• TABLE OF CONTENTS
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 Evaluation of irradiated Caribbean...
 Effectiveness of parasitoids of...
 Chemical analysis of female volatiles...
 Response of the predatory mite...
 Association between ants and a...
 The impact of temperature on biological...
 Temperature-dependent development...
 Impact of urbanization on tri-trophic...
 Parasitoids attacking the emerald...
 Description of the larva of Lophodiplosis...
 The parasitoid fly Ormia Ochracea...
 Epizootic of Acalitus vaccinii(Acari:...
 Diaphorina citri (Hemiptera: Psyllidae)...
 Blood meal identification from...
 A modified method to visualize...
 Effectiveness of reduced rates...
 Natural selection and maternal...
 Status of Coccobius nr. fulvus...
 Wolcottia (=Isohydnocera) aegra...
 Extraordinarily quick visual startle...
 Rearing Hylobius transversovittatus...
 Sampling riparian arthropods with...
 Steinernema diaprepesi (Nematoda:...
 An improved method for rearing...
 Efficacy of the suterra biolure...
 Asiatic garden beetle Maladera...
 Observation on Asphondylia websteri...
 Book reviews
 Volume 92 author index
 Presidential address: Fostering...
 Back Matter














Group Title: Florida Entomologist
Title: The Florida entomologist
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Permanent Link: http://ufdc.ufl.edu/UF00098813/00362
 Material Information
Title: The Florida entomologist
Uniform Title: Florida entomologist (Online)
Abbreviated Title: Fla. entomol. (Online)
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Language: English
Creator: Florida Entomological Society
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Publication Date: December 2009
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Subject: Entomology -- Periodicals   ( lcsh )
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periodical   ( marcgt )
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Additional Physical Form: Also issued in print.
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Language: In English; summaries in Spanish.
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.
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 Related Items
Preceded by: Florida buggist (Online)

Table of Contents
    Evaluation of irradiated Caribbean fruit fly (Diptera: Tephritidae) larvae for laboratory rearing of doryctobracon areolatus (Hymenoptera: Braconidae)
        Page 535
        Page 536
        Page 537
    Effectiveness of parasitoids of Bemisia tabaci (Hemiptera: Aleyrodidae) on cotton cultivars differing in leaf morphology
        Page 538
        Page 539
        Page 540
        Page 541
        Page 542
        Page 543
        Page 544
        Page 545
        Page 546
        Page 547
    Chemical analysis of female volatiles and field response of the coffee leafminer moth (Lepidoptera: Lyonetiidae) to stereoisomers of its major sex pheromone component
        Page 548
        Page 549
        Page 550
        Page 551
        Page 552
        Page 553
    Response of the predatory mite Phytoseiulus macropilis (Acari: Phytoseiidae) to pesticides and kairomones of three spider mite species (Acari: Tetranychidae), and non-prey food
        Page 554
        Page 555
        Page 556
        Page 557
        Page 558
        Page 559
        Page 560
        Page 561
        Page 562
    Association between ants and a leafhopper (Cicadellidae: Idiocerinae) in the central Brazilian cerrado
        Page 563
        Page 564
        Page 565
        Page 566
        Page 567
        Page 568
    The impact of temperature on biological aspects and life table of Rhopalosiphum padi (Hemiptera: Aphididae) fed with signal grass
        Page 569
        Page 570
        Page 571
        Page 572
        Page 573
        Page 574
        Page 575
        Page 576
        Page 577
    Temperature-dependent development of the cycad aulacaspis scale, Aulacaspis yasymatsui (Hemiptera: Diaspididae)
        Page 578
        Page 579
        Page 580
        Page 581
    Impact of urbanization on tri-trophic interactions in an endemic scrub community
        Page 582
        Page 583
        Page 584
        Page 585
        Page 586
        Page 587
    Parasitoids attacking the emerald ash borer (Coleoptera: Buprestidae) in western Pennsylvania
        Page 588
        Page 589
        Page 590
        Page 591
        Page 592
    Description of the larva of Lophodiplosis trifida, an Australian gall midge (Diptera: Cecidomyiidae) and biocontrol agent of paperbark in Florida, USA
        Page 593
        Page 594
        Page 595
        Page 596
        Page 597
    The parasitoid fly Ormia Ochracea (Diptera: Tachinidae) can use juvenile crickets as hosts
        Page 598
        Page 599
        Page 600
    Epizootic of Acalitus vaccinii(Acari: Eriophyidea) caused by Hirsutella thompsonii on southern highbush blueberry in north-central Florida
        Page 601
        Page 602
        Page 603
        Page 604
        Page 605
        Page 606
        Page 607
    Diaphorina citri (Hemiptera: Psyllidae) infection and dissemination of the entomopathogenic fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae) under laboratory conditions
        Page 608
        Page 609
        Page 610
        Page 611
        Page 612
        Page 613
        Page 614
        Page 615
        Page 616
        Page 617
        Page 618
    Blood meal identification from Florida mosquitoes (Diptera: Culicidae)
        Page 619
        Page 620
        Page 621
        Page 622
    A modified method to visualize infection sites of spores of the entomopathogen Beauveria bassiana (Deuteromycotina: Hyphomycetes) on the exoskeleton of citrus root weevil Diaprepes abbreviatus (Coleoptera: Curculionidae) adults
        Page 623
        Page 624
        Page 625
        Page 626
        Page 627
        Page 628
    Effectiveness of reduced rates of insecticides for the control of Melanotus communis (Coleoptera: Elateridae) in sugarcane
        Page 629
        Page 630
        Page 631
        Page 632
        Page 633
        Page 634
    Natural selection and maternal effects in life history traits of Brevicoryne brassicae (Homoptera: Aphididae) on two sympatric closely related hosts
        Page 635
        Page 636
        Page 637
        Page 638
        Page 639
        Page 640
        Page 641
        Page 642
        Page 643
        Page 644
    Status of Coccobius nr. fulvus (Hymenoptera: Aphelinidae), a parasitoid of euonymus scale (Hemiptera: Diaspididae), 12-16 years after it release in Massachusetts
        Page 645
        Page 646
        Page 647
    Wolcottia (=Isohydnocera) aegra (Coleoptera: Cleridae): association with grasses (Poaceae), native Spartina ssp. and the introduced Eragrostis curvula, and resemblance to co-occurring pseudomyrmecine ants
        Page 648
        Page 649
        Page 650
        Page 651
        Page 652
    Extraordinarily quick visual startle reflexes of skipper butterflies (Lepidoptera: Hesperiidae) are among the fastest recorded in the animal kingdom
        Page 653
        Page 654
        Page 655
        Page 655a
    Rearing Hylobius transversovittatus and Cyphocleonus achetes larvae on artificial diets (Coleoptera: Curculionidae)
        Page 656
        Page 657
    Sampling riparian arthropods with flight-interception bottle traps
        Page 658
        Page 659
        Page 660
    Steinernema diaprepesi (Nematoda: Steinernematidae): its occurrence in western Mexico and susceptibility of engorded cattle ticks Boophilus microplus (Acari: Ixodidae)
        Page 661
        Page 662
        Page 663
    An improved method for rearing western flower Thrips frankliniella occidentalis
        Page 664
        Page 665
        Page 666
    Efficacy of the suterra biolure individual female fruit fly attractant packages vs. the unipak version
        Page 667
        Page 668
        Page 669
    Asiatic garden beetle Maladera castanea (Coleoptera: Scarabaeidae) grubs found in damaged turf in Alabama
        Page 670
        Page 671
        Page 672
    Observation on Asphondylia websteri (Diptera: Cecidomyiidae) infesting hass avocados in Guatemala
        Page 673
        Page 674
    Book reviews
        Page 675
        Page 676
        Page 677
        Page 678
        Page 679
        Page 680
        Page 681
        Page 682
    Volume 92 author index
        Page 683
        Page 684
        Page 685
        Page 686
        Page 687
        Page 688
    Presidential address: Fostering collaboration and linkages with our Latin American and Caribbean neighbors
        Page 689
        Page 690
    Back Matter
        Page 691
        Page 692
Full Text



Palenchar et al.: Rearing the Parasitoid D. areolatus


EVALUATION OF IRRADIATED CARIBBEAN FRUIT FLY
(DIPTERA: TEPHRITIDAE) LARVAE FOR LABORATORY REARING OF
DORYCTOBRACONAREOLATUS (HYMENOPTERA: BRACONIDAE)

JESSICA PALENCHAR', TIM HOLLER AMY MOSES-ROWLEY ROBERT McGOVERN1 AND JOHN SIVINSKI3
'University of Florida, Plant Medicine Program, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL 32611

2Center for Plant Health Science and Technology Plant Protection and Quarantine,
Animal Plant Health Inspection Service, United States Department of Agriculture, Gainesville, FL 32604

'Center for Medical, Agricultural and Veterinary Entomology, United States Department of Agriculture,
Agricultural Research Service, 1700 SW 23rd Dr., Gainesville, FL 32608

ABSTRACT

We report here that it is possible to rear D. areolatus on irradiatedA. suspense larvae with-
out adversely affecting sex ratio and overall parasitoid emergence and with no adult A. sus-
pense emergence. There was no difference in emergence of D. areolatus adults from
irradiated versus non-irradiated hosts (72.4 1.9% vs. 73.0 1.9%), and no difference in sex
ratio of parasitoids obtained from irradiated and non-irradiated hosts (50.0 1.6 and 47.0
1.4% female, respectively). The successful use of A. suspense larval hosts can greatly ease
the process of rearing, transporting, and releasing fruit fly parasitoids while eliminating the
need to separate flies from parasitoids. Further improvements in the laboratory rearing pro-
cess of D. areolatus, including irradiating late A. suspense larvae at a lower dosage and ir-
radiatingA. suspense as egg or early instars, are discussed.

RESUME

Reportamos que es possible criar D. areolatus sobre larvas deA. suspense irradiadas sin afec-
tar adversamente la proporci6n de machos y hembras y la emergencia total de los parasitoi-
des y sin desarrollo de adults deA. suspense. No hubo diferencia en la emergencia de adults
de D. areolatus de hospederos irradiados versus no irradiados (72.4 1.9% vs. 73.0 1.9%),
y no hubo diferencia en la proporci6n de machos y hembras obtenida de hospederos irradiados
versus no irradiados (50.0 1.6 y 47.0 1.4% hembras, respectivamente). El uso exitoso de
la larva hospedera A. suspense puede facilitar much el process de cria, transport y libera-
ci6n de parasitoides de moscas de la fruta con la eliminaci6n en la necesidad de separar las
moscas de sus parasitoides. Se discuten otras mejoras en el process de criar D. areolatus en
el laboratorio incluyendo la irradiaci6n de los ultimos estadios de las larvas deA. suspense a
una dosis menor y la irradiaci6n los huevos y estadios tempranos de A. suspense.


Mass-rearing and augmentative release of hy-
menopterous parasitoids has been a component of
area-wide management programs for several te-
phritid fruit flies, including pestiferous species of
the genusAnastrepha (Cancino & Montoya 2008).
Laboratory rearing of Doryctobracon areolatus
(Szepligeti), a braconid larval-prepupal parasi-
toid ofAnastrepha fruit flies, was first done in the
United States in Florida in the late 1960s as part
of an effort to biologically control the Caribbean
fruit fly, Anastrepha suspense (Loew) (Bara-
nowski et al. 1993; Cancino et al. 2008). Although
releases of D. areolatus for management of the A.
suspense in Florida have ended, laboratory rear-
ing of D. areolatus is needed to produce parasi-
toids for establishment in Caribbean locations
with pest fruit flies (Holler, unpublished data).
The process of rearing, transporting, and re-
leasing parasitoids can be simplified if irradiated


fruit fly larvae are used as hosts (Sivinski & Smit-
tle 1990). Larvae irradiated at an appropriate
dose will not develop into adult flies, but are capa-
ble of supporting the development of a number of
fruit fly-specific braconid parasitoids, including
Doryctobracon crawfordi (Viereck) (Aluja et al.
2008; Cancino et al.2008). In addition, parasitoids
can be moved as pupae without transferring the
pest. Therefore, tests were conducted to deter-
mine the effects of gamma irradiation of host lar-
vae at a single dose on D. areolatus production
and sex ratio, and on the ability of the host to com-
plete development to adult.

MATERIALS AND METHODS

Laboratory tests were initiated 18 Jun 2007
and all studies were completed 23 Aug 2007 when
parasitoid and fly emergence had ceased. Dorycto-







Florida Entomologist 92(4)


bracon areolatus adults were hand aspirated from
F26 or F27 generation stock cages maintained by
the USDA-APHIS-PPQ-CPHST Station, in
Gainesville, FL. Fifty females and 15 males per
cage were placed into 10 oviposition Plexiglas
cages (30 cm3), with fine mesh fabric (organza)
tops and provisioned with a water source and a
food source (honey on a moistened paper towel).
Food was replaced weekly, water was replaced ev-
ery 2 weeks and cages were washed after 4 weeks.
To replace parasitoids that had died, 50 females
and 5 males were added to each cage after 2
weeks, 50 females and 15 males were added after
4 weeks, and 8 females and 6 males were added af-
ter 5 weeks. Positions of the cages in the room
were rotated weekly. All studies were conducted in
a room maintained at 24.4-26.7C, 60-85% relative
humidity, and with a 12:12 h light:dark cycle.
Host larvae were raised on a sterilized corn-
cob-grit-based diet at the Biological Control Rear-
ing Facility, FDACS-Division of Plant Industry
in Gainesville, Florida. Weekly, 140 g of diet con-
taining second instars were placed in 550 mL ir-
radiation tubes and irradiated at 70 Gray for 7.5
min with a cesium source (Isomedix Gamma Cell
1000) (Sivinski & Smittle 1990;Aluja et al. 2008).
After 5-6 d, irradiated and non-irradiated larvae
were placed separately in sting rings and were
presented to the parasitoids (Eitam et al. 2003).
The sting rings contained 13.5 g of larvae and diet
(approximately 107 insects) sandwiched between
a piece of organza on the inside bottom of an em-
broidery ring and a piece of Parafilm on top. One
sting ring with either irradiated or non-irradiated
larvae was set in each cage on the top of an upside
down cup for 24 h. The entire procedure was re-
peated the following day with fresh larvae. This
procedure began 1 week after the oviposition
cages were set up and continued for 4 weeks, with
2 oviposition periods tested per week for a total of
10 host exposure periods.
When removed from a cage, the sting ring was
disassembled and the contents were placed in a
plastic cup (215 mL) filled with fresh diet. The cup
was placed in a plastic container (650 mL) with
vermiculite (60 g) moistened with 1% sodium hy-
pochlorite. The plastic containers were placed in a
plastic storage box (25.5 L, Sterilite Corp., Bir-
mingham, AL) with 2 organza-covered holes in
the lid to provide ventilation. After 5-7 d, the pu-
pae were sieved from the vermiculite and moved
to fresh vermiculite, again moistened with 1% so-
dium hypochlorite, and kept for approximately 1
week. At 24- 48 h prior to emergence, the pupae
were transferred to 10 x 10-celled emergence lids
with louvered florescent light cover with a solid
white acrylic bottom and clear acrylic top. After
12 d, approximately 25 d after the oviposition pe-
riod, numbers of adult flies and parasitoids were
recorded. Adult emergence was complete by that
time.


