, Dryininae). Acta Zootax. Sin-
ica 23 (3): 309-312.
XU, Z., M. OLMI AND, J. HE. 2006. A contribution to the
knowledge of Dryininae and Gonatopodinae of
China (Hymenoptera: Dryinidae). Oriental Insects
40: 93-98.
YANG, C. 1994. Descriptions of Dryinus lycormae sp. n.
and its biological notes (Hymenoptera: Dryinidae),
pp. 37-42 In X. Shen and Z. Shi [eds.], Fauna and
Taxonomy of Insects in Henan, Vol. I (1994). Chinese
Press Agric. Sci. & Tech., Beijing. 351 pp.
YANG, C., AND Z. MA. 1994. Hymenoptera: Dryinidae,
pp. 263-265 In T Zhu [ed.], Insects and Macrofungi
of Gutianshan, Zhejiang. Zhejiang Sci. & Tech. Pub-
lishing House, Hangzhou. 327 pp.







MacGown et al.: Brachymyrmex patagonicus, an Emerging Pest


BRACHYMYRMEX PATAGONICUS (HYMENOPTERA: FORMICIDAE),
AN EMERGING PEST SPECIES IN THE SOUTHEASTERN UNITED STATES

JOE A. MACGOWN1, JOVONN G. HILL1 AND MARK A. DEYRUP2
'Mississippi Entomological Museum, Mississippi State University, Box 9775, MS 39762-9775

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

ABSTRACT

Brachymyrmexpatagonicus Mayr is a recently introduced species that is well established in
the Gulf Coast region of the United States. This species is abundant in Georgia, Florida, Al-
abama, Mississippi, and Louisiana and has spread into other states. It has become a nui-
sance pest with occasional large infestations occurring in homes, hospitals, and other
businesses. Brief descriptions and illustrations of all castes, biological and economic impor-
tance, and known distribution in the United States are given.

Key Words: exotic insects, ants, distribution, dark rover ant

RESUME

Brachymyrmex patagonicus Mayr es una especie introducida recientemente que esta bien
establecida en la region de la Costa del Golfo de los Estados Unidos. Esta especie es abun-
dante en los estados de Georgia, Florida, Alabama, Mississippi y Louisiana y se ha esparcido
a otros estados. Esta hormiga ha convertido en una plaga fastidiosa con infestaciones oca-
sionales grandes en hogares, hospitals y otros negocios. Se provee una descripci6n breve
con ilustraciones de todas las castas, su importancia biol6gica y econ6mica, y la distribuci6n
conocida en los Estados Unidos.


Members of the genus Brachymyrmex Mayr
(Hymenoptera: Formicidae: Formicinae) are
small, soft-bodied ants that are only 2.5 mm or
less in length (workers of most species are
smaller), range in color from pale yellow to black-
ish-brown, and possess distinctive nine-seg-
mented antennae. Most species nest in soil or in
rotting wood, although a few are arboreal. The ge-
nus includes 38 species worldwide, with most oc-
curring in the Neotropical region (Bolton 1995).
However, due to their minute size, there are likely
many more undescribed species. Currently, at
least eight distinct species, four of which appear
to be undescribed (from the southeastern United
States), are known to occur in the United States.
This genus needs revision because most species
descriptions are brief, and type specimens of
many species are lost or are poorly preserved,
some being shriveled. Although the genus was re-
vised by Santschi (1923), the morphological char-
acters he used to define species were ill-defined
and, in many cases, were useless for differentiat-
ing species. For these reasons, species epithets
and identifications in the genus are suspect (Dey-
rup 2003). Compounding this problem, there are
several known undescribed species, and undoubt-
edly more to be discovered. However, revisions of
the genus (Quiran 2005; Quiran et al. 2004) have
resulted in six species being redescribed, includ-
ing the type species, B. patagonicus Mayr, for
which the original types are lost.


Brachymyrmex patagonicus is native to Argen-
tina (Quiran et al. 2004). This species was first re-
ported from the United States as Brachymyrmex
musculus Forel from St. Tammany Parish, Louisi-
ana in 1978 from a single colony collected in 1976
from sawdust beneath a recently cut live oak tree
(Wheeler & Wheeler 1978). Wheeler & Wheeler
(1978) note that they identified the species by us-
ing Santschi's key to species (1923). They specu-
lated that it could have been introduced into the
United States through nearby New Orleans,
which is a reasonable entry point for tropical spe-
cies, although other localities, such as Mobile, Al-
abama, or Pensacola, Florida, are just as likely. In
his unpublished dissertation Naves (1976) also
reported the presence of this species, which he re-
ferred to as B. patagonicus from the Southeast. In
his monograph of Pheidole of Florida, Naves
(1985) again mentioned B. patagonicus as occur-
ring in the southeastern United States. Although
Naves (1985), whose research was conducted in
Florida, implied that this species occurred in
Florida, subsequent faunal lists of ants for the
state failed to mention B. patagonicus (Deyrup
2003; Deyrup et al. 1989; Deyrup et al. 2000). In
2000 this species, then referred to asB. musculus,
was reported from Florida (Deyrup et al. 2000).
The name B. patagonicus again reappeared in the
literature from collections in Louisiana when
Hooper-Buii et al. (2000) mentioned it as an un-
welcome house pest. A subsequent thesis on ants







Florida Entomologist 90(3)


of Louisiana (Dash 2005) did not mention B. pat-
agonicus, but referred to B. obscurior Forel and
B. musculus, occurring in 3 parishes.
The first goal was to determine whether the
southeastern species named B. musculus and
B. patagonicus were actually 1 species. Specimens
identified as B. musculus and B. patagonicus, and
on which the Louisiana records are based, were
borrowed from the Louisiana State University Ar-
thropod Collection (LSUC) and were compared
with specimens identified as B. musculus from
Georgia, Florida, Alabama, Mississippi, Louisi-
ana, and Texas. All of these specimens appeared to
be the same species. Additionally, specimens from
LSUC identified as B. obscurior also appeared to
be this species, not B. obscurior. The next goal was
to assign the proper name to this species. The orig-
inal descriptions of both B. musculus as a race of
B. tristis Mayr by Forel (1899) and B. patagonicus
by Mayr (1868) are brief, and of little use for iden-
tification. However, the redescription ofB. patag-
onicus (Quiran et al. 2004), which includes all
castes, is much more detailed, and appears to
closely match our southeastern species. To verify
this tentative identification, representatives of all
castes were sent to Estela Quiran, who identified
them as B. patagonicus, the name that we use in
this publication. We suggest, therefore, that all
references to B. musculus in North America be re-
ferred to as B. patagonicus. This has no bearing on
the taxonomic status ofB. musculus, originally de-
scribed from Costa Rica (Bolton 1995).
Although B. patagonicus recently was re-
described from Argentinian specimens (Quiran et
al. 2004), a few errors in the publication concern-
ing measurements give an impression that the
overall lengths of workers and queens are less
than they actually are. Additionally, the castes
have not been illustrated except for the heads of
the male and worker, mesosoma of worker, and
some male genitalic structures. Therefore, a brief
diagnosis and illustration of each caste is given
here to aid in identifying this species in the
United States. A common name of "dark rover
ant" is proposed for the species.

MATERIALS AND METHODS

Ants for this study were collected as part of
larger surveys of Formicidae in Florida (Deyrup
2003), Alabama (MacGown & Forster 2005), and
Mississippi (MacGown et al. 2005; MacGown &
Brown 2006), with additional collecting trips
made to southern Georgia, Arkansas, Louisiana,
and Texas. Specimens were collected in 90% etha-
nol, and representatives were pinned and labeled.
Vouchers are deposited in the Archbold Biological
Station Collection (ABSC) and the Mississippi
Entomological Museum (MEM).
The gaster of B. patagonicus is soft and often
shrivels when pinned. Consequently, mesosomal


lengths (measured from the anterior edge of the
pronotum to the posterior edge of the metapleural
gland) are given in lieu of overall lengths. Head
width was measured in full frontal view at the
widest point on the head including the eyes, and
head length was measured in full frontal view
from the anterior edge of the clypeus to the poste-
rior border of head. Eye length refers to the long-
est measurement of the compound eye. Measure-
ments were made with a micrometer mounted in
a 10x eyepiece on a Leica MZ16 stereomicroscope
at 50x (for females) and 100x (for males and work-
ers). Drawings were made with a drawing tube
mounted on a Leica MZ16 stereomicroscope.

RESULTS

Diagnosis of the Worker (Figs. 1 and 4)

Size minute, mesosomal length 0.43-0.51 mm
(n = 10). Head and mesosoma medium brown to
blackish-brown, gaster usually blackish-brown,
often darker than head and mesosoma, tarsi and
mandibles pale, and antennae brownish-yellow.
Head slightly longer than wide, covered with fine
pubescence, and with a few longer erect hairs; an-
tennal scapes surpassing occipital border of head
by 1/5 their total length; eyes relatively large,
about as long as length of malar space and placed
at approximately the middle third of side of head;
3 tiny, barely visible ocelli present. Promesono-
tum with 3-9 (usually 4-6) stout, erect hairs
present dorsally, with fine pubescence that does
not obscure the shiny sheen of integument.
Gaster with scattered, long, erect hairs, especially
along the edges of the tergites, and with sparse,
decumbent hairs, separated by about 1/3 to 2/3
their length.

Diagnosis of Female (Figs. 3 and 6)

Mesosomal length 1.24-1.42 mm (n = 10). Con-
colorous light brown. Head wider than long, with
abundant, fine pubescence, and with long erect
hairs present; large compound eyes located at
middle of side of head; 3 large ocelli present; fron-
tal lobes well developed; scapes surpassing occip-
ital border by 1/4 their length. Mesosoma with
moderately dense, fine pubescence, and 30-40
long erect hairs (about 3-4 times length of fine pu-
bescence); anepisternum and katepisternum sep-
arated by a distinct suture, with erect hairs
present. Forewing with pterostigma; hind wing
with 7 hammuli. Gaster with moderately dense,
fine pubescence, and erect hairs along apical
edges of sternites and tergites.

Diagnosis of Male (Figs. 2 and 5)

Mesosomal length 0.8 mm (n = 2). Head dark
brown to blackish-brown, rest of body, including


September 2007






MacGown et al.: Brachymyrmex patagonicus, an Emerging Pest 459





















2.














3


Figs. 1-3. Profile views of Brachymyrmex patagonicus: (1) worker, (2) alate male, and (3) dealate female. Scale
bar equals 1.0 mm.







Florida Entomologist 90(3)


Figs. 4-6. Full-face views of Brachymyrmex patagonicus: (4) worker, (5) male, and (6) female. Scale bar equals
0.5 mm.


appendages, very light brown. Head wider than
long, with fine, sparse pubescence, lacking erect
hairs except on mouthparts, and with smooth,
shiny integument; frontal lobes reduced; scapes
surpassing occipital border by more than 1/5 their


length, first segment of funiculus enlarged, al-
most globular, wider than succeeding segments;
eyes large, about 1/2 length of head, and located
on lower half of head; 3 large, prominent, raised
ocelli present. Mesosoma with sparse pubescence


September 2007







MacGown et al.: Brachymyrmex patagonicus, an Emerging Pest


and shiny integument, lacking erect hairs. Hind
wing with 5 or 6 hammuli. Gaster shiny, lacking
pubescence, with scattered erect hairs on last few
sternites and tergites.

Similar Species

In the United States, B. patagonicus is most
similar to B. obscurior, another exotic species.
Workers differ in the size of the eye, which is about
the length of the malar space in B. patagonicus,
and conspicuously shorter than the malar space
in B. obscurior. Additionally, the gaster ofB. pata-
gonicus has scattered pubescence, giving it a more
shiny appearance, whereas B. obscurior has more
dense pubescence. An undescribed species of Bra-
chymyrmex recorded from Florida (referred to as B.
brevicornis Emery in some publications-Deyrup
2003; Deyrup et al. 2000) is also dark brown in
color, but lacks erect hairs on the body. Another un-
described, brown colored species, known only from
2 queens from Arkansas, differs from B. patagoni-
cus in that the queens are tiny, approximately the
size of typical workers (pers. comm., Lloyd Davis).
Brachymyrmex heeri Forel is another similar spe-
cies that could be potentially found in the United
States, but has not been found here yet. Workers of
this species are brownish-yellow and lack ocelli.
Other species of Brachymyrmex found in the
United States are yellowish in color.

Biology and Economic Importance

This species nests in a variety of habitats, both
natural and disturbed. Natural habitats include
pine forests (with nests often in loose bark at the
bases of the tree trunks), beaches (with nests at
the bases of plants), mixed forests (nests in soil,
dead wood, and litter), and prairie remnants
(nests in soil, accumulations of organic litter, and
grass thatch). In disturbed areas, nests of B. pat-
agonicus are especially frequent in landscaping
mulch, a habitat that is increasing exponentially
throughout the Southeast, and which positions
colonies to make forays into buildings. In dis-
turbed areas it also nests in soil under objects on
the ground (stones, bricks, railroad ties, lumbers,
or a variety of other objects), under grass at edges
of lawns and parking lots, in leaf litter, at the
bases of trees, in rotting wood, in piles of dead
wood, and in accumulations of trash. Colonies
may contain many hundreds of workers packed
into a small sheltered area. Where this species is
found, colonies are often abundant and even may
be found within a few centimeters from one an-
other. The social structure of B. patagonicus has
not been studied, but apparently separate colo-
nies show considerable mutual tolerance.
In many sites the occurrence ofB. patagonicus
appears to be centered around urban areas or
places frequented by people, such as the more in-


tensive recreation areas of state parks, gas sta-
tions, restaurants, grocery stores, and along high-
way edges. As is the case with many other pest
plants and animals, this species appears to act as
an invasive organism that is likely to return
quickly whenever there are attempts to control it
or other species of ants. It has been reported that
this species may be found in higher numbers after
imported fire ant suppression has taken place
(Dash et al. 2005). This does not imply that popu-
lations of this species are excluded in areas where
large populations of fire ants are present, as re-
cent surveys of ants in the Southeast by the MEM
have found this species to be abundant in areas
with high numbers of imported fire ants. We have
observed this species nesting side-by-side and
freely roaming about with both Solenopsis invicta
Buren and S. invicta x richteri on numerous occa-
sions and have seen no obvious correlation of its
abundance to that of the fire ants' presence and
abundance.
This species is considered a nuisance pest spe-
cies, as both alates and foraging workers may en-
ter houses, hospitals, schools, or other man-made
structures to forage and/or nest. Occasionally
these infestations may be quite large, with nests
being found in the structure of the buildings, espe-
cially in bathrooms and kitchens, in light sockets
and in electrical outlets, inside cinder blocks of ex-
terior walls, and under shingles. During recent
years (2005-2006), the Department of Entomology
and Plant Pathology at Mississippi State Univer-
sity has had more requests by pest control opera-
tors in Mississippi about this species than all other
ant species totaled together. Pest control operators
have found this species in very high numbers in
hospitals and other businesses, especially in met-
ropolitan areas, and have expressed difficulty in
controlling it. This may be partly due to the fact
that many indoor infestations of foraging workers
may be coming from outdoor nests some distance
from control efforts. As in the case ofB. obscurior
and other ants whose alates fly into openings in
buildings or clutter up swimming pools, there may
be no effective control of alate patagonicus where
the ambient population is high, except by restrict-
ing access. The attention received by this ant may
be more associated with its novelty than with any
harm it causes, as it does not cause structural
damage, bite, sting, or transmit disease, nor has it
been shown to invade stored foods. As a nuisance
species, however, invading buildings and causing
annoyance, patagonicus shows considerable po-
tential, perhaps comparable to the effects of Tapi-
noma melanocephalum (Fabricius) in tropical and
subtropical regions.
The diet of Brachymyrmex patagonicus is
thought to consist largely of honeydew from vari-
ous insects, especially subterranean homopterans
(Dash et al. 2005). They undoubtably supplement
their diet with other food sources and will readily







Florida Entomologist 90(3)


come to sweet baits such as honey or cookies.
Workers of these ants can be commonly seen scur-
rying about during the day as they forage. Female
and male alates have been collected from mid
May through early Aug.

Distribution (Fig. 7)

Since its first report in the United States,
B. patagonicus has become extremely common
and abundant in the Gulf Coast states, and in the
last few years its range in the southeastern
United States has grown considerably. In a study
(Storz & Tschinkel 2004) of the spread of another
exotic South American ant species, Pheidole ob-
scurithorax Naves, B. patagonicus (referred to as
B. musculus in their publication) was also re-
ported. They made collections along a transect
through 46 counties and parishes in Georgia,
Florida, Alabama, Mississippi, and Louisiana and
found this species in Thomas County, Georgia;
Liberty and Santa Rosa Counties, Florida; Es-
cambia and Baldwin Counties, Alabama; Pike
and George Counties, Mississippi; and did not
find it in Louisiana. It is now known to occur in 27
counties in Georgia (MEM; Ipser et al. 2004-re-


ported as B. musculus), 23 counties in Florida, but
reported to be widespread throughout (Deyrup
2003-reported as B. musculus), 27 counties in Al-
abama (MEM; MacGown and Forster 2005-re-
ported as B. musculus), 31 counties in Mississippi
(MEM), and 15 parishes in LA (MEM; Wheeler &
Wheeler 1978).
In May of 2007, two transects were made
across central Georgia by the MEM, and B. patag-
onicus was found to be abundant. It is likely that
this species is abundant throughout the entire
southern half of Georgia, although the northern
limit of its distribution in the state is not known.
The easternmost record from Georgia, in
Chatham County, borders South Carolina, and it
is probable that this species occurs there as well.
In Florida, B. patagonicus appears to be much
more common in the northern portions of the
state, whereas in Alabama and Mississippi it is
most common in the southern halves of the states,
with scattered northern records. The earliest
known collection date ofB. patagonicus from Mis-
sissippi is 1977, which is only 1 year later than
the earliest published record of this species in the
United States. Surprisingly, that record is from
Holly Springs, Marshall County, located in ex-


Fig. 7. Map of the southeastern United States showing the known distribution of Brachymyrmex patagonicus.


September 2007







MacGown et al.: Brachymyrmex patagonicus, an Emerging Pest


treme north Mississippi and bordering Tennessee
near the Memphis area. It has not yet been re-
ported from Tennessee, but it will likely expand
its range to this state, if it is not already found
there. In a recent thesis documenting the ants of
Louisiana (Dash 2005), this species was only re-
ported in Louisiana from 3 parishes (reported as
B. musculus and B. obscurior). Recent collections
by the MEM have revealed that this species is
now widespread in that state. The MEM also col-
lected this species in 2006 in 2 counties in south-
ern Arkansas. In a paper documenting the distri-
bution of ants of Texas (O'Keefe 2000), B. patag-
onicus was not reported from the state. However,
during a collecting expedition in Jul, 2006 by the
MEM, which traversed Texas from east to west, it
was collected at one locality in Smith County, in
the eastern portion of the state. This species was
also collected in 2004 on the grounds of a hotel
near the Tucson Airport in Pima County, Arizona
(M.A.D., unpublished data). Considering the cli-
matic regimes under which this species thrives in
southeastern North America and southern South
America, there is no obvious reason why B. patag-
onicus should not extend its range through the
entire Gulf Coast, and through the states border-
ing Mexico, at least in irrigated urban and subur-
ban areas.
The distribution by county and parish in the
southeastern United States of B. patagonicus
based on specimens examined and literature
records is given below (also see Fig. 7). Gaps in
the distribution map do not imply absence of this
species, but may reflect a lack of collecting. Geor-
gia: Appling, Brooks, Chatham, Chattahoochee,
Crisp, Decatur, Dodge, Early, Emanuel, Grady,
Houston, Jeff Davis, Laurens, Muscogee, Peach,
Stewart, Sumter, Talbot, Tattnall, Taylor, Telfair,
Thomas, Toombs, Treutlen, Webster, and Wilcox
Counties (MEM); Seminole County (Ipser et al.
2004-reported as B. musculus). Florida: Ala-
chua, Bradford, Calhoun, Clay, Dade, Escambia,
Franklin, Gadsden, Highlands, Hillsborough,
Holmes, Jackson, Leon, Liberty, Madison, Mon-
roe, Okaloosa, Osceola, Putnam, Santa Rosa, Tay-
lor, Wakulla, and Walton Counties (ABSC, MEM).
Alabama: Baldwin, Bibb, Butler, Choctaw,
Clarke, Coffee, Conecuh, Covington, Dale, Dallas,
Escambia, Geneva, Houston, Lee, Lowndes, Ma-
con, Marengo, Marion, Mobile, Monroe, Mont-
gomery, Russell, Shelby, Sumter, Tuscaloosa,
Washington, and Wilcox Counties (MEM;
MacGown & Forster 2005-reported as B. muscu-
lus). Mississippi: Clarke, Copiah, Covington,
Forrest, Franklin, George, Greene, Hancock, Har-
rison, Hinds, Jackson, Jasper, Jefferson Davis,
Lauderdale, Leake, Lowndes, Madison, Marion,
Marshall, Newton, Oktibbeha, Pearl River, Perry,
Pike, Rankin, Smith, Scott, Stone, Warren,
Wayne, and Wilkinson Counties (MEM). Arkan-
sas: Ashley and Union Counties (MEM). Louisi-


ana: Caddo, East Baton Rouge, Jackson, Lincoln,
Madison, Natchitoches, Orleans, Quachita,
Rapides, Richland, Sabine, Tangipahoa, Vernon,
and Winn Parishes (MEM); St. Tammany Parish
(Wheeler and Wheeler 1978). Texas: Smith
County (MEM).

DISCUSSION

Although B. patagonicus is a relatively recent
introduction to the United States, it is now well
established and abundant in both natural and
disturbed areas throughout much of the South-
east, especially in Georgia, Florida, Alabama,
Mississippi, and Louisiana. To give an indication
of how common B. patagonicus is within its range
is the fact that the authors have usually been able
to find workers of this species at new localities
within 5 minutes by simply stopping at gas sta-
tions, motels, restaurants, and other such busi-
nesses, and searching at the edges of parking lots,
at edges of grass areas, on tree trunks, exteriors
walls of buildings, or on bare ground. Random
stops at highways and rural roadsides have re-
vealed similar abundance and ease of detecting
this species.
A major reason for the success ofB. patagoni-
cus in the United States may be its ability to
thrive in a variety of habitats, especially dis-
turbed sites. Other contributing factors could be
its ability to coexist with a variety of other domi-
nant ant species, such as Dorymyrmex bureni
(Trager), S. invicta, Pheidole moerens Wheeler,
and P obscurithorax. This is similar to other spe-
cies of Brachymyrmex, which also usually occur
where there are many other ant species that are
larger, faster, more hard-bodied, and armed with
stingers and more powerful mandibles. It is diffi-
cult to avoid the conclusion that this species may
be protected by potent chemicals.
Because colonies can fit into a small space,
they easily could be transported by man from site
to site, making it likely that this species will in-
crease its range further. This hypothesis is sup-
ported by isolated collections in the northern
parts of both Alabama and Mississippi where this
species was collected in landscaped areas of state
parks, campuses, or other public areas, but not
yet found in more natural areas. Several isolated
populations in Mississippi have been found in
mulch, which may have been transported from ar-
eas where this species was already common.
It is unclear what affect, if any, this species will
have on native species in the area, but based on
its abundance where it is now established, it
might have some negative impact. In many areas
in the southern portions of Alabama and Missis-
sippi, this species, along with several other intro-
duced species including Linepithema humile
(Mayr), Pyramica membranifera (Emery), Cy-
phomyrmex rimosus (Spinola), S. invicta, P moe-







Florida Entomologist 90(3)


rens, and P. obscurithorax, are now the most com-
monly found ants. Its effect, if any, on honeydew-
producing sap-sucking insects is also unknown. It
also will be interesting to see how B. patagonicus
interacts with the related, exotic species, B. ob-
scurior, over the next few years. Enormous popu-
lations ofB. obscurior are found in southern Flor-
ida, and this ground nesting species might com-
pete with B. patagonicus for food resources, and to
a lesser extent, nesting sites. Brachymyrmex pat-
agonicus shows the explosive increase and spread
that is typical of some recently imported species,
especially those that thrive in the ever-expanding
zone of habitats profoundly disturbed by human
activities. It is not known whether this will lead
to permanently high population levels, or
whether some form of biotic resistance will even-
tually catch up with this species.

ACKNOWLEDGMENTS

We thank Linda Hooper-Bui and Lee Womack (LSU)
for the loan of Louisiana specimens and Estela Quiran
for verification of specimens and useful comments. Spe-
cial cooperation has been provided by State Parks, Na-
tional Forests, National Wildlife Refuges, the Natchez
Trace Parkway, and from various private landowners in
the Southeast. This research was supported by Missis-
sippi Agricultural and Forestry Experiment Station
State Project MIS-311080, the USDA-ARS Areawide
Management of Imported Fire Ant Project (Richard L.
Brown, P.I.), and the Georgia Department of Natural Re-
sources (JoVonn G. Hill and Joe A. MacGown, Co-P.I.s).

REFERENCES CITED

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of the World. Harvard University Press, Cambridge,
MA, USA. 504 pp.
DASH, S. 2005. Species Diversity and Biogeography of
Ants (Hymenoptera: Formicidae) in Louisiana, with
Notes on Their Ecology. M.S. Thesis, Louisiana State
University, Baton Rouge, LA. v + 290 pp.
DASH, S. T., L. M. HOOPER-BfI, AND M. A. SEYMOUR.
2005. The Pest Ants of Louisiana: A Guide to Their
Identification, Biology and Control. Louisiana State
University Agricultural Center Communications,
Baton Rouge. 24 pp.
DEYRUP, M. 2003. An updated list of Florida ants (Hy-
menoptera: Formicidae). Florida Entomol. 86: 43-48.
DEYRUP, M., L. DAVIS, AND S. COVER 2000. Exotic ants
in Florida. Trans. American Entomol. Soc. 126: 293-
326.
DEYRUP, M., C. JOHNSON, G. C. WHEELER, AND J.
WHEELER 1989. A preliminary list of ants of Florida.
Florida Entomol. 72: 91-101.