Statistical Analyses

Analysis of variance with PROC ANOVA (SAS
Institutel989) was used to test the effect of radi-
ation treatment, host exposure period and cage on
parasitoids. The numbers of host pupae per cage
per d varied due to availability, so emergence data
for both flies and parasitoids were converted to
proportion parasitized, which were arcsine
(square root)-transformed prior to analysis.
Transformed data were compared by Wilcoxon
paired-sample test. Sex ratio, as indicated by per-
centage of the offspring that were female, was
compared by Student's t-test (SAS Institute
1989). All summary statistics are presented as
mean and standard error, and emergence rates as
percentages.

RESULTS

There was a significant effect of day of expo-
sure on emergence rates (F = 19.5; df = 1, 9; P <
0.0001) but no effect of cage (F = 1.6;df= 1, 4;P =
0.19). Parasitoid emergence in the last 2 exposure
periods was lower than in previous exposure peri-
ods, most likely because fewer parasitoids were
added to the oviposition cages prior to these peri-
ods. There was no difference in emergence ofD.
areolatus adults from irradiated versus non-irra-
diated hosts (72.4 1.9% vs. 73.0 1.9%; t = 21.5;
n = 10; P > 0.25). Nor was there a difference in sex
ratio of parasitoids obtained from irradiated and
non-irradiated hosts (50.0 1.6 and 47.0 1.4%
female, respectively; t = 1.18; df= 1, 88;P = 0.24).
Anastrepha suspense were affected by irradia-
tion, with 0% emergence from irradiated larvae
versus 15.3 1.9% emergence from non-irradi-
ated, parasitized larvae, (t = 0; n = 10; P < 0.003).
It was assumed that the mortality rates of the
parasitoids in oviposition cages that were pro-
vided with non-irradiated larvae versus irradi-
ated larvae were similar over time. The actual
numbers ofparasitoid females per cage at host ex-
posure period were not recorded, so data on off-
spring per female were not available for analysis.
Parasitoid numbers in the last 2 exposures, fol-
lowing a smaller than usual addition of parasi-
toids, were lower than in previous exposures,
which resulted in lower parasitoid emergence and
higher fly emergence.
Larvae ofA. suspense irradiated at the dose
used herein can be utilized to rear D. areolatus
successfully in the laboratory. No A. suspense
adults emerged from irradiated larvae, and
there was no difference in the percent parasit-
ism of irradiated and non-irradiated hosts Use
of irradiated larvae as rearing hosts would
streamline the rearing process and result in
both increased savings and greater safety when
D. areolatus are shipped overseas for biological
control programs.


December 2009








Palenchar et al.: Rearing the Parasitoid D. areolatus


Future studies might further improve use of ir-
radiated host larvae for production of D. areola-
tus. Cancino et al. (2008) showed that irradiating
A. ludens larvae at dosages as low as 20 Gray was
just as effective as higher dosages in preventing
the emergence of adults. Earlier irradiation
might be more convenient and could expedite par-
asitoid production. Anastrepha suspense that are
used for rearing parasitoids are placed as eggs on
artificial diet several days prior to stinging. It
would be desirable to irradiate A. suspense at the
egg or earliest larval instar because it would not
require handling later instars to obtain hosts for
oviposition, which would avoid any concomitant
mechanical damage. Preliminary tests by the au-
thors suggest that irradiating the entire pan of
diet shortly after the egg strips are added is suc-
cessful in preventing emergence of A. suspense
adults, but will not prevent A. suspense from pu-
pating. Further work is needed to determine if ir-
radiating A. suspense as eggs or early instars
could be incorporated into the rearing of D. are-
olatus.


ACKNOWLEDGMENTS

The authors thank the U.S. Department of Agricul-
ture and Dr. Robert J. McGovern for funding the intern-
ship that allowed this work to take place, Morgan
Scarborough for assistance in preparation and sanita-
tion, Carl Gillis for larval irradiation, Nancy Epsky for
valuable manuscript critique, Valerie Malcolm and
Shelley Olson for manuscript preparation, Dr. Mirian
Medina Hay-Roe for the RESUMEN, and the Palenchar
family.


REFERENCES CITED
ALJUA, M., SIVINSKI, J., OVRUSKI, S., GUILLEN, L., LO-
PEZ, M., CANCINO, J., TORRES-ANAYA, A., GALLEGOS-
CHAN, G., AND RUIZ, L. 2008. Colonization and domes-
tication of seven species of native New World hy-
menopterous larval-prepupal and pupal fruit fly
(Diptera: Tephritidae) parasitoids. Biocont. Sci. Tech-
nol. http://dx.doi.org/10.1080/09583150802377373
BARANOWSKI, R., GLENN, H., AND SIVINSKI, J. 1993. Bi-
ological Control of the Caribbean fruit fly
(Diptera:Tephritidae). Florida Entomol. 76: 245-251.
CANCINO, J., AND MONTOYA, P. 2008. Advances and per-
spectives in the mass rearing of fruit fly parasitoids in
Mexico, pp. 133-142 In R. L. Sugayama, R. A. Zucchi, S.
M. Ovruski, and J. Sivinski [eds.], Fruit Flies of Eco-
nomic Importance: From Basic to Applied Knowledge,
Proc. 7t Intl. Symp. on Fruit Flies of Economic Impor-
tance, 10-15 Sept. 2006, Salvador, Brazil.
CANCINO J., RUIZ, L., SIVINSKI, J., GALVEZ, F. 0., AND
ALUJA, M. 2008. Rearing of five hymenopterous lar-
val-prepupal (Braconidae, Figitida) and three pupal
(Diapriidae, Chalcidoidea, Eurytomidae) native par-
asitoids of the genus Anastrepha (Diptera: Tephriti-
dae) on irradiated A. ludens larvae and pupae. Bio-
control Sci. Techn. http://dx.doi.org/10.1080/
09583150802377423
EITAM, A., HOLLER, T., SIVINSKI, J., AND ALUJA, M.
2003. Use of host fruit chemical cues for laboratory
rearing of Doryctobracon areaolatus (Hymenoptera:
Braconidae), a parasitoid of Anastrepha spp. (Diptera:
Tephritidae). Florida Entomol. 86: 211-216.
SAS INSTITUTE. 1989. Users Guide, SAS Institute. Cary,
NC, USA.
SIVINSKI, J., AND SMITTLE, B. 1990. Effects of gamma
radiation on the development of the Caribbean fruit
fly (Anastrepha suspense) and the subsequent devel-
opment of its parasite Diachasmimorpha longicau-
data. Entomol. Exp. Appl. 55: 295-297.







Florida Entomologist 92(4)


December 2009


EFFECTIVENESS OF PARASITOIDS OF BEMISIA TABACI
(HEMIPTERA: ALEYRODIDAE) ON COTTON CULTIVARS
DIFFERING IN LEAF MORPHOLOGY

RAMAZAN CETINTAS AND HEATHER MCAUSLANE
University of Florida, Department of Entomology and Nematology, Gainesville, FL 32611

ABSTRACT

Field and laboratory experiments were conducted to determine resistance of cotton Gossyp-
ium hirsutum L. cultivars differing in leaf morphology (shape and pubescence) to the B bio-
type of sweetpotato whitefly Bemisia tabaci (Gennadius) and impacts on its parasitoids. Five
cotton cultivars were evaluated in a field-plot experiment at Hastings, Florida. The pubes-
cent cultivars, DP DES 119 and Stoneville 453, were significantly hairier than moderately
hairy okra-leaf cultivar, Gumbo 500, and smooth-leaved cultivars NU COTN 33B and DP 51.
There were significant differences among cultivars in eggs laid on the second and fifth node
leaves. DP DES 119 and Stoneville 453 had greater numbers of whitefly eggs than did the
glabrous cultivars. The okra-leaf cultivar, Gumbo 500, had greater numbers of eggs than the
2 glabrous varieties. There were significant differences among cultivars in numbers of first
and second instars (young instars), third instars, unparasitized fourth instars and red-eyed
nymphs on fifth node leaves, with higher populations occurring on pubescent cultivars and
lower populations on glabrous cultivars. The abundant parasitoids were Encarsia pergandi-
ella Howard, Encarsia nigricephala Dozier and Eretmocerus spp., however parasitism did
not differ among the cultivars. The responses of Eretmocerus rui Zolnerowich & Rose and
Encarsia formosa Gahan (Nile Delta strain) (Hymenoptera: Aphelinidae) as a function of
density of the host were investigated in laboratory experiments on 3 cotton cultivars differ-
ing in leaf pubescence and shape (DP 51, DP DES 119, and Gumbo 500). Eretmocerus rui
showed a type II functional response to second instars of the host with the mean number of
parasitized hosts increasing as host density increased on all 3 cultivars. Encarsia formosa
showed a type II functional response to fourth instars, where the number of hosts parasit-
ized increased up to a density of 16 but remained constant for 32 and 64 hosts. Significantly
more whiteflies were parasitized by both E. formosa and E. rui on DP 51, the smooth-leaved
cotton cultivar, than on the hairy cotton cultivars. We conclude that glabrous cotton cultivars
are likely to support lower whitefly populations than pubescent cultivars because of reduced
whitefly oviposition and increased parasitoid foraging efficiency.

Key Words: Bemisia tabaci, biological control, functional response, parasitoid, whitefly, Eret-
mocerus, Encarsia

RESUME

Se realizaron experiments en el campo y en el laboratorio para determinar la resistencia de
variedades de algod6n, Gossypium hirsutum L., con diferencias en la morfologia de la hoja
formaa y pubescencia) hacia el biotipo B de la mosca blanca de camote, Bemisia tabaci (Gen-
nadius) y el impact sobre sus parasitoides. Se evaluaron cinco variedades de algod6n en un
experiment de parcelas de campo en Hastings, Florida. Las variedades con hojas pubescen-
tes, DP DES 119 y Stoneville 453, fueron significativamente mas peludas que las variedades
de hojas de okra moderadamente peludas, Gumbo 500 y variedades con hojas lisas NU
COTN 33B y DP 51. Hubo diferencias significativas entire las variedades en cuanto a los hue-
vos puestos sobre las hojas del segundo y quinto n6dulo. La DP DES 119 y Stoneville 453 tu-
vieron un mayor numero de huevos de mosca blanca que las variedades con hojas glabrosas.
La variedad de hoja de okra, Gumbo 500, tuvo un mayor numero de huevos que las 2 varie-
dades glabrosas. Hubo diferencias significativas entire las variedades en cuanto al numero
de los estadios de primero y segundo estadio (estadios j6venes), tercer estadio, cuarto estadio
no parasitados y de las ninfas de ojos rojos sobre hojas del quinto n6dulo, con poblaciones
mas altas sobre las variedades pubescentes y poblaciones menores sobre las variedades gla-
brosas. Los parasitoides mas abundantes fueron Encarsia pergandiella Howard, Encarsia
nigricephala Dozier y Eretmocerus spp., sin embargo el nivel del parasitismo no fue dife-
rente entire las variedades. Se investigo la respuesta de Eretmocerus rui Zolnerowich & Rose
y Encarsia formosa Gahan (cepa Nile Delta) (Hymenoptera: Aphelinidae) como una funci6n
de la densidad de hospedero en experiments del laboratorio sobre 3 variedades de algod6n
con diferencias en la forma y la pubescencia de la hoja (DP 51, DP DES 119 y Gumbo 500).
Eretmocerus rui mostr6 una respuesta funcional de tipo II hacia el segundo estadio del hos-
pedero con un promedio del numero de hospederos parasitados incrementandose con el au-
mento en la densidad del hospedero en todas las 3 variedades. Encarsia formosa mostr6 una







Cetintas & McAuslane: Parasitism ofB. tabaci on Cotton


respuesta funcional de tipo II hacia el cuatro estadio, donde el nmmero de hospederos para-
sitados aumento hasta una densidad de 16 pero se mantuvo constant para los 32 y 64 hos-
pederos. Significativamente un mayor numero de moscas blancas fueron parasitadas por
ambos E. formosa y E. rui en DP 51, la variedad de algod6n con hojas lisas, que en las varie-
dades de algod6n con hojas peludas. Concluimos que las variedades glabrosas de algod6n son
mas propensas de apoyar poblaciones menores de la mosca blanca que las variedades pub-
escentes por la oviposici6n reducida de la mosca blanca y el aumento en la eficiencia del pa-
rasitoide para forrajear.


The B biotype of the sweetpotato whitefly, Be-
misia tabaci (Gennadius) (Hemiptera: Aley-
rodidae), also known as the silverleaf whitefly, Be-
misia argentifolii Bellows & Perring, is a world-
wide tropical and subtropical pest of many crops,
including agronomic field crops such as cotton.
The A biotype of the sweetpotato whitefly had
been present in the United States since 1894
(Quaintance 1900) and was seen on cotton as
early as 1929 (Mound & Halsey 1978). After intro-
duction into Florida in the late 1980s, and subse-
quent spread throughout the United States, out-
breaks of the B biotype of B. tabaci in cotton and
other crops caused widespread economic losses
(Choen et al. 1992; Perring et al. 1993). Huge in-
festations of whiteflies on cotton reduce the vigor
of young plants, leading to reduced yields, and
produce copious quantities of sticky honeydew.
Cotton fibers contaminated with honeydew and
stained with sooty mold are unacceptable to buy-
ers because this "sticky cotton" is difficult to pro-
cess (Hendrix et al. 1995). Whiteflies have been
reported to vector many important viral diseases,
including crumple cotton and cotton leaf curl
(Costa 1976; Butler & Henneberry 1994). All of
these problems can cause plant stunting, prema-
ture leaf drop, defoliation, boll shed, and reduced
yields in cotton.
The use of resistant cultivars may be an effec-
tive tactic in the management ofB. tabaci. Cotton
cultivars which have smooth leaves have been
shown to support smaller whitefly populations
than highly pubescent cultivars (Butler & Hen-
neberry 1984; Butler at al. 1986). Plants with an
okra-leaf shape and an open canopy have been
shown to exhibit resistance to B. tabaci (Ozgur &
Sekeroglu 1986; Chu at al. 2002). Recent observa-
tions have shown that cotton cultivars with lower
numbers of stellate trichomes on the abaxial leaf
surface supported lower numbers of B. tabaci
eggs, nymphs, and adults (Chu at al. 2001).
In addition to the potential reduction in white-
fly populations due to use of less susceptible culti-
vars, considerable mortality can be imposed by
natural enemies, such as parasitoids, predators,
and pathogens (Carruthers et al. 1993). Of these
agents, most research on biological control has fo-
cused on parasitoids, particularly parasitic wasps
in the Encarsia and Eretmocerus genera (Carru-
thers et al. 1993). Apparent parasitism levels
ranging from 70 to 80% have been reported in cot-


ton (Horowitz 1993) and kenaf (Legaspi et al.
1997). A study by Chu et al. (1998) indicated that
whitefly colonization could be reduced signifi-
cantly by planting cotton cultivars that are at-
tractive to parasitoids.
However the success of parasitoids is highly
variable, depending on the host plant, climate,
presence of competing natural enemies, use of
nonselective insecticides, and a number of envi-
ronmental factors (Butler & Henneberry 1994;
Hoelmer 1995). In addition, the parasitism ability
of whitefly parasitoids may be directly affected by
plant resistance traits (McAuslane et al. 1995;
van Roermund & van Lenteren 1995; van Len-
teren et al. 1995; McAuslane et al. 2000). Leaf pu-
bescence has received the most study for its po-
tential effect on whitefly parasitoids. An early
study showed the negative effect of leaf hairs on
cucumber varieties on walking speed and walking
pattern of Encarsia formosa Gahan (Hulspas-
Jordaan & van Lenteren 1978). Consequently, the
rate of parasitism of the greenhouse whitefly, Tri-
aleurodes vaporariorum Westwood, by E. formosa
decreased linearly with increasing hair density
on cucumber hybrids (van Lenteren et al. 1995). A
greenhouse study by Gruenhagen & Perring
(2001) showed that among 5 host plants studied,
including cotton, the plants with higher tri-
chomes levels resulted in lower parasitism of sil-
verleaf whitefly by Eretmocerus eremicus Rose &
Zolnerowich. This was, in part, attributed to exu-
date from glandular trichomes, which entrapped
parasitoids.
The objective of the experiments reported here
was to determine the susceptibility to B. tabaci of
cotton cultivars suitable for production in Florida
and the southeastern United States and the effec-
tiveness of parasitoids of B. tabaci on these culti-
vars. We chose 5 cultivars that differed in leaf
morphology (okra-leaf or normal-leaf) and pubes-
cence. Whitefly populations and parasitism were
assessed in a replicated field trial of all cultivars.
In a second experiment, we evaluated the func-
tional responses of 2 whitefly parasitoids, E. for-
mosa (MCB 92030, Nile Delta strain) and Eret-
mocerus rui Zolnerowich & Rose on 3 of the culti-
vars. We chose 1 species of each genus because of
differences in the oviposition behavior of the gen-
era. Encarsia formosa oviposits directly into the
body of third, fourth, and prepupal stages of the
whitefly (Nell et al. 1976). Eretmocerus species







Florida Entomologist 92(4)


lay eggs under earlier stages of the host, prefera-
bly the second nymphal stage (McAuslane &
Nguyen 1996). Ease of inserting the ovipositor
underneath the whitefly host may be affected by
how well the host can seal itself to the leaf with
wax. Leaf hairs may reduce the ability of the
whiteflies to seal themselves to the leaf.

MATERIALS AND METHODS

Field Experiment and Design

The field experiment was conducted in 1997 in
Hastings, Florida. Five commercial cotton culti-
vars (Gossypium hirsutum L.) with different leaf
pubescence and shape were evaluated for their
susceptibility to B. tabaci and the effectiveness of
parasitoids ofB. tabaci on these cultivars. The ex-
periment was designed as a randomized block de-
sign with 5 replications. Plots were 12.2 m long by
6 rows wide and plots were separated by a break
of 1.2 m. Delta Pine 51 (DP 51) and NU COTN
33B (genetically modified to express to Bt toxin
CrylAc) were both smooth (i.e., glabrous) normal-
leaf cultivars. Delta Pine DES 119 (DP DES 119)
and Stoneville 453 were hairy (i.e., pubescent)
normal-leaf cultivars and Gumbo 500 was a mod-
erately hairy okra-leaf cultivar. Cotton cultivars
were planted on 23 May 1997; however, due to
poor germination, Gumbo 500 was replanted 2, 9,
16, and 23 Jun. Sampling began approximately 9
weeks after first planting.

Sampling

Sampling was carried out for 7 weeks, from 27
Jul to 12 Sep. Each week 40 plants were sampled
randomly in each replicate. On the first day of the
week, a leaf from the second node (counting down
from the top) of 20 plants, and on the third day of
the week a leaf from the fifth node of another 20
plants were sampled. In the laboratory, the area
of each leaf was measured with a LiCor 3000 leaf
area meter (LiCor, Lincoln, NE). Leaf hairs were
counted within an area of the abaxial leaf surface
contained within a No. 3 cork borer impression
(area = 0.384 cm2). Numbers of whitefly eggs,
young nymphs (first and second instars com-
bined), third instars, unparasitized fourth in-
stars, red eyed nymphs, exuvia (as parasitized
and unparasitized separately), and parasitized
fourth instars (as Encarsia parasitized, Eret-
mocerus parasitized or as unidentifiable) were
counted within an area (located between the cen-
tral main vein and the medial vein (Ellsworth et
al., 1996) on the abaxial leaf surface contained
within a No.13 cork borer impression (area = 3.14
cm2). All counts were made with a dissecting mi-
croscope at 20x magnification.
Beginning with the fourth week of sampling,
the leaf disks sampled from the fifth node were


kept in 450-mL cardboard cartons so that all par-
asitoids emerging from parasitized nymphs could
be counted and identified to species 5 weeks later.
Cotton cultivars were monitored for insect
pests other than whiteflies by standard cotton
scouting procedures. When pests reached eco-
nomic threshold levels, insecticides were used in
order to prevent economic loss. Whenever feasi-
ble, the plots were treated with selective insecti-
cides that would have the least effect on whiteflies
and beneficial insects. Dimilin 2L (diflubenzuron,
(N-(4-chlorophenyl) amino) carbonyl)-2, 6-difluo-
robenzamide (224 giA) (Chemtura, Middlebury,
CT), and Dipel 2X ((6.4% w/w), Bacillus thuring-
iensis var. kurstaki (450 giA)) (Valent BioSciences,
Libertyville, IL) were applied twice, on Aug 8 and
12. Other agronomic practices used were stan-
dard for the area.
The experiment was designed as a randomized
complete block design with 5 replications, how-
ever data for replications 1 and 2 were dismissed
because of hurricane damage to plant stand; con-
sequently data for the remaining 3 replications
were evaluated. Data were pooled across sam-
pling dates (over a 7-week period) and analyzed
for whitefly numbers, trichome densities, and pro-
portional parasitism on the 5 cotton cultivars
with Proc GLM (SAS Institute, 2001) at a signifi-
cance level ofP < 0.05. Proportional parasitism on
the fifth leaf node was calculated with the follow-
ing equation:

FPER+FPEN+FPAR/FPER+FPEN+FPAR+FNPA,

where FPER = Fourth instar parasitized by Eret-
mocerus sp., FPEN = Fourth instar parasitized by
Encarsia sp., FPAR = Fourth instar parasitized
(parasitoid unknown), FNPA = Fourth instar non-
parasitized. Means were separated by Waller-
Duncan's multiple-range test.

Laboratory Experiment and Insects Used

Whiteflies used in the laboratory study came
from a colony reared on DP 50 cotton and collards,
Brassica oleraceae L., in an indoor rearing room
maintained at the Department of Entomology
and Nematology, University of Florida, Gaines-
ville, Florida. The photoperiod was 14:10 (L: D) h,
temperature averaged 27 1VC during the day
and 24 1VC at night, and relative humidity
ranged from 40 to 60 percent. Encarsia formosa
(Nile Delta strain, MCB 92030) was acquired
from USDA-APHIS-PPQ, Mission Plant Protec-
tion Center (Mission, Texas). This species was
reared on B. tabaci in Mission and is considered a
Bemisia-adapted, rather than a Trialeurodes-
adapted, strain. A subcolony of this parasitoid
was maintained in an indoor rearing room at the
University of Florida on B. tabaci on hibiscus, Hi-
biscus rosa-sinensis L., (red single flower) under


December 2009







Cetintas & McAuslane: Parasitism of B. tabaci on Cotton


the same conditions used to rear the whitefly
host. Eretmocerus rui originated from a separate
indoor colony rearing room with the same condi-
tions as E. formosa. Eretmocerus rui is a thelytok-
ous species and was imported into the United
States from Hong Kong in Jul 1992 (McAuslane &
Nguyen 1996). Parasitoids used in this experi-
ment were obtained from the colony and were
standardized as 2 1 d old.

Functional Responses of Parasitoids

The purpose of this study was to determine the
functional responses of 2 whitefly parasitoids, E.
rui and E. formosa, on 3 different cotton cultivars
differing in leaf shape and trichome density. The
experiment was designed as a randomized com-
plete block with a factorial design. The factors
were 3 cotton cultivars [DP DES 119 (normal-leaf,
hairy), Gumbo 500 (okra-leaf), DP 51 (normal-
leaf, smooth)], four different whitefly nymph den-
sities (8, 16, 32 and 64 per plant), and 2 parasitoid
species (E. rui and E. formosa). The experiment
was conducted in small cylindrical cages, in the
laboratory, 3 times for E. rui (5 individual females
at a time for a total of 15 replicates) and 4 times
for E. formosa (5 individual females at a time for
a total of 20 replicates), under a 14:10 light/dark
cycle and 25C temperature conditions. The cages
measured 18.5 cm in diameter and 61.5 cm in
height and were formed of 0.02-mm-thickness Vi-
vak plastic. The measured average numbers of
hairs for DP DES 119, Gumbo 500 and DP 51
were 182.5, 68.1 and 7.0, respectively, within an
area of 0.384 cm2.
The 3 cotton cultivars were grown in a green-
house in 15-cm-diameter pots containing Metro-
Mix 220 (Grace Sierra, Milpitas, CA). Plants were
watered as necessary and fertilized twice a month
with liquid fertilizer (20:20:20, N: P: K, Peters, W.
R. Grace, Fogelsville, PA). Plants were grown in
the greenhouse until the Gumbo 500 plants had
developed 2 fully extended okra-shaped leaves.
All other leaves were removed from the 3 culti-
vars in order to standardize the number of leaves
on the plants. Following this, male-female pairs of
adult whiteflies (5, 10, 20, and 40) were released
onto each plant to obtain an appropriate infesta-
tion. After 24 h, the adult whiteflies were re-
moved. Extra nymphs were removed after 5 d
with a minute pin to obtain the desired 4 differ-
ent densities of immature whitefly instars of 8,
16, 32, and 64 per plant.
A single female parasitoid was introduced onto
each plant when the correct whitefly stage was
available for parasitism. This appropriate stage
was determined as fourth instar for E. formosa,
and second instar for E. rui. The parasitoid was
removed after 24 h. Parasitism of the host was re-
corded as pupae of the parasitoids became evident
within the host after 15 d for E. formosa and after


11 to 12 d for E. rui. The number of whiteflies par-
asitized was analyzed using Proc GLM (SAS In-
stitute, 1989) at a significance level of P < 0.05.
Means were separated by Waller-Duncan's multi-
ple-range test. Functional response types for the
2 parasitoids were determined visually.

RESULTS

Field Experiment

Pubescence on the second (F = 1297.2; df = 4,
24; P < 0.0001) and fifth node leaves (F = 1303.7;
df = 4, 24;P < 0.0001) differed significantly among
the cultivars (Table 1). The mean number of hairs
was greatest in Stoneville 453 (second leaf) and
DP DES 119 (fifth leaf) and was lowest in DP 51
and NU COTN 33B (for second and fifth leaves).
Pubescence was intermediate on the okra-leaf va-
riety, Gumbo 500.
There were significant differences among culti-
vars for number of eggs on both the second node
leaf (F = 489.2; df = 4, 24; P < 0.0001) and on the
fifth node leaf (F = 29.2; df = 4,24;P < 0.0001) (Ta-
ble 2). DP DES 119 and Stoneville 453 had the
greatest number of eggs on the second node leaf
and fifth node leaf, respectively, whereas NU
COTN 33B and DP 51 had the least number of
whitefly eggs.
There were significant differences among culti-
vars for young instars (F = 107.8; df = 4, 24; P <
0.0001) and third instars (F = 15.8; df = 4, 24; P <
0.0001) on the second node leaf (Table 2). DP DES
119 sustained the greatest numbers of these in-
stars among cultivars. The mean numbers of
other whitefly stages varied among plant culti-
vars on the second node leaf. There were signifi-
cant differences among cultivars for young in-
stars (F = 88.87; df = 4, 24; P < 0.0001), for third
instars (F = 89.75; df = 4, 24; P < 0.0001), for
fourth unparasitized instars (F = 98.72; df= 4, 24;
P < 0.0001), and for red-eyed nymphs (last instar)
(F = 81.92; df= 4, 24; P < 0.0001) for fifth node

TABLE 1. MEAN ( SD) NUMBER OF HAIRS PER 0.384 CM2
ON THE ABAXIAL SURFACES OF THE SECOND
AND FIFTH NODE LEAVES OF 5 COTTON CULTI-
VARS DIFFERING IN LEAF MORPHOLOGY AND
PUBESCENCE GROWN IN HASTINGS, FLORIDA.

Second Fifth
Cultivar node leaf node leaf

DP DES 119 182.8 67.3 b 104.2 37.3 a
Stoneville 453 194.7 68.1 a 98.3 33.6 b
Gumbo 500 68.1 36.4 c 37.0 24.2 c
NU COTN 33B 9.0 11.2 d 4.3 7.2 d
DP 51 7.0 8.3 d 3.0 4.7 d

Data were pooled across sampling dates. Means within a col-
umn with the same letter did not differ significantly (Waller-
Duncan's multiple-range test; a = 0.05). (n = 320).



















TABLE 2. MEAN ( SD) NUMBER OF WHITEFLY LIFE STAGES PER 3.14 CM2 ON THE ABAXIAL SURFACES OF SECOND AND FIFTH LEAVES OF 5 COTTON CULTIVARS DIFFERING IN
LEAF MORPHOLOGY AND PUBESCENCE GROWN IN HASTINGS, FLORIDA.