FOREL, A. 1899. Hymenoptera. III. Formicidae. Biol.
Centrali-America. H. R. Porter. Dulau & Co., Lon-
don. 160 pp., 4 pl.
HOOPER-BUI, L. M., H. M. STORY, AND A. M. PRAN-
SCHKE. 2000."Brachymyrmex patagonicus andB. de-
pilis: Unwelcome Housepests in Louisiana". Proc.
National Conference on Urban Entomol. Fort Lau-
derdale, FL.
IPSER, R. M., M. A. BRINKMAN, W. A. GARDNER, AND
H. B. PEELER 2004. A survey of ground-dwelling
ants (Hymenoptera: Formicidae) in Georgia. Florida
Entomol. 87: 253-260.
MACGOWN, J. A., AND J. A. FORSTER 2005. A prelimi-
nary list of the ants (Hymenoptera: Formicidae) of
Alabama. Entomol. News 116: 61-74.
MACGOWN, J. A., R. L. BROWN, AND J. G. HILL. 2005.An
annotated list of the Pyramica (Hymenoptera: For-
micidae: Dacetini) of Mississippi. J. Kansas Ento-
mol. Soc. 78: 285-289.
MACGOWN, J. A., AND R. L. BROWN. 2006. Survey of
Ants (Hymenoptera: Formicidae) of the Tombigbee
National Forest in Mississippi. J. Kansas Entomol.
Soc. 79: 325-340.
MAYR, G. L. 1868. Formicidae novae americannae col-
lectae a Prof. P. de Strobel. Ann. Soc. Nat. Modena 3:
161-178.
NAVES, M. A. 1976. A Monograph of the Genus Pheidole
in Florida (Hymenoptera: Formicidae). Ph.D. Disser-
tation, University of Florida. 120 pp.
NAVES M. A. 1985. A monograph of the genus Pheidole
in Florida (Hymenoptera: Formicidae). Insecta
Mundi 1: 53-90.
O'KEEFE, S. T., J. L. COOK, T. DUDEK, D. F. WUNNEBUR-
GER, M. D. GUZMAN, R. N. COULSON, AND S. B. VIN-
SON. 2000. The distribution of Texas ants.
Southwestern Entomol. 22: 1-92.
QUIRAN, E. M. 2005. El G6nero Neotropical Brachy-
myrmex Mayr (Hymenoptera: Formicidae) en la Ar-
gentina. II: Redescripci6n de las species B. admotus
Mayr, B. brevicornis Emery y B. gaucho Santschi.
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QUIRAN, E. M., J. J. MARTINEZ, AND A. O. BACHMANN.
2004. The Neotropical genus Brachymyrmex Mayr,
1868 (Hymenoptera: Formicidae) in Argentina. Re-
description of the type species, B. patagonicus Mayr,
1868; B. bruchi Forel, 1912 and B. oculatus Santschi,
1919. Acta Zool6gica Mexicana (n.s.) 20: 273-285.
SANTSCHI, F. 1923. Revue des fourmis du genre Brach-
ymyrmex Mayr. Annals del Museo Nacional de Histo-
ria de Buenos Aires 31: 650-678 + 4 plates.
STORZ, S. R., AND W. R. TSCHINKEL. 2004. Distribution,
spread, and ecological associations of the introduced
ant Pheidole obscurithorax in the southeastern
United States. J. Insect Sci. 4: 1-11.
WHEELER, G. C., AND J. WHEELER. 1978. Brachymyrmex
musculus, a new ant in the United States. Entomol.
News 89: 189-190.


September 2007







Jeon & Ahn: Larvae of Littoral Genus Cafius


DESCRIPTIONS OF LATE INSTARS OF THREE LITTORAL CAFIUS
SPECIES (COLEOPTERA: STAPHYLINIDAE) BY ASSOCIATION
OF LIFE STAGE WITH DNA SEQUENCES

MI-JEONG JEON AND KEE-JEONG AHN
Department of Biology, Chungnam National University, Daejeon 305-764, Republic of Korea

ABSTRACT

Late instars of 3 littoral Cafius Curtis species [Cafius fucicola Curtis, C. nauticus (Fair-
maire), C. vestitus (Sharp)] are described for the first time by using sequences of mitochon-
drial gene. In total, 13 unknown larvae were collected in association with adults from the
Nearctic and Palearctic regions. The partial cytochrome oxidase II (CO II) gene was se-
quenced from the unknown larvae and several identified adult specimens for adults-larvae
association. The range of intraspecific p-distance was from 0 to 2.30% and the minimum in-
terspecific p-distance was 14.46% between Cafius nauticus and C. vestitus. Parsimony and
distance analyses were conducted and individuals of a single species were always grouped
closely together. Based on these results, the unknown larvae were identified and diagnostic
characters of the species are provided, with illustrations of features.

Key Words: DNA identification, larval description, C. fucicola, C. nauticus, C. vestitus

RESUME

Los iltimos instares de 3 species litorales del g6nero Cafius Curtis (Cafius fucicola Curtis,
C. nauticus (Fairmaire) y C. vestitus (Sharp)) son descritas por primera vez basadas sobre se-
cuencias del gene mitocondrial. En total, 13 larvas desconocidas fueron recolectadas en aso-
ciaci6n con adults de la region nearctica y palearctica. El gene citocromo oxidasa II (CO II)
fue secuenciado de larvas desconocidas y various especimenes adults para relacionar dichos
adults con las larvas. El rango de la distancia-p intra-especifico fue de 0 a 2.30% y la dis-
tancia-p minima fue 14.46% entire Cafius nauticus y C. vestitus. El andlisis de parsimonia y
distancia fue realizado e individuos de la misma especie siempre fueron agrupadas estrecha-
mente juntas. Basado en estos resultados, las larvas desconocidas fueron identificadas y ca-
racteres diagn6sticos de las species e ilustraciones de las caracteristicas son proveidas.


The family Staphylinidae includes over 47,744
described species in 3,847 genera and 31 subfami-
lies in the world (Herman 2001; Thayer 2005). The
high diversity of the Staphylinidae is a result of
remarkable radiations in diverse habitats. Most of
the staphylinids are found in terrestrial habitats
such as leaf litter, plant debris, and fungi. How-
ever, 442 species in 102 genera and 7 subfamilies
are known to be confined to seashore habitats
(Ahn & Ashe 1996, 2004; Hammond 2000; Moore
& Legner 1976). One of the 7 subfamilies with
coastal representatives is Staphylininae.
Most coastal staphylinine larvae live in
stranded accumulations of decaying seaweed. The
seaweed forms the basis of a food chain. Larvae
and adults are predators that feed on amphipods,
larvae of seaweed flies (Fucellia Robineaux-des-
voidy) or enchytraeid worms (Lumbricillus
Orsted and Enchytraeus Henle) (Moore & Legner
1976; Topp & Ring 1988) that in turn feed on the
seaweed.
The immature stages of a few staphylinids
were studied early, but taxonomic studies of them
remained fragmentary (Smetana 1995). Struc-
tures of larvae are diverse and present many sim-


ilarities to those of related families. No single
structure characterizes larvae of a family, many
genera, and some tribes (Frank 1991). Larvae of
staphylinids provide important information for
phylogenetic and evolutionary studies. However,
very few immature staphylinids have been de-
scribed because of the difficulty of making larval-
adult associations (Ashe & Watrous 1984; Frank
1991). Larvae can be reared through pupae to
adults in the laboratory, allowing larval identifi-
cation and association with adults. However,
rearing of larvae is time-intensive, and it is diffi-
cult to achieve the appropriate rearing conditions.
Recently, DNA sequencing has become straight-
forward and inexpensive and therefore represents
an obvious alternative for the identification of im-
mature insects (Caterino & Tishechkin 2006; He-
bert et al. 2003; Tautz et al. 2003; Blaxter 2004).
A partial sequence of the cytochrome oxidase II
(CO II) gene is sufficient to make a confident as-
sociation between life stages of staphylinid bee-
tles (Jeon & Ahn 2005).
Fifty five species of the genus Cafius Curtis are
now recognized. It is the most speciose genus of
the coastal staphylinid subfamily Staphylininae.







Florida Entomologist 90(3)


Most are from temperate regions, although sev-
eral species are widespread in the tropics (Orth &
Moore 1980). To date, 9 late instar larvae of Caf-
ius species have been identified from the Neotro-
pical and Palearctic regions (Coiffait 1974; James
et al. 1971; Jeon & Ahn 2005; Moore 1975; Orth &
Moore 1980; Paulian 1941). In spite of the previ-
ous studies, most of the Cafius still have been in-
sufficiently described. In this study, we first de-
scribe larvae of three Cafius species [C. fucicola
Curtis, C. nauticus (Fairmaire), C. vestitus
(Sharp)] through the association of larvae-adults
based on DNA sequences. We also provide a key to
the Cafius larvae and diagnostic characters with
illustrations of features and discuss differences
between species.

MATERIALS AND METHODS

In total, 25 specimens including 13 larval spec-
imens were used in this study (Table 1). Unknown
larvae and adult staphylinids were collected to-
gether, and we attempted association by gene se-
quencing of individuals. The partial cytochrome
oxidase II gene (375 bp) was sequenced from the
unknown larvae and identified adult specimens to
confirm that the unknown larvae and adult were
conspecific. Two or more specimens of several spe-
cies were sequenced in order to examine intraspe-
cific variation.
Preparations of permanent microscopic slides
for late-instar larvae were made by the tech-
niques described by Ashe (1986). Specimens pre-


pared for study of microscopic structures were ex-
amined under a compound microscope. Terms for
larvae follow James et al. (1971) and Paulian
(1941). Materials for this study were deposited in
the Chungnam National University Insect Collec-
tion (CNUIC, Daejeon), Korea.

DNA Extraction, Amplification, and Sequencing

For adults, total genomic DNA was extracted
from muscles in the head and pronotum to pre-
vent contamination with DNA from parasites in
food remains. Genitalia were preserved to confirm
the species identification. After grinding the spec-
imens in liquid nitrogen, we followed the manu-
facturer's protocol for the DNeasy Tissue Kit
(QIAGEN, Hilden, Germany). For larvae, DNA
was extracted from muscles in the head and
pronotum. The remaining cuticle was used as a
voucher specimen of the sample.
The cytochrome oxidase II region examined in
this study was amplified by primers C2J 3400
(Simon et al. 1994) and TKN 3782 (Brent et al.
1999). PCR was performed in 50 pL with 1-10 pL
of the genomic DNA and 1 or 2 units of Taq-poly-
merase, 3 mmol MgC12, 1.5 mmol dNTPs, and 50
pmol of each primer. The amplification involved 2
min of denaturation at 94C, followed by 35 cycles
of 30 s at 94C, 30 s of primer annealing at 45C-
55C, and 1 min of extension at 72C, followed by
a final 4-min extension at 72C. PCR products
were examined by gel electrophoresis. They were
cleaned of enzymes and remaining primers with a


TABLE 1. SPECIES AND SPECIMENS, COLLECTION LOCALITIES, AND GENBANK ACCESSION NUMBERS FOR CYTOCHROME
OXIDASE II (CO II) SEQUENCES USED IN THIS STUDY.

Species Collection locality GenBank

Cafius fucicola [1]* England: Devon, Plymouth, Mount Batten Point EF450174**
[2] Same as above EF450175**
C. nauticus [1]* USA: Hawaii, Oahu, Honolulu, Kawailoa beach EF450198**
[2]* Same as above EF450199**
[3] Same as above EF450200**
[4] Australia: Queensland, Daintree N.P., Wonga beach EF450201**
[5] Japan: Okinawa, Iriomote Isl., Uibaru EF450202**
[6] Philippines: Cebu, Bohol, Panglao Isl., Alona beach EF450203**
[7] Vietnam: Hai Phong, Do Son EF450204**
C. vestitus [1]* Korea: Gangwon Prov., Donghae City, Daejin beach EF450210**
[2]* Same as above EF450211**
[3]* Same as above EF450212**
[4]* Korea: Jeju Isl., Bukjeju-gun, Gujwa-eup, Hado-ri EF450213**
[5] Korea: Jeonnam Prov., Jangheong-gun, Hoejin-myeon DQ069324
[6] Korea: Jeju Isl., Moseulpo EF450214**
[7] Japan: Hokkaido, Nemuro City, Onneto EF450215**
[8] Japan: Honshu, Nagasaki EF450216**
Philonthus nudus [1] Korea: Gangwon Prov., Gangreung City, Gyeongpo EF450227**
[2] Japan: Hokkaido, Shibetsu-shi, River mouth EF450228**

*Indicates larval specimen.
**Indicates new sequence data.


September 2007







Jeon & Ahn: Larvae of Littoral Genus Cafius


PCR Product Purification Kit (Roche, Indianapo-
lis, Indiana, USA) and recovered in 20 pL of H2O.
Amplified DNA was sequenced with a Perkin
Elmer ABI377 Automated Sequencer (Applied
Biosystems Inc., Foster City, California, USA) and
was confirmed with both sense and anti-sense
strands. Partial cytochrome oxidase II sequences
of larval specimens and related adult species have
been deposited in GenBank under accession num-
bers (Table 1).

Parsimony and Distance Analyses for Identification

As the cytochrome oxidase II gene is a protein
coding region, it is length invariant and easily
aligned. Alignment was performed by using Seq-
Pup (Gilbert 1995). Parsimony and distance anal-
yses were carried out in PAUP* (Swofford 2003).
Parsimony analysis was conducted with a branch
and bound tree search option. Branch support
values were estimated by 100 bootstrap repli-
cates, each comprising 2 heuristic random addi-
tion searches. Distance analysis was conducted by
Neighbor-Joining method and branch support
values were also obtained by bootstrap analysis.
Philonthus nudus Sharp was selected to root the
resulting trees.


RESULTS

Sequences for 7 larval specimens were com-
pared with those for 4 adult species collected from
the same regions and habitats. The maximum in-
traspecific pairwise distance was 2.30% between
C. nauticus Hawaii populations (range 0-2.30%)
and the minimum interspecific distance was
14.46% between C. nauticus-Hawaii and C. ues-
titus-Donghae, Korea (range 14.46-20.83%). The
intraspecific and interspecific congeneric pairwise
distances are presented in Table 2.
The analysis resulted in 30 equally parsimoni-
ous cladograms with a length of 157, a consis-
tency index of 0.85, a retention index of 0.95, and
strict consensus tree as shown in Fig. lB. The
Neighbor-joining analysis showed that specimens
of a single species formed cohesive assemblages
(Fig. 1A). Every terminal branch at the species
level is supported by 100% of branch support val-
ues. The unknown larvae grouped unambiguously
with the adult specimens of 3 coastal Cafius spe-
cies in both analyses. Individuals of a single spe-
cies were always grouped closely together, regard-
less of where they were collected. Therefore, we
identified and described the unknown larvae as
probable late instars of the genus Cafius.


KEY TO THE KNOWN LARVAE OF CAFIUS

1. Urogomphus with a single article, spherical; maxillary palpomere 4th as long as 3rd ...... C. seminitens Horn
1'. Urogomphus with 2 articles, slender or cylindrical; maxillary palpomere 4th shorter than 3'd ............... 2
2. Urogomphus shorter than tergite X, cylindrical; conical ligula much longer than 1"
labial palpomere; mala with 3 setae on apex .................. .............. C. canescens (Maklin)
2'. Urogomphus longer than tergite X, slender; conical ligula subequal or shorter than 1"
labial palpomere; mala with 1 seta on apex ................................................... 3
3. Nasale teeth blunted, central teeth (LT4 and 5) shorter than lateral teeth; tergite X
longer than 1" article of urogomphi ......................................... C. mimulus (Sharp)
3'. Nasale teeth stout or acute, central teeth (LT4, 5) subequal or longer than lateral teeth;
tergite X shorter or subequal to 1" article of urogomphi ......................................... 4
4. Conical ligula as long as 1" labial palpomere .................................................... 5
4'. Conical ligula shorter than 1" labial palpomere .................................................. 8
5. Tergite X longer than 1" article of urogomphi; 3rd labial palpomere as long as 2nd ....... C. rufescens (Sharp)
5'. Tergite X shorter than 1" article of urogomphi; 3rd labial palpomere much shorter than 2nd. ................ 6
6. Maxillary palpus longer than stipes; mandible with serration ................. C. lithocharinus (LeConte)
6'. Maxillary palpus shorter than stipes; mandible without serration ................................... 7
7. Mala as long as lacinia; nasale LT4 longer than LT3 ....................... C. xantholoma (Gravenhorst)
7'. Mala longer than lacinia; nasale LT4 as long as LT3 (Figs. 2B, C) ..................... C. fucicola Curtis
8. Central three teeth distinct with side teeth, LT5 as long as LT4; 2nd article of urogomphi
less than 1/3 of 1" (Figs. 4B, F) ............................................... C. uestitus (Sharp)
8'. Central three teeth continued with side teeth, LT5 smallest; 2nd article of urogomphi
more than 1/2 of 1 ................................................................... 9
9. Tergite X as long as 1" article ofurogomphi; mandible without serration ............... C. histrio (Sharp)
9'. Tergite X shorter than 1" article of urogomphi; mandible with fine serration
(Figs. 3D, F) ......................................................... C. nauticus (Fairm aire)


















TABLE 2. PAIRWISE DISTANCES BETWEEN TAXA IN ANALYSIS EXPRESSED AS A PERCENTAGE OF NUCLEOTIDE DIFFERENCES (P-DISTANCES). INTRASPECIFIC PAIRWISE DISTANCES
INDICATED IN BOLD.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

C. fucicola [1]
C. fucicola [2] 0.000
C. nauticus [3] 0.176 0.176
C. nauticus [4] 0.179 0.176 0.021
C. nauticus [5] 0.179 0.176 0.016 0.011
C. nauticus [6] 0.171 0.171 0.011 0.021 0.011
C. nauticus [7] 0.176 0.176 0.019 0.013 0.003 0.013
C. nauticus [1] 0.168 0.168 0.003 0.023 0.017 0.014 0.017
C. nauticus [2] 0.170 0.170 0.003 0.023 0.017 0.014 0.017 0.000
C. vestitus [5] 0.152 0.157 0.152 0.160 0.152 0.158 0.150 0.149 0.148
C. vestitus [6] 0.152 0.157 0.152 0.160 0.152 0.158 0.150 0.149 0.148 0.000
C. vestitus [7] 0.152 0.157 0.152 0.160 0.152 0.158 0.150 0.149 0.148 0.000 0.000
C. vestitus [8] 0.155 0.159 0.152 0.160 0.158 0.163 0.155 0.149 0.148 0.005 0.005 0.005
C. vestitus [1] 0.163 0.159 0.149 0.157 0.155 0.160 0.154 0.146 0.145 0.003 0.003 0.003 0.003
C. vestitus [2] 0.159 0.159 0.151 0.162 0.159 0.162 0.159 0.145 0.145 0.006 0.006 0.006 0.006 0.003
C. vestitus [3] 0.152 0.152 0.155 0.166 0.157 0.155 0.157 0.154 0.156 0.009 0.009 0.009 0.015 0.012 0.012
C. vestitus [4] 0.158 0.162 0.152 0.160 0.158 0.163 0.155 0.149 0.148 0.005 0.005 0.005 0.005 0.003 0.006 0.009
P. nudus [1] 0.163 0.161 0.203 0.200 0.198 0.198 0.200 0.206 0.208 0.174 0.174 0.174 0.176 0.180 0.185 0.178 0.179
P. nudus [2] 0.163 0.162 0.198 0.195 0.198 0.198 0.200 0.200 0.203 0.174 0.174 0.174 0.171 0.174 0.179 0.178 0.174 0.005







Jeon & Ahn: Larvae of Littoral Genus Cafius


Caofus nauticus Hawaii (EF450200)
62J C. nauticus Hawaii (EF450198)
pairwis instance C nauticus Hawaii (EF4S0199)
-- C nauticus AU. (EF450201)
C. nauticus JP (EF450202)
L C nauticus Vt. (EF450204)
I C nauticus PH. (EF450203)
C vestitus KOR. (DQ069324)
SC. vestitus KOR. (EF450214)
C vestius JP. (EF450215)
pairwise distance
0-0.023% r c vestitus JP. (EF450216)
i I E C vestitus KOR. (EF450211)
0 ~_ C vestitus KOR. (EF450210)
o C vestitus KOR. (EF450213)
pairs dince C. vestitus KOR. (EF450212)


S oo C. fucicola (EF450175)
1 C fucicola (EF450174)
100oo Philonthus nudus KOR. (EF450227)
0 P. nudus JP. (EF450228)
- 0.01 substitutionslsite


* Cafius nauticus Hawaii (EF450200)
S] C nauticus Hawaii (EF450198)
- C nauticus Hawaii (EF450199)
m C nauticus PH. (EF450203)
SC. nauticus AU. (EF450201)
* C. nauticus JP. (EF450202)
* C nauticus Vt. (EF450204)
- C vestitus KOR. (DQ069324)
. C vestitus KOR. (EF450214)
- C vestitus JP. (EF450215)
- m C vestitus JP. (EF450216)
- C. vestitus KOR. (EF450211)
SC. vestitus KOR. (EF450210)
- a C. vestitus KOR. (EF450213)
S[ C. vestitus KOR. (EF450212)
SC. fucicola (EF450175)
SC. fucicola (EF450174)


C nauticus









C vestitus






C fucicola


S100 Philonthus nudus KOR. (EF450227)
I P. nudus JP. (EF450228)

Fig. 1. A, Neighbor-Joining tree based on cytochrome oxidase II sequences; B, strict consensus tree of 30 equally
parsimonious trees (Tree length = 157, CI = 0.85, RI = 0.95). Every terminal branch at the species level is supported
by 100% bootstrap values.


06


H


C. nouticus







C. vestitus




C. fucicola







Florida Entomologist 90(3)


Late Instars of Cafius fucicola Curtis

Description: Length 6.5~7.0 mm. General body
shape elongate, flattened, parallel-sided. Body
pale, head pale ferruginous.
HEAD: Sub-quadrate, almost equally wide
from apical to basal margin; about 1.2 times as
long as wide. Four stemmata present. Ecdysial
sutures distinct and complete from near antenna
insertion. Antennae (Fig. 2A): Four-articled.
Length of articles 1t < 4th < 2nd = 3rd; article 1


A


longer than wide, transverse; one campaniform
sensillum present on middle of article 2; article 3
with 2 solenidia (III S1 and III S2) and 1 campan-
iform sensillum, 1 corn-type sensory appendage
present; article 4 with 4 solenidia (IVS1~IVS4);
article 3 and 4 each with 3 setae. Mentum with 3
pairs of setae. Gular suture constricted in middle
of head, divergent to apex.
MOUTHPARTS: Nasale (Fig. 2B): Nine teeth
present; central 3 teeth pointed (LT4 and LT5),
central tooth (LT5) smallest, penultimate teeth


Tergite IX


Fig. 2. Cafius fucicola. A, Antenna; B, nasale; C, maxilla; D, mandible; E, labium; F, urogomphi, dorsal view.
Scale = 0.1 mm.


September 2007







Jeon & Ahn: Larvae of Littoral Genus Cafius


(LT2) very weak. Eight setae present along outer
line. Maxilla (Fig. 2C): Stipes almost as long as
maxillary palpus; articulated mala with small
seta at apex, small, elongate-ovoid; maxillary pal-
pus with 4 articles, a separate sclerotization form-
ing a short ring at base in form of an extra seg-
ment present, 1 seta and 1 campaniform sensil-
lum present; length ratio of palpomeres 1"t : 2nd :
3rd : 4th = 1:2.8:2.8:0.6; width of palpomeres 1st =
2nd > 3rd = 4th Mandible (Fig. 2D): Two macrosetae
present along outer surface, falciform, undivided
at acute apex, almost symmetrical. Labium (Fig.
2E): Labial palpus with 3 articles; length ratio of
palpomeres 1t : 2d : 3rd = 1:0.91:0.49, conical ligula
reaching end of labial palpomere 1, pubescent at
base.
THORAX: Pronotum: Transverse, lightly scle-
rotized, setae scattered at sides and on disc.
Length of pronotum about 2x length of mesono-
tum. Mesonotum length subequal to metanotum,
both slightly broader than posterior margin of
pronotum. Legs: Coxa, trochanter, femur, tibia
and tarsungulus distinguishable, tarsungulus
with 3 articulated spines.
ABDOMEN: Abdominal tergite I-VIII trans-
verse, parallel sided, slightly narrowed to apex.
Tergites and sternites divided by midlongitudinal
line; Tergite X (Fig. 2F) about 3 times longer than
wide. Urogomphi (Fig. 2F): Two-articled, much
longer than tergite X; article 1 slender and with 4
pairs of macrosetae, as long as tergite X; article 2
with 2 small setae and 1 large seta arising from
apex, much narrower than article 1.
SPECIMENS EXAMINED: England: Devon,
Plymouth, Mount Batten point, 7 VIII 2004, K.-J.
Ahn and J.-S. Park, ex under seaweeds (CNUIC, 1).
REMARKS: This species can be distinguished
from other Cafius species by the combination of
the following features: 2nd antennomere as long as
3rd, mala longer than lacinia, 2 articled urogom-
phi, 1" article of urogomphi as long as tergite X,
conical ligula subequal to 1" labial palpomere,
and mandible without serration.

Late Instars of Cafius nauticus (Fairmaire)

Description: Length 8.3~9.0 mm. General body
shape elongate, flattened, parallel-sided. Body
pale, head pale ferruginous.
HEAD: Sub-quadrate, almost equally wide
from apical to basal margin. About 0.8 times as
long as wide. Four stemmata present. Ecdysial
sutures distinct and complete from near antenna
insertion. Antennae (Fig. 3A): Four-articled.
Length of articles 1" = 4th < 2nd = 3rd; article 1
longer than wide, transverse; 1 campaniform sen-
sillum present on middle of article 2; article 3
with 2 solenidia (III S1 and III S2) and 2 campan-
iform sensilla, 1 corn-type sensory appendage
present; article 4 with 4 solenidia (IVS1~IVS4);
article 3 and 4 each with 3 setae. Mentum with 3


pairs of setae. Gular suture converged in middle
of head, divergent to apex.
MOUTHPARTS: Nasale (Fig. 3B): Nine teeth
present; central 3 teeth pointed (LT4 and LT5),
central tooth (LT5) smallest, penultimate teeth
(LT2) very weak. Eight setae present along outer
line. Maxilla (Fig. 3C): Stipes almost as long as
maxillary palpus; articulated mala with small
seta at apex, small, elongate-ovoid; maxillary pal-
pus with 4 articles, a separate sclerotization form-
ing a short ring at base in form of an extra seg-
ment present, 1 seta and 1 campaniform sensil-
lum present; length ratio of palpomeres 1"t : 2d :
3rd : 4th = 1:2.6:2.6:1.1; width of palpomeres 1st =
2nd > 3rd > 4th Mandible (Fig. 3D): Two macrosetae
present along outer surface, falciform, undivided
at acute apex, almost symmetrical and molar area
saw-like. Labium (Fig. 3E): Labial palpus with 3
articles; length ratio of palpomeres 1t : 2nd : 3rd =
1:0.8:0.4, conical ligula a little shorter than pal-
pomere 1, pubescent at base.
THORAX: Pronotum: Transverse, lightly scle-
rotized, setae scattered at sides and on disc.
Pronotum about 1.6 times longer than mesono-
tum. Mesonotum length subequal to metanotum,
both as long as posterior margin of pronotum.
Legs: Coxa, trochanter, femur, tibia and tarsun-
gulus distinguishable, tarsungulus with 3 articu-
lated spines.
ABDOMEN: Abdominal tergite I-VIII trans-
verse, parallel sided, slightly narrowed to apex.
Tergites and sternites divided by midlongitudinal
line; Tergite X (Fig. 3F) about 3 times longer than
wide. Urogomphi (Fig. 3F): Two-articled, much
longer than tergite X; article 1 slender, as long as
tergite X; article 2 with 2 small setae and 1 large
seta arising from apex, much narrower than arti-
cle 1.
SPECIMENS EXAMINED: USA: Hawaii,
Oahu, Honolulu, Kawailoa beach, 1 VIII 1992, K.-
J. Ahn, ex under seaweeds (CNUIC, 8).
REMARKS: This species can be distinguished
from other Cafius species by combination of the
following features: 2nd antennomere as long as 3rd,
mala as long as lacinia, 2 articled urogomphi, 1t
article of urogomphi as long as tergite X, conical
ligula subequal to 1" labial palpomere, and man-
dible with serration.