Mean SD per 3.14 cm2

Whitefly life stages DP DES 119 Stoneville 453 Gumbo 500 NU COTN 33B DP 51

Second node leaf

Eggs 224.2 23.5 a 97.7 135.0 a 280.8 131.0 b 101.4 59.0 c 44.6 36.0 d
Young instars" 54.5 + 34.5 a 8.7 11.5 b 6.7 + 8.6 b 4.4 + 6.2 c 4.2 + 5.7 c
Third instars 1.5 + 2.8 a 0.7 1.7 b 0.3 + 0.7 b 0.4 + 0.85 b 0.3 + 0.82 b
Fourth (unparasitized) 0.5 0.2 a 0.4 0.2 a 0.1 + 0.8 b 0.2 0.8 b 0.1 + 0.1 b
Red-eyed nymphs 0.1 + 0.2a 0.1+ 04 a 0 b 0.1 0.1 a 0 b
Fifth node leaf

Eggs 14.0 16.7 b 17.8 + 22.4 a 9.2 12.4 c 3.6 7.1 d 5.0 7.9 d
Young instars 31.5 32.5 a 35.1 + 30.8 a 13.0 14.9 b 6.7 7.6 c 6.3 7.8 c
Third instars 7.7 + 7.9 a 7.3 6.5 a 3.1 + 3.3 b 1.3 1.9 c 1.6 1.8 c
Fourth (unparasitized) 6.7 7.9 a 6.8 6.5 a 2.7 3.3 b 1.3 1.9 c 1.3 1.8 c
Red-eyed nymphs 6.6 8.7 a 5.2 5.8 b 1.5 2.3 c 0.9 1.3 c 0.9 1.5c

"Sum of first and second instars.
Data were pooled across sampling dates. Means within a row within a leaf node followed by the same letter did not differ significantly (Waller-Duncan's multiple-range test; a = 0.05) (n = 320).







Cetintas & McAuslane: Parasitism ofB. tabaci on Cotton


leaves. The mean numbers of all whitefly stages
were always greater on the pubescent cultivars,
DP DES 119 and Stoneville 453, than on the gla-
brous cultivars, DP 51 and NU COTN 33B.
There were no significant differences in pro-
portional parasitism among cultivars (Fig. 1). Al-
though one pubescent cultivar, DP DES 119, had
higher peak parasitism than the others, there
was only a slight trend toward higher parasitism.
DP 51, a glabrous cultivar, and Gumbo 500, the
intermediate hairy cultivar, apparently had the
lowest peak parasitism. Proportional parasitism
increased as the season progressed to a peak in
August, after which parasitism steadily declined
(Fig. 1). This is probably because parasitism fol-
lows the availability of hosts to parasitize and
there was a new generation of susceptible white-
flies available at that time (Fig. 2).
The parasitoids emerging from parasitized
whiteflies were identified as Encarsia pergandi-
ella Howard, Encarsia nigricephala Dozier and
Eretmocerus californicus Howard. Encarsia per-
gandiella comprised 93.3% (68.5% Y and 25.0%
6) of all the parasitoids that emerged from sam-
pled leaves. DP DES 119 had the highest number
of parasitoids emerging from collected leaves
(149, comprising 47.75% of all parasitoids)
(Table 3). NU COTN 33B had the lowest number
of parasitoids emerging (17, 5.44%).

Laboratory Experiment (Functional Responses of
Parasitoids)

There was an increase in mean number of sec-
ond instars parasitized by E. rui as host density
increased from 8 to 64 hosts on all 3 cotton culti-
vars (Fig. 3). A maximum mean of 27.7 hosts par-
asitized in 24 h was reached when the parasitoid
was offered 64 whitefly second instars. Curves ob-
tained from E. rui on all 3 cultivars visually ap-
proximated a type II functional response (Fig. 3).


0.9-
-- - umbo 500
0.8 --IVP I
0.7 IP DS 119
o 0 s n 'i S I Ilk-. 03
0.6


0.4 .


0.1 -
0




S 2 3 4 5 6 7
Weeks

Fig. 1. Proportional parasitism of B. tabaci over the
season (7-week period) for the fifth leaf of 5 cotton culti-
vars differing in leaf morphology and pubescence.


300
250
200



15
100
;s
18
12
9
6


0 50
45
40
35
30
25
20
15
10
I0
,


Second node lear --- I ",
.............. un








... ....... .........

2 3 4 5 6 7


1 2 3 4
Weeks


5 6 7


Fig. 2. Seasonal trend in overall mean number of dif-
ferent life stages ofB. tabaci over the season (7-week pe-
riod) for the second and fifth node leaves of 5 cotton
cultivars differing in leaf morphology and pubescence in
field trials.


Cultivar (F = 23.61; df = 2, 154; P < 0.0001) and
host density (F = 449.47; df = 3, 154; P < 0.0001)
significantly affected parasitism of second instars
by E. rui. The interaction of cultivar and host den-
sity also significantly influenced the number of
second instars parasitized by E. rui (F = 5.01; df=
6, 154; P < 0.0001). On all 3 cultivars, the number
of hosts parasitized increased with increasing
host densities. However, the increase in parasit-
ism on the smooth-leaved DP 51 was greater than
the increase on the hairy cultivars, DP DES 119
and Gumbo 500 (Fig. 3), thus accounting for the
significant density by cultivar interaction.
Parasitism of fourth instar whitefly nymphs
by E. formosa also visually approximated a type
II functional response (Fig. 3). However, the
maximum mean number of whiteflies parasit-
ized by E. formosa, 11.7 in 24 h, was only half
that of E. rui. Cultivar was a significant source
of variation (F = 5.78; df = 2, 209; P = 0.0036)
with more whiteflies parasitized on DP 51 than
on DP DES 119 and Gumbo 500. Density of
whitefly hosts significantly influenced parasit-
ism (F = 95.3; df = 3, 209; P < 0.0001); only half
as many whiteflies were parasitized at the 8-
nymph density as at the other densities, but the
number parasitized did not differ at the 16, 32,
or 64 nymph densities. The interaction of culti-


Fith node rlaf
------- thirI NMt
,...- I huid irj.itin
Rcd,,
Il i- ','"I


I







Florida Entomologist 92(4)


TABLE 3. PARASITOID EMERGENCE FROM LEAF DISKS OF 5 COTTON CULTIVARS DIFFERING IN LEAF MORPHOLOGY AND
PUBESCENCE GROWN IN HASTINGS, FLORIDA.

Parasitoids

Encarsia Encarsia Eretmocerus
Cotton cultivars (leaf morphology) nigricephala pergandiella californicus Total

No. %, No. % No. % No. %
Gumbo 500 (okra) 4 1.3 35 11.2 7 2.2 46 14.7
DP 51 (smooth) 3 1.0 16 5.1 2 0.6 21 6.7
NU COTN 33B (smooth) 2 0.6 15 4.8 0 0 17 5.4
DP DES 119 (hairy) 1 0.3 147 47.1 1 0.3 149 47.8
Stoneville 453 (hairy) 1 0.3 78 25.0 0 0 79 25.8
Sum Total 11 3.5 291 93.3 10 3.2 312 100

"Percentage of total parasitoids emerged from leaves collected during 7 weeks.
Data were pooled across sampling dates (n=320).


var and host density did not significantly influ-
ence the number of fourth instars parasitized
by E. formosa.

DISCUSSION
Our field study results indicated a preference
for oviposition of female B. tabaci on pubescent
cotton cultivars over glabrous cultivars. Corre-
spondingly, the density of B. tabaci nymphs was
higher on cultivars with higher leaf hair density.
Several other studies have indicated increased
whitefly populations on pubescent cotton culti-
vars relative to glabrous cultivars (Butler & Hen-
neberry 1984; Butler et al. 1986; Wilson et al.
1993; Chu et al. 2000, 2001). The intermediately
hairy, okra-leaf cultivar, Gumbo 500, had an in-
termediate number of whitefly instars. Because
Gumbo 500 has an okra-leaf shape and has re-
duced number of hairs compared to the pubescent
cultivars, it is unclear which factor may have con-
tributed to reduced whitefly oviposition. However,
supporting our results, several previous studies
have shown that okra-leaf cotton cultivars were
colonized with fewer whitefly adults, eggs and
nymphs compared to normal-leaf cultivars, indi-
cating the potential of okra-leaf genetic traits for
reducing the population of this pest (Ozgur & Se-
keroglu 1986; Chu et al. 1999, 2002; Raghuraman
et al. 2004). On the other hand, NU COTN 33B, a
cultivar genetically modified to express the Bt
toxin CrylA(c), had the lowest number of whitefly
numbers and lowest parasitoids emerging (17,
comprising 5.44% of all parasitoids). Higher sus-
ceptibility of parasitized hosts to intoxication by
NU COTN 33B could contribute to the observed
differences between parasitoid and its host (Baur
& Boetel 2002). The parasitoid species that
emerged from whitefly nymphs on cotton were the
common species that have been collected in Flor-
ida on wild and cultivated hosts (Bennett et al.
1990) and on the agronomic crops, peanut and


SImst Densitics
Fig. 3. Mean ( SD) number of second instars of B.
tabaci parasitized by E. rui and mean ( SD) number of
fourth instars of B. tabaci parasitized by E. formosa on
3 cotton cultivars differing in pubescence and leaf mor-
phology: DP 51 (smooth normal-leaf), DP DES 119 (pu-
bescent normal-leaf), and Gumbo 500 (intermediate
okra-leaf) in laboratory trials.

soybean (McAuslane et al. 1995). Parasitism was
similar among all 5 cotton cultivars tested in this
study. However, in soybean fields in Florida,
whiteflies on a glabrous isoline suffered more par-
asitism than whiteflies on a pubescent isoline
(McAuslane et al. 1995).


32 64


December 2009







Cetintas & McAuslane: Parasitism ofB. tabaci on Cotton


Our laboratory studies indicated a higher daily
fecundity for E. rui than for E. formosa on white-
fly nymphs. The average maximal daily fecundity
of 31 nymphs for E. rui corresponds well with that
indicated previously by McAuslane & Nguyen
(1996) of approximately 30 eggs in 24 h. There
have been a number of studies on the fecundity of
Encarsia species and E. formosa, in particular,
parasitizing whitefly species on various plant cul-
tivars (Vet & van Lenteren 1981; Bethke et al.
1991; Heinz & Parrella 1994; Qiu et al. 2004). Vet
& van Lenteren (1981) recorded a maximal daily
fecundity rate of 12 for Encarsia species in gen-
eral attacking Trialeurodes vaporariorum West-
wood. Qiu et al. (2004) estimated a maximum
daily fecundity of 14.5 for E. formosa strain D at
25C attacking B. argentifolii on poinsettia, al-
though the mean daily parasitism was quite low,
being 11.2 and 9.88 for temperatures of 20C and
25C, respectively. The maximum daily fecundity
for E. formosa at 25C obtained in our study was
15.8, which was slightly greater than that ob-
tained by Qiu et al. (2004). Our study indicated
that the number of hosts parasitized by E. for-
mosa remained stable at around the maximum
daily fecundity even when plenty of hosts were
available. This study showed that E. rui parasit-
ized almost twice as many instars as E. formosa.
It is unclear which factors) resulted in the low
maximal daily fecundity of E. formosa compared
to E. rui. It is likely that the E. formosa strain we
used in this study has an innately low maximum
daily fecundity or is not very well adapted to B.
tabaci. We can conclude that E. rui might be more
effective than E. formosa in biological control ofB.
tabaci on cotton.
In this study, E. rui showed lower parasitism
on pubescent cotton cultivars than on smooth and
okra-leaf shape cotton cultivars. However, para-
sitism by E. formosa did not show any cultivar ef-
fect. The reason for this might be that E. formosa
and E. rui have different oviposition behavior or
preference on different plants. Results from these
evaluations suggest that the probability of
achieving successful biological control will likely
be greater on cotton cultivars with fewer tri-
chomes. Previously, a number of studies were con-
ducted to understand the potential effect of plant
trichomes on whitefly parasitoids (Hulspas-
Jordaan & van Lenteren 1978; Heinz & Parrella
1994; van Lenteren et al. 1995; Hoddle et al. 1998;
Gruenhagen & Perring 2001). An early study by
Hulspas-Jordaan & van Lenteren (1978) showed
the negative effect of leaf hairs on cucumber vari-
eties on walking speed and walking pattern of E.
formosa. Similarly, the rate of parasitism of the
greenhouse whitefly, T vaporariorum, by E. for-
mosa decreased linearly with increasing hair den-
sity on cucumber hybrids (van Lenteren et al.
1995). A greenhouse study of parasitism of silver-
leaf whitefly by E. eremicus on 5 host plants, in-


cluding cotton, indicated that fewer whiteflies
were parasitized on plants with higher trichome
densities (Gruenhagen & Perring 2001). These 3
studies corroborate our results where we found a
significantly greater mean number of nymphs
were parasitized on glabrous-leaf DP 51 than on
hairy DP DES 119 and Gumbo 500 by both E. for-
mosa and E. rui.
Contrary to the laboratory studies, parasitism
rates in the field were similar, regardless of the
pubescence of the cotton cultivars. There are sev-
eral potential reasons for this. The higher popula-
tions of whiteflies on the pubescent varieties
could have represented higher quality host
patches than the lower population density
patches on the glabrous cultivar so that the para-
sitoids stayed and searched for hosts on the pu-
bescent plants, despite the trichome abundance.
This could cause a numerical response not ob-
served in the lab when individual parasitoids
were confined on each plant. There could be para-
sitoid species-specific responses to trichomes; for
example, E. pergandiella, the most abundant par-
asitoid in the field, was not tested in the labora-
tory study. In addition, parasitism in the field
could have been affected by other plant-related
factors and/or abiotic factors (Butler and Hen-
neberry 1994; Hoelmer 1995) that were not
present in the laboratory study.
These results indicate that there may be a dis-
advantage to planting pubescent cultivars of cot-
ton in Florida if whiteflies are expected to be
abundant; whitefly females prefer to oviposit on
pubescent cultivars. Consequently, there will be
more eggs laid and more adults emerging on pu-
bescent cultivars than on glabrous cultivars. By
planting a glabrous cotton cultivar, a grower may
be able to avoid severe infestation of whitefly due
to reduced whitefly oviposition and also due to in-
creased performance by parasitoids resulting in
an increase in parasitism and decrease in white-
fly numbers. Additional studies to aid in the un-
derstanding of the effectiveness of parasitoids of
B. tabaci and their dynamics, and to determine
the mechanisms behind differences in both white-
fly and parasitoid oviposition on cotton cultivars
differing in leaf morphology are warranted.