Late Instars of Cafius vestitus (Sharp)

Description: Length 7.0~9.0 mm. General body
shape elongate, flattened, parallel-sided. Body
pale, head pale ferruginous.
HEAD: Sub-quadrate, almost equally wide
from apical to basal margin. About 0.74 times as
long as wide. Four stemmata present. Ecdysial
sutures distinct and complete from near antenna
insertion. Antennae (Fig. 4A): Four-articled.
Length of articles 1" = 4th < 3rd < 2nd; article 1
longer than wide, transverse; 1 campaniform sen-







Florida Entomologist 90(3)


Fig. 3. Cafius nauticus. A, Antenna; B, nasale; C, maxilla; D, mandible; E, labium; F, urogomphi, dorsal view.
Scale = 0.1 mm.


sillum present on middle of article 2; article 3
with 2 solenidia (III S1 and III S2) and 2 campan-
iform sensillia, 1 corn-type sensory appendage
present; article 4 with 4 solenidia (IVS1~IVS4);
article 3 and 4 each with 3 setae. Mentum with 3
pairs of setae. Gular suture converged in middle
of head, divergent to apex.
MOUTHPARTS: Nasale (Fig. 4B): Nine teeth
present; central 3 teeth pointed (LT4 and LT5),
central tooth (LT5) smallest, penultimate teeth
(LT2) very weak. Eight setae present along outer


line. Maxilla (Fig. 4C): Stipes a little shorter than
maxillary palpus; articulated mala with small
seta at apex, elongate-ovoid; maxillary palpus
with 4 articles, a separate sclerotization forming
a short ring at base in form of an extra segment
present, 1 seta and 1 campaniform sensillum
present; length ratio ofpalpomeres 1 : 2nd : 3rd : 4th
= 1:2:2:1; width of palpomeres 1st = 2nd > 3rd > 4th
Mandible (Fig. 4D): Two macrosetae present
along outer surface, falciform, undivided at acute
apex, almost symmetrical. Labium (Fig. 4E): La-


September 2007







Jeon & Ahn: Larvae of Littoral Genus Cafius


vestitus. A, Antenna; B, nasale; C, maxilla; D, mandible; E, labium; F, urogomphi, dorsal view.


bial palpus with 3 articles; length ratio of pal-
pomeres 1 : 2nd : 3rd = 1:0.8:0.5, conical ligula a lit-
tle shorter than palpomere 1, pubescent at base.
THORAX: Pronotum: Transverse, lightly scle-
rotized, setae scattered at sides and on disc.
Pronotum about 1.5 times longer than mesono-
tum. Mesonotum length subequal to metanotum,
both as long as posterior margin of pronotum.
Legs: Coxa, trochanter, femur, tibia and tarsun-
gulus distinguishable, tarsungulus with 3 articu-
lated spines.


ABDOMEN: Abdominal tergite I-VIII trans-
verse, parallel sided, slightly narrowed to apex.
Tergites and sternites divided by midlongitudinal
line; Tergite X (Fig. 4F) about 3 times longer than
wide. Urogomphi (Fig. 4F): Two-articled, longer
than tergite X; article 1 slender, longer than terg-
ite X; article 2 with 2 small setae and 1 large seta
arising from apex, much narrower than article 1.
SPECIMENS EXAMINED: Korea: Gangwon
Prov., Donghae City, Daejin beach, 21 VII 2004,
M.-J. Jeon, ex under seaweeds (CNUIC, 3); Jeju


D








Fig. 4. Cafius
Scale = 0.1 mm.











Prov., Bukjeju-gun, Gujwa-eup, Hado-ri, 11 VI
2005, M.-J. Jeon and K.-J. Ahn, S.-J. Park, ex un-
der seaweeds (CNUIC, 1).
REMARKS: This species can be distinguished
from other Cafius species by a combination of the
following features: 2nd antennomere longer than
3rd, mala as long as lacinia, 2 articled urogomphi,
1' article of urogomphi longer than tergite X, con-
ical ligula shorter than 1t labial palpomere, and
mandible without serration.

ACKNOWLEDGMENTS

We thank Clive Turner (Devon, England) and Jong-
Seok Park (Daejeon, Korea) for assistance in collecting
littoral staphylinids. We are grateful to an anonymous
reviewer for helpful comments on the manuscript. This
research was supported by the Korean Institute of En-
vironmental Science and Technology, Ministry and En-
vironment Grant (KIEST 052-041-029, 2007) awarded
to K.-J. Ahn.

REFERENCES CITED

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axy of Aleocharinae (Staphylinidae) based on a de-
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BALXTER, M. L. 2004. The promise of a DNA taxonomy.
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BRENT, C. E. PEDRO, AND G. M. HEWITT. 1999. MtDNA
phylogeography and recent intra-island diversifica-
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tles) in the British Isles, with special reference to


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Florida Entomologist 90(3)







McKern & Szalanski: Molecular Diagnostics of Clearwing Moths



MOLECULAR DIAGNOSTICS OF ECONOMICALLY IMPORTANT
CLEARING MOTHS (LEPIDOPTERA: SESIIDAE)

JACKIE A. MCKERN AND ALLEN L. SZALANSKI
Department of Entomology, University of Arkansas, Fayetteville, AR, USA


ABSTRACT

Larvae of many species of Sesiidae, the clearing moths, are important pests in commercial
nurseries, urban landscapes, timber stands, vineyards, and orchards. They cause economic
loss by larval boring in stems and roots of herbaceous and woody plants. Researchers and
growers often monitor for the presence of economically important sesiid adults with phero-
mone traps. These traps often attract more than one species of Sesiidae and specimens often
degrade making identification difficult or impossible. This can cause problems in monitoring
programs where species-specific control programs are used. Polymerase chain reaction
(PCR) was used to amplify a 606-bp region of the mitochondrial DNA cytochrome oxidase I
(COI), tRNA leucine, and COII gene. This region exhibited 7.7-19.5% genetic variability
among 8 species of Sesiidae. Samples were sequenced and restriction sites identified. PCR-
restriction fragment length polymorphism (PCR-RFLP) analysis was conducted on 8 species
of Sesiidae, Melittia satyriniformis, Paranthrene simulans, Pennisetia marginata, Synanthe-
don pictipes, S. exitiosa, S. scitula, S. rileyana, and Vitacea polistiformis, with 2 restriction
enzymes, Dra I and HinfI. This method is time efficient requiring less than 8 h to perform
and cost efficient with each sample about $1. PCR-RFLP provides an accurate method to dif-
ferentiate 8 species of adult clearing moths commonly found in traps baited with commer-
cially available pheromone lures.

Key Words: COI, PCR-RFLP, Melittia, Paranthrene, Pennisetia, Synanthedon, Vitacea

RESUME

Las larvas de muchas de las species de la familiar Sesiidae, palomillas de alas claras, son
plagas importantes en viveros comerciales, areas urbanas, arboles maderables, viias y
huertos. Ellas causan perdidas econ6micas al barrenar los tallos y raices de plants herba-
ceas y lehosas. Investigadores y agricultores a menudo realizan un monitoreo para la pre-
sencia de los adults sesiidos de importancia econ6mica usando trampas con feromonas.
Estas trampas a menudo atraen mas de una especie de Sesiidae y los especimenes a menudo
se degradan y con ello se hace dificil o impossible su identificaci6n. Esto puede causar proble-
mas en los programs de monitoreo donde se usan programs de control para la especie es-
pecifica. La reacci6n en cadena por la polimerasa (RCP) fue usada para amplificar la region
de 606-pb de la citocromo-c-oxidasa I (COI) de la ADN mitocondrial, tRNA de leucina y del
gene COII. Esta region exhibit una variabilidad gen6tica de 7.7-19.5% entire 8 species de Se-
siidae. Las muestras fueron secuenciadas y los centros de restricci6n identificados. Se rea-
liz6 un andlisis del polimorfismo de la longitud de los fragments de restricci6n (PCR-RFLP)
para las 8 species de Sesiidae, Melittia satyriniformis, Paranthrene simulans, Pennisetia
marginata, Synanthedon pictipes, S. exitiosa, S. scitula, S. rileyana y Vitacea polistiformis,
con 2 enzimas de restricci6n Dra I y Hinf I. Este m6todo es eficiente en cuanto a que re-
quiere menos de 8 horas de tiempo para ejecutar y eficiente en cuanto a su costo de $1 por
muestra. El PCR-RFLP provee un m6todo precise para diferenciar los adults de estas 8 es-
pecies de palomillas de alas claras encontradas frecuentemente en trampas cebadas con se-
Auelos de feromonas comercialmente disponsibles.


The Sesiidae are a well defined lepidopteran
family with over 1000 described species worldwide
(Eichlin & Duckworth 1988). In North America
north of Mexico 123 species in 20 genera are rep-
resented (Eichlin & Duckworth 1988). Geographic
distribution and abundance at all taxonomic lev-
els are poorly known. The Sesiidae are quite in-
conspicuous and difficult to collect due to their
mimicry of wasps and diurnal flight. This has re-
sulted in very poor representation in collections.
Sesiid species in the genera Paranthrene (Hiibner)


and Synanthedon (Hiibner) cause economic loss to
commercial nurseries and timber producers in the
United States (Solomon et al. 1982). If not con-
trolled, Synanthedon exitiosa (Say) and S. pictipes
(Grote & Robinson) can destroy entire orchards of
fruit trees (Nielson 1978). Vitacea polistiformis
(Engelhardt) is a common pest in vineyards
(Johnson et al. 1981); Melittia satyriniformis
(Hiibner) is a pest of squash (Klun et al. 1990), and
Pennisetia marginata (Harris) will damage black-
berry and raspberry plants (Raine 1962).







Florida Entomologist 90(3)


Pheromone wing traps are a popular method of
monitoring for the adult moths in areas at risk for
sesiid damage. These traps are economical and
easy to use. The insect becomes entangled in
sticky glue on the trap bottom and can survive
several days, losing scales and limbs while trying
to break free. Unless these traps are checked on a
daily basis, moths trapped on the sticky bottoms
can become impossible to identify to species. Sesi-
ids are often cross-attracted to pheromones and
different species are commonly found in traps
baited with species-specific pheromone lures
(McKern 2005). Even when specimens are prop-
erly preserved, species can be difficult to differen-
tiate morphologically. A molecular diagnostics
protocol could be very helpful in distinguishing
sesiids that are target pests from non-pest species.
Polymerase chain reaction-restriction frag-
ment length polymorphism (PCR-RFLP) is a tech-
nique that is inexpensive, simple, reliable, repeat-
able, and can be used on the insect during any de-
velopmental stage, including eggs, larvae, pupae
and adults (Taylor & Szalanski 1999). The mito-
chondrial region has proven useful in other molec-
ular diagnostic protocols. Brown et al. (1999) used
the cytochrome oxidase region of mtDNA to differ-
entiate 6 moth species in the genus Wiseana
(Viette) (Lepidoptera: Hepalidae) and Lewter et
al. (2006) developed molecular diagnostics using
mtDNA to distinguish 7 noctuid species com-
monly found in Spodoptera frugiperda (J.E.
Smith) pheromone traps. The purpose of this
study was to identify PCR-RFLP diagnostic char-


acters with mtDNA marker for 8 species of Sesi-
idae commonly found in wing traps baited with
commercially available pheromone lures.

MATERIALS AND METHODS

Samples

Sesiids were collected in 2004 with Trece
Pherocon IC wing traps (Trece, Inc., Adair, OK)
baited with commercially available pheromone
lures. Moths were collected from 4 counties in Ar-
kansas as follows: blackberry planting and vine-
yard in Faulkner County; vineyard in Madison
County, peach and apple orchard in Carroll
County; and forest, squash planting, and apple
orchard in Washington County (Table 1). Two
P marginata larvae were collected from black-
berry crowns for analysis. All other samples con-
sisted of adults. Samples were identified morpho-
logically with the key "The Moths of America
North of Mexico" (Eichlin & Duckworth 1988) and
placed separately in 1.5-mL Eppendorf tubes and
stored at -20C until processing. Voucher speci-
mens were deposited in the University of Arkan-
sas Arthropod Museum Fayetteville, AR.

DNA Extraction, Amplification, and Purification

DNA was extracted from the thoraces of indi-
vidual adult specimens or the head capsules of
larval specimens by using the Puregene DNA iso-
lation kit D-5000A (Gentra, Minneapolis, MN).


TABLE 1. SAMPLE COLLECTION DATA, AND SAMPLES SUBJECTED TO DNA SEQUENCING AND PCR-RFLP.

Sample ID County, AR Manufacturer, lure No. sequenced No. PCR-RFLP

1. Paranthrene simulans Washington Scentry, BC 3 7
Carroll 2 3
Faulkner 2 5
Madison 1 1
2. Vitacea polistiformis Faulkner Tr6c6, GRB 2 3
3. Melittia satyriniformis Washington 2 8
Faulkner 2 2
Madison 2 1
4. Pennisetia marginata Faulkner APTIV, RCB 4 8
5. Synanthedon rileyana Washington APTIV, RCW 2 3
Carroll 2 3
Faulkner 1 4
6. Synanthedon exitiosa Faulkner Tr6c6, GPTB 2 10
7. Synanthedon pictipes Washington Tr6c6, LPTB 2 10
Carroll 2 2
8. Synanthedon scitula Washington Tr6c6, DWB 4 7
Carroll 2 3
Faulkner 1 1

Abbreviations: BC: Oak, Rhododendron, Ash, Banded Ash, Lilac borer; GRB: Grape Root Borer; SVB: Squash Vine Borer; RCB:
Raspberry Crown Borer; RCW: Raspberry Clearwing; GPTB: Greater Peach Tree Borer; LPTB: Lesser Peach Tree Borer; DWB:
Dogwood Borer.
Manufacturer Locations: Scentry (Billings, Montana); Tr6ec (Adair, Oklahoma); APTIV (Portland, Oregon).


September 2007







McKern & Szalanski: Molecular Diagnostics of Clearwing Moths


Extracted DNA was resuspended in 50 pL of Tris:
EDTA and stored at -20C.
DNA PCR was conducted with primers C1-J-
2797 (5'-CCTCGACGTTATTCAGATT ACC-3')
(Simon et al. 1994) and C2-N-3400 (5'-TCAATAT-
CATTGATGACCAAT-3') (Taylor et al. 1997).
These primers amplify approximately 606 bp of
the mtDNA cytochrome oxidase I gene (COI),
tRNA-leu and cytochrome oxidase II gene (COII).
PCR reactions were conducted with 2 pL of the ex-
tracted DNA. The thermal cycler profile for the
mtDNA COII gene consisted of 35 cycles of 94C
for 45 s, 46C for 45 s, and 72C for 45 s per Sza-
lanski et al. (2000).
Amplified DNA from individual sesiids was pu-
rified and concentrated with minicolumns accord-
ing to the manufacturer's instructions (Wizard
PCRpreps, Promega) (Table 1). Samples were
sent to the University of Arkansas Medical School
Sequencing Facility (Little Rock, AR) for direct se-
quencing in both directions. DNA sequences were
aligned with Clustal W (Thompson et al. 1994)
and consensus sequences obtained with BioEdit
5.89 (Hall 1999). Sequence data were deposited in
GenBank with accession numbers DQ205539-
DQ205573.

Digests

Restriction sites were predicted from the DNA
sequence data with BioEdit 5.89 (Hall 1999). Am-
plified DNA from unknown specimens of each spe-
cies (P. simulans, V polistiformis, M. satyrinifor-
mis, P marginata, S. rileyana, S. pictipes, and S.
scitula) was digested according to manufacturer's
(New England Biolabs, Ipswich, MA) recommen-
dations following Cherry et al. (1997) with the re-
striction enzymes Dra I and Hinf I (Table 2).
Fragments were separated by 2% agarose gel
electrophoresis per Taylor et al. (1996). Gels were
photographed with a UVP BioDoc-it documenta-
tion system (Upland, CA).


RESULTS AND DISCUSSION

DNA sequencing of the mtDNA amplicon from
38 sesiids resulted in an average amplicon size of
606 bp. Interspecific genetic variation ranged be-
tween 7.1-19.5% among the 8 species of Sesiidae.
Two restriction enzymes, which did not have any
intraspecific variation, were selected for PCR-
RFLP. The Dra I digest had 6 restriction patterns
among the 8 sesiid moths (Table 2 and Fig. 1), and
the Hinf I digest produced four restriction pat-
terns (Table 2 and Fig. 2). From the 81 moths sub-
jected to PCR-RFLP no intraspecific variation
was observed for the 2 restriction enzymes. By
combining the restriction patterns from the 2 di-
gests, the 8 sesiid species in this study can be
readily differentiated. This is the first time PCR-
RFLP has been utilized to distinguish sesiid pests
commonly found in pheromone traps.
This technique is cost efficient and useful for
identification of degraded or badly damaged adult
specimens and hard to identify larvae. Each reac-
tion costs <$1.00 per sample and the use of a
small amplicon facilitates the use of specimens
that are slightly degraded (Taylor et al. 1996).
Other advantages to the PCR-RFLP technique in-
clude reliability and time efficiency. Because the
restriction patterns are based on specific DNA se-
quences, there are no false positives obtained
(Roehrdanz 1997) and the whole procedure can be
performed in under 8 h.
An important advantage in using the COI,
COII mtDNA region for this PCR-RFLP tech-
nique is that most fragments created by the di-
gests are relatively large and clearly separated
with 2% agarose gel during electrophoresis, which
eliminates the need for polyacrylamide-gel elec-
trophoresis or high resolution agarose gels. This
increases cost efficiency when dealing with large
numbers of samples.
The results described here demonstrate that
PCR-RFLP of mtDNA provides a simple and reli-


TABLE 2. RESTRICTION SITES, FRAGMENTS, AND PATTERNS FOR SESIID PCR-RFLP USING RESTRICTION ENZYMES Hinf I
AND Dra I.

Hinf I Dra I

Species Restriction site Fragments Pattern Restriction site Fragments Pattern

Paranthrene simulans -606 A 256, 312, 582 256, 270 A
Vitacea polistiformis 203 203, 403 B 252, 309 252,297 A
Melittia satyriniformis 102 102, 504 C 139, 253, 309 139, 114, 297 B
Pennisetia marginata 102 102, 504 C 253, 558, 579 253,305 A
Synanthedon rileyana 15, 102, 270 87, 168, 336 D 252,306 252,300 A
S. exitiosa 15, 102 87, 504 C 252 252,354 C
S. pictipes 15, 102 87, 504 C 139, 252 139, 113, 354 D
S. scitula 15, 102 87, 504 C 606 E

-= "no cut".







Florida Entomologist 90(3)


A B A A C D E

Fig. 1. Agarose gel depicting PCR-RFLP Dra I digest patterns of PCR amplified mtDNA for 8 sesiid species.


A B C D C

Fig. 2. Agarose gel depicting PCR-RFLP Hinf I digest patterns of PCR amplified mtDNA for 8 sesiid species.


September 2007







McKern & Szalanski: Molecular Diagnostics of Clearwing Moths


able method to distinguish 8 species of Sesiidae.
Monitoring and identification of these pests is es-
sential to apply control tactics at crucial points in
their life cycle before they have bored too deep
within plants for insecticide penetration (McKern
et al. in press). The PCR-RFLP procedure can be
used by researchers not only for identification of
sesiids but also to monitor seasonal and geo-
graphical differences. Very little is known about
sesiid distribution, abundance, and gene flow, and
this procedure will allow us to address these is-
sues in the future.

ACKNOWLEDGMENTS

This work is part of the Ph.D. project of J. McKern.
We thank Dr. D. T. Johnson for reviewing the manu-
script. We thank B. Lewis for assistance with the collec-
tion of sesiid samples. Funding for this research was
provided by the University of Arkansas Agricultural Ex-
periment Station.

REFERENCES CITED

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markers for Wiseana (Lepidoptera: Hepalidae) spe-
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POWERS. 1997. The internal transcribed spacer re-
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EICHLIN, T. D., AND W. D. DUCKWORTH. 1988. The
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JOHNSON, D. T., R. L. MEYERS, AND P. A. GRAY. 1981.
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idae) management and feasibility of control of dis-
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Entomol. Soc. Am. 12: 1-7.
KLUN, J. A., M. SCHWARZ, B. A.LEONHARDT, AND W. W.
CANTELO. 1990. Sex pheromone of the female squash
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MEAGHER, JR., C. B. OWENS, AND R. G. LUT'RELL.
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MCKERN, J. A, D. T. JOHNSON, AND B. A. LEWIS. 2007.
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berry crown borer, Pennisetia marginata (Lepi-
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RAINE, J. 1962. Life history and behavior of the rasp-
berry crown borer Bembecia marginata (Harr.) (Lep-
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SIMON, C., F. FRATI, A. BECKENBACH, B. CRESPI, H. LIU,
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R. E. DOOLITTLE. 1982. Occurrence of clearing bor-
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FRITZ. 2000. Population genetics and phylogenetics
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J. J. PETERSON. 1997. Mitochondrial DNA variation
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4673-4680.







Florida Entomologist 90(3)


September 2007


SEASONAL DISTRIBUTION AND EVALUATION OF TWO TRAP TYPES
FOR MONITORING GRAPE ROOT BORER VITACEA POLISTIFORMIS
(LEPIDOPTERA: SESIIDAE) IN FLORIDA VINEYARDS


SCOTT W. WEIHMAN AND OSCAR E. LIBURD
Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611

ABSTRACT

Sixteen vineyards from 4 grape-growing regions across Florida were evaluated for seasonal
distribution and presence of grape root borer (GRB), Vitacea polistiformis Harris (Lepi-
doptera: Sesiidae), during 2003 and 2004. Vineyards consisted of both muscadine (Vitis ro-
tundifolia Michx.) and bunch grapes (Euvitis spp.). Grape root borer males were caught in
all vineyards with Universal Moth Traps baited with female GRB pheromones, with higher
captures in the northern and southern counties. Grape root borers began emerging in late
Jun and early Jul in the Panhandle and southern regions. Emergence occurred in late Jul in
the north-central region and in mid-Aug in the central region. Weekly trap catches indicated
that peak GRB flights occurred during mid-to late Aug for the Panhandle region. In the
north-central, central, and southern regions, peak flights occurred in the second and third
week of Sep, coinciding with the period of grape harvest for muscadine grapes. The emer-
gence period ceased with the onset of colder temperatures as the grapevines approached dor-
mancy. Wing-style sticky traps were compared with Universal Moth Traps (bucket traps) in
2003 and 2004. Bucket traps were more effective and caught significantly more GRB than
wing traps during both years of the study.

Key Words: grape root borer, Vitacea polistiformis, seasonal distribution, monitoring, pher-
omones, trapping

RESUME

Se evaluaron diecis6is vifias en cuatro regions productoras de uva en Florida para determi-
nar la presencia del perforador de la raiz de la uva, Vitacea polistiformis Harris (Lepidop-
tera: Sesiidae), en el 2003 y 2004, en vifias con uvas de vino (Vitis rotundifolia Michx.) y en
uvas de mesa (Euvitis spp.). Los machos del perforador de la raiz de la uva fueron capturados
en trampas universales para lepidopteras (Universal Moth Trap) usando la feromona pro-
ducida por la hembra. El mayor numero de polillas fue capturado en los condados localizados
en el norte. El perforador de la raiz de la uva comenz6 a merger a finales de Junio y princi-
pios de Julio en el Panhandle y en las regions del sur. La emergencia de los adults ocurri6
a finales de Julio en la region norte-central y a mediados o finales de Agosto en la region cen-
tral. Capturas semanales indican que entire mediados y finales agosto se alcanz6 el pico de
la poblaci6n en la region del Panhandle. En la region norte-central, central, y sur, la maxima
poblaci6n fue capturada durante la segunda semana o tercera semana de septiembre, coin-
cidiendo con los periods de cosecha de las uvas muscadine. El final del period de emergen-
cia coincidi6 con las temperatures frias, y el comienzo de la dormancia de las viias. Trampas
adhesivas fueron comparadas con las trampas universales en el 2003 y 2004. Las Trampas
universales capturaron un numero de polillas significantemente alto comparado con las
trampas adhesivas en ambos aios del studio.


Translation provided by the authors.


Grape acreage has increased significantly over
the past several years in Florida. The 2 most
widely planted species are muscadine grapes,
Vitis rotundifolia Michx. and hybrid bunch
grapes, Euvitis spp. (Vitaceae). Despite an in-
crease in the production of grapes, the grape root
borer (GRB), Vitacea polistiformis (Harris) (Lepi-
doptera: Sesiidae) is the greatest insect deterrent
to grape growing in the southeastern United
States. It is the key pest of grapes in several
states, including Georgia (All et al. 1989), North


Carolina (McGiffen & Neunzig 1985) and Florida
(Liburd et al. 2004).
Large infestations of GRB result in the death
of vines, and smaller infestations weaken plants
and reduce yields. Currently, chlorpyrifos (Lors-
ban 4E), applied as a soil drench, is one of the few
chemical control tactics available to growers
(Weihman & Liburd 2006). In order for chlorpyri-
fos to be effective, it must be applied during the
period in which it can kill the most GRB. Under-
standing the seasonal distribution of the GRB, its







Weihman & Liburd: Traps for Monitoring Vitacea polistiformis


peak emergence periods, data on trap effective-
ness, and information on monitoring, are essen-
tial for coordinating efforts and development of
control tactics for GRB.
Snow et al. (1991) and Webb et al. (1992) inves-
tigated the seasonal distribution of GRB and
found that, in Florida, GRB generally begin to
emerge in Jun and continue until the onset of
colder temperatures. They found that peak emer-
gence was generally bimodal. Since the findings
were published, new muscadine and bunch grape
hybrids have been planted (Gray 2003). In order to
detect any changes that may have occurred in
GRB flight patterns over a 10-year period (1993-
2003), populations of GRB were monitored in the
major grape-growing regions of Florida during
2003 and 2004. The overall goals of this study were
twofold. Our first objective was to monitor vine-
yards across Florida to determine seasonal trends
of GRB, specifically the initiation of emergence,
peak flight periods, and the cessation of GRB ac-
tivity. We increased the scope of the studies previ-
ously conducted by Snow et al. (1991) and Webb et
al. (1992) by using 16 vineyards in key production
areas as opposed to 4 and 9, respectively.
Our second goal was to compare the overall ef-
fectiveness of traditional wing-style sticky traps
with Universal Moth Traps (bucket traps) for use
in monitoring GRB. The wing-style sticky trap
has been used for monitoring in all the previous
GRB pheromone and mating disruption studies
(Johnson et al. 1986; Johnson et al. 1991; Snow et
al. 1987; Snow et al. 1991; Webb 1991; Webb et al.
1992). The problem with wing traps is that they
become inundated with insects and debris and
need to be replaced on a regular basis to be effec-
tive. These 2 trap styles have been compared in
previous studies with other lepidopteran pests,
and in each study, the bucket traps caught signif-
icantly more moths than the wing traps (Shaver
et al. 1991; Schmidt & Roland 2003). A better
trapping device would alleviate some of the prob-
lems discussed previously.