ACKNOWLEDGMENTS

We thank Debbie Boyd for support and guidance in
the conduct of these experiments. We are appreciative of
the help offered by Drs. Howard Frank and Jon Allen,
who reviewed this manuscript.

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Florida Entomologist 92(4)


December 2009


CHEMICAL ANALYSIS OF FEMALE VOLATILES AND FIELD RESPONSE OF
THE COFFEE LEAFMINER MOTH (LEPIDOPTERA: LYONETIIDAE) TO
STEREOISOMERS OF ITS MAJOR SEX PHEROMONE COMPONENT

EDI A. MALO, JULIO C. ROJAS, GUILLERMO LOPEZ-GUILLEN AND JUAN F. BARRERA
Departamento de Entomologia Tropical, El Colegio de la Frontera Sur (ECOSUR), Apartado Postal 36,
Carretera Antiguo Aeropuerto Km. 2.5 C.P. 30700 Tapachula, Chiapas, M6xico

ABSTRACT

We analyzed the volatiles emitted by Mexican populations of the female coffee leafminer
moth Leucoptera coffeella that were collected in coffee plantations located in the Soconusco
region of Chiapas. Our results confirmed previous results that L. coffeella females emitted
5,9-dimethylpentadecane as the major and 5,9-dimethylhexadecane as the minor compo-
nent. Field response of the coffee leafminer moth to stereoisomers of the major component
showed that delta traps baited with (5S,9R)-dimethylpentadecane caught a significantly
greater number of coffee leafminer males when compared to those captured by traps baited
with (5R,9R)-dimethylpentadecane, (5R,9S)-dimethylpentadecane and the control. The
number of coffee leafminer captured by traps baited with (5S,9S)-dimethylpentadecane was
not significantly different from the number captured by traps baited with (5R,9R)-dimeth-
ylpentadecane. The results are discussed in view of the possibility of using stereoisomers as
bait for monitoring the leafminer moth.

RESUME

Nosotros estudiamos los volatiles emitidos por hembras del minador de la hoja del caf6, Leu-
coptera coffeella, colectados en plantaciones de caf6 de la region del Soconusco, Chiapas,
M6xico. Nuestros resultados confirman los resultados previous que la hembra de L. coffeella
emite (5,9)-dimetilpentadecano como compuesto mayoritario y (5,9)-dimetilhexadecano
como minoritario. Los resultados de la evaluaci6n en campo de los estereois6meros del com-
puesto mayoritario mostraron que las trampas delta cebadas con (5S,9R)-dimetilpentade-
cano presentaron captures significativamente mayores comparadas con las captures de
trampas cebadas con (5R,9R)-dimetilpentadecano, (5R,9S)-dimetilpentadecano y el control.
El numero promedio de minadores capturados en trampas cebadas con (5S,9S)-dimetilpen-
tadecano fue similar al capturado con (5R,9R)-dimetilpentadecano. Los resultados de este
trabajo son discutidos considerando la posibilidad que algunos de estos estereois6meros pue-
dan ser usados como cebo para el monitoreo del minador de la hoja del caf6.

Translation provided by the authors.


The coffee leafminer, Leucoptera coffeella
(Guerin-Meneville), is an important and widely
distributed pest among the main coffee producing
countries in Latin America (Sanchez-De Leon
1984; Souza & Reis 1992; Barrera 2008). Eggs are
laid individually or in small clusters of up to 7
eggs, and total fecundity varies between 30-80
eggs. Upon hatching the larva makes a semi-cir-
cular cut at the leaf base and penetrates rapidly
into the leaf, where it moves around, mining the
palisade parenchyma tissue (Barrera 2008). Sev-
eral small mines may run together, causing brown
spots to appear and in severe attacks major loss of
leaf tissue and premature leaf drop occurs, reduc-
ing plant vigor and yield (Souza & Reis 1992). In
Brazil, this insect is considered a key pest of the
coffee plant and causes losses of 50% in produc-
tion (Souza & Reis 1992). In Mexico, the leaf-
miner is frequently found attacking coffee plants
in Chiapas (Segura et al. 2004; De la Mora et al.
2008) and Veracruz (Nestel et al. 1994) but infes-


station levels are generally low. Nevertheless, the
intense use of pesticides has reduced many of the
natural enemies of this insect, allowing increased
L. coffeella populations in some municipalities of
Chiapas (Barrera et al. 2003). A sampling made
in the Soconusco region of Chiapas revealed that
the coffee leafminer is present all year around but
principally preceding the rainy season during
Mar and Apr (Barrera et al. 2006). Currently, the
main method of coffee leafminer control in tropi-
cal America is the application of insecticides.
However, the use of insecticides increases the
likelihood of the coffee beans containing residual
pesticides affecting human health and the envi-
ronment, and consistent use may induce resis-
tance in the coffee leafminer (Fragoso et al. 2002).
The use of sexual pheromones of the coffee leaf-
miner may help reduce the use of insecticides, re-
ducing toxic residues in the coffee fruit and pre-
serving its natural enemies in the agro-ecosystem
(Michereff et al. 2007). Furthermore, the sexual







Malo et al.: Field Response of Coffee Leafminer to Stereoisomers


pheromone of the coffee leafminer may be used for
monitoring populations of this pest, allowing
identification of the main areas of infestation so
the control can be specifically directed to these
more heavily infested areas. This would increase
efficiency and decrease costs and non-target im-
pact (Baca et al. 2008).
Francke et al. (1988) identified 5,9-dimethyl-
pentadecane as major component and 5,9-dimeth-
ylhexadecane as minor component in the female
produced pheromone and reported that both com-
ponents are EAD active. However, the absolute
configuration of both natural pheromone compo-
nents still remains to be determined. Lima (2001)
found that traps baited with a racemic mixture of
5,9-dimethylpentadecane captured more males
than traps baited with pure stereoisomers. The
catches of traps baited with the single pure stere-
oisomers were not significantly different among
compounds. Zarbin et al. (2004) reported that
(5S,9S)-dimethylpentadecane elicited higher an-
tennal responses when compared with 3 other pos-
sible isomers. Insect pheromones, especially in
moths, may vary between populations of the same
species in different geographic locations (e.g.,
Hansson et al. 1990; Battista-Pereira et al. 2000).
In this study, we determined the chemical compo-
sition of the volatiles released by female coffee leaf-
miner moths present in the Soconusco region of
Chiapas, Mexico, and then we evaluated the 4 ste-
reoisomers of the major sex pheromone component
as attractants of the coffee leafminer in the field.

MATERIAL AND METHODS

Insects

Insects were reared in the laboratory as de-
scribed elsewhere (Reis et al. 2000). Larvae were
reared on green coffee leaves, where the larvae
develop and pupate, and maintained at 25-27C,
60-70% RH, and a photoperiod of 12:12 (L:D). At
emergence, moths were kept separately by sex
and fed with 10% honey solution.


Pheromone Collection


For volatile collection ten 1-2-day-old virgin fe-
males were placed in a cylindrical glass aeration
chamber (20 cm long x 15 cm i.d.). A charcoal-fil-
tered airstream (1 L/min) was maintained
through the glass aeration chamber. The female
volatiles were collected with Porapack Q (50-80
meshes, Water Associates, Inc., Milford, MA)
packed between silanized glass wool plugs in a
Pasteur pipette during 24 h. The collected vola-
tiles were eluted from the absorbent with 500 pL
of HPLC grade hexane (Sigma-Aldrich, Toluca,
Mexico), and concentrated to 200 pL by a slow
stream of nitrogen. The extract was stored at 20'C
until chemical analysis.


Chemical Analysis

The gas chromatography-mass spectrometry
(GC-MS) analysis was conducted with a Varian
Star 3400 CX chromatograph linked to a Varian
Saturn 4D mass spectrometer. The samples were
analyzed with a factor four capillary column VF-
5MS, 30 m x 0.25 mm i.d. x 0.250 pm film thickness,
Varian) and two chiral columns: Cyclosilb-B (30 mx
0.25 mm i.d. x 0.25 pm film thickness, Agilent Tech-
nologies, Santa Clara, CA), or Cyclodex-B (30 m x
0.25 mm i.d. x 0.25 pm film thickness, Agilent Tech-
nologies, Santa Clara, CA) that were programmed
at 50'C for 1 min, then 10C/min until 200'C, and
held for 10 min. The carrier gas was helium (1 mL/
min). The injector port temperature was held at
250'C. Ionization was by electron impact at 70 eV.
Identifications of the volatiles emitted by the coffee
leafminer females were based on retention time,
mass spectrum compared with the mass spectrum
of synthetic standards, or tentatively identified
based on the fragmentation pattern suggested by
Pomonis et al. (1980). The retention index was cal-
culated based on a standard of hydrocarbons (C7-
C30) (Sigma-Aldrich, Toluca, Mexico).

Compounds

The synthesis of (5R, 9R)-dimethylpentade-
cane, (5R, 9S)-dimethylpentadecane, (5S, 9R)-
dimethylpentadecane, (5S, 9S)-dimethylpentade-
cane, the 4 stereoisomers (Fig. 1) of the major sex
pheromone components of the coffee leafminer,
was reported by Kuwahara et al. (2000). The com-










,'^-^ '~ ^.-^ N, B




N ^




D

Fig. 1. Structures of stereoisomers of the major sex
pheromone component of the coffee leafminer tested in
the field. A, (5R,9R)-dimethylpentadecane; B, (5R,9S)-
dimethylpentadecane; C, (5S,9R)-dimethylpentade-
cane; and D, (5S,9S)-dimethylpentadecane.







Florida Entomologist 92(4)


pounds used in this study were supplied by Dr. S.
Kuwahara (Laboratory of Applied Bioorganic
Chemistry, Division of Life Sciences, Graduate
School of Agricultural Science, Tohoku University,
Japan). The purity of the compounds was deter-
mined by GC-MS as described below.

Study Area

The trial was performed on a coffee farm
(14'57'N, 9211'W, altitude 385 m above sea level)
at Manuel Lazos, Municipality of Tuxtla Chico in
the Soconusco region of SW Chiapas, Mexico. The
experiment was performed in an 8-ha area culti-
vated with approximately 8-year-old Coffea ara-
bica L. cv Bourbon, Coffea canephora Pierre ex
Froehner and Robusta coffee trees combined with
a variety of shade trees including Cedrela odorata
L., Citrus sinensis (L.) Osbeck, Enterolobium cy-
clocarpum (Jacq.) Griseb, Inga micheliana
Harms, Musa sapientum L. and Pouteria sapota
(Jacq). This area has a humid tropical climate
with heavy rain commencing in Mar and ending
in Nov, with an annual rainfall of 4160 mm in
2003 and an average annual temperature of
25.50C.

Field Test

We used white Delta plastic traps (Phero-
tech, Delta, BC) with a sticky plastic sheet be-
low to capture the insects. Traps were arranged
in a randomized plot design with 3 replicates of
each treatment. The replicate plots were ar-
ranged in parallel lines separated by 30 m. The
stereoisomers were dissolved in hexane (HPLC
grade) and a solution of 10 pg/pL of each com-
pound was prepared. The lures tested were (5R,
9R)-dimethylpentadecane, (5R, 9S)-dimethyl-
pentadecane, (5S, 9R)-dimethylpentadecane,
(5S, 9S)-dimethylpentadecane, and 500 pg of
each stereoisomer was loaded in a rubber sep-
tum dispenser (Thomas Scientific, Swedesboro,
NJ). Hexane (50 pL) was used as a control. Due
to non-availability, the racemic mixture was not
included in the experiment. The traps baited
with the lures were suspended from the coffee
tree branches at a height of approximately 1.5
m between 13 Mar and 25 Apr 2003 and the cof-
fee leafminer male moths captured were re-
corded every 3 d, giving a total of 11 observation
dates. The plastic sticky sheet containing the
insects from each trap was labeled and trans-
ported to the laboratory where the coffee leaf-
miner was identified under a stereomicroscope.
Traps were rotated after each observation date
between plots and the lures were replaced with
freshly treated septa at 3 weeks. Voucher speci-
mens were placed in the insect collection held at
El Colegio de la Frontera Sur, Unidad Tapa-
chula, Mexico.


Statistical Analysis

The data analyses were performed with the
computer package Statistica (StatSoft 2003). The
number of the coffee leafminer captured per trap
was transformed by (1 + 0.5)-12 by the Box & Cox
(1964) transformation for normalizing the data
and analyzed by one-way analysis of variance
(ANOVA). Significant ANOVAs were followed by a
posthoc Tukey test for multiple comparisons of
means (P < 0.05).

RESULTS

Chemical Analysis

GC-MS analyses of volatiles emitted by the cof-
fee leafminer females showed the presence of 2
saturated branched hydrocarbons identified as
5,9-dimethylpentadecane (RI = 1588) and 5,9-
dimethylhexadecane (RI = 1696). The mass spec-
trum of the 5,9-dimethylpentadecane matched
the synthetic compound (Fig. 2A). The purity of
stereoisomer synthetics was 99%, determined by
GC-MS with traces of hydrocarbons possibly
formed during the synthesis (Fig. 2B).

Field Study

The number of coffee leafminer males caught
was significantly affected by treatment (F = 7.93;
df = 4, 55; P < 0.001) (Fig. 3). Traps baited with
(5S,9R)-stereoisomer captured more males than
traps baited with (5R,9R)-stereoisomer, (5R,9S)-
stereoisomer and control. The catches of traps
baited with (5S,9S)-stereoisomer were intermedi-
ate and not significantly different from those cap-
tured by traps baited with (5S,9R)-stereoisomer
and (5R,9R)-stereoisomer. There were no differ-
ences in the catches of traps baited with (5R,9R)-
stereoisomer, (5R,9S)-stereoisomer and control.