MATERIALS AND METHODS

Monitoring

Pheromone monitoring of GRB was carried out
in 16 vineyards in 2003 and 2004, representing 4
distinct grape-growing regions of Florida (Fig. 1).
Sixteen privately owned vineyards were chosen in
4 areas: the Panhandle [including one vineyard
each in Washington (1), Calhoun (2), Leon (3), and
Jefferson (4) Counties], north-central Florida [1
vineyard in Alachua County (5) and 3 vineyards
in Putnam County (6-8)], mid-Florida [four vine-
yards in Lake County (9-12)], and south-central
Florida [including 1 vineyard in Hillsborough
County (13), 2 in Manatee County (14,15), and 1
in Highlands County (16)]. Vineyards were cho-


sen with similar cultural practices and grape
types (muscadine and bunch grapes).
Four green Universal Moth Traps (bucket
traps) (Great Lakes IPM, Vestaburg, MI) baited
with female GRB pheromone (99% (E,Z)-2,13-oc-
tadecadienyl acetate, 1% (Z,Z)-3,13-octadecadie-
nyl acetate) (1 mg of pheromone per septum)
(Great Lakes IPM, Vestaburg, MI) were placed in
each vineyard. Traps were distributed evenly
throughout each vineyard, at least 30 m apart.
The baits in the bucket traps were changed once
per season, at the midpoint of the GRB emergence
period for the specific region (approximately 2
months). A Vaportape (Hercon Environmental,
Emigsville, PA) treated with 2,2-dichlorovinyl
dimethyl phosphate was affixed to the bottom of
each trap within the bucket to kill the GRB as
they became entrapped. Traps were hung from
the trellis wire approximately 1 to 1.5 m above
the ground near the vine trunk.
Monitoring began in Jun each year, according
to Webb et al. (1992), who demonstrated that the
first emergence of GRB in our regions began in
Jun or later. Trap contents were collected weekly
into labeled plastic resealable bags and taken to
the Small Fruit and Vegetable IPM Lab at the
University of Florida in Gainesville for analysis.
The number of GRB, along with other insects col-
lected in each trap, was recorded each week.
Traps were continuously monitored until no more
borers were caught.

Evaluation of Traps

This study was carried out at 3 sites in 2003
and expanded to 5 sites in 2004. In 2003, traps
were placed in 3 vineyards representing different
areas of Florida: the Panhandle (vineyard #2)
(Calhoun County), mid-Florida (vineyard #10)
(Lake County), and the south-central grape-grow-
ing region of Florida (vineyard #13) (Hillsborough
County) (Fig. 1). For 2004, 2 vineyards in the
north-central region in Alachua County (vineyard
#17) and Putnam County (vineyard #18) were
added to the original 3 locations (Fig. 1).
The 2 traps compared for this experiment were
Universal Moth Traps dimensions ~13.2 x 20.4
cm, and wing-style sticky traps ~20.4 x 16.8 cm
(dimensions of sticky surface). Both traps were
baited with GRB pheromone, 99% (E, Z)-2,13 oc-
tadecadienyl acetate and 1% (Z, Z)-3,13 octadeca-
dienyl acetate (Great Lakes IPM, Vestaburg, MI).
Pheromone lures were changed once per season,
after 8 weeks.
Traps were hung from the trellis wires at ap-
proximately 1.5 m above the ground, and spaced
at least 30 m apart. The experimental design was
a Completely Randomized Block with 6 and 10
replicates during 2003 and 2004, respectively.
Traps were set out at the beginning of the GRB
emergence period for each region, late Jun for the







Florida Entomologist 90(3)


Panhandle


COUNTIES
Wahington- #1
Calhoun- #2
Leon- #3
Jefferson- #4
Alachua- #s 5 and 17
Putnam- #s 6-8,18
Lake- #s 9-12
Hillsborough #13
Manatee #s 14 and 15
Highlands #16


Fig. 1. Map of Florida showing the locations of the 16 vineyards involved in the grape root borer survey (vine-
yards 1-16). Vineyards 2, 10, 13, 17, and 18 were used in the trapping study.


Panhandle and south, and early Jul for the cen-
tral regions. Traps were serviced weekly.

Statistical Analysis

Monitoring Studies. Trap catches were counted
and recorded weekly for the entire emergence pe-
riod (16 weeks) during 2003 and 2004 and the
mean number of GRB per vineyard per week was
reported.
Evaluation of Traps. Trap catches were
counted and recorded weekly for the entire emer-
gence period. In 2003 there were 18 weekly sam-
ples and in 2004 there were 16. Total number of
GRB weekly captures was analyzed by Repeated
Measures Analysis of Variance (ANOVA) and dif-
ferences were evaluated by using a paired t-test
(a = 0.05) (SAS Institute 2004).

RESULTS

Monitoring

Table 1 shows the first emergence, peak emer-
gence, and last captures of GRB in all vineyards
for the entire season in 2003 and 2004. Overall,


the timing of the peaks of emergence generally co-
incided for vineyards within the same region.
In the Florida Panhandle, activity began in
late Jun-early Jul (Table 1). Male GRB moth peak
flight occurred in mid- to late Aug in both 2003
and 2004, except for 2 vineyards, whose peaks oc-
curred in early Sep in 2004. Grape root borer
ceased activity during the first week of Oct for
most Panhandle vineyards.
In the north-central region, GRB emerged in
mid- to late Jul for most vineyards, with a few ex-
ceptions: Jul 2 (vineyard 6, 2004) and Aug 13
(vineyard 7, 2003) (Table 1). Peak flights occurred
mostly during the second week of Sep in 2003. In
2004, the peak flights occurred at the beginning of
Oct, later than the previous year. Grape root
borer adults stopped emerging the third and
fourth weeks of Oct.
In the mid-Florida region (Lake County), GRB
mostly emerged in mid- to late Aug (Table 1). This
was later than all other areas monitored. Peak flight
occurred mostly in the second and third weeks of
Sep. Generally, GRB activity ceased in Oct.
In the south-central grape-growing region of
Florida, GRB began emerging at the end of Jun
and early Jul (Table 1). Emergence peaked in the


North-
central
Florida


Mid-
Florida


South -
central
Florida




South-
Florida


September 2007






Weihman & Liburd: Traps for Monitoring Vitacea polistiformis


140 -
120
S100
Ia
0 80-
g 60

202
0-


Panhandle Vineyards

2003 -
2004


/,C q#5~ q"' fl p"~~~," Pt~~ ee9 C1~~~
Ncf' fl" t'*1 *


140
120
S 100
80
60
S 40
1 20
0-


North-Central Vineyards

2003 -
2004 -


N b 'Il ly' OC> & 4

140
g 120 -
S100 -
E 80
g 60
S 40
a 20
0


Central Florida Vineyards

2003 -
72004


140
120 D Southern Vineyards
o 100
Soo 2003 -
80 80
S 60 27004
S 40
S 20 .





Fig. 2. Seasonal distribution of GRB in 4 regions of Florida: (A) Panhandle, (B) North-Central, (C) Central, and
(D) Southern. The data from 4 vineyards within each region were pooled for 2003 and 2004.


1







Florida Entomologist 90(3)


September 2007


TABLE 1. FIRST AND PEAK GRAPE ROOT BORER EMERGENCE IN 16 FLORIDA VINEYARDS BY REGION, 2003-2004.


First
emergence
Vineyard 2003


Jun 25
Jun 25
Jul 2
Jun 18
Jul 23
Jul 16
Aug 13
Jul 30
Aug 13
Aug 27
Aug 6
Aug 13
Jul 2
Jul 2
Jul 2


Peak First
emergence Last capture emergence
2003 2003 2004


Aug 20
Aug 13
Aug 27
Aug 27
Sep10
Sep 10
Sep 10
Aug 27
Sep 10
Sep 10
Sep 24
Sep 24
Sep 17
Sep 10
Sep 10
Oct 8


Oct 1
Oct 22
Oct 8
Oct 1
Oct 22
Oct 22
Oct 15
Oct 15
Oct 22
Oct 1
Oct 8
Oct 29
Nov 5
Nov 5
Dec 10
Nov 12


Jul 2
Jul 2
Jul 2
Jul 2
Jul 23
Jul 2
Jul 30
Jul 30
Aug 27
Jul 9
Sep 4
Jul 1
Jun 25
Jun 25
Aug 6


Peak
emergence
2004


Aug 20
Aug 27
Sep 3
Sep 3
Oct 1
Oct 1
Oct 1
Sep 10
Oct 1
Sep 24
Sep 24
Sep 24
Sep 17
Sep 14
Aug 27
Sep 24


Last capture
2004


Oct 1
Oct 1
Oct 8
Oct 1
Oct 28b>
Oct 21
Oct 28b>
Oct 28b>
Oct 26b>
Oct 26b>
Oct 26b>
Oct 26b>
Nov 5b>
Nov 5b>
Nov 5b>
Nov 5b>


a<, GRB were caught the first week of monitoring and may have emerged sooner than data indicates.
>, GRB were caught on the last day of monitoring and additional GRB may have emerged after specified date.


second and third weeks of Sep at the vineyards
nearer to the coast and in the first week of Oct at
the inland vineyard (vineyard 16). In 2004, the
peaks occurred from late Aug to mid-Sep, with the
inland peak (vineyard 16) slightly later than the
coastal peaks (vineyards 13, 14, 15) in the third
week of Sep. The GRB flight period ended for the
south-central region in the first few weeks of Nov
for most vineyards.
Overall, adult GRB activity lasted for approxi-
mately 3.5 months [late Jun to early Oct (Fig. 2A)]
in the Panhandle region, 4 months in North-cen-
tral Florida [Jul through Oct (Fig. 2B)], 3-3.5
months in mid-Florida [Aug through Oct (Fig. 2C)],
and 5-5.5 months in the south-central region [Jun-
Nov (Fig. 2D)]. Peaks were higher in the 2 northern
areas (Fig. 2A, B) than in the central and southern
regions. Overall, the central region had the short-
est GRB flight period (Fig. 2C) and the southern re-
gion had the longest (Fig. 2D). The GRB flight sea-
son ended earliest in the Panhandle (Fig. 2A).
The mean (+ S.E.M) trap captures of GRB for
each vineyard for 2003 and 2004 are reported in
Fig. 3. In 2003, the mean ( SEM) weekly GRB
trap catch for all vineyards was 20.3 + 6.5, and in
2004, it was 16.1 4.9. Overall trap catches varied
among vineyards, with the central region (vine-
yards 9-12) having the lowest populations. Fewer
GRB were caught in 2004 compared with 2003,
but this reduction was not significantly different.

Evaluation of Traps

For the 2003 GRB emergence period, the mean
( SEM) GRB trap catch per week was 10.2 2.6


(84%) for the bucket trap and 1.9 0.6 GRB (16%)
for wing traps, more than 5-fold difference (Table
2). Bucket traps caught significantly more GRB
than the wing traps on a weekly basis (t = 2.00;
df = 1, 106; P = 0.0020) (Table 2). In 2004, bucket
traps caught 11.1 2.3 GRB (66%) and wing traps
caught 5.7 1.1 (34%) (Table 2). Overall, bucket
traps captured significantly more GRB per week
than wing traps (t = 1.98; df = 1,158; P = 0.0392)
(Table 2). Additionally, the percentage of zero trap
captures was lower in bucket traps than in wing
traps (Table 2).

DISCUSSION

Monitoring

The results of the monitoring experiments in-
dicate that GRB were present in all of the vine-
yards studied. Our study corroborates the earlier
work of Snow et al. (1991) and Webb et al. (1992).
However, significantly more vineyards were eval-
uated in the current work, indicating that the
problem of GRB infestation in Florida vineyards
is more severe than previously thought. In addi-
tion, newer vineyards (1993-2003) with grape hy-
brids did not show a significant change in the po-
tential to be infested with GRB. The results were
useful in showing the general emergence patterns
and peak flight activities for GRB in these 4 re-
gions of Florida. Overall, the results show that
GRB begin emerging in late Jun or early Jul in
the Panhandle and south-central region of the
state, but later for the north-central and central
regions. The peak flights occur in late Aug in the


Region


Panhandle



North-central



Central



South-central







Weihman & Liburd: Traps for Monitoring Vitacea polistiformis


E 2003

U2004


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Vineyards

Fig. 3. Mean ( SEM) grape root borer trap catch in 16 Florida vineyards, 2003-2004.


Panhandle and generally occur around mid- to
late Sep for the rest of the state. In contrast to
previous research, which suggested a bimodal
GRB peak emergence in the southern end of the
GRB range, most of the peaks observed in the cur-
rent study were single, although a few bimodal
peaks did occur. Differences in seasonal distribu-
tion between our work and earlier findings may
be attributed to changes in weather conditions, as
opposed to the occurrence of a true 'two-phase'
emergence. During weeks of unseasonably cold
temperatures, fewer GRB were usually caught.
Another factor may be the use of bucket traps in
this study versus wing traps used in the previous
work. Wing traps are incapable of capturing the
volume of moths we caught in bucket traps on a
weekly basis during peak emergence.
The Panhandle had the highest GRB trap
catch during the 2 years of this study, which may
be due to its having a richer, more moisture-reten-
tive soil than that found lower in the peninsula of


Florida. There was a slight reduction in regional
total trap catches from 2003 to 2004 as well as
mean ( SEM) GRB trap catch from 2003 to 2004.
The number of GRB decreased in 10 of the 16
vineyards monitored. In 2004, a dry spring re-
sulted in slower growth of the vines and set har-
vest back several weeks for most areas. It is inter-
esting to note that grape harvest occurred 2
weeks later in 2004, and the GRB peak emer-
gence occurred 2 weeks later than the previous
year as well. This suggests that the GRB life cycle
may be highly synchronized with grape plant phe-
nology, as proposed by Dutcher & All (1978).
The results of our monitoring study have di-
rect applications. The GRB emergence informa-
tion can help growers to optimize the timing of
their chlorpyrifos application or other tactics for
the control of GRB in grapes. For example, in the
Panhandle, the GRB peak coincides with the
muscadine grape harvest, around the third and
fourth weeks of Aug. Our results show that if


TABLE 2. TOTAL GRAPE ROOT BORER CATCHES, MEAN (+ SEM) WEEKLY CAPTURES, AND FREQUENCY OF WEEKS WITH
ZERO CAPTURES FOR TWO TRAP TYPES, 2003-2004.

Trap catch Weekly mean Zero frequency (%)

Trap type 2003 2004 2003 2004 2003 2004

Bucket 553 884 10.24 + 2.55 A 11.05 2.33 a 33.7% 41.6%
Wing 104 455 01.93 0.63 B 05.69+ 1.11 b 62.2% 53.2%

Weekly mean trap catch for 2003 (t = 1.99; df= 1, 106; P = 0.002) and 2004 (t = 0.98; df= 1,158; P = 0.0392) with t-pairwise com-
parison (a = 0.05). Means followed by upper-case letters represent differences for 2003 and lower-case letters represent differences
for 2004.







Florida Entomologist 90(3)


farmers were to apply chlorpyrifos 35 days pre-
harvest (as suggested by the post harvest interval
[PHI] label), they would only be able to affect a
small percentage of the GRB population. Accord-
ing to our study, it would be best to apply it di-
rectly after harvest to maximize the potential
number of GRB killed, which could affect subse-
quent generations. Similarly, in the 3 more south-
central regions, GRB peaks occur mostly 1 to 2
weeks after muscadine grape harvest begins, dur-
ing the third and fourth weeks of Aug, so chlorpy-
rifos applications would be optimized if applied
after harvest.
Bunch grapes, however, are ready for harvest
at the end of Jun and throughout Jul in Florida,
depending on the region. As with muscadine
grapes, our study suggests that the application of
chlorpyrifos would be best timed after harvest
and slightly after the peak GRB emergence for
that area.

Evaluation of Traps

Bucket traps caught significantly more GRB
than did wing traps during both seasons. Also, the
wing trap treatments had higher percentages of
zero weekly counts (62.2 and 53.2%) than did the
bucket traps (33.7 and 41.6%). The poorer perfor-
mance of the wing traps may be due to the fact
that they become saturated with GRB moths,
other insects, and plant debris over the period of
their use. This reduces the sticky surface and di-
minishes the effectiveness of the trap over time.
Some studies suggest that the wing trap may
be more effective at low population densities, but
Schmidt & Roland (2003) showed the opposite to
be true for forest tent caterpillar, Malacosoma
disstria Hubner (Lepidoptera: Lasiocampidae).
Most importantly, in our monitoring experiment,
we often had weekly trap catch numbers of 40 to
70 in the bucket traps with highs in the 150-170
range during peak emergence. Wing traps are in-
capable of capturing this volume of moths. The
wing trap can be an effective monitoring device,
but would need to be changed daily during peri-
ods of such high activity. Clearly, the bucket trap
is the superior GRB trap for use by growers.
Several factors may contribute to the greater
effectiveness of bucket traps. The bucket trap has
a more open design than the wing trap. The pher-
omone cage at the top allows the plume to be dis-
pensed relatively uninterrupted whereas the
wing trap is closed on 2 sides, and this may distort
the plume structure. The bucket trap's success
may also be attributable to its trapping method.
The moths are trapped within a lower enclosure
and killed quickly by the Vaportape inside the
traps. Moths trapped on sticky boards have
greater opportunity to escape via other debris, au-
totomy, or perhaps being near the edge of the
sticky surface.


In addition to its greater efficiency, the bucket
trap may be more cost-effective in the long run.
The bucket trap is more expensive initially, at
$8.95 per trap (Great Lakes IPM, Vestaburg, MI),
whereas the wing trap is only $2.32 per trap (IPM
Tech, Portland, OR). However, bucket traps are
more durable and can be used year after year.
They are easy to assemble and are placed in the
field once per season. Due to the frequency with
which wing traps need to be changed, they would
be more costly per season than bucket traps, in
addition to their higher labor demands.
For monitoring purposes, the bucket trap is
easier to use and gives a more accurate insect
count, as shown by the current data. A saturated
sticky board is often difficult to count; exposure to
the elements leads to rapid deterioration. Preda-
tors, such as lizards and frogs, are often observed
on the sticky boards, along with partially eaten
GRB moths.
The bucket traps used in this study were green
in color. There are no previous studies indicating
a trap color preference for GRB, or other sesiid
moths. Shaver et al. (1991) caught significantly
more Mexican rice borers Eoreuma loftini (Dyar)
with green-yellow-white traps than with all
green. One possibility for the higher number of
zero counts for the wing traps may be a different
behavioral response due to the trap design or
color. Future studies should focus on trap colors
as well as optimal trap density.


ACKNOWLEDGMENTS

We thank the grape growers who participated in this
research and contributed their farms and time, espe-
cially Byron Biddle (3 Oaks Winery), Roger and Marcia
Price (Harmony Vineyard), Dr. Jiang Lu, Garry Ford,
Mr. Ren and Mr. Inyang (Florida A&M University),
Cynthia Connolly (Ladybug Farms), Robert Henderson
(Grandma's Vineyard), Jay Pemberton (Florigon Vine-
yard), Felicity Trueblood (Meadowmere Vineyard),
George Comer and Jerry Mason (Comer and Mason
Nursery), John Sirvent (Sirvent's Vineyard), Opal Lillie
(Lillie's Vineyard), Robert, RuthAnn and Kellie Thropp
(Log Cabin Vineyard), Marsha Stephany (Grape Expec-
tations), Gary Salzman and Bob Stevens (Palatlakaha
Environmental and Agricultural Reserve), James and
Lois Hangar (Orange Blossom Vineyard), Brian
Johnson and Gary Cox (Lakeridge Winery), Robert and
Bonniejean Paulish (Blue Heron Vineyard), Lawrence
and Heidi May (Old Mission Vineyard), Antonio and
Rosa Fiorelli (Rosa Fiorelli Winery), and Joanne Lauch-
man and Christopher King (Henscratch Farms and
Winery). We thank the Florida Grape Growers Associa-
tion for funding this research for 3 consecutive years.
We appreciate the assistance of the staff from the Small
Fruit and Vegetable IPM Lab at the Department of En-
tomology and Nematology at the University of Florida,
especially Gisette Seferina and Alejandro Ar6valo for
assistance with the Spanish translation. We thank
Dr. Robert McSorley and Dr. Susan Webb for reviewing
earlier drafts of this manuscript.


September 2007







Weihman & Liburd: Traps for Monitoring Vitacea polistiformis


REFERENCES CITED

ALL, J. N., D. L. HORTON, AND J. D. DUTCHER. 1989. A
pest management program for the grape root borer
using Lorsban. Proc. Vit. Sci. Sym. Florida A&M
University, Tallahassee. pp. 60-61.
DUTCHER, J. D., AND J. N. ALL. 1978. Predictive models
for the summer activity of Vitacea polistiformis in Con-
cord grape vineyards. Environ. Entomol. 7: 456-460.
GRAY, D. 2003. New Plants for Florida: Grapes! EDIS,
University of Florida, Gainesville. Circular 1440.
http://edis.ifas.ufl.edu
JOHNSON, D. T., J. R. MEYER, AND R. L. MAYES. 1986.
Evaluation of Hercon laminated dispensers baited
with Z,Z-3,13-octadecadien-1-ol acetate for suppres-
sion of grape root borer, Vitacea polistiformis (Har-
ris) (Lepidoptera: Sesiidae), populations in grapes.
J. Entomol. Sci. 21: 231-236.
JOHNSON, D. T, B. A. LEWIS, AND J. W. SNOW. 1991. Con-
trol of grape root borer (Lepidoptera: Sesiidae) by
mating disruption with 2 synthetic sex pheromone
compounds. Environ. Entomol. 20: 931-934.
LIBURD, O. E., G. SEFERINA, AND S. WEIHMAN. 2004. In-
sect pests of grapes in Florida. IFAS EDIS Ext. Pub.
713. University of Florida, Gainesville.
MCGIFFEN, K. C., AND H. H. NEUNZIG. 1985. A guide to
the identification and biology of insects feeding on
muscadine and bunch grapes in North Carolina.
North Carolina Agric. Res. Ser. Bull. 470: 50-51.
SAS INSTITUTE. 2004. Statistical Analysis System SAS
V9.0. Cary, NC.
SCHMIDT, B. C., AND J. ROLAND. 2003. Developing tech-
niques for monitoring forest tent caterpillar popula-


tions using synthetic pheromones. Can. Entomol.
135: 439-448.
SHAVER, T. N., H. E. BROWN, J. W. BIRD, T. C. HOLLER,
AND D. E. HENDRICKS. 1991. Field evaluations of
pheromone-baited traps for monitoring Mexican rice
borer (Lepidoptera: pyralidae). J. Econ. Entomol. 84:
1216-1219.
SNOW, J. W., M. SCHWARTZ, AND J. A. KLUN. 1987. The
attraction of the grape root borer, Vitacea polistifor-
mis (Lepidoptera: Sesiidae) to (E,Z)-2,13 octadeca-
dien-1-ol acetate and the effects of related isomers
on attraction. J. Entomol. Sci. 22: 371-374.
SNOW, J. W., D. T. JOHNSON, J. R. MEYER, D. G. PFEIF-
FER, R. F. MIZELL, M. C. SAUNDERS, J. N. ALL, C. S.
GORSUCH, J. D. DUTCHER, R. N. WILLIAMS, S. R.ALM,
J. H. JARRATT, J. C. KILLIAN, W. C. ADLERZ, W. TAFT,
D. SMITLEY, S. R. RACE, H. G. TOWNSEND, G. L.
JUBB, M. SCHWARZ, AND T. D. EICHLIN. 1991. The
seasonal occurrence of grape root borer Vitacea polis-
tiformis (Harris) in the eastern United States. J. En-
tomol. Sci. 26: 157-168.
WEBB, S. E. 1991. Management of grape root borer in
Florida with a pheromone. Proc. Fla. State Hort. Soc.
104: 3-5.
WEBB, S. E., R. K. SPRENKEL, AND J. L. SHARP. 1992.
Seasonal flight activity of grape root borer (Lepi-
doptera: Sesiidae) in Florida. J. Econ. Entomol. 85:
2161-2169.
WEIHMAN, S. W., AND O. E. LIBURD. 2006. Mating dis-
ruption and attract-and-kill as reduced-risk strate-
gies for control of grape root borer, Vitacea
polistiformis (Lepidoptera: Sesiidae) in Florida vine-
yards. Florida Entomol. 89: 245-250.







Florida Entomologist 90(3)


September 2007


RED OAK BORER (COLEOPTERA: CERAMBYCIDAE) FLIGHT TRAPPING
IN THE OZARK NATIONAL FOREST, ARKANSAS

MELISSA K. FIERKE AND FRED M. STEPHEN
Department of Entomology, University of Arkansas, AGRI Rm. 319, Fayetteville, AR 72701

ABSTRACT

High population densities of red oak borer, Enaphalodes rufulus (Haldeman), are believed
to be a major contributor to recent widespread oak mortality in the Ozark Mountains of Ar-
kansas. The intent of this research was to expand our knowledge on specific aspects of this
insect's biology, life history, and distribution by trapping flying adults in the Ozark National
Forest during emergence of 2001, 2003, and 2005 cohorts. Passive flight intercept trap
catches revealed that preferred flight height and thus optimal trap placement for monitor-
ing populations was close to the base of the dominant/co-dominant northern red oak, Quer-
cus rubra L., canopy. Flight periods and peak flight were different in 2001 and 2003.
Emergence occurred a week earlier in 2001 vs. 2003. Peak flight occurred over a 3-week pe-
riod from mid Jun to early Jul in 2001 in contrast to 2003 peak flight, which occurred the
first week of Jul. Male to female ratios were 1.9:1 and 1.4:1 for 2001 and 2003, respectively.
In 2003, sex ratios varied significantly among 5 topographic positions evaluated, north,
south, east, and west-facing benches and ridges. Total numbers of red oak borers caught var-
ied both spatially and temporally from cohort to cohort in traps placed in 3 different areas
on 5 topographic positions. This research should facilitate efficient future monitoring of
adult red oak borer and form a basis for investigating stand and landscape-level factors af-
fecting population densities throughout the forest.

Key Words: Enaphalodes rufulus, insect trapping, Quercus, native insect pest, oak decline

RESUME

Se cree que las altas densidades de la poblaci6n del barrenador del roble rojo, Enaphalodes
rufulus (Haldeman), es un contribuidor important en la reciente mortalidad extensive de
los robles en las Montaias de Ozark en el estado de Arkansas (EEUU). El prop6sito de esta
investigaci6n fue para ampliar el conocimiento de aspects especificos de la biologia, historic
de vida y distribuci6n de este insecto por medio de la capture de los adults volantes en el
Bosque Nacional de Ozark durante la emergencia de los cohortes de los aios 2001, 2003 y
2005. Los individuos volantes interceptados y capturados en trampas pasivas de revelaron
que la altura de vuelo preferida y la colocaci6n 6ptima de las trampas para el monitoreo de
la poblaci6n fue cerca del base de la copa (parte superior del arbol) del arbol dominante/co-
dominante roble rojo del norte, Quercus rubra L. Los periods de vuelo y el pico en el numero
de individuos volantes fue diferente en los aios 2001 y 2003. La emergencia ocurri6 una se-
mana mas temprana en 2001 versus el 2003. El pico en numero de individuos volantes ocu-
rri6 en el period que va desde el medio de junio hasta la primera parte del julio en el 2001,
a diferencia del el pico de individuos volantes en 2003 que ocurri6 en la primera semana de
julio. La proporci6n del numero de machos a hembras fue 1.9:1 y 1.4:1 para los aios 2001 y
2003 respectivemente. En el 2003, la proporci6n de machos a hembras varia significativa-
mente entire las 5 posiciones topograficas evaluadas, los bancos y los lomos dirigidos hacia
el norte, sur, este y oeste. El numero total de los barrenadores del roble rojo capturados varia
espacialmente y temporalmente de cohorte a cohorte en trampas colocados en 3 areas dife-
rentes en 5 posiciones topograficas. Esta investigaci6n facilitard la eficiencia del monitoreo
de los adults del barrenador de roble rojo en el future y da una base para investigar los fac-
tores a nivel de las areas uniforms manejadas del bosque y terreno afectando la densidad
de la poblaci6n a travez del bosque.


An oak decline event occurring in the Ozark ring only in odd-numbered years (Hay 1969).
National Forest of northern Arkansas (Starkey et These beetles normally occur at low population
al. 2000) is unique as red oak borer, Enaphalodes densities reported at fewer than 1 adult emerging
rufulus (Haldeman) (Coleoptera: Cerambycidae), per tree from mature oaks in Ohio (Hay 1974).
has been implicated as a major contributor to tree Populations associated with the current oak mor-
mortality (Stephen et al. 2001; Fierke et al. tality event are many times higher, averaging 45
2005a, b). Red oak borer is a native long-horned late-stage red oak borers per tree, range 0-130, in
wood-boring beetle that exhibits a rare synchro- 24 whole trees dissected just before adult emer-
nous 2-year life cycle with adult emergence occur- gence in 2003 (Fierke et al. 2005a, b).







Fierke & Stephen: Red Oak Borer Flight Trapping


The life cycle of red oak borer has been de-
scribed previously in Ohio and Kentucky oaks
(Hay 1969) and is similar in Arkansas (Fierke et al.
2005a). Reports of economic losses associated with
wood degrade due to red oak borer are numerous
(Hay & Wootten 1955; Morris 1964; Donley et al.
1974; Feicht & Acciavatti 1985). "Black check"
caused by larvae of large beetles was described in
Ozark Mountain oaks as a very common defect in
the early 1920s (Snyder 1927). Red oak borer at-
tack and emergence holes also serve as entryways
for decay fungi (Berry 1978) and other organisms,
e.g., carpenter worms (Lepidoptera: Cossidae), and
oak timber worms (Coleoptera: Brentidae), which
cause additional wood degrade and further de-
crease timber values (Donley & Acciavatti 1980).
Research objectives were to determine adult
flight height, flight period, peak flight, and ratio
of adult males to females. Determination of pre-
ferred flight height as well as peak flight period
should enhance trapping of adult beetles in future
research as well as being valuable for monitoring
population densities in high risk areas. Addi-
tional objectives were to investigate distribution
and densities of flying adults on 5 topographic po-
sitions, north, south, east, and west-facing
benches and ridges, in 3 areas of the Ozark Na-
tional Forest that recently experienced high lev-
els of red oak borer-induced tree mortality. Eluci-
dation of adult red oak borer abundance and dis-
tribution should enhance efforts to understand
the recent outbreak and to predict stands/areas
that may be susceptible to future outbreaks.


MATERIALS AND METHODS

Study Areas

Study areas were located in northern Arkan-
sas within the Boston Mountain physiographic
section of the Ozark Plateau. Boston Mountain el-
evations range from 370 to 700 m and the land-
scape is characterized by deep valleys, steep
ledges, and cliffs with rock formations of lime-
stone, sandstone, and shale (Fenneman 1938; Ad-
amski et al. 1995). Passive flight intercept traps
were located in 3 general geographic areas expe-
riencing high levels of tree mortality (UTM Zone
15-N NAD83: Fly Gap-0431660, 3954978, White
Rock-412668, 3949429 and Oark-0450792,
3952369) in the Boston Mountain, Pleasant Hill,
and Buffalo Ranger Districts of the Ozark Na-
tional Forest (Fig. 1). Flight was monitored for 3
red oak borer emerging adult cohorts during the
summers of 2001, 2003, and 2005.

Traps

Handmade passive flight intercept traps had flat
60 x 60 cm plywood tops mounted to 56 x 3-cm slot-
ted wood centerpieces within which four 25 x 56-cm
rectangles of clear plexiglass fit. Fifty cm diameter
handmade galvanized steel funnels were attached
to the bottom of traps and emptied into 0.95-L glass
jars. Collecting jars were filled to 1/3 of volume with
a 1/3 dilution of propylene glycol/water as a preser-
vative. These traps were used only in 2001.


Fig. 1. Research sites were located in 3 general areas of the Ozark National Forest in Northwest Arkansas ex-
periencing oak mortality. Circles indicate 5 stands in each area on 5 topographic positions; north, south, east, and
west-facing benches, and ridges. Ridge, south, and west plots at Fly Gap appear to overlap due to their proximity
and scale of map.







Florida Entomologist 90(3)


Commercially available black passive flight-
intercept traps (IPM Technologies, Portland, OR)
were made of corrugated plastic with 81 x 30.5-cm
folding-interlocking vanes. Clear plastic 1-L hold-
ing cups were attached to the bottom of traps and
contained ~300 mL of a 1/3 dilution of propylene
glycol/water as the liquid preservative. This type
of trap was used as black cross-vane traps with
cylindrical silhouettes and wet cups have been
shown to be more effective than other methods of
trapping for large wood-boring insects (McIntosh
et al. 2001; Morewood et al. 2002; de Groot & Nott
2003; Sweeney et al. 2004).

2001

Ten hand-made passive flight-intercept traps
and 1 IPM trap were placed in mixed hardwood
forests experiencing elevated levels of oak mortal-
ity in the Fly Gap area. Three traps were installed
on 1 Jun, 4 on 12 Jun, and 4 more on 15 Jun. Adult
flight was monitored weekly or more often from 8
Jun through 24 Aug to determine 2001 flight pe-
riod, peak flight, and sex ratios. Ultraviolet lights
were installed in two traps on 31 Jul and in a
third trap on 17 Aug. Sex, based on antennal
length, was noted for all beetles. Female antennae
are about body length and male antennae are
about twice body length (Solomon 1995). Prelimi-
nary data on this flight period were reported by
Stephen et al. (2001).

2003

To determine optimal trap placement, 5 verti-
cal trapping systems were constructed with 25
IPM traps placed just below the northern red oak
canopy layer in stands located on 5 topographic
positions in the Fly Gap area. This allowed evalu-
ation of differences in trends to catch beetles rel-
ative to the base of the dominant/co-dominant
northern red oak canopy on the different topo-
graphic positions. The trapping system was hung
from the lowest large branches of northern red
oaks that allowed access to raise and lower traps
without undue interference from mid and under-
story trees. The base of the canopy was chosen as
the point of origin as field observations indicated
that these insects spend their day within the can-
opy with nocturnal flight being initiated from
there. This made a ground-based point of origin il-
logical considering beetles likely never encounter
the forest floor throughout their entire life cycle.
Height of limbs ranged from approximately 17-
20 m for the 5 stands and was assumed equiva-
lent to the base of the dominant/co-dominant
northern red oak canopy layer. Base of the canopy
layer was defined as an imaginary horizontal line
across tree boles to represent the average live
dominant/co-dominant crown base (USDA FS
2001).


Vertical trapping systems were constructed
with 5 interconnected IPM traps with 2-m spacing
intervals totaling about 15 m in length. A pulley
was installed at the top of the system, the system
was raised to the base of the canopy, and the bot-
tom trap was tied off to stabilize the system. Insect
catch data for this experiment are reported rela-
tive to the height of the limb on which traps were
hung, e.g., 1 m below the limb. Height of the trap-
ping systems above the forest floor varied and spe-
cific heights relative to the forest floor in the differ-
ent stands were not measured. Vertical trap sys-
tems were installed in ridge, south, and west plots
on 22 Jun, the east plot on 24 Jun, and the north
plot on 25 Jun. Data were collected weekly or more
often beginning 27 Jun and ending 28 Aug.
To determine spatial distribution and abun-
dance among 5 topographic positions, 15 IPM
flight intercept traps were hung 2-4 m above the
forest floor (approximately 15-18 m below tree
canopy) in 15 stands on 5 topographic positions in
the 3 geographic areas. Trap data were collected
weekly beginning 13 May through 28 Aug. Indi-
vidual traps in Fly Gap plots were incorporated
into vertical trapping systems and data from the
bottom trap in the vertical systems (2-4 m above
the forest floor) were used in data analyses as
those traps were comparable to traps hung 2-4 m
above the forest floor in other areas. All trap data
from 2003 were used to evaluate flight period,
peak flight, and sex ratios.

2005

To compare number of adult red oak borers
trapped on ridges among the 3 areas, 3 IPM flight
intercept traps were hung just below the northern
red oak canopy (approximately 16-19 m above the
ground) in each of the 3 ridge stands. Trap data
were collected beginning 5 Jul and ending 18 Aug.

Data Analysis

Data were analyzed with JMP 6.0 (SAS 2006).
ANOVA, LSD Means Comparison Student's t-
test, and regression analyses were used to test for
significance at a = 0.05. Distribution and abun-
dance trapping data from 2003 were blocked by
geographic area to accommodate environmental
heterogeneity (Potvin 2001) so that differences in
trap catches among the 15 plots on the 5 topo-
graphic positions provided a measure of topo-
graphic position effect. Data were checked for nor-
mality with the Shapiro-Wilk W test and then, if
necessary, square root transformed to improve as-
sumptions of normality (McCune and Grace
2002). Chi square analysis was used to test for dif-
ferences in sex ratios among trap heights and to-
pographic positions.
Some data were lost early in the summer of
2003 in the White Rock and Oark east plots due to


September 2007







Fierke & Stephen: Red Oak Borer Flight Trapping


damage from black bears, Ursus americanus Pal-
lus, and the bottom 2 traps in the Fly Gap ridge
plot were blown down once by high winds. Bear
damage occurred before adult flight and traps
that were blown down were after peak flight.
Traps were replaced and it was unlikely that
losses appreciably impacted results.

RESULTS

Flight Period and Sex Ratios

One hundred and seventy-two adult red oak
borers were caught in 11 traps in 2001. The flight
period began in mid-Jun with the first beetles
caught the week of 15 Jun (Fig. 2A). The last bee-
tle caught in traps without lights was 7 Aug. Nine
of the last 10 beetles trapped after 31 Jul were in
traps with UV lights. Peak flight was over a 3-
week period from the third week of Jun through
the first week of Jul. Average male to female ratio
was 1.9:1 (111 to 52) for traps without lights.
One hundred and seventy-six beetles were
caught in 35 traps in 2003 with flight beginning
late Jun and ending mid Aug (Fig. 2B, C). Peak
flight was the first week of Jul with 68 beetles or
38% of the total catch occurring within that week.
Average male to female ratio was 1.4:1 (103 to 73).
Considering beetles caught in vertical systems in
Fly Gap, sex ratios were significantly different
from 1:1 on 2 of the 5 topographic positions (Table
1), and there were significant differences among
the different topographic positions (X2 < 0.0001).
There were no differences in sex ratios among dif-
ferent trap heights (X2 = 0.1994) and no interac-
tion effect occurred between trap height and topo-
graphic position (X2 = 0.5926).

Flight Height

One hundred forty-five red oak borers were
caught in 25 vertical flight traps in 2003. Vertical
trapping systems on the ridge caught 83 beetles,
26 were caught on east-facing, 22 on south-facing,
9 on west-facing, and 5 on north-facing plots.
There was a significant negative linear trend in
numbers of beetles caught in traps down from the
base of the forest canopy for trapping systems in
the ridge, north, south, and west-facing stands
(F, > 10, P < 0.05) (Fig. 3). Trap catches from the
east stand did not exhibit a significant trend with
linear, transformed, or second degree polynomial
trendlines.

Abundance and Distribution

Thirty-two red oak borers were caught in traps
placed 2-4 m off the ground in 2003. Trap catches
varied among topographic positions based on
analysis of transformed data (F,14 = 4.2, P = 0.04).
More beetles were caught in traps hung in south


11









15,lun 22Jun 30-Jun 6Jiul


SMale 0 Female


7 11 B 11
r-{J Vb = 1n
13-Jul 20-Jul 27-Jul 3-Au 10-Aug
2001


25 25
-A n1
6-Aug 14-Aug


15 15 15
28-Jul 4-Auf 11-Aug


2003
Fig. 2. Number of beetles caught by sex in (A) 2001
traps at Fly Gap, (B) 2003 vertical traps at Fly Gap, and
(C) 2003 abundance and distribution traps in three ar-
eas. Number of traps given above bars. 2001 traps mon-
itored 8 Jun to 24 Aug (data from traps with lights were
not included). 2003 vertical traps monitored 25 Jun to
28 Aug and distribution traps from 13 May to 28 Aug.


(14) and ridge (10) plots than those hung on east
(1) and north-facing (1) benches. West-facing
benches had intermediate beetle catches (6). The
area blocking term was significant (F214 = 13.1,
P = 0.003) with White Rock traps catching 22 bee-
tles, Oark 9, and Fly Gap 1.
Twenty-seven beetles (16 males, 10 females,
and 1 unknown (broken antennae)), were caught
in 9 traps hung on ridge plots in the three areas in
2005. Trap catches varied among ridges in the
three areas based on analysis of transformed data
(F,,8 = 13.4, P = 0.0061). Traps placed in the Oark
ridge plot caught 19 beetles, Fly Gap 3, and White
Rock 5.

DISCUSSION

Flight periods in 2001 and 2003 were similar at
9 and 8 weeks, respectively, but the beginning of
the flight period started a week later in 2003. The
2001 distribution is also different from 2003 as
numbers increased sharply, remained high for 3
weeks and dropped off abruptly the second week of


.Jun 1-Ju| @Jul flf-J, W
2S

Is




274-Jun 1Jul a-Jul 154-Jul 23-Jul4
Is


El 1w 1 1
23-Jun 30Jun 7-Jul 14-Jul 21-Jul







Florida Entomologist 90(3)


TABLE 1. NUMBER OF RED OAK BORER CAUGHT BY SEX IN VERTICAL TRAPS ON 5 TOPOGRAPHIC POSITIONS IN THE FLY
GAP AREA OF THE OZARK NATIONAL FOREST.

Ridge East South West North

Male 63 11 6 4 1
Female 20 15 16 5 4
Sex ratio 3.2:1 0.7:1 0.4:1 0.8:1 0.3:1
X2 <0.0001 0.4319 0.0299 0.7389 0.1650

%' < 0.05 indicates sex ratios significantly different from 1:1.


Jul. In 2003 the first males were caught the third
week of Jun, numbers peaked the first week of Jul
and decreased steadily to the second week of Aug.
Temperature controls developmental rates of
many organisms, including insects, many of
which require a certain amount of heat to develop
from one point in their life cycle to another (Shel-
ford 1927; Howe 1967; Galford 1974; Wagner et al.
1987). Red oak borer late-stage larvae pupate in
late May to early Jun with adult eclosion in mid-
Jun to early Jul in Arkansas oaks (Fierke et al.
2005a). An investigation of accumulation of de-
gree-days with a base temperature of 65 from
2 nearby weather stations, Hunstville and Deer,
AR, revealed that >30% more degree days accu-
mulated by end of May 2001 than by the same
time in 2003. The developmental threshold is un-
known for red oak borer; however these calcu-
lated differences in degree-day accumulation may
be an explanation for earlier flight in 2001.
Adult emergence of 3 red oak borer cohorts
from Ohio oaks occurred over 5-6 weeks starting
the 3rd or 4th week of Jun and lasting through the
3rd or 4th week of Jul (Hay 1972). This emergence
period corresponds nicely to our flight period as
beetles normally live 2 to 3 weeks (Donley 1978).
A 1:1 sex ratio was observed in Ohio beetles with
males outnumbering females in the first 2 weeks


SRidge pe -o l.P tofl
*soun (t.sQ&.P- O.2)
wA Y Vf TSn.P ao05
Eas (le a U&Pa19
*NOeS (W LS&P-1o04


0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Trap Location (m below base of tree canopy)

Fig. 3. Trap data from passive flight intercept traps
located vertically down from the base of the tree canopy
in the Fly Gap area of the Ozark National Forest, Ar-
kansas. Data collected from 25 Jun to 28 Aug 2003.


and females outnumbering males in the last 2
weeks of the emergence period (Hay 1972). This is
lower than the 1.4:1 ratios seen in Arkansas bee-
tles in 2003, and much lower than the 1.9:1 ratio
in 2001. This indicates that there may be other
unexplained factors involved in the skewed sex
ratios documented in Arkansas forests.
One possible explanation may be that males
fly more than females in search of mates. Hay
(1972) installed cages on active attack sites and
so ratios were for emerging rather than flying
adults. Another explanation may be increased
male flight associated with higher red oak borer
population densities. Numbers of red oak borer
caught in the Fly Gap area in 2001 (174) were
high compared with 2003 catches in the same
area (145). There were fewer traps in 2001 vs.
2003 (11 vs. 25, respectively) and although hand-
made traps had a little more functional surface
area (2,800 vs. 2,470 cm2), all 2001 traps were
hung about 2 m from the ground while some of
the 2003 traps were hung at more "optimal"
heights relative to the dominant/co-dominant oak
canopy. Field personnel also noted abundant red
oak borer adults on understory vegetation in
2001, an observation not made during 2003.
Ridge plots have higher within-tree beetle densi-
ties (Fierke et al. 2007) and sex ratios based on
trap data on the different topographic positions
appeared to be very different with much higher
male to female sex ratios in ridge traps (3.2:1)
compared with traps on other topographic posi-
tions (0.3-0.8:1). Flight period and sex ratios were
not specifically reported for 2005 as some traps
were not in place until the end of Jun, however,
considering the data gathered, a 1.6:1 male to fe-
male ratio was found on ridge plots.
Installation of black lights in traps late in the
field season in 2001 indicated that doing so likely
increased number of beetles caught. This sug-
gested that flying adults are attracted to light and
was corroborated by lab personnel, who used
black lights and white sheets to capture large
numbers of adults for genetic analyses and lab-
rearing of eggs and neonates. This knowledge
should prove useful trapping adults in low den-
sity areas to ascertain presence/absence.


September 2007







Fierke & Stephen: Red Oak Borer Flight Trapping


Previous research has shown that changing
trap height in both agricultural and forested sys-
tems results in varying catches of different organ-
isms (Moeed & Meads 1984; Schulze et al. 2001;
Su & Woods 2001; Botero-Garces & Isaacs 2003).
There were significant linear trends in number of
red oak borers caught relative to vertical trap lo-
cation below the dominant/co-dominant tree can-
opy with 4 out of 5 trapping systems having in-
creased catches closer to the canopy base. This
suggests that optimal trap placement for monitor-
ing adult red oak borer populations should be
close to the base of the tree canopy. This supports
field observations that beetles likely spend their
days in or near the canopy with nocturnal beetle
flight initiating from there and remaining just be-
low the canopy until beetles land on other trees.
Nocturnal field observations revealed beetles fre-
quently walked down and up tree boles with fe-
males spending a great deal of time probing bark
crevices with their ovipositors and males moving
relatively rapidly along tree boles with antennae
held out in front of their bodies.
Adult red oak borer population densities var-
ied both spatially and temporally in study stands.
In 2003, more adult beetles were captured in
traps located on ridges and south-facing benches
compared with traps on east and north-facing
benches. These data were corroborated by vertical
flight trapping data as there were obvious differ-
ences in y-intercepts of linear trendlines for
height models on different topographic positions
(Fig. 3). Adult flight data presented here on differ-
ences in topographic population densities also are
supported by other lab research showing higher
red oak borer population densities on ridge topo-
graphic positions (Fierke et al. 2007).
Flight catches in 2005 were much lower than
expected. This was likely due to a precipitous de-
crease in population densities. This decrease was
documented through intensive sampling of whole
trees conducted within the same plots (unpub-
lished data) but may also be inferred from trap-
ping data presented here as there was a large de-
crease in the number of borers caught in the Fly
Gap ridge plot between 2003 and 2005. In 2003,
one trap just below the canopy in the vertical
trapping system caught 25 beetles whereas only 2
borers were caught in 3 traps similarly hung in
the same plot in 2005.
This research should enable efficient trap place-
ment and timing of trapping efforts for future
monitoring efforts. More information is needed to
understand influences of population densities on
variation in male to female flight ratios. In-
creased knowledge of relative distribution and
population densities in different areas and in dif-
ferent forest stands should provide insight re-
garding important factors influencing population
densities and may prove helpful in predicting
future outbreaks.


ACKNOWLEDGMENTS

The authors thank Stephen Wingard for ideas and
field assistance as well as Dana Kinney, Vaughn Salis-
bury, Brent Kelley, Leah Chapman, Larry Galligan,
Josh Jones, and Jarrett Bates. We thank Brent Kelley
and Vanessa Ware for helpful comments during manu-
script preparation and Andy Mauromoustakos for sta-
tistical advice. Funding was provided by the University
of Arkansas Agricultural Experiment Station and
grants from the USDA Forest Service, Southern Re-
search Station, and USDA Forest Service, Forest Health
Protection STDP and FHM programs.

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Florida Entomologist 90(3)







Nava et al.: Biology of Zaprionus indianus in Brazil


BIOLOGY, THERMAL REQUIREMENTS, AND ESTIMATION OF THE NUMBER
OF GENERATIONS OF ZAPRIONUS INDIANUS (DIPTERA: DROSOPHILIDAE)
FOR THE MAIN FIG PRODUCING REGIONS OF BRAZIL


DORI E. NAVA', ADRIANA M. NASCIMENTO2, CESAR P. STEIN3, MARINEIA L. HADDAD2,
JOSE M. S. BENTO2 AND JOSE R. P. PARRA2
'Embrapa Clima Temperado, Rodovia Br 392 Km 78, 9 Distrito, Caixa Postal 403, 96001-970;
Fone +55 53 3275 8198, Pelotas-Rio Grande do Sul, Brazil;
Corresponding author: nava@cpact.embrapa.br

2Depto. de Entomologia, Fitopatologia e Zoologia Agricola, ESALQ/USP, Caixa Postal 9, 13418-900;
Fone +55 19 3429 4199, Piracicaba-Sao Paulo, Brazil

'Instituto Agronomico, C.P.D. de Fitossanidade-Entomologia, Caixa Postal 28,
13001-970-Campinas, Sao Paulo, Brazil

ABSTRACT

The African fig fly, Zaprionus indianus, was first recorded in fig orchards in Brazil in 1999,
at the Valinhos region, in the State of Sao Paulo. In view of the scarcity of biological data on
this insect in Brazil, the objective of this research was to study the biology, temperature re-
quirements, and to estimate the number of Z. indianus annual generations for the main fig
producing regions of Brazil, aimed at controlling the pest. The insects were reared on artifi-
cial diet at constant temperatures of 18, 20, 22, 25, 28, 30, and 32 C, relative humidity 80
10%, and 14-h photophase. Duration of the development stages was variable with tempera-
ture, being longer at the lower temperatures. Temperature also influenced viability during
the larval and pupal stages and the egg-adult biological cycle, with significant differences
between 32 C and the other temperature conditions. The lower temperature development
threshold (TT) and thermal constant (K) values for the egg, larval, and pupal stages were 9.7
and 10.5; 9.2 and 148.6, and 10.7 C and 66.25 DD, respectively, for a total thermal constant
of 262.2 DD for the egg-adult biological cycle. Based on a lower temperature development
threshold of 7.9 C., Z. indianus may have up to 16.6, 12.4, and 12.7 annual generations in
the regions of Valinhos-SP, Pelotas-RS, and Sao Sebastido do Paraiso-MG, respectively,
which are traditionally known as fig producing regions in the country.

Key Words: fig fly, temperature requirements, exotic pest, bioecology

RESUMO

A mosca-africana-do-figo Zaprionus indianus, foi registrada, pela primeira vez, em pomares
de figo no Brasil em 1999, na regido de Valinhos, Sao Paulo. Tendo em vista a escassez de da-
dos biol6gicos sobre o inseto no Brasil, o objetivo da pesquisa foi estudar a biologia e estimar,
a partir das exig6ncias t6rmicas, o numero de geracoes anuais de Z. indianus para as prin-
cipais regioes produtoras de figo do Brasil, com vistas ao seu control. Foram criados insetos
em dieta artificial e nas temperatures constantes de 18, 20, 22, 25, 28, 30 e 32 C, umidade
relative de 80 10% e fotofase de 14 h. A duracao das fases do desenvolvimento foi variavel
com a temperature, sendo maior nas temperatures menores. A temperature tamb6m in-
fluenciou a viabilidade das fases de larva e pupa e do ciclo biol6gico ovo-adulto, havendo di-
ferencas significativas entire 32 C e as demais condigoes t6rmicas. O limiar t6rmico inferior
de desenvolvimento (Tb) e a constant t6rmica (K) para as fases de ovo, larva e pupa foram
de 9,7 e 10,5; 9,2 e 148,6; 10,7 C e 66,25GD, respectivamente, totalizando para o ciclo biol6-
gico (ovo-adulto) uma constant t6rmica de 262,2GD, a partir de um limiar t6rmico inferior
de desenvolvimento de 7,9 C. Z. indianus poderd dar at6 16,6; 12,4 e 12,7 geracoes anuais
para as regioes de Valinhos-SP, Pelotas-RS e Sao Sebastido do Paraiso-MG, respectivamente,
tradicionalmente conhecidas como produtoras de figo no pals.