DISCUSSION

In this study, we confirmed that the Mexican
coffee leafminer population emitted 5,9-dimethyl-
pentadecane and 5,9-dimethylhexadecane such
as was reported by Francke et al. (1988). We also
obtained a chromatogram with a similar reten-
tion time to that reported recently by Lima et al.
(2008), who reported 5,9-dimethylpentadecane as
the major compound extracted from pheromone
glands of virgin females. Our results agree with
those of Lima et al. (2008) in that 5,9-dimethyl-
pentadecane is the major component released by
L. coffeella females. The Mexican population of L.
coffeella released 5,9-dimethylpentadecane and
5,9-dimethylhexadecane.
The absolute configuration of the sex phero-
mone components of the coffee leafminer has not
been reported. We were not able to separate the


December 2009








Malo et al.: Field Response of Coffee Leafminer to Stereoisomers


kCoimts


mautes


Fig. 2. Trace of the GC-MS (from a factor four capillary column VF-5MS) of pheromone volatiles from virgin fe-
males of the coffee leafminer showing both sex pheromone components (A), and 100 ng of synthetic (5S,9R)-dime-
thylpentadecane (B).


isomei
elected
with 2
tiomei
not pc
rivatiz


Fig.
caught
ylpetai
ters ar


rs present in the pheromone volatiles col- arated by chromatographic methods or by spec-
from females or synthetic stereoisomers troscopic techniques such as Nuclear Magnetic
different chiral columns because the enan- Resonance (Meierhenrich et al. 2003). However,
rs of branched aliphatic hydrocarbons do the enantiomers of a few methyl-substituted al-
ssess a functional group that permits de- kanes have been separated with enantioselective
sed into diastereoisomers that could be sep- gas chromatography. For example, Chow et al.
(2004) separated stereoisomers of 7,11-dimethyl-
heptadecane under enantiosoelective GC condi-
a tions, with a modified cyclodextrin phase. Further
studies will be necessary to determine the abso-
lute configuration of the female-produced sex
pheromone by using techniques such as enanti-
oselective (ciclodextrin) gas chromatography and
ra gas chromatography coupled electroantennogra-
phy.
be c We report for the first time that the coffee
leafminer was primarily captured with traps
aSB ass s.R 5E.ss cGr" baited with the isomers (5S,9R)-dimethylpenta-
decane and (5S,9S)- dimethylpentadecane. The
3. Mean number of coffee leafminer males number of coffee leafminer captured in the
with delta traps baited with the (5,9)-dimeth- present study was very low compared to that re-
idecane stereoisomers. Bars with the same let- ported in Brazil (Lima 2001; Michereff et al.
e not significantly different (Tukey test,P > 0.05). 2007). One possibility is that population levels of







Florida Entomologist 92(4)


the coffee leafminer in Mexico are lower than in
Brazil. A second possibility is that a mixture of
major component isomers might be necessary to
improve male captures. Lima (2001) evaluated
the effect of the following ratios of the binary
blend of 5R,9S-/ 5S,9S- dimethylpentadecane:
0:100, 20:80, 50:50, 80:20, and 100:0 on male
catches. He found that traps baited with the sin-
gle 5S, 9S- isomer captured more than traps
baited with any other treatments. The least male
captures were obtained by traps baited with the
single 5R,9S-isomer in the ratio 80:20, which
suggests that this isomer inhibited the attrac-
tion of males to the 5S,9S-isomer. Furthermore,
he found that traps baited with a racemic mix-
ture of dimethylpentadecane captured more L.
coffeella males than traps baited with single
pure stereoisomers. There were no significant
differences in the number of males captured by
traps baited with the single pure stereoisomers,
which is somewhat unexpected because it seem
likely that males would at least be attracted
more to the stereoisomer emitted by females. In
contrast, we found that males were mostly at-
tracted to the 5S, 9S-isomer, but whether this is
the isomer released naturally by L. coffeella fe-
males remains to be investigated. However, be-
cause the racemic mixture was not included in
our study, it is difficult to compare our results
with those reported by Lima (2001). A third pos-
sibility is that we only used the major phero-
mone component, and the presence of the minor
component might be necessary to improve male
attraction. To our knowledge nobody has evalu-
ated the biological activity of the minor compo-
nents released by L. coffeella females, but in
some moth species the presence of the minor
components in the blend are critical for male at-
traction (Christensen 1997).
In conclusion, the results of this study confirm
that 5,9-dimethylpentadecane and 5,9-dimethyl-
hexadecane are released by the coffee leafminer
moth present in the Soconusco region of Chiapas,
Mexico. Field tests showed that coffee leafminer
males responded significantly to 2 stereoisomers
of the major component: (5S,9R)-dimethylpenta-
decane, and (5S,9S)-dimethylpentadecane.


ACKNOWLEDGMENTS

We thank S. Kuwahara ((Laboratory of Applied
Bioorganic Chemistry, Division of Life Sciences,
Graduate School of Agricultural Science, Tohoku Uni-
versity, Japan) for supplying the stereoisomers used
in this study, Joel Herrera-Mufioz, Antonio Satieste-
ban, and Armando Virgen for technical assistance,
Javier Valle-Mora for statistical advice (ECOSUR),
and 2 anonymous reviewers for suggestions to the
manuscript. This study was part of the project: "Bio-
ecologia y manejo de plagas del caf6 en el Soconusco y
Sierra de Chiapas" supported by Fundaci6n Produce
Chiapas.


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239-241.








Florida Entomologist 92(4)


December 2009


RESPONSE OF THE PREDATORY MITE PHYTOSEIULUS MACROPILIS
(ACARI: PHYTOSEIIDAE) TO PESTICIDES AND KAIROMONES OF THREE
SPIDER MITE SPECIES (ACARI: TETRANYCHIDAE), AND NON-PREY FOOD

MUHAMMAD M. AMIN', RUSSELL F. MIZELL2, III AND R. WILLS FLOWERS'
'Center for Biological Control, Florida A&M University, Tallahassee, FL 3230

2NFREC-Quincy, University of Florida, 155 Research Rd, Quincy, FL 32351

ABSTRACT

The predatory mite Phytoseiulus macropilis Banks (Acari: Phytoseiidae) is native to Florida.
Some biology and ecology of this phytoseiid have been documented, but its potential as a bi-
ological control agent of phytophagous mites (Acari: Tetranychidae) has received less atten-
tion. The response of P. macropilis to 12 acaricides, 3 tetranychid mite species and 5
potential alternate foods was evaluated in laboratory bioassays. Pesticide residual effects on
P. macropilis were evaluated by a double-disk leaf residue method. The synthetic pyre-
throids Tame (fenpropathrin), Cymbush (cypermethrin) and Mavrik (fluvalinate) were
highly toxic. Tolerance was observed to the acaricides, Omite (propargite), and Avid (abam-
ectin), while Vendex (hexakis), Pentac (dienochlor), and Kelthane (dicofol) were highly toxic.
The insecticides Orthene (acephate) and Diazinon and the fungicides, Domain (thiophanate-
methyl) and Cleary (thiophanate) were not toxic to P. macropilis. Field efficacy tests of fen-
propathrin and dicofol indicated that these chemicals lose toxicity to P. macropilis 21 and 7
d after application, respectively. In olfactometer bioassays, female predators were attracted
to kairomones produced by their rearing host Tetranychus urticae Koch on bean leaves but
not to kairomones of the tetranychids Oligonychus ununguis (Jacobi) and T evansi Baker
and Pitchard on their respective host plants. Predators did not respond significantly to se-
lected alternate foods: pollen from the hybrid daylily Hemerocallis spp., Phylloxera spp. lar-
vae, eggs of false oleander scale Pseudaulacaspis cockerelli (Cooley), a sugar-water solution
and water. This study identified several pesticides that could be integrated with use of P.
macropilis as a biological control. Results also indicate that the predator may have a narrow
prey range and require specific species of mite prey for survival and oviposition.

Key Words: predatory mite, insecticide, attractant, spider mite

RESUME

El acaro depredador Phytoseiulus macropilis Banks (Acari: Phytoseiidae) es native de la
Florida. Algunos aspects de su biologia y ecologia han sido documentados, pero su poten-
cial como un agent de control biol6gico de acaros fit6fagos (Acari: Tetranychidae) ha reci-
bido menor atenci6n. La respuesta de P. macropilis a 2 acaricidas, 3 species de acaros
tetraniquidos y 5 classes de comida alternative potential fue evaluada en bioensayos en el
laboratorio. Los efectos de los residues de pesticides sobre P. macropilis fueron evaluados
utilizando el m6todo de residues sobre doble-discos de hojas. Los piretroides sint6ticos
Tame (fenpropatrin), Cymbush (cypermetrin) y Mavrik (fluvalinate) fueron altamente
t6xicos. Tolerancia fue observada hacia los acaricidas Omite (propargite) y Avid (abamec-
tin), mientras Vendex (hexakis), Pentac (dienochlor) y Kelthane (dicofol) fueron altamente
t6xicos. Los insecticides Orthene (acefate) y Diazinon y los fungicidas, Domain (thiofa-
nate-metil) y Cleary (thiofanate) no fueron t6xicos a P. macropilis. Las pruebas de eficacia
en el campo para feopropatrin y dicofol indicaron una perdida de toxicidad a P. macropilis
de los 21 y 7 dias despu6s de aplicaci6n, respectivamente. En los bioensayos utilizando el
olfactometro, depredadores hembras fueron atraidas a las kairomonas producidas por su
hospedero Tetranychus urticae Koch sobre hojas de frijol sobre lo cual fueron criados pero
no fueron atraidas a kairomonas de los tetraniquidos Oligonychus ununguis (Jacobi) y Te-
tranychus evansi Baker y Pritchard sobre sus plants hospederas respectivas. Los depre-
dadores no respondieron significativamente de las classes de comida alternative
seleccionadas incluyendo polen de lirio hibrido Hemerocallis spp., larvas de Phylloxera
spp., huevos de la escama diaspidido Pseudaulacaspis cockerelli (Cooley), una soluci6n de
agua-azucar, y solo agua. Este studio identifico various pesticides que pueden ser integra-
dos con el uso de P. macropilis como un agent de control biol6gico. Los resultados indican
que el depredador posiblemente tiene un rango de presa estrecho y require species de
presa especificas para sobrevivir y ovipositar.







Amin et al.: Response of the Predatory Mite Phytoseiulus macropilis


The economic losses caused by phytophagous
mites (Acari: Tetranychidae) in various agricul-
tural crops in most areas of the world is well doc-
umented (Huffaker et al. 1970). The genera Tet-
ranychus, Oligonychus, Eotetranychus and
Panonychus are some of the world's most impor-
tant pests of agricultural crops (Jeppson et al.
1975). Tetranychid mites, including the twospot-
ted spider mite Tetranychus urticae Koch, the
southern red spider mite Oligonychus ilicis
(McGreger), and the spruce spider mite 0. unun-
guis (Jacobi), are important pests of ornamental
nursery crops in the southern United States, with
T urticae being the predominant pest species.
Nursery and landscape plants have an "aesthetic"
economic threshold with low tolerance for pest
damage. Therefore, nurseries frequently use pes-
ticides to combat pest outbreaks (Mizell & Schiff-
hauer 1991). Nursery production requires inten-
sive labor and high volumes of water, both subject
to high risk of pesticide exposure. Rapidly in-
creasing pesticide costs, social clamor against
hazardous effects of chemicals, legal regulations
regarding worker safety, and the risk of pesticide
resistance development among pest species indi-
cate a need to develop alternate integrated pest
management strategies that reduce or eliminate
pesticide use and its side effects in nurseries. In-
tegration of natural enemies with selective pesti-
cides could be a viable strategy for some pest sit-
uations in nurseries.
Although natural enemies of phytophagous
mites have been reported from several acarine
families (Lord 1949; Knavel & Salheime 1967),
the majority of the well known predatory mites
belong to the family Phytoseiidae. Phytoseiids as
biological control agents of phytophagous mites
are effective in many agricultural systems (Fla-
herty & Huffaker 1970; Pickett & Gilstrap 1986;
Hamlen & Lindquist 1981; Mizell & Schiffhauer
1991). Phytoseiids locate their prey through
chemical cues known as "herbivore induced plant
volatiles" (van Wijk et al. 2008) emitted from host
plants as a result of spider mite feeding activity
(Dicke et al. 1990; Takabayashi & Dicke 1992;
Takabayashi et al. 1994; van Wijk et al. 2008).
The perception of kairomones increases the prob-
ability of prey finding by phytoseiids (Hislop &
Prokopy 1981; Dicke et al. 1990; Takabayashi et
al. 1994; van Wijk et al. 2008). Rosen & Huffaker
(1982) regard searching ability as the most impor-
tant attribute of an effective predator. Some phy-
toseiids also feed on non-prey foods, such as pol-
len, honeydew, and plant juices (McMurtry 1982),
which may help to sustain the predators through
periods when prey are at low densities (Huffaker
& Flaherty 1966).
Despite many documented successes against
tetranychid mites in diverse agricultural sys-
tems, the family Phytoseiidae has only a few well-
known species. Thus there is a need to research


and develop new, particularly native, predators to
suppress phytophagous mite outbreaks. The
predatory mite, Phytoseiulus macropilis Banks,
indigenous to Florida (Muma & Denmark 1970),
has shown its efficacy against phytophagous
mites in cotton (Saba 1971) and glasshouse crops
(Hamlen 1978; Hamlen & Lindquist 1981) and
may have excellent potential as a biological
control agent of tetranychid mites of nursery or-
namentals. Aspects of the biology and ecology of
this phytoseiid are well documented (Smith &
Summers 1949; Prasad 1967; Shih et al. 1979;
Ball 1980; Hislop & Prokopy 1981; McMurtry &
Badii 1989; Mesa et al. 1990), but P macropilis
response to common pesticides and volatiles of
tetranychid mites-host plant species (except T ur-
ticae) is still unknown. In addition, potential use
of alternate food by P. macropilis has not yet been
determined. This study evaluated the response of
P macropilis to (1) pesticides commonly used in
nurseries, (2) volatiles from potential tetranychid
prey-host plant species, and (3) selected alternate
food to determine the predator's potential use in
an integrated pest management program against
tetranychid mites in nurseries.

MATERIALS AND METHODS

Predator Rearing

Phytoseiulus macropilis (Banks) were sub-cul-
tured from a laboratory colony established in
1987 by R. F. Mizell, III from a wild population
feeding on twospotted spider mites at Monticello,
Florida. Mite identification was performed by H.
A. Denmark and vouchers specimens are housed
in the Florida Collection of Arthropods, Gaines-
ville, Florida. Predators were reared in the labo-
ratory during 1993 and 1994 at 27-34C, 60 5%
relative humidity (R.H.) and a photoperiod of 16:8
light:dark (L:D). Rearing units consisted of a rect-
angular sponge (length = 23.5 mm, width = 16.5,
thickness = 2 mm) placed in a metal tray (length
= 27 mm, width = 17.5, height = 3.5 mm). The tray
was filled with tap water until the sponge was
saturated. A black plastic plate (length = 22 mm,
width = 12.3 mm, thickness = 0.2 mm) was seated
on the sponge as the rearing arena.
Lima beans, Phaseolus uulgaris L.,'Henderson',
were grown in a greenhouse (25C and 60% 5%
R.H. under fluorescent lights) at the North Florida
Research and Education Center in Monticello,
Florida. Twospotted spider mites were reared on 1-
2-week-old lima bean seedlings at 27-31C, 60 +
5% R.H. under fluorescent lights in the laboratory.
Predators were fed on the rearing units every 24 h
by adding 2-4 lima bean leaves infested with all
stages of T urticae. The old dried leaves from rear-
ing units were discarded after removing predator
eggs, nymphs, and adults. Adult female predators
3-5 d old were used in all bioassays.








Florida Entomologist 92(4)


Residue Bioassays

Residue bioassays were used to determine the
toxic effects of selected registered pesticides on P.
macropilis by a modified double-leaf-disk method.
The double-leaf-disk method described by Schiff-
hauer & Mizell (1988) was modified so that the
ring was closed with a treated leaf instead of
cloth. Rings cut from standard PVC pipe (diame-
ter = 20 mm; thickness = 2.5 mm) were attached
to a 7-d-old cotyledon leaf of lima bean with con-
tact cement (DAP Inc., Dayton, Ohio). The seed-
lings with the PVC rings were dipped in the test
concentrations for 5 s. After drying seedlings in a
hood, eggs and other life stages of T urticae were
placed inside the ring arena with a fine brush. Ten
to 15 female predators from the colony were aspi-
rated into a small straw with a vacuum device.
The straw was sealed with parafilm and held in
ice for about 5 min to slow down predator move-
ment to prevent their escape. Predators were
transferred into the ring arena, and the latter was
closed on top by the second cotyledon leaf with
contact cement. Treated and untreated control
seedlings were held in beakers with their roots
immersed in water. The number of concentrations
tested varied by chemical, but most were tested at
5 serial dilutions of the label rates (Table 1). Six
replicates of 10 predators were tested for each
concentration of 12 acaricides. Untreated control
groups (6 replicates of 10 predators), were in-
cluded with each test. After 72 h, the top leaf from
each ring was removed and mortality was as-
sessed under a steromicroscope by a gentle touch
to the predators with a fine probe. Predators that
could not walk one body length when probed were
counted as dead. These bioassays were carried out
at 27-34C and 50+ 15% R.H. under fluorescent
lighting. Toxicity was rated as in IOBC/WPRS
(Hassan et al. 1987). Dose-response lines, LC5s
values and slopes were calculated for 5 acaricides
by the Probit Procedure in SAS (SAS 1994).

Field Efficacy of Fenpropathrin and Dicofol Residues

The pesticides, fenpropathrin and dicofol, were
applied to Euonymus japonica Thunb to runoff at
the Ix rate (Table 1) with a backpack sprayer.
Seven to 10 cm long shoots were cut from treated
plants and leaf replicates and numbers of preda-
tors tested were as described in the lima bean bio-
assays at 0, 3, 7, 14, and 21 d in order to deter-
mine the mortality of the residues to P. macropi-
lis. The final mortality was adjusted for untreated
control mortality by Abbott's formula (Abbott
1925).

Olfactometer and Response to Prey-host Emitted Volatiles

A glass Y-tube olfactometer of diameter 1.1 cm
with arms 8.5 cm long (Mizell & Schiffhauer


1991) was used to observe the behavioral re-
sponse of gravid female P macropilis to plant-
and prey-emitted volatiles. The central arm of the
Y-tube was connected to an air outlet and a Nal-
gene water vacuum pump (11.5 L/minute capac-
ity, NO. 6140-0010) on a water spigot via a plastic
tube. Each end of the Y-tube was connected to ad-
justable plastic containers that contained the test
treatments. Air flowed through a canister of acti-
vated charcoal into the adjustable plastic contain-
ers and through the Y-tube. Air flow was adjusted
with flowmeters at 100 mL/min in both arms of
the Y-tube. Treatments were placed upwind in the
plastic containers. The Y-tube was placed horizon-
tally on a wooden block over white paper to im-
prove observation of predator movement. Adult
female predators were placed in the main arm of
the olfactometer with a fine probe.

Predatory Mites

Adult female P macropilis reared on T urticae
Koch on 'Henderson' lima bean were starved for
24 h before the tests. Thirty-six predators (used
only once) were tested individually for each treat-
ment. Each predator was observed for a maxi-
mum of 10 min. After 10 predators were tested,
the treatments in the olfactometer arms were ro-
tated to remove possible positional effects) and
the olfactometer was washed with acetone. Pred-
ators that did not choose one of the arms of the ol-
factometer after 10 min were removed and scored
as no response. Predators were scored '+' or '-'
when they reached the upper end of one of the Y-
arms of the olfactometer. The tests were carried
out at 27-34C and 60-80% R.H., under fluores-
cent light from 1130-1800 EDT. The following
treatments were tested to observe the response of
P macropilis to kairomonal cues from the prey
and host plant: (1) blank versus blank (to test the
validity and potential positional bias of the olfac-
tometer); (2) blank us uninfested lima bean
leaves; (3) T urticae infested lima bean leaves us
blank; (4) T urticae-infested bean leaves us unin-
fested lima bean leaves; (5) lima bean leaves pre-
viously infested by T urticae but with all traces of
mites removed with a light brushing us blank; (6)
blank us uninfested juniper leaves; (7) 0. unun-
guis infested juniper leaves us uninfested juniper
leaves; (8) T urticae on lima bean leaves us 0. un-
unguis on juniper leaves; (9) T evansi infested to-
mato leaves us blank; (10) T evansi on filter paper
us blank; and (11) T urticae on lima bean leaves
us T evansi on tomato leaves. The results were
statistically analyzed by the Sign-test at P = 0.05
(Conover 1971).

Alternative Food Bioassays

Selected non-prey food: water, water and sugar
solution (16:1), daylily pollen Hemerocallis sp.,


December 2009




















TABLE 1. PESTICIDES TESTED FOR THEIR EFFECT ON ADULT PHYTOSEIULUS MACROPILIS.

Label' Serial dilution2
Pesticide trade name rate (1X)
(active ingredient) Manufacturer mg a.i./L 3X 2X 0.1X 0.01X 0.001X 0.0001X 0.00001X

1. Abamectin (Avid) Merck Sharps Dhome3 5.8 + + + + +
2. Vendex (Hexakis) E.I. Du Pont De Ne Mours & Co. (Inc.) Wilm. 450.6 + + + + + +
3. Kelthane (Dicofol) Rohm & Haas 478.4 + + + + +
4. Pentac (Dienochlor) Sandoz 480.0 + + + + +
5. Mavrik (Fluvalinate) Zoecon Corp. 139.2 +
6. Cymbush Cypermethrin) ICI Americas, Inc. 208.54
7. Tame (Fenpropathrin) Chevron Chemical Co. 138.0
8. Omite (Propargite) Uniroyal Chemical, Inc. 360.0 + + + + + + +
9. Orthene (Acephate) Chevron Chemical Co. 1132.53
10. Domain (Thiophanate- methyl) Grace Sierra Corp. 626.0'
11. Cleary (Thiophanate) Cleary Chemical 1198.43
12. Diazinon Ciba-Geigy Corp. 1764.03

'Rates recommended by manufacturers for spider mite control on nursery plants.
2Concentration was tested in addition to the label rate.
3Manufacturers for some compounds have changed since the data collection, but these were the origin of the compounds in the experiments.
Only label rate was tested.







Florida Entomologist 92(4)


eggs of false oleander scale Pseudaulacaspis cock-
erelli (Cooley), and Phylloxera sp. nymphs were
presented to adult female predators to study their
effect on survival and fecundity. The experimen-
tal units consisted of an inverted plastic Petri
dish (5.3 mm dia.) closed with silicone (Alex Plus,
DAP Inc. Dayton, Ohio 45401). A moistened filter
paper placed at the bottom retained humidity and
also served as the arena for the subjects. The in-
verted dish had 2 holes at the base; one was used
for predator and food placement (fitted with a
cork) and the other was fitted with a stub of filter
paper to supply moisture. Food was provided ev-
ery 24 h. The number of eggs and live predators
were recorded per 24 h. Five replicates of 6 sati-
ated, gravid female P macropilis were used for
each treatment. The treatments were continued
until the tested predators died. A control group of
5 replicates of 6 predators with all life stages of T
urticae as food was included with each treatment.
Bioassays were conducted at 27-34oC and 60-80%
R.H. Results were analyzed by the randomization
test at P = 0.05 (Conover 1971).

RESULTS AND DISCUSSION

Residue Bioassays

Synthetic pyrethroids were highly toxic as per-
methrin, fluvalinate, and fenpropathrin atlX la-
bel rates caused 100% mortality (Table 2). How-
ever, in the field efficacy test, fenpropathrin toxic-
ity decreased to 30% at 21 d after application (Ta-
ble 3). Other studies of synthetic pyrethroid
toxicity to phytoseiids reported similar results
(Croft et al. 1983; Riedl & Hoying 1983; Mizell &
Schiffhauer 1991). Fenpropathrin under field con-
ditions showed high toxicity during the 2 weeks
after application, but its toxicity declined to 30%
after 21 d (Table 3). Thus, P. macropilis might be
released into nurseries 3-4 weeks following treat-
ment with fenpropathrin.
Dienochlor at the IX concentration caused
high mortality, but at lower concentrations mor-
tality declined sharply (Table 3). These results


agree withreports of Mizell & Schiffhauer (1991)
for N. college De Leon, and Malezieux et al.
(1992) for N. fallacis (Garman). The LC5, value
was 335.54 mg a.i./L, which is more than half of
the registration field rate (480 mg a.i./L). Phy-
toseiulus macropilis is relatively unharmed by ex-
posure to dienochlor, which is an organochlorine
compound no longer available for use (Table 4).
The insecticide Diazinon surprisingly caused
only 18% mortality whereas acephate caused only
5% mortality at lx rate and was the safest of all
pesticides tested (Table 2). Nevertheless, Hassan
et al. (1987) reported acephate as highly toxic to
Phytoseiulus spp. Similarly, Diazinon has been
observed as highly toxic to A. hibisci (Bartlett
1964) andA. fallacis (Croft & Nelson 1972). How-
ever, results similar to ours were reported by Bab-
cock & Tanigoshi (1988) on T occidentalis and Za-
charda & Hluchy (1991) on T pyri Scheuten. Ap-
parently both insecticides could be used in nurs-
eries targeted to pest species other than mites
and cause little harm to P macropilis.
The acaricide dicofol at the lx rate caused
100% mortality and the LC5, was very low 0.083
mg a.i./L in comparison to the label rate. Under
field conditions, however, residual toxicity de-
clined 7 to 14 d after application to 12% and 1%,
respectively (Table 3). Heretofore, this chemical
has been reported as nontoxic (Rock & Yeargan
1971; Theiling & Croft 1988), slightly toxic
(MacPhee & Sanford 1961), moderately toxic
(Bartlett 1964; Zacharda & Hluchi 1991) and
highly toxic to phytoseiids (Van de Vrie 1962;
Hassan et al. 1987; Mizell & Schiffhauer 1991).
Based on our observation of dicofol in the field, it
appears that P macropilis could be safely inte-
grated with this chemical for tetranychid sup-
pression in nurseries 7-14 d after its application.
The acaricides abamectin and hexakis at the
IX rate caused high P. macropilis mortality (Ta-
ble 4). The LC5, value for hexakis was 74.54 mg
a.i./L and for abamectin 0.7513 mg a.i./L. Pro-
pargite at the IX rate caused low mortality
(Table 4). The acaricide propargite was harmless,
both at the label rate and at concentrations 2, 3,


TABLE 2. RESPONSE OF PHYTOSEIULUS MACROPILIS FEMALES AFTER 72 H TO COMMERCIAL PESTICIDES AT LABEL
RATES IN A LABORATORY BIOASSAY.

Pesticide Label rate'(1X) mg ai. L' Mean % mortality SE Classification2

Fluvalinate 139.2 100 0 4
Fenpropathrin 138.0 100 0 4
Cypermethrin 208.0 100 0 4
Acephate 1132.5 5.0 0.84 1
Thiophanate-methyl 626.0 35.0 0.26 1
Thiophanate 1198.4 48.0 0.84 1
Diazinon 1764.0 18.0 0.76 1

'Rates recommended by manufacturers for spider mite control on nursery plants.
2Toxicity rating after the convention of IOBC/WPRS, 1 = harmless (<50%), 4 = harmful (99%).


December 2009








Amin et al.: Response of the Predatory Mite Phytoseiulus macropilis


TABLE 3. RESIDUE TOXICITY OF 5 ACARICIDES TO PHYTOSEIULUS MACROPILIS.

Total no. Label LC5o
Acaricide of predators tested rate (lx)'mg a.i./L (95% CI mg a.i./L) Slope SE

Dienochlor 420 480.0 335.54 (37.86 451.36) 1.46 6.80
Hexakis 540 450.6 74.54 (10.21 303.85) 0.87 2.47
Abamectin 420 5.8 0.7513 (0.444 588.71) 0.77 1.69
Dicofol 480 478.4 0.083 (0.000248 1.561) 2.10 4.39
Propargite 420 360.0 8917.0 (423.6 8.30413 x 1016) 12.20 40.30

'Rates (middle of range) recommended by manufacturers for spider mite control on nursery plants.


TABLE 4. RESPONSE OF P. MACROPILIS TO RESIDUES OF
FENPROPATHRIN AND DICOFOL (AT LABEL RATES)
UNDER COMMERCIAL NURSERY CONDITIONS.

Days after treatment

Pesticide 0 3 7 14 21
Mean % mortality'
Fenpropathrin 100 73 100 100 30
Dicofol 100 100 12 1

Mortality corrected by Abbott's formula.


and 6-fold of the label rate. In contrast, propargite
was reported slightly toxic to A. hibisci (Bartlett
1964) andA. fallacis (Croft & Nelson 1972). How-
ever, these results are similar to those reported by
Rock & Yeargan (1971) on T occidentalis, Babcock
& Tanigoshi (1988) on N. fallacies, and Mizell &


Schiffhauer (1991) onN. college. The LC50 was re-
corded as 8917 mg a.i/L, more than 10 times the
label rate.
The fungicides thiophanate and thiophanate-
methyl at the 1X rates caused 58% and 35% mor-
tality, respectively (Table 2). The predator's toler-
ance to tested fungicides is important relative to
its use in nurseries, as fungicides are frequently
applied for disease control.
In this study a double-leaf-disk method was
used to avoid 'runoff' mortality. The method used
was similar to Schiffhauer & Mizell (1988), with
the difference that the ring was closed with a
treated leaf instead of a cloth. This modification
provided more exposure of the treated surface to
the predators. Phytoseiulus macropilis, like many
other phytoseiids, has a wandering tendency, and
is difficult to confine in a treated arena during ex-
periment set up, often trying to escape the treated
arena and is killed in the contact glue. This prob-


TABLE 5. RESPONSE OF ADULT FEMALE PHYTOSEIULUS MACROPILIS TO ODORS IN AN OLFACTOMETER PRODUCED BY
TETRANYCHID MITE SPECIES. STATISTICAL SIGNIFICANCE IS SCORED WITH RESPECT TO PREFERENCE FOR THE
FIRST TREATMENT IN THE COUPLETS. ZERO AND N.S. APPLIES TO NO SIGNIFICANT DIFFERENCE BETWEEN THE
TREATMENTS, ++ EXPLAINED AS COMMENTS.