Translation provided by the authors.



The African fig fly, Zaprionus indianus Brazil in 1999, causing yield losses of up to 50%
(Diptera: Drosophilidae) was first reported on fig (Vilela 1999). This drosophilid measures between
plants (Ficus carica L., Moraceae) in Valinhos, 2.5 and 3.0 mm in length, and is brown in color,







Florida Entomologist 90(3)


with red eyes. The dorsal region of the head and
thorax has two longitudinal silvery-white stripes,
between which run narrow black stripes. The eggs
are milky and contain a filament, and generally
are laid in small masses by more than one female
into the ostiole of figs near maturation (Raga
2002).
Z. indianus is a semicosmopolitan fly which,
apparently by human mediation, has spread
across tropical regions in recent decades (Vilela et
al. 2000; Bachli 1999-2005; van der Linde et al.
2006). It is probably of African origin, where it has
been recorded in fruits of 74 plant species (Vilela
1999). In Brazil it has been recorded in several
hosts (Souza Filho et al. 2000), and it became a
pest in fig and required the use of chemical con-
trol by fig growers. The vast majority of droso-
philid species are associated with bacteria and
yeasts; Candida tropicalis has been identified in
figs infested with Z. indianus collected in Valin-
hos, SP. This yeast causes increasing fig deprecia-
tion during production and trade, and attracts
adults of the pest for feeding and oviposition
(Raga 2002).
Chemical control was initially adopted but
proved to be ineffective and brought about a sig-
nificant increase in production costs. In addition,
products registered for this pest are nonexistent,
and there are barriers imposed by importing
countries, especially from Europe, preventing im-
port of fruits that present residues of unregis-
tered chemical products (Vilela et al. 2000).
Within this context, several other manage-
ment techniques are being adapted and devel-
oped, such as the use of fig ostiole protectors,
which make it difficult for Z. indianus to oviposit
and feed (Raga et al. 2003), use of attractive
traps, and elimination of food substrates in the
area and surroundings (Raga & Souza Filho
2003). Additionally, the collection and identifica-
tion of natural enemies may facilitate the devel-
opment of applied biological control (Marchiori &
Silva 2003).
An important measure to improve pest control
effectiveness is to establish prediction models for
their occurrence based on temperature because it
directly influences insect biology, metabolism, re-
production, and longevity (Sharpe & DeMichele
1977). Ecological zoning, based on temperature
requirement values, indicates the most favorable
regions for development of the biological potential
of pests. In turn, population monitoring of the
pest in the regions that are most favorable and
with the fastest development rates increases the
chance of determining when a pest outbreak will
occur (Cividanes 2000).
The goals of this work were to study the devel-
opment ofZ. indianus at different temperatures,
determine its temperature requirements in the
laboratory, and estimate the number of genera-
tions for the main fig producing regions of Brazil,


which may facilitate the establishment of pest
management strategies.

MATERIALS AND METHODS

Stock Rearing

Zaprionus indianus started from insects that
came from the Entomology laboratory at Instituto
Agron6mico de Campinas. One-liter glass cages
were used for rearing, with lids adapted with a
nylon screen to allow aeration. The diet used for
larval development and adult feeding consisted of
brewer's yeast (7.5 g), banana (205 g), agar (5 g),
nipagin (methyl parahydroxybenzoate) (1 g), Te-
trex (tetracycline) (0.05 g), and water (500 mL),
and was adapted from diets for drosophilids
(Singh 1977). The insects were maintained in the
laboratory at a temperature of 27 + 2C, relative
humidity of 80 10%, and 14 h photophase.

Biology at Different Temperatures

In order to obtain eggs, approximately 300
adults were placed in a plastic cage (50 x 40 x 60
cm). An acrylic plate (2 x 7 cm) was placed inside
the cage containing liquefied mango pulp to serve
as substrate for oviposition. Every 15 min, the
plate was replaced with a fresh one, and the sub-
strate was inspected with a stereoscopic micro-
scope to remove the eggs, which were transferred
to Petri dishes (2 x 10 cm). In order to retain mois-
ture and facilitate egg visualization on the plates,
a piece of black filter paper (dyed with Guarany
fabric dye) was placed on the bottom of the Petri
dish and moistened as needed. Twenty eggs were
transferred to each Petri dish, for a total of 200
eggs. Each set of eggs was placed in climatic
chamber (Fanem, Camara Incubadora BOD.
Mod. 347 CD, Sao Paulo, Brasil) held at 18, 20, 22,
25, 28, 30, and 32C, 80 + 10% relative humidity,
and 14-h photophase. Evaluations were per-
formed hourly (12 h), and duration and viability
were recorded by counting the number of larvae
hatched at each temperature.
Vilela's diet (1999) inside glass vials (2 x 8 cm)
was used to study development at the larval
stage. Ten larvae with ages up to 30 min after
hatching were transferred to each vial, totaling
300 larvae at each temperature. Evaluations
were performed daily, and the duration of the lar-
val stage was recorded.
Acrylic plates (6.0 x 2.0 cm) containing moist-
ened filter paper on the bottom were used for the
development of the pupal stage. Pupae up to 24 h
of age were used, totaling 200 insects at each tem-
perature.
Bioassays for the egg, larval, and pupal stages
were conducted in a completely randomized de-
sign, constituted of 7 treatments (temperatures)
with 20, 30, and 20 replications, for egg, larval


September 2007







Nava et al.: Biology of Zaprionus indianus in Brazil


and pupal stages, respectively. The duration and
viability data were submitted to analysis of vari-
ance (ANOVA) and the means were compared by
Tukey test (P < 0.05).

Determination of Temperature
Requirements and Number of Generations

Based on the duration data for the egg, larval,
and pupal stages and for the egg-adult period, the
lower temperature development thresholds (TT)
and thermal constants (K) were determined by
the hyperbole method (Haddad & Parra 1984).
Duration at 32C was not taken into consider-
ation, since it was not statistically different from
duration at 30C (P < 0.05), thus avoiding the lin-
earity of the hyperbolic curve that represented
duration x temperature.
The likely number of annual Z. indianus gen-
erations was estimated based on the number of
degree-d required for development of the insect as
determined in the laboratory, and the tempera-
ture normals of the main fig producing regions
of Brazil (Valinhos-SP, Pelotas-RS, and Sao
Sebastiao do Paraiso-MG).

RESULTS AND DISCUSSION

The biological cycle of the insect (egg-adult)
lasted less than 1 month at the 7 temperature
conditions studied, creating conditions for the oc-
currence of several generations of the pest
throughout the year. It was observed that the du-
ration and viability of the different stages of the
biological cycle varied according to the tempera-
ture (Tables 1 and 2). During the embryonic stage,


there was a shortening of duration with increase
in temperature from 18C (1.23 d) to 30C (0.49
d); at 32C there were no changes in the incuba-
tion stage, showing a tendency of unsuitability of
the egg stage to temperatures higher than 30C
although this value was statistically similar for
28C, 30C and 32C. A high viability was ob-
served at all temperatures except at 32C, with a
much lower value than the values recorded at the
other temperatures (Table 2).
The tendency for negative effect at higher tem-
peratures was observed in the larval stage. The
duration at 32C (9.3 d) was longer than at 30C
(8.6 d) and at 28C (7.8 d), although it was only
statistically different from 28C and not from
30C (Table 1). The negative effect of higher tem-
peratures on larval development can be observed
in the viability as 71.1% of the insects died at
32C (Table 2).
For the pupal stage, a variation from 9.7 d at
18C to 3.2 d at 32C was observed, and the dura-
tion was shorter in the range from 28 to 32C, al-
though no significant differences were observed.
Possibly, temperatures higher than 32C are
harmful to Z. indianus development as the viabil-
ity at this temperature was significantly lower
than the other temperatures, which showed val-
ues higher than 88.6%.
The total duration of the cycle (egg-adult) in-
creased with the decrease in temperature, vary-
ing from 28.8 d at 18C to 12.7 d at 28C (Table 1).
At 30C (13.3 d) and 32C (13.0 d), the duration of
the cycle was longer than at 28C (12.7 d), sug-
gesting that high temperatures are harmful to
development although there were no significant
differences observed (Table 1). For total viability,


TABLE 1. MEAN DURATION AND VARIATION INTERVAL (BETWEEN PARENTHESES) FOR THE EGG, LARVAL, AND PUPAL
STAGES AND BIOLOGICAL CYCLE (EGG-ADULT) OF Z. INDIANUS ON ARTIFICIAL DIET AND AT DIFFERENT TEM-
PERATURES, 80 + 10% RH, AND 14-H PHOTOPHASE.

Duration

Temperature (C) Egg stage (d) Larval stage (d) Pupal stage (d) Biological cycle (d)

18 1.23 + 0.07 a 17.9 + 0.41 a 9.7 0.08 a 28.8 0.68 a
(2.05-0.83) (19.6-13.7) (10.5-6.6) (30.7-23.1)
20 1.00 0.04 b 13.0 0.38 b 6.9 0.19 b 20.9 0.66 b
(1.17-0.53) (15.5-10.8) (8.1-4.2) (25.5-16.1)
22 0.84 0.03 c 11.4 0.20 c 5.4 0.07 c 17.6 0.23 c
(1.01-0.58) (13.5-10.6) (6.4-4.2) (22.3-15.7)
25 0.73 0.03 d 9.9 0.20 d 4.8 0.12 c 15.4 + 0.26 d
(0.88-0.35) (11.3-8.2) (5.5-3.4) (18.3-13.1)
28 0.60 0.03 e 7.8 0.16 e 4.3 0.07 d 12.7 0.21 e
(0.76-0.35) (8.6-6.8) (5.0-3.7) (15.2-11.0)
30 0.49 0.03 e 8.6 0.28 de 3.2 0.11 d 13.3 0.47 e
(0.69-0.22) (10.00-7.30) (3.6-3.0) (14.1-10.9)
32 0.51 0.04 e 9.3 0.29 d 3.2 0.07 d 13.0 0.53 de
(0.74-0.17) (10.0-7.3) (3.4-3.0) (15.0-10.6)

Means followed by the same letter in the columns are not statistically different by the Tukey test (P < 0.05).







Florida Entomologist 90(3)


TABLE 2. MEAN VIABILITY AND VARIATION INTERVAL (BETWEEN PARENTHESES) FOR THE EGG, LARVAL, AND PUPAL
STAGES AND BIOLOGICAL CYCLE (EGG-ADULT) OF Z. INDIANUS ON ARTIFICIAL DIET AND AT DIFFERENT TEM-
PERATURES, 80 + 10% RH, AND 14-H PHOTOPHASE.

Temperature (C) Viability (%)

Egg stage Larval stage Pupal stage Biological cycle

18 86.8 + 4.26 a 74.6 4.6 a 91.4 3.40 a 57.1+ 2.61 a
(100.0-40.0) (90.0-30.0) (100.0-80.0) (64.8-46.7)
20 87.8 + 3.12 a 76.0 + 4.12 a 90.0 5.8 a 65.3 7.5 a
(100.0-70.0) (100.0-40.0) (100.0-60.0) (80.0-36.0)
22 90.3 + 2.01 a 66.0 3.62 a 91.4 2.60 a 59.3 4.8 a
(100.0-70.0) (100.0-50.0) (100.0-80.0) (75.0-43.2)
25 82.5 + 3.3 a 72.0 4.3 a 88.6 3.40 a 49.3 + 9.1 a
(93.0-50.0) (100.0-30.0) (100.0-70.0) (81.0-18.0)
28 84.0 2.7 a 84.6 4.47 a 93.3 4.22 a 62.0 7.7 a
(100.0-66.7) (100.0-50.0) (100.0-70.0) (90.0-32.0)
30 84.2 4.0 a 67.0 7.61 a 91.7 3.07 a 46.0 8.8 ab
(100.0-20.0) (100.0-30.0) (100.0-80.0) (72.9-18.9)
32 25.3 3.22 b 28.9 4.2 b 66.7 8.43 b 15.3 4.8 b
(36.0-3.0) (100.0-50.0) (40.0-10.0) (80.0-30.0)

Means followed by the same letter in the columns are not statistically different by Tukey test (P < 0.05).


it was observed that temperatures higher than
32C are harmful to the development (Table 2).
In general, it can be said that temperatures
near to 28C is the thermal optimum to egg-adult
development of Z. indianus, allowing shorter de-
velopment time and high viability. This tempera-
ture is similar to the findings by Silveira Neto et
al. (1976), who reported that temperatures within
the range of 22 and 28C are optimal for insect de-
velopment in tropical conditions. Furthermore,
the duration and viability results indicate that,
although the diet employed was simple and con-
sisted of components easily purchased in the do-
mestic market, it was suitable for rearing this
pest in laboratory conditions.
In Brazil, Stein et al. (2003) studied the biology
of the insect at 25C, and obtained a duration of
17.1 d for the egg-adult period. In other countries
(Saudi Arabia), Amoudi et al. (1993) verified a
biological cycle of 23 d at 20C for Z. indianus,
higher than the value obtained in the present re-
search (Table 1). Amoudi et al. (1991) recorded
values of 8.1 and 6.9 d at 250 and 30C for the lar-
val stage and 6.9 and 4.7 d for the pupal stage of
this species. These small variations from results
obtained in the present work are probably due to
the different diet used and to different photo-
period conditions. The authors in Saudi Arabia
reared the insect with a 24-h photophase, while in
the present research the insect was reared with a
14-h photophase; in addition, different popula-
tions were studied. It is convenient to highlight
that variations during the egg stage may be re-
lated to the interval between observations, which
was short in the present research (1 h) and longer
in the other studies (Amoudi et al. 1991).


The artificial diet used in this work was pre-
pared with yeast and banana and was suitable for
development for Z. indianus, although 75% via-
bility recognized by Singh (1983) as the percent-
age above which the artificial diet can be consid-
ered suitable was not achieved. The results ob-
tained by C. P. Stein (pers. inform.), who main-
tains laboratory populations since 1999,
demonstrated that the insect does not become
adapted to the diet as verified with other labora-
tory species. In this case, such alterations might
be associated with the effect of high tempera-
tures, causing greater genetic variability in the
population, while temperatures near the opti-
mum reduce variability, increasing viability
(David et al. 1983). In the present case, such effect
was evident for the egg and pupal stages (Table
3). In this research viability ranged from 15.3%
(32C) to 65.3% (20C) (Table 2). The highest mor-
tality occurred in the larval stage in general, with
low mortality values in the egg and in the pupal
stage. Handling adjustments and even replace-
ment of diet components might improve the in-
sect's performance during the larval stage. Stein
et al. (2003) also observed greater mortality in the
larval stage when they reared the pest at 25C;
Amoudi et al. (1991) reared the insect at 35C and
obtained low larval viability (26%). They did not
record pupal development at that condition and
no adults of the species were obtained. The un-
suitability of higher temperatures was clearly
demonstrated for the egg and larval stages and
for the entire cycle ofZ. indianus.
The lower temperature development threshold
(TT) and thermal constant (K) values for the egg,
larval, and pupal stages were 9.7 and 10.5; 9.2


September 2007







Nava et al.: Biology of Zaprionus indianus in Brazil


TABLE 3. TEMPERATURE THRESHOLD (TT), THERMAL CONSTANT (K), REGRESSION EQUATION (1/D), AND COEFFICIENT
OF DETERMINATION (R2) FOR THE EGG, LARVAL, AND PUPAL STAGES AND FOR THE EGG-ADULT PERIOD OF Z.
INDIANUS, UNDER LABORATORY CONDITIONS.

Stages/period TT (C) K (DD) Regression equation R2 P

Egg 9.7 10.5 1/D = 0.095276 t 0.923945 0.9742 0.05
Larva 9.2 148.6 1/D = 0.006727 t 0.062075 0.9804 0.05
Pupa 10.7 66.25 1/D = 0.015094 t 0.161966 0.9392 0.05
Egg-adult 7.9 262.2 1/D = 0.003814 t 0.030169 0.9754 0.05

DD = Growing degree-days.
D = Duration.


and 148.6; and 10.7C and 66.25 DD, respectively.
Therefore, the Tb and K values for the biological
cycle (egg-adult) were 7.9C and 262.2 DD (Table
3, Fig. 1). These TT values differ from those re-
corded for Z. indianus in Saudi Arabia, as 7.7C
for the egg stage, 11.7C for the larval stage, and
8.0C for the pupal stage, totaling 10.1C for the
biological cycle (egg-adult) (Amoudi et al. 1993).
Probably the geographic distribution (Honek
1996) and/or food used in the larval stage (Zeiss
et al. 1996) may have contributed toward these
differences, in addition to the photoperiod and
insect strain used.
These results may contribute to implement
control measures aimed at fig exports. Adults lay
their eggs into the ostiole, and these eggs will orig-
inate larvae in a very short period of time. Thus,
depending on the condition, the larvae will hatch
before arriving at the importing country. Placing
figs at temperatures below the egg stage TT
(9.7C) may kill the embryos and prevent hatching
and damage. According to C. V. Rombaldi (pers.


cu 1.4

1 .2


S0.8
0.
0 0.6
S0.4


comm.), the Roxo-de-Valinhos fig cultivar can
withstand temperatures from 2 to 5C, with a
shelf life of 2 d. Research must be conducted in
this direction, making fig storage compatible with
the mortality of eggs laid, although due to the
short embryonic development period, such mea-
sure only finds application for freshly-laid eggs.
The number of annual Z. indianus generations
varied according to the mean monthly tempera-
ture of each region. Estimated values for the
Valinhos-SP, Pelotas-RS, and Sao Sebastiao do
Paraiso-MG regions were 16.6, 12.4, and 12.7
generations, respectively (Table 4). Although a
succession of generations may occur throughout
the year, in some regions figs are only available
during a certain season. In the other months, Z.
indianus probably feeds on other hosts. Souza
Filho et al. (2000) mentioned that like all droso-
philids, the pest feeds on decaying fruits and
plants, including acerola, banana, cashew, star
fruit, citrus, guava, jaboticaba, mango, peach,
purple mombin, tomato, persimmon, and others.


OEgg Tb-= 9.7; K= 10.5GDD
O Larva Tb = 9,2C K = 1487GDD

APupa Tb = 10.C K = 663GDD

[ Cycle (egg-adult) Th 79C.; K = 262.2GDD


5 10 15 20 25 30
Temperature (C)

Fig. 1. Relation between developmental speed and temperature for the egg, larval, and pupal stages and biolog-
ical cycle (egg-adult) ofZ. indianus reared on artificial diet, RH: 80 + 10%, and 14-h photophase. TT = Temperature
thresholds; K = Thermal constant.







Florida Entomologist 90(3)


September 2007


TABLE 4. TEMPERATURE NORMALS FOR THREE FIG PRODUCING REGIONS IN BRAZIL, CUMULATIVE DEGREE-DAYS, AND
ESTIMATES FOR THE NUMBER OF GENERATIONS OF Z. INDIANUS AS DETERMINED FROM TEMPERATURE RE-
QUIREMENTS.

Sao Sebastiao do Paraiso
Valinhos (SP)1 Pelotas (RS)2 (MG)3

Temperature Cumulative Temperature Cumulative Temperature Cumulative
(C) degree-d (C) degree-d (C) degree-d

Jan 23.5 458.8 23.2 449.5 19.0 319.3
Feb 23.6 417.2 23.0 400.4 18.3 268.8
March 23.0 443.3 21.7 403.0 18.5 303.8
Apr 20.8 363.0 18.5 294.0 17.5 264.0
May 18.4 300.7 15.1 198.0 16.6 244.9
Jun 17.1 252.0 12.4 111.0 15.4 201.0
Jul 16.9 246.0 12.3 105.0 15.3 198.0
Aug 18.5 303.8 13.4 145.7 17.8 282.1
Sep 20.0 339.0 14.9 186.0 18.9 306.0
Oct 21.2 387.5 15.5 210.8 19.7 341.0
Nov 22.0 399.0 19.6 327.0 18.8 303.0
Dec 22.7 434.0 22.0 412.3 18.7 310.0

Total 4,344.3 3,242.7 3,341.9

Thermal constant for Z. indianus 262.2 262.2 262.2

Number of annual generations 16.6 12.4 12.7

Data obtained from 'Departamento de Ciencias Exatas, ESALQ/USP; 'Embrapa Clima Temperado; Epamig.


Therefore, local control measures, including or-
chard cleaning, with elimination of fallen fruit,
may contribute to reduce the population of the
pest. In regions where late prunings are per-
formed, and production occurs almost throughout
the year, problems with the pest are even greater,
such as in the Valinhos region.
Nonetheless, the temperature requirement-re-
lated results obtained are a head start for studies
on the prediction of occurrence of the pest, based on
alert system to be installed in fig producing areas.

ACKNOWLEDGMENTS

We thank agronomist Amauri A. Frizzas and Andr6
G.C. Signoretti and interns Danilo J. Libardi and Andr6
H. Costa for helping conduct the experiments.

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A. RAGA. 2000. Host plants ofZaprionus indianus in
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Florida Entomologist 90(3)


September 2007


PRESENTATION OF ARTIFICIAL DIET: EFFECTS OF COMPOSITION
AND SIZE OF PREY AND DIET DOMES ON EGG PRODUCTION
BY ORIUS INSIDIOSUS (HETEROPTERA: ANTHOCORIDAE)

STEPHEN M. FERKOVICH1, THIRUVENGADAM VENKATESAN2, JEFFREY P. SHAPIRO1 AND JAMES E. CARPENTER3
'Center for Medical, Agricultural, and Veterinary Entomology, USDA, ARS,
1700 SW 23rd Dr., P.O. Box 14565, Gainesville, FL 32604

2Biotechnology Laboratory, Project Directorate of Biological Control,
P. B. No. 2491, H. A. Farm Post, Bellary Road, Bangalore 560024, Karnataka, India

3Crop Protection and Management Research Unit, Agricultural Research Service,
U.S. Department of Agriculture, Tifton, GA 31793-0748

ABSTRACT

The size of prey is critical to the feeding success of any given predator, but the effects of diet
packet size have not been studied. We examined the effects of size of packets (Parafilm
domes) of artificial diet and the size of prey eggs on oviposition and mortality rates of Orius in-
sidiosus (Say). Artificial diet was presented to adult female 0. insidiosus in 10-, 25- and 50-IL
domes for 6 d and rate of oviposition was measured for 24 h. Oviposition was highest after
feeding on the 10-pL domes, decreased slightly on the 25-pL domes, and was significantly re-
duced on the 50-pL domes. The effect of capsule size was negated on the 25-pL and 50-pL diet
domes when the diet was supplemented with E. kuehniella egg protein. Predators were also
fed eggs of 4 species of Lepidoptera, Ephestia kuehniella Zeller, Plodia interpunctella (Hiib-
ner), Helicoverpa zea (Boddie), Spodoptera frugiperda (J. E. Smith), and Heliothis virescens
(Fabricius), ranging in volume from 19 1.7 nL (mean SD) for P. interpunctella to 108 21.3
nL for H. virescens eggs. Oviposition was highest and comparable on the E. kuehniella, P. in-
terpunctella and H. virescens eggs and significantly less on S. frugiperda and H. virescens eggs.
Oviposition positively correlated and mortality negatively correlated with weight-specific pro-
tein contents of the eggs, but neither correlated with egg volume. When all species of eggs were
extracted and combined with diet in domes of constant size and constant protein content, only
extracts ofE. kuehniella and P. interpunctella eggs were more active than diet alone.

Key Words: predator, artificial diet, egg size, prey egg proteins, predator, egg production, nu-
trition, oviposition, diet presentation, diet acceptance

RESUME

El tamaio de la presa es critic para una alimentaci6n exitosa por parte de cualquier depre-
dador, pero los efectos por el tamano de los paquetes de dieta no han estudiado. Examinamos
los efectos del tamano de los paquetes (domos de Parafilm) de dieta artificial y el tamano de
los huevos de la presa sobre las tasas de oviposici6n y de mortalidad de Orius insidiosus (Say).
Una dieta artificial fue presentada a hembras adults de 0. insidiosus en domos de 10-, 25- y
50-pL por 6 dias y la tasa de oviposici6n fue media por 24 horas. La oviposici6n fue mas alta
despu6s de alimentar en domos de 10-pL, en domos de dieta de 25-pL fue menor y fue signi-
ficativamente reducida en domos de dieta de 50-pL. El efecto del tamano de la capsula fue
anulado en domos de dieta de 25-pL y 50-pL cuando la dieta fue suplementada con la adicion
de protein de huevos de E. kuehniella. Los depredadores tambien fueron alimentados con
huevos de 5 species de Lepidoptera, Ephestia kuehniella Zeller, Plodia interpunctella (Hiib-
ner), Helicoverpa zea (Boddie), Spodoptera frugiperda (J. E. Smith) y Heliothis virescens (Fa-
bricius), que varian en cuanto al volume de los huevos de 19 1.7 nL (mean SD) para
P. interpunctella hasta 108 21.3nL para H. virescens. La tasa de oviposici6n fue mas alta y
comparable en huevos de E. kuehniella, P. interpunctella y H. virescens y significativamente
menos en huevos de S. frugiperda yH. virescens. Hubo una correlaci6n positive de oviposici6n
y una correlaci6n negative de mortalidad con el contenido de protein especifico del peso de
los huevos, pero no hubo una correlaci6n en cuanto del volume de huevo en ninguna de las
dos. Cuando los huevos de todas las species fueron extraidos y combinados con la dieta en los
domos de tamaio constant y el contenido de protein constant, solamente los extractos de
huevos de E. kuehniella y P. interpunctella fueron mas activos que sola la dieta artificial.


Artificial diets for predators have been pre- size of the packaged diet. Semi- or fully liquid
sented in several forms without any regard for the diets have been presented within paraffin wax







Ferkovich et al.: Artificial Diet Presentation to Orius insidiosus


capsules (1-2 mm diameter) (Hagen & Hassan
1965; Martin et al. 1978) or encapsulated in Para-
film (Cohen & Smith 1998) for the green lacew-
ing, CL., .... .,I. rufilabris (Stephens). Other di-
ets for C. carnea have been presented on cellulose
sponge (Vanderzant 1969; Hoosegow et al. 1989),
and as uncovered capsules containing petroleum
jelly and paraffin (Venkatesan et al. 2000). Four
methods for presentation of liquid artificial diet
for C. rufilabris (Burmeister) employed capillary
tubes, cellulose sponge cubes, agarose-based jelly
on coverslips, or artificial eggs (paraffin/petrola-
tum droplets on microscope slides) (Greenberg et
al. 1994). The artificial eggs and agarose-based
jelly showed the most promise. Diets for preda-
ceous coccinellids were presented in gelled cubes
(Atallah & Newsom 1966), as a powder, or as dry
pellets (Smirnoff 1958; Niijima et al. 1977; Mat-
suka et al. 1982). Cohen (1985, 1992) and Cohen
& Smith (1998) used Parafilm to seal a semi-
solid diet in the form of a sachet containing 50 mg
of diet for C. rufilabris, and Grenier et al. (1989)
used Parafilm to seal foam cubes soaked with
diet for the polyphagous predator Macrolophus
caliginosus (Wagner). Arijs & De Clercq (2004)
prepared a meat-based diet in cylindrical Para-
film packets (1 cm long, 0.3 cm dia.).
In other studies, the form and materials used
in diet presentation to predators were compara-
ble, while size varied. With Parafilm encapsula-
tion, diet dome-size ranged from 50 pL/diet-dome
for Orius insidious (Say) (Ferkovich & Shapiro
2004) to 40-500 pL/dome for Podisus maculiven-
trus (Say) (Shapiro et al. 2001; Whittmeyer &
Coudron 2001). However, none of these studies
specifically controlled for the effect of capsule
size. We therefore varied the size of Parafilm
diet domes to test its effect.
In nature, prey size has been reported to im-
pact the efficiency of predators (Evans 1976;
Meiracker & Sabelis 1999; Anderson et al. 2001;
De Clercq et al. 2002; Obrycki et al. 2005; Roger
et al. 2000). Because 0. insidiosus preys on the
egg stage of numerous Lepidoptera, we examined
the effect of the size of several species of prey-egg
on oviposition (and presumptive feeding). In addi-
tion, because nutritional or phagostimulatory
value of prey eggs may vary and differentially
affect feeding and fecundity, we determined the
effect of extracts of those eggs by adding them to
artificial diet contained in domes of constant con-
trolled size.

MATERIALS AND METHODS

Insect Rearing

Insects for rearing and experiments were held
in a growth chamber at 25.5 + 1C, 75 5% RH,
and a photoperiod of 15:9 (L:D). A Florida strain
of O. insidious was reared on eggs ofE. kuehniella


(Beneficial Insectary, Redding, CA). The eggs
were received frozen and held at -80C until used.
Insects were held in 1 pint-canning jars covered
with ripstop nylon. The insects were provided wa-
ter every other day as 1.2 mL of Hydrocapsules
(Analytical Research Systems, Gainesville, FL),
0.6 mL (3 mg) of E. kuehniella eggs, and 1 fresh
green bean for oviposition. Green beans with eggs
were removed every other day and placed in new
jars with water beads, E. kuehniella eggs and 2
grains of bee pollen (purchased locally) for first-
instars. Crumpled strips of wax paper were used
as substrates. Following adult eclosion, the green
bean was replaced and all green beans were
checked for eggs every other day. Eggs in the
beans were counted under a stereomicroscope,
and beans with fewer than 400 eggs were added
to each new jar to reduce cannibalism. A camel
hair brush was used to remove the nymphs from
the old green beans and to transfer the adults.

Prey Eggs

Eggs of E. kuehniella Zeller were purchased
from Beneficial Insectary, Redding, CA and
shipped on dry ice. Eggs of Plodia interpunctella
(Hiibner) were obtained from a laboratory colony
of P interpunctella reared as described by Sil-
hacek & Miller (1972). Eggs of Helicoverpa zea
(Boddie), Spodoptera frugiperida (J. E. Smith),
and Heliothis virescens (Fabricius) were reared as
described by Burton (1969).

Artificial Diet

Diet was prepared under aseptic conditions in
a clean room and encapsulated in stretched Para-
film with a diet encapsulation apparatus (Ana-
lytical Research Systems, Gainesville, FL) as de-
scribed by Ferkovich & Shapiro (2004). We refer
to resulting capsules as domes, because they are
hemispherical in shape. The original diet was pre-
pared as described by Weiru & Ren (1989) for
rearing Orius sauteri (Poppius) and consisted of
397 mg brewers yeast, 39.7 mg sucrose, 208 mg
soy protein acid hydrolysate, 5.0 mg of 99% palm-
itic acid (all from Sigma, St. Louis, MO), 49.5 mg
chicken egg yolk, and 99.2 mg honey brought to
1.2 mL with distilled water. Palmitic acid was
mixed with the egg yolk component before adding
it to the diet. The resulting diet contained 273 mg
protein/ml of diet as calculated from protein com-
position of the components (Souci et al. 1989).

Preparation of Egg Protein Extract

Soluble proteins were isolated from eggs of the
4 species of insects as described by Ferkovich &
Shapiro (2004). Briefly, the eggs were homoge-
nized in ammonium acetate buffer (pH 7.5), cen-
trifuged at 20,200g, and the soluble fraction be-







Florida Entomologist 90(3)


tween the upper lipid layer and pelleted debris
was collected. This fraction was then run through
a centrifugal desalting column to remove com-
pounds <5,000 MW, freeze-dried, and stored at -
80C. The freeze-dried desalted powder was then
added to distilled water and analyzed for the
quantity of soluble protein by the Lowry proce-
dure (Protein Assay Kit, Sigma, St. Louis, MO)
before mixing it into diet.

Bioassay of Diets

Adults that had closed within 24 h were se-
lected from the colony for use in the bioassay.
Each replicate consisted of 6 females and 4 males
in a 100-mL plant tissue culture jar (Sigma, St.
Louis, MO), with 4 replicates per treatment. In
the first experiment, each jar contained 0.6 mL of
Hydrocapsules, either 3 mg ofE. kuehniella eggs
or two domes each containing 10, 25, or 50 pL of
artificial diet or diet plus E. kuehniella egg extract
(36 mg protein/ml), and 3 crumpled strips of wax
paper (5 mm x 80 mm) as substrates. E. kueh-
niella eggs, Hydrocapsules, and treatment diets
(artificial or insect eggs) were replaced daily and
mortality was recorded. At the end of 6 d, one 7-cm
section of green bean pod was placed in each jar
for 24 h as an oviposition substrate. The number
of females alive at the end of the oviposition pe-
riod was recorded, eggs deposited in the green
beans were counted under a stereomicroscope,
and the number of eggs oviposited per 5 females
was calculated. In the second experiment, the
setup was the same, except that the treatment di-
ets were made up of 3 mg of eggs of each species of
prey, E. kuehniella, P interpunctella, H. zea,
S. frugiperda, and H. uirescens added to each of
the jars. The setup for the third experiment was
identical except that each treatment diet con-
sisted of protein (18 or 36 mg protein/mL of diet)
extracted from one of 5 species of prey eggs and
added to artificial diet in two 25-pL domes per jar.

Egg Measurements

Measurements of the eggs were made with a
stereomicroscope with a 10x ocular micrometer
calibrated with a 1-mm stage micrometer. The
volumes of the eggs of H. zea, S. frugiperida, and
H. uirescens were calculated by the formula for a
sphere, 4/3 n r3. Egg volumes for P interpunctella
and E. kuehniella were calculated by the formula
for a prolate spheroid, 4/3 nab where a = the
semi-major axis length and b = the semi-minor
axis length.

RESULTS

Oviposition was affected by feeding females on
increasing sizes of diet domes, with or without
added Ephestia egg protein (Fig. 1). Oviposition


"" r Diet+Egg Potein
E a a
S6- ab
0) 21% b
b
"4-

2- c

0
10 25 50
Dome Volume (pl)
Fig. 1. Effect of size of diet domes on rate of oviposi-
tion (mean + SEM, n = 4) by predators fed artificial diet
with and without E. kuehniella egg protein extract (29
mg protein/mL diet). Percentages indicated on bars rep-
resent mortalities. Bars with the same letter are not sig-
nificantly different by ANOVA and multiple range test
(Newman-Keuls method, P > 0.05).


was highest in females fed on the 10-pL domes,
24% lower on the 25-pL domes (N.S.), and 85%
lower on the 50-pL domes (P < 0.05). When domes
were supplemented with E. kuehniella egg pro-
tein, there was no decrease in oviposition between
10- and 25-pL domes, and a 47% decrease on 50-
pL domes (P < 0.05). Female mortalities following
the 7-d bioassay on the 10-, 25- and 50-pL diet
domes containing artificial diet were 29.1%,
33.3%, and 83.3%, respectively. On 10-, 25- and
50-pL diet domes with egg protein added to the
diet, mortalities were 20.8%, 49.9%, and 70.8%,
respectively. Female mortality on whole E. kueh-
niella eggs was 12.5%.
Fig. 2 shows the relative volumes of 5 species of
prey eggs, protein content of prey eggs per mg of
fresh weight, and resultant oviposition by
0. insidiosus females feeding on the eggs. Plodia
interpunctella and E. kuehniella eggs were the
smallest, 19 + 1.7 nL (mean SD) and 26.9 3.4
nL, respectively; S. frugiperda eggs were of inter-
mediate size, 61.3 10.0 nL; and H. zea and
H. uirescens were the largest, 102.5 10.6 nL and
107.6 21.3 nL, respectively. The concentrations
(pg/mg fresh wt) of protein in the eggs in were:
E. kuehniella, 162; P interpunctella, 122; H. uire-
scens, 119; H. zea, 89; and S. frugiperda, 69. Ovi-
position was highest and comparable on the
E. kuehniella, P interpunctella, and H. uirescens
eggs and significantly less on H. zea and S. fru-
giperda eggs. Female mortality on the prey eggs
was as follows: E. kuehniella, 8.3%; P interpunc-
tella, 8.3%; H. uirescens, 8.3%; H. zea, 21%; and
S. frugiperda, 29%. If protein extracted from
E. kuehniella eggs was added to diet, oviposition
of females fed on the 25- and 50-pL domes signif-
icantly increased over those without additional


September 2007







Ferkovich et al.: Artificial Diet Presentation to Orius insidiosus


T o-
T A? 9-
E
M R 8-

F 7-

CL
(om a p
*5:5-
0


Piey-Egg Species

Fig. 2. Oviposition rate of O. insidious females fed
whole prey eggs (mean SEM; n = 4), in relation to vol-
umes (mean SEM; n = 5) and weight-specific protein
content of prey eggs (iig/mg fresh weight). Prey species:
E. kuehniella (Ek), P. interpunctella (Pi), H. zea (Hz), S.
frugiperida (Sf), and H. virescens (Hv). Bars with the
same letter are not significantly different (Newman-
Keuls method, P > 0.05).


protein. However, oviposition was still reduced in
those fed on 50-pL domes relative to 10- or 25-pL
domes.
When the mean oviposition and mortality were
regressed against prey-egg volume and protein
content of eggs, neither oviposition nor mortality
correlated with egg volumes (Fig. 3A), while both
strongly correlated (mortality was inversely cor-
related) with protein content of eggs (Fig. 3B).
Only protein extracts from the E. kuehniella
and P interpunctella eggs significantly enhanced
egg production when added to artificial diet (Fig.
4). The E. kuehniella extract improved egg pro-
duction at both protein levels tested, whereas the
P interpunctella extract was only effective at the
higher level. Female mortality on the diets sup-
plemented with the egg protein from each species
was as follows: E. kuehniella whole eggs, 12.5%;
artificial diet, 37%; E. kuehniella extract (18 mg
protein), 25%;E. kuehniella extract (36 mg), 21%;
P interpunctella extract (18 mg), 37%; P. inter-
punctella extract (36 mg), 33.3%; H. virescens ex-
tract (18 mg), 46%; H. virescens extract (36 mg),
37%; H. zea extract (18 mg protein), 46%; H. vire-
scens extract (36 mg), 33%; and S. frugiperda ex-
tract (36 mg), 46%.

DISCUSSION

The mode of presentation of an artificial diet is
important in determining its acceptance by a
predator (Cohen & Staten 1993; Grenier et al.
1994; Cohen 2004). Important issues in diet pre-
sentation include phagostimulants, texture, liq-
uid or semi-solid state of the ingredients, and
methods of containment. Cohen and Staten
(1993) found that Geocoris punctipes fed longer
when the diet format was changed from cylindri-


E


0


12
0
tI


10 20 3 0 40 60 70 80 90 100110120
Egg Volume (nl)
40
a Oviposition
B ---- Mortay

oR = 0,850
0-
-20

5.. R2 = 0.767 -10
AA A


50 100 150 200
ProteintFresh Weight (pg/mg)


Fig. 3. Regressions on the means of oviposition and
mortality vs. (A) prey-egg volume; and (B) weight-spe-
cific protein content.


cal artificial "larvae" to flattened packets made
from stretched Parafilm. Carpenter & Greany
(1998) designed the encapsulation unit used in
the present study, which is capable of forming diet
domes of varied volumes from Parafilm. De
Clercq et al. (1998) evaluated 2 methods to
present a meat-based diet to Podisus maculiven-
tris (Say), and found that containing the diet in
stretched Parafilm sheets to form cylindrically
shaped artificial larvae yielded better results
than the gelled open form of the diet. They con-
cluded that inferior results from the gelled form of
the diet may be related to acceptability problems.
Methods of diet containment have focused mainly
on mechanized means of producing diet packets
for mass production of predators such as Chrysop-
erla rufilabris (Cohen 2004), and little consider-
ation has been given to the size and shape of the
diet capsules.
Diet dome size clearly affected oviposition. Fe-
males that fed on 50-pL domes produced fewer
eggs than those fed on 10- or 25-pL domes. One
possible reason for this is that the digestive en-
zymes that the feeding predators injected into the
prey eggs during feeding were diluted signifi-
cantly in the larger domes. Cohen (1989) observed
that G. punctipes varied their probing of artificial
larvae while feeding, in a manner similar to prob-


a1 Oviposition
A Moctahy


RI W=o143 A


R2=O.081
A A As







Florida Entomologist 90(3)


9 Ction and inversely with mortality (Fig. 3B). These
8 Low P rotein Does (18 results suggest that the protein contents of eggs
a i High Protein Domes (36 m rml played critical roles in stimulating egg develop-
7 ment and oviposition and in aiding survival. To
6 further examine the effect of the egg protein, a
Constant amount from each species of egg was
5 added to diet in domes of equal volume (Fig. 4).
S4 Only E. kuehniella and P interpunctella egg ex-
S3 tracts induced oviposition at a significantly
0 higher rate than the control diet, suggesting that
Something nutritionally active in those eggs was
0 1 lacking in other species. This relates to earlier
0 studies in which we reported oviposition-stimu-
Eggs Diet Ek Pi Hz Sf Hv lating activity in whole egg extracts of E. kueh-
niella and P interpunctella (Ferkovich & Shapiro
Species, Prey-Egg Extract 2004a, 2005a), in embryonic cell lines from the
Fig. 4. Effect of supplementing diet with protein ex- two species (Ferkovich & Shapiro 2004b, 2005b;
tracts from 4 species of prey eggs on oviposition rates Ferkovich & Lynn 2005), in specific fractions from
(mean SEM, n = 4). Only the higher concentration of extracts of E. kuehniella (Ferkovich & Shapiro
Sf was run. Dunnett's test was used to compare the 2005a), and fractions from a P. interpunctella cell
amended treatment diets against the Diet (control); as- line (Ferkovich & Shapiro 2007).
terisk indicates that the treatment means were signifi- Because we observed that O. insidious pro-
cantly different from Diet (control) (P < 0.05). duced the most eggs on the smallest diet domes,
we surmised that the predator would exhibit a
preference for smaller prey eggs. Egg production
ing different parts of their natural larval prey. was not only highest on the 2 smallest eggs
During this feeding behavior they injected saliva (E. kuehniella and P interpunctella) but also on
containing digestive enzymes, initiating a process the larger eggs ofH. virescens. It appeared that the
that he called extra oral digestion. Cohen and H. virescens eggs were either more attractive to
Smith (1998) attributed the success of their solid the predators or better met the nutritional needs
artificial diet to its accommodation of the extra for yolk production than the other 2 species of
oral digestive nature of predator feeding. large eggs, H. zea and S. frugiperda. Based on
The addition of protein extract from E. kueh- these results, we hypothesized that proteins ex-
niella eggs, notably in females fed on the 50-pL tracted from H. virescens eggs would have activity
diet domes, reduced the effect of the large dome similar to the E. kuehniella and P interpunctella
size (Fig. 1). One possibility for the reduced effect egg protein extracts when bioassayed (Ferkovich
is that phagostimulants may be present in prey- & Shapiro 2004, 2005). Bioassaying the 5 egg pro-
egg extracts. Chemoreception is an important tein extracts at similar protein concentrations,
means by which the insect detects food resources however, revealed that the H. virescens protein ex-
and oviposition sites (Nation 2002; Mowery et al. tract did not have significant activity in the large
2004). De Clercq et al. (1998) stated that a lack of diet domes (25 pL) as did the E. kuehniella and
feeding stimulants may be more important than a P interpunctella extracts. This indicated that ei-
lack or poor balance of nutrients in a predator's their nutrients other than proteins or some other
diet. There are numerous reports in the literature factors) was responsible for the increase in ovipo-
on chemoreception by phytophagous insects sition rate we observed with whole H. virescens
(Chapman 2002); however, little information is eggs. Numerous reports indicate that feeding dif-
available on predators and prey-associated pha- ferent prey to predators can affect their fecundity
gostimulants. Cohen & Staten (1994) found that and other biological characters of predators be-
G. punctipes were attracted to green beans and in- cause of differences in the nutritional values of the
duced the predators to aggregate and feed and prey (Chyziketal. 1995;Thompson & Hagen 1999;
drink for extended periods. Relatedly, chemical De Clercq et al. 1998; Roger et al. 2000; Torres et
cues derived from larval prey ofSpodoptera litura al. 2004; Venzon et al. 2001; Specty et al. 2003).
were found to elicit prey-locating behavior by the In conclusion, 0. insidious oviposited at a
predatory stink bug, Eocanthecona furcellata higher rate when control diet was presented to
(Yasuda 1997). them in small domes rather than larger domes,
In analogy to the experiment on the effect of suggesting higher consumption rates. This effect
dome size on oviposition, we examined the effect could be overridden by adding a protein extract
of prey-egg size on oviposition. There was no cor- from either E. kuehniella or P interpunctella eggs,
relation between prey-egg size and oviposition likely because the extract made the diet more
(Fig. 3A); however, weight-specific protein con- attractive to the predators. Alternatively, the
tents of prey eggs correlated directly with oviposi- extract may have contained enzymes that aided


September 2007







Ferkovich et al.: Artificial Diet Presentation to Orius insidiosus


digestion of the diet. Although females fed intact
eggs from H. virescens had oviposition rates simi-
lar to those fed eggs from E. kuehniella and P. in-
terpunctella, females fed diet with extracts from
H. virescens eggs did not show significantly higher
oviposition rates. We cannot explain this discrep-
ancy. Identification of the active substance in the
E. kuehniella and P interpunctella extracts
should provide a better understanding of why
these extracts are active. Once the active compo-
nents are identified they may prove to be useful in
improving artificial diets for Orius species.

ACKNOWLEDGMENTS

We thank Dr. Terry Arbogast for helpful review of an
earlier draft. We thank Dr. Patrick Greany for his re-
view and valuable insight into interpretation of some of
the data. We appreciate the excellent technical assis-
tance of Delaine Miller in this study.

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Florida Entomologist 90(3)







Boughton et al.: Mating Biology ofAustromusotima camptozonale



MATING BIOLOGY OF AUSTROMUSOTIMA CAMPTOZONALE
(LEPIDOPTERA: CRAMBIDAE), A POTENTIAL BIOLOGICAL
CONTROL AGENT OF OLD WORLD CLIMBING FERN,
LYGODIUM MICROPHYLLUM (SCHIZAEACEAE)

ANTHONY J. BOUGHTON1, JUDY WU'2 AND ROBERT W. PEMBERTON1
'USDA-ARS Invasive Plant Research Laboratory, 3225 College Avenue, Fort Lauderdale, FL 33314

2Student Conservation Association, c/o USDA-ARS Invasive Plant Research Laboratory,
3225 College Avenue, Fort Lauderdale, FL 33314

ABSTRACT

Austromusotima camptozonale (Hampson) is under investigation as a potential biological
control agent of Old World climbing fern, Lygodium microphyllum (Cav.) R. Br., which is a
serious invasive weed in southern Florida. Studies were conducted to investigate aspects of
the mating biology ofA. camptozonale with a view to improving field colonization efforts. A
laboratory colony ofA. camptozonale had a female-biased sex ratio of 1:1.3 male:female, with
females emerging slightly earlier than males. The majority of female moths mated only once,
even when they were confined with multiple males for several nights. Sex ratio had a signif-
icant effect on the percentage of females that were mated, with higher percentages of females
mated at high male sex ratios. However sex ratio had no effect on the number of times indi-
vidual females mated. Larval production was significantly higher in colony cages with high
male sex ratios, and this was likely due to the higher percentages of females that were mated
in these cages. Data suggest that A. camptozonale females are likely to be functionally mo-
nandrous under field conditions. Females produced a lifetime average of 61.2 9.7 larvae,
and were short-lived, surviving an average of 5.7 0.5 d. Females began oviposition on the
first night after mating and continued until the day of their death. However 90% of eggs were
deposited between the first and third night after mating. Due to the short lifespan of female
moths, adults may not be the best life stage for field release in a biological control program,
owing to likely disruption of critical mating and oviposition activities. Elevating the ratio of
males in colony mating cages is a strategy for maximizing female reproductive output.

Key Words: Austromusotima camptozonale, mating biology, Lygodium microphyllum

RESUME

Austromusotima camptozonale (Hampson) esta bajo investigaci6n como una agent poten-
cial de control biol6gico para el helecho trepador de Mundo Antiguo, Lygodium microphyl-
lum (Cav.) R. Br., que es una maleza invasora seria en el sur de la Florida. Se realizaron
studios para investigar la biologia del apareamiento de A. camptozonale con el prop6sito de
mejorar los esfuerzos de colonizaci6n de esta especie en el campo. Una colonia de laboratorio
deA. camptozonale tuvo una preferencia en la proporci6n de machos a hembras de 1:1.3 ma-
cho:hembra, con las hembras emergiendo un poco antes que los machos. La mayoria de las
palomillas adults se aparearon solamente una vez, aun cuando fueron confinadas con ma-
chos multiples para varias noches. La proporci6n de macho a hembra tuvo un efecto signifi-
cativo sobre el porcentaje de hembras que se aparearon, con un mayor porcentaje de
hembras apareadas cuando las proporciones de macho a hembra fueron mas altos. Sin em-
bargo, la proporci6n de macho a hembra no tuvo ningun efecto sobre el numero de veces que
las hembras individuals se aparearon. La producci6n de larvas fue significativamente mas
alta en las colonies en jaulas que tuvieron una proporci6n mas alta de machos a hembra, y
esto probablemente es debido a la proporci6n mas alta de hembras que fueron apareadas en
estas jaulas. Estos datos indican que las hembras de A. camptozonale son probablemente
funcionalmente mon6gamas bajo condiciones de campo. Las hembras produce un promedio
de 61.2 9.7 larvas por todo su vida, y vivieron por un tiempo corto, sobreviviendo por un
promedio de 5.7 0.5 dias. Las hembras empezaron la oviposici6n en la primera noche des-
pu6s de aparearse y continuaron hasta el dia de su muerte. Sin embargo, 90% de los huevos
fueron depositados entire el primer y tercer dia despues del apareamiento. Debido a la dura-
ci6n corta de vida de las palomilla hembras, los adults posiblemente no son el mejor estadio
para hacer liberaciones de esta especie en el campo en un program de control biologico, pro-
bablemente a causa de la disrupci6n critical en las actividades del apareamiento y oviposi-
ci6n. El aumento de la proporci6n de machos en las colonia usadas para el apareamiento en
jaulas es una estrategia para maximizar el rendimiento reproductive de las hembras.







Florida Entomologist 90(3)


A variety of different mating systems are
found within lepidopteran species (Drummond
1984; Arnqvist & Nilsson 2000). These systems
range from monandry, in which females mate
with only a single male during their lifetime, as
occurs in the gypsy moth (Giebultowicz et al.
1991), to systems where females exhibit a high
degree of polyandry and mate with many differ-
ent males, as occurs in the corn earworm (Raina
et al. 1986) and some arctiids (LaMunyon 1997).
Although a single mating is often sufficient to pro-
vide a female with a lifetime of sperm, polyandry
is common in Lepidoptera (Arnqvist & Nilsson
2000; Ridley 1990). Polyandry confers a variety of
benefits to females including full fertilization of
her egg complement, increased genetic diversity
of offspring, receipt of non-sperm nutritional ben-
efits, and avoidance of problems with old sperm
(Drummond 1984; Ramaswamy et al. 1997; Jime-
nez-Perez et al. 2003). However there are also
costs associated with polyandry including; ener-
getic costs of courtship, energetic costs of multiple
matings, reduced time for oviposition, increased
predation risk, and increased risk of pathogen
transfer (Svensson et al. 1998; Jimenez-Perez et
al. 2003).
Old World climbing fern, Lygodium microphyl-
lum (Cav.) R. Br., is native to tropical regions of
Africa, Australia and Southeast Asia (Pemberton
1998). Sometime during the latter half of the 20th
century, L. microphyllum became established in
southern Florida (Pemberton & Ferriter 1998),
and over recent decades has spread rapidly to in-
fest communities on moist soils on private lands,
parks, wildlife refuges, and even remote locations
in Everglades National Park (Volin et al. 2004).
Lygodium microphyllum grows rapidly under the
subtropical conditions of southern Florida, climb-
ing over native plants, denying them light, and
smothering them. Lygodium microphyllum
readily grows up trees, providing a conduit by
which brush fires may move into tree canopies,
where damage may be done even to mature trees
(Pemberton & Ferriter 1998; Volin et al. 2004).
Most natural areas in south central Florida are
vulnerable to invasion by L. microphyllum (Pem-
berton & Ferriter 1998) and consequently this
weed poses a substantial threat to native plants
and ecosystems (Lott et al. 2003).
Lygodium microphyllum has proven to be ex-
tremely difficult to manage in Florida (Hutchin-
son et al. 2006). Herbicide treatments are not ef-
fective at preventing re-growth and mechanical
removal is too expensive to be viable. In addition,
native insect and arthropod herbivores are rarely
encountered feeding on L. microphyllum, so the
fern is not subject to the feeding pressures that
typically help to regulate plant populations
within their home range. Biological control is be-
ing considered as a potential management solu-
tion (Pemberton 1998). The United States Depart-


ment of Agriculture (USDA), Agricultural Re-
search Service (ARS), Invasive Plant Research
Laboratory (IPRL) in Fort Lauderdale, Florida, in
cooperation with overseas collaborators has con-
ducted surveys in SE Asia and Australia to iden-
tify host-specific arthropod herbivores ofL. micro-
phyllum that could be introduced to southern
Florida to help control Old World climbing fern
(Goolsby et al. 2003). One potential biological con-
trol agent is a moth, Austromusotima camptozo-
nale (Hampson) (formerly Cataclysta camptozo-
nale) (Lepidoptera: Crambidae) (Yen et al. 2004).
Larvae ofA. camptozonale are defoliators ofL. mi-
crophyllum, and are thought to reduce the fern's
capacity for vegetative growth and reproduction.
In Dec 2004, the USDA Animal Plant Health
Inspection Service (APHIS) issued a release per-
mit for A. camptozonale, and field releases of
adult moths were made by USDA-ARS-IPRL dur-
ing the spring and summer of 2005. However sub-
sequent monitoring of release sites during fall
2005 failed to yield definitive evidence of moth es-
tablishment. In an effort to better understand the
biology of A. camptozonale and improve the suc-
cess of future field colonization efforts, studies
were conducted to elucidate aspects of the mating
biology and life history traits of adult A. campto-
zonale. Studies were conducted to investigate pat-
terns of adult emergence, sex ratio, male and fe-
male longevity, the timing of oviposition, and ef-
fect of different sex ratios on mating frequency
and female fertility.

MATERIALS AND METHODS

Insects

Insects used in studies were obtained from a
laboratory colony of A. camptozonale maintained
at the USDA-ARS Invasive Plant Research Lab in
Fort Lauderdale, FL. This Fort Lauderdale colony
was derived from a pre-existing USDA colony
maintained at the Florida Department of Agricul-
ture & Consumer Services, Quarantine facility in
Gainesville, FL. The Gainesville colony had been
established from insects collected in Apr 2000 from
Carbrook Creek, Brisbane, Queensland, Australia.

Colony Maintenance

Larvae in the Fort Lauderdale colony were fed
fresh L. microphyllum foliage collected weekly
from infested field sites in Palm Beach and Martin
counties in southern Florida. Immediately follow-
ing collection, plant material was soaked for 10
min in 10% (v/v) Clorox bleach in water, and sub-
sequently rinsed 5 times in clean water. Washed
foliage was drained and packaged in clean 18-liter
plastic bags, and stored at 4C until needed. Lar-
vae were reared in 739-mL, plastic sandwich
boxes (Glad Products Company, Oakland, CA)


September 2007







Boughton et al.: Mating Biology ofAustromusotima camptozonale


(25 larvae per box) on fresh-cut L. microphyllum
foliage, at 24C and a photoperiod of 12:12 (L:D).
Larvae were allowed to pupate in rearing contain-
ers, and following emergence, adults were col-
lected in a bench top light box, with a battery-pow-
ered, handheld aspirator (Hausherr's Machine
Works, Toms River, NJ). Male moths (prominent
dark brown stripes on wings) and female moths
(fewer, paler stripes, prominent wing spots) were
sexed visually (Yen et al. 2004) and transferred
into aluminum-framed mating cages (61 x 30 x 30-
cm) screened with insect-proof, metal window
screen. Mating cages were positioned on the
bench top, and were provisioned with an oviposi-
tion sprig, consisting of a bouquet ofL. microphyl-
lum stems wrapped in cotton balls and inserted
into a 150-mL plastic vial (Thornton Plastics, Salt
Lake City, UT) of water, and 2 cotton balls, 1
soaked in 9:1 Gatorade [Lemon-Lime]:honey, the
other in water. Gatorade contains sucrose, glu-
cose, and fructose. These sugars and honey have
frequently been used to supplement diets of adult
female Lepidoptera in captivity as a source of sug-
ars, amino acids, and vitamins they might other-
wise have obtained from extra floral nectaries or
homopteran honeydew in the environment
(Romeis & Wackers 2002; Tisdale & Sappington
2001). Moths were allowed to mate and oviposit,
and following their death, oviposition sprigs were
removed and incubated in clear plastic boxes (11 x
11 x 23-cm) (Pioneer Plastics, Inc., Dixon, KY) on
the bench top until larvae emerged. Newly
emerged neonate larvae were transferred with a
paintbrush onto fresh L. microphyllum foliage in
sandwich boxes to begin the next rearing cycle.
Austromusotima camptozonale is multivoltine
and so can be reared continuously in the lab. Typ-
ical durations for the egg, larval, and pupal stages
at 24C are 10, 10, and 11 d, respectively (A. J. B.,
unpublished data).

Emergence and Sex Ratio Studies

Sandwich boxes containing A. camptozonale
larvae were selected at random from the colony
and put aside for use in adult emergence and sex
ratio studies. Following pupation, pupae were
transferred into individual 25-mL plastic vials
(Thornton Plastics, Salt Lake City, UT), contain-
ing a small (1 x 1-cm) piece of moistened paper
towel. Vials were left on the bench top and
checked daily for emergence, and the numbers of
male and female moths recorded. Observations
were repeated over 5 larval cohorts, and a total of
347 pupae.

Colony Cage Studies

Experiments were conducted to investigate the
effect of the ratio of male to female moths inside
mating cages (henceforth "cage sex ratio") on


subsequent larval production by female moths.
Aluminum mating cages provisioned with ovipo-
sition sprigs and cotton balls as previously de-
scribed, were set up with 1 of 3 ratios of virgin
male to virgin female moths: (i) 5 male (6): 10 fe-
male (t); (ii) 10d:10 ; (iii) 206:10Y. Cages were
set up at the same time and were left undisturbed
on the bench top for several days to allow time for
female moths to mate and oviposit. When all fe-
male moths had died, oviposition sprigs were re-
moved from cages and transferred to individual
clear plastic boxes on the bench top until eggs
emerged. The number of neonate larvae on each
sprig was determined and used as a measure of
total female oviposition at that cage sex ratio.
Studies were set up according to a replicated com-
plete block design, with each of the 3 cage sex ra-
tios replicated across 5 generations of the colony.
In 2 of these replicates, female moths were dis-
sected after death. The numbers of spermato-
phores present in the bursa copulatrix of each
female was used to infer how many times she
had mated (Drummond 1984).

Individual Female Studies

Experiments with a single female moth per
mating cage were performed to more precisely in-
vestigate the effect of male to female sex ratio on
the frequency of female matings and subsequent
fertility. Large, clear-plastic mating cages (41 x 41
x 41-cm) with access sleeves on either side, provi-
sioned with an oviposition sprig and food-soaked
cotton balls as previously described, were set up
on the bench top with newly emerged virgin fe-
male and virgin male moths to achieve 1 of 3 cage
sex ratios: (i) 16:12 ; (ii) 2 :12 ; (ii) 36:12. Cages
were left overnight for females to mate, and on
the morning of the second day, male moths were
removed from cages and oviposition sprigs were
replaced. Oviposition sprigs were replaced daily
until females died. Oviposition sprigs were main-
tained in plastic boxes on the bench top until eggs
hatched, at which point the number of larvae on
each sprig was determined. Following death, fe-
male moths were dissected and the number of
spermatophores present within the bursa copula-
trix was noted. Experiments were set up accord-
ing to a complete block design, with treatments
replicated 10 times.

Individual Male Studies

Experiments were set up to assess the propen-
sity of male moths to mate with more than 1 fe-
male moth. A single, newly emerged virgin male
moth together with 2-5 newly emerged virgin fe-
male moths were added to each of 5 plastic insect
rearing cages (30 x 30 x 30-cm) (BioQuip Prod-
ucts, Rancho Dominguez, CA). Each cage was pro-
visioned with an oviposition sprig and cotton balls






Florida Entomologist 90(3)


soaked in liquid food, as previously described.
Moths were left in cages overnight to mate and
the next day females were removed and replaced
with new virgin females. Females were replaced
daily until male moths died. Upon removal, fe-
male moths were dissected and the number of
spermatophores in each female was recorded.
Statistical Methods
Data were analyzed according to the General
Linear Models for analysis of variance (ANOVA).
Data were checked for conformity to ANOVA's un-
derlying assumptions of normality of error and
homogeneity of variance by examining plots of re-
siduals and predicted values, and when necessary
data were transformed to fix departures from
these assumptions. Data on percentage of females
mated, spermatophore counts, mean larval pro-
duction and adult longevity data were analyzed
by using the univariate ANOVA procedure of
SPSS statistical software (SPSS, Inc., Chicago,
IL) with post-hoc comparison of means with
Tukey's means separation test. Data on daily ovi-
position across sex ratios were analyzed by the re-
peated measures ANOVA procedure of SPSS.
Emergence data for male and female moths were
combined across replicates, and used to construct

45


I


Kaplan Meier survival curves for cumulative
daily emergence. Dead pupae were excluded from
consideration. Survival curves were then com-
pared by a log-rank test to check for statistical dif-
ferences in the timing of emergence between male
and female moths.
RESULTS
Emergence and Sex Ratio Studies
Adults generally started emerging 10 to 11 d
after pupation, but considerable variability was
present in the timing of emergence of moths
across cohorts. For this reason, daily emergence
data for the different cohorts was synchronized
relative to the first day on which adults started to
emerge (Fig. 1). Moths emerged over a period of
7 d, with peak emergence occurring on the sec-
ond, third and fourth days. Both sexes were
present from the first day of emergence, but fe-
males showed a small but statistically significant
tendency to emerge earlier than males (log rank
statistic = 11.67; df = 1; P = 0.0006). Sex ratios of
males to females across the 5 cohorts ranged from
1:0.9 to 1:1.5, with an overall ratio of 1:1.3
male:female, respectively (Table 1). Overall adult
emergence from the pupal stage was 88.4%.


o Male moths
* Female moths


K


1 2 3 4 5 6 7 8 9
Day of emergence
Fig. 1. Timing of emergence of male A. camptozonale moths relative to female moths. Graph shows percentage of
total emergence for each sex occurring on the first through ninth d of the emergence. Figure constructed by averaging
percent daily emergence data for males and females from 5 pupal cohorts (n = 155 6; n = 192 ). Emergences across
cohorts synchronized relative to the first d on which adults emerged. Bars show standard errors.


September 2007







Boughton et al.: Mating Biology ofAustromusotima camptozonale


TABLE 1. SEX RATIO AND PERCENTAGE EMERGENCE OF MOTHS.

Number Number Number moths Sex ratio % Adult
Cohort 6 moths Y moths that died 6: emergence'

1 10 14 1 1:1.4 96.0
2 10 15 0 1:1.5 100.0
3 9 12 4 1:1.3 84.0
4 91 118 31 1:1.3 87.1
5 35 33 23 1:0.9 74.7

Overall 1:1.3 88.4

'Percentage of pupae from which adult moths emerged.


Colony Cage Studies

Cage sex ratio had a significant effect on mean
percentage of females that were mated (F = 35.95;
df = 2, 6; P = 0.03) (Table 2). Tukey's test indicated
that a significantly higher percentage of females
were mated in cages at the 206:109 ratio, than
were mated in cages with ratios of 56:109 or
106:10 9. However, cage sex ratio failed to explain
a significant amount of the variation seen in mean
number of spermatophores per mated female (F =
1.26; df = 2, 36; P = 0.30) (Table 2). Mean number
of spermatophores per mated female in cages at
the 206:109 ratio was not significantly higher
than mean number of spermatophores per female
observed in either the 56:109 or the 106:109
cages. Colony cage larval production data were
transformed by squaring to stabilize the variance,
and analyzed according to a blocked ANOVA de-
sign. Sex ratio was shown to have a significant ef-
fect on mean larval production (F = 5.77; df2, 15;
P = 0.03). The back-transformed mean and stan-
dard error for cages receiving the 56:10 ratio
(367.2 larvae/cage 222.0) was not statistically
different from cages receiving the 106:10 (337.8
larvae/cage 149.0) ratio. Mean larval production
for cages with a sex ratio of 20X:102 (498.1 lar-


vae/cage 250.0) was substantially higher than
mean larval productions observed in cages with
106:10 9 or 56:10 although this difference was
only statistically significant between the 206:10 9
and 106:10 treatments.

Individual Female Studies

Although there was a trend towards increasing
percentages of mated females at high male-biased
sex ratios, the effect was not statistically signifi-
cant (F = 2.10; df = 2, 27; P = 0.14) at the sample
sizes used in these studies (Table 3). Sex ratio did
not have a significant effect on mean number of
spermatophores per mated female (F = 0.79; df =
2, 23;P = 0.47) (Table 3). Based on 10 replicates of
the single-female studies, mean larval production
values at the 16:19, 26:19, and 36:19 sex ra-
tios were 73.0 17.8, 56.0 + 20.0, and 54.6 13.6
larvae/female, respectively, and mean female lon-
gevity at these sex ratios were 5.8 + 0.7, 5.5 + 0.9,
5.9 0.9 d, respectively. Cage sex ratio did not
have a significant effect on mean larval produc-
tion per female (F = 0.287; df = 2, 24; P = 0.76) or
mean female longevity (F = 0.048; df = 2, 21; P =
0.95) and means were not significantly different
across treatments by Tukey's means separation


TABLE 2. DISSECTION DATA SHOWING EFFECT OF SEX RATIO ON FREQUENCY OF MATING AND SPERMATOPHORE TRANS-
FER IN FEMALES MAINTAINED IN COLONY CAGES.

Percentage of females (Mean S.E.)

Spermatophores/
Cage ratio No. Mated once mated female
(6: ) females' Unmated Once mated Twice mated or more2 (Mean S.E.)3

5:10 16 31.3 + 6.3 68.7 + 6.30 0.0 0.0 68.7 6.3 a 1.00 0.00 a
10:10 19 36.7 + 3.3 57.8 2.20 5.6 5.6 63.4 3.3 a 1.08 0.08 a
20:10 17 5.0 + 5.0 80.0 + 20.0 15.0 15.0 95.0 5.0 b 1.19 + 0.10 a

'Females from 2 replicates of the colony cage studies (10 Y per treatment, per replicate) were dissected. In a small number of
cases, dissections were ambiguous revealing the presence of material in the bursa copulatrix that could not be definitively identified
as a spermatophore. These females were excluded from consideration.
Means followed by different letters are significantly different by Tukey's means separation test (P < 0.05).
'Mean calculated as "total number of spermatophores" divided by "number of mated females".







Florida Entomologist 90(3)


TABLE 3. DISSECTION DATA SHOWING EFFECT OF SEX RATIO ON FREQUENCY OF MATING AND SPERMATOPHORE TRANS-
FER IN STUDIES WITH SINGLE FEMALES.

Percentage of females (Mean S.E.)

Spermatophores/
Cage ratio No. Mated once mated female
(6: ) females' Unmated Once mated Twice mated or more2 (Mean S.E.)

1:1 10 30.0 + 15.3 70.0 + 15.3 0.0 0.0 70 15.3 a 1.00 0.00 a
2:1 10 10.0 10.0 90.0 + 10.0 0.0 + 0.0 90 10.0 a 1.00 0.00 a
3:1 10 0.0 + 0.0 90.0 + 10.0 10.0 10.0 100 0.00 a 1.10 + 0.10 a

Females from 10 replicates of the single female studies (1 Y per treatment, per replicate).
'Means followed by different letters are significantly different by Tukey's means separation test (P < 0.05).
'Mean calculated as "total number of spermatophores" divided by "number of mated females".


test (P > 0.05). When data were pooled across sex
ratios, mean number of spermatophores per
mated female was 1.04 + 0.04, mean larval pro-
duction was 61.2 + 9.7, and mean female longev-
ity was 5.7 0.5 d.
Repeated measures ANOVA identified "days
after mating" as a factor explaining significant
amounts of variation in nightly oviposition totals
(F = 27.28;df= 1, 21;P < 0.01). However, repeated
measures ANOVA failed to identify sex ratio as a
factor explaining significant variation in nightly




30 1


oviposition totals (F = 0.35; df=2, 21; P = 0.71), so
oviposition data from females at the 3 different
sex ratios were combined. Data showed that no
eggs were laid during the first day/night that fe-
male and male moths were together (Fig. 2). Ovi-
position began and was highest on the second day/
night after the female and male moth(s) were put
together, averaging 20.8 eggs, decreasing through
18.5, 16.1, 3.8, 1.7, and 0.3 eggs per day/night dur-
ing the third, fourth, fifth, sixth, and seventh
days/nights, respectively.


1 2 3 4 5 6 7 8

Number of nights after males first introduced to cage

Fig. 2. Timing of oviposition by female A. camptozonale moths. Male moths were present in cages on the first
night, but were removed early on the second d. Studies used 1 female moth per cage. Plant oviposition bouquets
changed daily. Means based on observation of n = 24 female moths. Bars show standard errors.


September 2007







Boughton et al.: Mating Biology ofAustromusotima camptozonale


Individual Male Studies

Male moths were never observed to mate with
more than 1 female per night (Table 4). Mean
number of lifetime matings per male was 2.0 +
0.32, with a maximum of 3 lifetime matings and a
minimum of 1 lifetime mating. Five male moths,
paired with a total of 74 virgin females, only man-
aged to mate with 10 of these females during their
combined lifetimes. Mean longevity of male moths
was 6.6 1.2 d.

DISCUSSION

Adult A. camptozonale emerged from pupae
over a period of about 7 d from the day the first
adult emerged, with peak emergence occurring on
the second day after emergence began. Males and
females emerged on every day of the emergence
cycle, but the bulk of female emergence occurred
earlier than for males, as has been documented
for other lepidopteran species (Uematsu & Mori-
kawa 1997). The laboratory colony ofA. camptozo-
nale was female-biased with an overall sex ratio
of 1:1.3 (6:9).
In colony studies, significant differences were
seen in the percentages of females that were
mated at the 3 different sex ratios. A significantly
higher percentage of females were mated at high
male sex ratios (206:109) than were mated at
lower male sex ratios (106:109 and 56:109).
This same trend towards higher percentages of
mated females in cages with high male sex ratios
was visible in studies of individual females, al-
though the trend was not statistically significant.
No significant differences in spermatophore num-
bers were found in mated females across the dif-
ferent sex ratios, in either the colony or individual
studies. This indicates that male sex ratio had an
effect only on the proportion of females that
mated within a cage and not on the number of
times that each female mated. The vast majority
of mated females in the colony studies (90%) and


the individual female studies (96%), had mated
only once. This finding together with the sper-
matophore data suggest thatA. camptozonale fe-
males are likely to be functionally monandrous
under field conditions, because mating frequen-
cies observed under field conditions tend to be
lower than those observed in the lab (Sadek 2001).
Cage sex ratio was found to have a significant
effect on larval production in the colony studies.
Mean larval production per cage at the 206:109
ratio was significantly higher than mean larval
productions observed in cages at the 56:10 or
106:109 ratios. However, because there was no
evidence that females in cages at the highest male
sex ratio (206:109) mated a greater number of
times than females maintained at lower male sex
ratios, the observed increase in larval production
must have been due to the higher percentage of
females that mated and subsequently went on to
oviposit in these cages.
In the individual female studies, high percent-
ages of once-mated females were found at each of
the 3 cage sex ratios. Because mating frequencies
were essentially the same for females across each
of these sex ratios, it was not surprising that cage
sex ratio didn't explain significant amounts of the
variation seen in mean larval production per fe-
male, mean female longevity or the timing of ovi-
position. Female longevity in these studies aver-
aged 5.7 d. Daily oviposition data showed that fe-
males continued to lay eggs up until the day of
their death, although 90% of eggs were laid be-
tween the second and fourth nights. These find-
ings indicate that A. camptozonale females are
relatively short lived compared with females of
other lepidopteran species (Bento et al. 2006;
Hughes et al. 2000; Svensson et al. 1998). In addi-
tion, the pre-ovipositional period was short and
there was little if any post-ovipositional period.
As such, the majority of the lifespan of female A.
camptozonale is occupied directly with aspects of
reproduction, and any events that delay mating
or otherwise occupy her time, are likely to nega-


TABLE 4. MATING BEHAVIOR OF INDIVIDUAL MALE MOTHS SUPPLIED DAILY WITH EXCESS VIRGIN FEMALE MOTHS.

Number of female moths mated/number exposed to males on night2

Male Night Night Night Night Night Night Night Night Night Night
moth' 1 2 3 4 5 6 7 8 9 10

#1 1/5 1/3 0/3 1/3 0/3 0/1 0/1 0/1 0/1 0/1
#2 1/5 1/3 0/3 0/3 0/3 -
#3 1/5 0/3 0/3 -
#4 1/5 1/3 0/3 0/3 0/3 0/1
#5 1/2 1/2 0/1 0/1 -

'Data based on multi-night observations of 5 male moths.
Virgin female moths added daily into cages with each male moth. Females recaptured and dissected the next day to see whether
they had mated overnight (indicated by presence of spermatophores in the bursa copulatrix).











tively impact her reproductive output. This may
explain why A. camptozonale recapture rates
were low and no evidence of field establishment
was found following field releases of moths in
2005. Stresses on newly emerged moths associ-
ated with collection and transportation to release
sites at a critical time in the life cycle when they
would ordinarily be focused on mating and ovipo-
sition, could easily have a negative impact on
moth reproduction in the field and reduce the
likelihood of successful establishment.
Austromusotima camptozonale females seldom
(1 of 29 females) mated more than once per night
even when they were confined with an excess of
virgin male moths. Similarly, virgin male moths
were never observed to mate with more than 1
female a night, even when surplus virgin female
moths were present. This pattern of behavior in
A. camptozonale is consistent with the general
pattern observed in Lepidoptera, where male and
female moths typically mate only once per night
(Gadenne et al. 2001). Reductions in female re-
ceptivity following mating are associated with
male factors transferred during copulation
(Raabe 1986; Foster & Ayers 1996), and are tem-
porary for polyandrous species, or permanent for
monandrous species (Gadenne et al. 2001). The
low propensity of males to mate more than once
per night appears to be due to energetic and time
constraints associated with replacement of a
large spermatophore (Hughes et al. 2000).
In conclusion, female A. camptozonale moths
are short-lived and heavily preoccupied with ovi-
position during their adult lifespan. As such,
adult A. camptozonale may not be the best life
stage to use in a biological control release pro-
gram, and future field colonization efforts will fo-
cus on larval releases. Released larvae will be
able to feed and pupate in the field, and emerging
female moths will be free to focus on mating and
oviposition without enduring the stresses associ-
ated with handling and transportation to field
sites. Meanwhile, male-biased sex ratios were
shown to increase larval production in laboratory
colonies by reducing the percentage of unmated
female moths. By shedding light on these 2 impor-
tant aspects of the reproductive biology of
A. camptozonale the research detailed herein sug-
gests a strategy for maximizing colony production
and suggests a possible approach for improving
A. camptozonale field colonization efforts.

ACKNOWLEDGMENTS

The authors thank Luke Kasarjian, Senior Horticul-
tural Technician, USDA-ARS Invasive Plant Research
Lab, for weekly collection of L. microphyllum foliage
used to maintain the A. camptozonale colony. This re-
search was funded by financial support to develop bio-
logical controls for L. microphyllum from the Florida
Department of Environmental Protection and the South
Florida Water Management District.


September 2007


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Florida Entomologist 90(3)


A CHECKLIST OF THE APHIDS OF HONDURAS (HEMIPTERA: APHIDIDAE)

GREGORY A. EVANS' AND SUSAN E. HALBERT2
1USDA/APHIS/NIS, BARC-West, Building 005, Beltsville, MD 20705

2Florida State Collection of Arthropods, Division of Plant Industry, P.O. Box 147100, Gainesville, FL 32614

ABSTRACT

Forty-six species, belonging to 30 different genera of aphids, are reported from Honduras. Of
these, 26 represent new distribution records for Honduras. A list of the plant hosts in Hon-
duras on which the aphid species were collected is included.

Key Words: Aphididae, aphids, Honduras, survey, host plants, citrus tristeza

RESUME

Cuarenta y seis species de afidos en 30 g6neros diferentes estan informados de Honduras.
De estos g6neros, 26 representan nuevos registros para Honduras. Se incluye una lista de las
plants hospederas de estos afidos en Honduras.


Translation provided by the authors.


Honduras comprises 18 political units called
Departmentos (Departments) representing a
wide variety of habitats and ecosystems. Very few
studies have examined the aphid fauna of Hondu-
ras. Evers (1968) reported 14 aphid species asso-
ciated with banana and nearby ground cover
plants in the banana plantations of the Ulua Val-
ley in northern Honduras. Smith & Cermeli
(1979) reported 3 additional species from Hondu-
ras in their catalog of aphids of the Caribbean Is-
lands, and South and Central America.
From 1987 to 1988, the senior author (GAE)
collected aphids and other insects on plants in
several departments of Honduras, but primarily
in the central and western region. Host plants
were identified by Dr. Antonio Molina of the Pan-
american Agricultural School (EAP), Zamorano,
Honduras. In addition, we have included unpub-
lished records of aphid specimens present in the
United States National Museum (USNM) collec-
tion and those intercepted by the Animal Plant
Health Inspection Service (APHIS, USDA). from
1988 to 2007 at U.S. ports of entry on shipments of
products from Honduras. Readers are cautioned
that species that are only known from specimens
intercepted at a U.S. port of entry on plant mate-
rial exported from Honduras may not necessarily
be established in Honduras, and must be con-
firmed by collections made on plants grown
within the country.
Forty-six species of aphids, belonging to 30 dif-
ferent genera, are reported in Honduras based
upon these collections, literature records, and the
species intercepted at U.S. ports of entry from
Honduras; of these, 26 represent new distribution
records. A host plant list for the aphids collected


in or on shipments from Honduras is included.
Several of the records of aphids occurring on
plants that we have noted are unlikely to be the
aphid's "true" host. Aphids often will land on a
non-host plant, but will not feed and develop on
the plant. The following format is used to present
the collection and record data. Each record begins
with the name of the Department of Honduras
written in capital letters followed by the city, date
of collection, collector and host plant. The Depart-
ment of Francisco Morazan and the Escuela Agri-
cola Panamericana are abbreviated as FCO.
MORAZAN and EAP, respectively.

COLLECTION DATA AND RECORDS
FOR APHID SPECIES OF HONDURAS

Aphis coreopsidis (Thomas): EL PARAISO, Rio
Chiquito near Repaco, 10-XII-1987, G. Evans, ex
Baccharis salicifolia. FCO. MORAZAN, Lizapa,
26-VI-1991, K. Andrews, ex Bidens pilosa. Evers
(1968) listed this species on Bidens pilosa, Chap-
talia nutans and Vernonia scorpioides.
Aphis craccivora Koch: FCO. MORAZAN, El
Zamorano, EAP, 14-IV-1988, G. Evans, ex Caja-
nus cajan and 18-V-1988, G. Evans, ex Kall-
stoemia maxima; El Zamorano, 23-III-1988, G.
Evans, ex Gliricidia sepium. Intercepted at a U.S.
port of entry from Honduras on Cucurbita sp. and
at Miami, Florida on "golden shower" [Cassia fis-
tula?], 19-1-1963, Buff from Honduras. Evers
(1968) listed this species on Gliricidia sepium,
Kallstroemia maxima and Vigna sinensis.
Aphis gossypii Glover: FCO. MORAZAN, El
Zamorano, EAP, 22-II-1988, G. Evans, ex Cypho-
mandra betacea; 10-X-1987, G. Evans, ex Calli-


September 2007




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