Outcome

Treatment No. (+) (-) Probability Comment

1. Blank vs Blank 36 0 0 n.s.' Bioassay is valid
2. Tetranychus urticae on lima bean leaves 36 22 0 <0.05 attraction
vs uninfested lima beans.
3. T urticae infested lima bean leaves vs 36 25 2 <0.05 attraction
blank.
4. Uninfested lima bean leaves vs blank. 36 0 0 n.s. no attraction
5. Previously T urticae infested lima bean 36 20 3 <0.05 attraction
leaves vs blank.
6. Oligoncyhus ununguis on juniper leaves 36 3 1 n.s. no attraction
vs blank.
7. Uninfested juniper leaves vs blank. 36 2 3 n.s. no attraction
8. T evansi on tomato leaves vs. blank. 36 0 1 n.s. no attraction
9. T evansi on filter paper vs blank. 36 0 0 n.s. no attraction
10. T urticae on lima bean leaves vs 0. unun- 36 19 4 <0.05 attraction to T. urticae
guis on juniper leaves.
11. T urticae on lima bean leaves vs T evansi 36 20 0 <0.05 attraction to T urticae
on tomato leaves.
'Determined using Sign test, P = 0.5.







Florida Entomologist 92(4)


lem was countered by drawing predators into
straws, and putting the latter in ice for about 5
min. This cooling technique (Mizell & Schiffhauer
1991) facilitated predator transfer into the
treated arena. However, the double-leaf-disk
method, like any other residue testing technique,
has some tradeoffs. For example, behavioral re-
sponse of subjects to pesticide residue cannot be
observed. The possible fumigation effect inside
the arena may cause some mortality among con-
fined subjects that may not occur on an open
treated surface. However, possible fumigation ef-
fects in this study were deemed negligible in com-
parison to the problems with predator escapes
from other arenas.

Olfactometer Bioassays

Of the 38 P macropilis tested, 22 responded
significantly (P < 0.05) to lima bean leaves in-
fested with T urticae, whereas none of the P mac-
ropilis responded to uninfested lima bean leaves
(P < 0.05). The response was also significantly dif-
ferent (P < 0.05) when 1 treatment was lima bean
leaves infested with T urticae and the other
blank. The P. macropilis response to T urticae-in-
fested lima bean versus a blank (25:2) and to lima
bean leaves that were previously infested by T ur-
ticae (20:0) to a blank were both significantly dif-
ferent (P < 0.05) (Table 5). The P. macropilis did
not respond significantly to 0. ununguis on juni-
per leaves both versus uninfested juniper leaves
and versus the blank (Table 5). Phytoseiulus mac-
ropilis were not attracted significantly to T.
evansi either on tomato leaves or on filter paper
(Table 5).
The significant response of adult P macropilis
to lima bean leaves infested with T urticae Koch,
both versus blank and uninfested lima bean
leaves, was similar to the results reported by His-
lop & Prokopy (1981). Predators did not respond
significantly to uninfested lima bean leaves. Sa-
belis et al. (1984) also reported that P persimilis
did not respond to uninfested lima bean leaves.
However, P macropilis responded significantly to
infested lima bean leaves from which T urticae
were removed.
Takabayashi & Dicke (1992) observed that
phytoseiids reared on twospotted spider mite on
lima bean leaves respond to volatiles from lima
bean leaves but the predators reared on the
twospotted spider mites from cucumber leaves
did not respond to lima bean volatiles. Phytoseiu-
lus macropilis used in this study were reared on
'Henderson' lima beans for about 7 years. Thus,
the significant response of P macropilis to the
kairomones of T urticae on lima bean leaves ver-
sus the negative response to 0. ununguis and T.
evansi on juniper and tomato leaves, respectively,
may possibly be explained by the rearing history.
The response of P macropilis to T evansi was not


significant, even when the latter were offered on
filter paper, which removed the effect of the to-
mato leaves. Thus, it appears odors produced by T
evansi on tomato do not attract P macropilis, or
perhaps volatile(s) carried over by T evansi from
feeding on tomato leaves made them repellent.
Results regarding the response of P macropilis to
these 2 tetranychid species warrant further in-
vestigation (van Wijk et al. 2008). The significant
response of P macropilis to one of the predomi-
nant phytophagous pest of nursery ornamentals
T urticae indicates that P. macropilis has poten-
tial as a biological control agent for twospotted
spider mite and warrants further study.

Alternate Food Bioassays

There was no significant difference between
the survivorship and fecundity (P < 0.05) of pred-
ators fed on alternate food and the controls after
24 h (Table 6). However, after 48 hr, mortality in-
creased and fecundity decreased sharply for P.
macropilis in the alternate food treatments,
whereas the P. macropilis controls fed spider
mites remained alive and continued ovipositing
(Table 6). These results suggest that P. macropilis
cannot survive or oviposit when fed only on non-
prey food. The rate of mortality, however, varied
among predators fed on different alternate foods
(Table 6). Interestingly, the highest observed sur-
vivorship for predators after 48-72 h was for those
fed on the solution of sugar and water. The preda-
tors provided with sugar-water solution survived
up to 120 h, which was the maximum survival pe-
riod in any of the alternate food treatments. Sim-
ilarly, egg production decreased rapidly for treat-
ments after 24 h except for the predators that
were fed on the eggs of false oleander scale P.
cockerelli and on sugar-water solution where they
oviposited for up to 72 h. Apparently, the de-
creased egg production among treated predators
indicates either that the predators' food reserves
were exhausted after 24 h or foods other than T
urticae are nutritionally deficient for egg produc-
tion. These results are similar to Kennett & Ho-
mai (1980), who reported that P persimilis and T
occidentalis provided with food other than tet-
ranychid prey did not oviposit.

CONCLUSIONS

The results from the 3 study types suggest that
P macropilis may have potential for use in an in-
tegrated biological and chemical control program
for tetranychid mites in nurseries. The discrepan-
cies involved in double-leaf-disk residue method
such as prevention of behavioral response by
predators and fumigation effect inside the treated
arena could have affected the residual efficacy of
tested pesticides. Thus, there is a need to field test
these and other chemicals for their possible inte-


December 2009








Amin et al.: Response of the Predatory Mite Phytoseiulus macropilis


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gration with P. macropilis to control spider mites
in nurseries. Nurseries use overhead irrigation
that over time may reduce residues on plants.
This was evident in the field test of dicofol, where
the chemical lost considerable toxicity to P. mac-
ropilis 7-14 d after application. For fenpropath-
rin, mortality in the field decreased from 100% to
30% three weeks after application. Because P.
macropilis apparently does not use alternate
food, chemicals potentially integrated with the
predator must not kill all the prey, thereby starv-
ing the predator. The response by P macropilis to
T urticae on lima beans suggests that the preda-
tor uses host kairomones to find its prey. There-
fore, further olfactometer studies with other spe-
cies of ornamental plants that are T urticae hosts
are warranted.

ACKNOWLEDGMENTS

Thanks to Dr. Michael D. Hubbard, Peter C. Ander-
sen, and Brent V. Brodbeck for improvements to a pre-
vious draft of the manuscript. Special thanks to Ms.
Cheryl Manasa for technical support.


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Florida Entomologist 92(4)







Maravalhas & Morais: Ants and Leafhopper Association in Brazil


ASSOCIATION BETWEEN ANTS AND A LEAFHOPPER
(CICADELLIDAE: IDIOCERINAE) IN THE CENTRAL BRAZILIAN CERRADO

JONAS MARAVALHAS AND HELENA C. MORAIS
Departamento de Ecologia, Instituto de Biologia, Universidade de Brasilia 70910-900 Brasilia, DF, Brasil

ABSTRACT

Associations between ants and Hemiptera are common in the families Membracidae, Coc-
coidea and Aphidoidea, but there are only a few cases of ants using honeydew of Cicadellidae
species. We surveyed plants of Roupala montana Aubl. (Proteaceae) in the cerrado area (sa-
vanna like vegetation) in the Federal District (Brazil) during the rainy season of 2006 and
2007. In this period the plants produce new leaves, which are consumed by the nymphs of a
probable new species of Rotundicerus (Cicadellidae: Idiocerinae). The nymph groups are of
variable size, with a mean of 20 individuals. We found 83% of them being tended by ants. A
total of 21 ant species was documented, with predominance of Camponotus spp. and Ceph-
alotes spp. There is a clear species turnover of ants on the nymph groups and attendance is
not continuous in most of the cases, suggesting an opportunistic association between ants
and Rotundicerus sp.

Key Words: Formicidae, Hemiptera, honeydew, Roupala, Rotundicerus, sap-sucking

RESUME

Associacoes entire formigas e Hemiptera sao comuns em Membracidae, Coccoidea e Aphidoi-
dea, por6m poucos sao os casos de formigas utilizando exsudado em esp6cies da familiar Ci-
cadellidae. Acompanhamos individuos de Roupala montana Aubl. (Proteaceae) em area de
cerrado no Distrito Federal (Brasil) durante a estacao chuvosa de 2006 e 2007. Neste period
a plant produz novas folhas utilizadas pelas ninfas de uma provavel esp6cie nova de Ro-
tundicerus (Cicadellidae: Idiocerinae). Os grupos de ninfas t6m tamanhos variados, com m6-
dia de 20 ninfas por grupo. Em 83% deles foram encontradas formigas explorando o
exsudado das ninfas, num total de 21 esp6cies de formigas, com predominancia de Campo-
notus spp. e Cephalotes spp. Ocorre uma variacgo temporal nas esp6cies de formigas que uti-
lizam os grupos de ninfas e o atendimento nao 6 continue na maioria dos casos, sugerindo
uma associacgo oportunista entire formigas e ninfas de Rotundicerus sp.


Translation by the authors.


Foliage-foraging ants use a wide range of plant
resources (Rico-Gray et al. 1998) and hemipteran
honeydew is the most commonly used food re-
source (Davidson et al. 2003). Ants play a major
role in arboreal ecosystems, being highly biodi-
verse, resource-dominant and the primary arthro-
pod predator (Hdlldobler & Wilson 1990; Floren et
al. 2002; Styrsky & Eubanks 2007).
Ant-hemipteran associations are mostly found
within treehoppers (Auchenorrhyncha: Mem-
bracidae), coccids and aphids (Sternorrhyncha:
Coccoidea and Aphidoidea) (Hdlldobler & Wilson
1990; Delabie 2001). Associations with leafhop-
pers (Auchenorrhyncha: Cicadellidae) are not
very common (Delabie 2001), and can occur indi-
rectly, without contact between ants and the leaf-
hoppers (Steiner et al. 2004), or directly, with
changes in the leafhopper's behavior and honey-
dew release after ant request (Larsen et al. 1992;
Moya-Raygoza & Nault 2000).
The Idiocerinae subfamily is one of the less
studied among the Cicadellidae with 20 described
genera, many of which are monotypic. The genus


Rotundicerus Maldonado-Capriles has three de-
scribed species (Lozada-Robles 2004) and R.
minutus Dietrich has been found associated with
ants (Dietrich & McKamey 1990).
In the cerrado vegetation of Central Brazil, Ro-
tundicerus sp., probably a new species (G. Me-
jdalani, personal communication), uses Roupala
montana Aubl. (Proteaceae) as host plants (Seyf-
farth 1996). Here we present information about
this leafhopper's biology as well as the associated
ant species.

MATERIALS AND METHODS

Study Area

The field work took place at the Fazenda Agua
Limpa (1530'S-4725'W), an experimental and
protected farm owned by the University of
Brasilia, in the Federal District, Brazil. The alti-
tude at the area varies between 1048 and 1160 m,
with an annual average rainfall of 1416.8 mm (Co-
efficient of Variation = 19.9%; series from 1980 to







Florida Entomologist 92(4)


2004; RECOR Meteorological Station, www.re-
cor.org.br) and mean annual temperature of
22.3C. The region is characterized by a very defi-
nite dry season, with the months between May to
Sep receiving less than 7% of annual rainfall. The
farm's natural vegetation has different phyto phys-
iognomies (Oliveira Filho & Ratter 2002) with the
predominance of cerrado sensu sticto, where the
studies were done. Detailed information about the
farm's vegetation can be found in Ratter (1980),
Eiten (1984), and Felfili et al. (2000).

Host Plant

Roupala montana is an evergreen shrub,
reaching up to 3 m high, and very common at the
study area (Franco 1998). There is foliar asyn-
chrony between plants of the same population, so
there is flush and loss of leaves throughout the
year, with a peak of leaf production in the transi-
tion between dry and rainy season (Sep and Oct)
(Lenza & Klink 2006). Roupala montana also oc-
curs in the borders of gallery forests at the Fa-
zenda Agua Limpa (Felfili & Abreu 1999).

Sampling

Weekly observations were conducted approxi-
mately between 9-11 am from Sep to Oct 2006 and
Apr to Oct 2007, with the examination of R. mon-
tana individuals for the presence of Rotundicerus
sp. and tending ants.
The presence of nymphs and the number of in-
dividuals per group was recorded. The presence of
tending ants was recorded and the specimens
were collected with an entomological aspirator
and kept in plastic vials containing 70% alcohol.
In Nov 2006 and 2007 a 24-h monitoring was
done on 7 chosen nymph groups of Rotundicerus


SRlants wth new leaves
0 Plants with Rlundicerus sp.


Apr May Jun


Jul Aug Sep


Fig. 1. Percentage of Roupala montana (Proteaceae)
individuals, examined monthly in 2007, with the pres-
ence of new leaves and of Rotundicerus sp. (Cicadel-
lidae, Idiocerinae) nymphs in a cerrado area of the
Federal District, Brazil.


sp., in order to record the ant species turnover. We
selected 1 group per plant and the groups con-
tained from 5 to 20 individuals.
Adults and nymphs of Rotundicerus sp. were
sent to Dr. Gabriel Mejdalani (Museu Nacional,
Universidade Federal do Rio de Janeiro) for iden-
tification. The ants collected were identified by
Dr. Jaques H. C. Delabie (Laborat6rio de Mirme-
cologia UESC/CEPLAC, Bahia).

RESULTS AND DISCUSSION

Between Sep and Oct 2006, at the weekly sam-
ples of the beginning of the rainy season, 56 indi-
viduals of R. montana were examined of which
28.6% were infested by Rotundicerus sp., with a
total of 116 groups of nymphs. Between Apr and
Oct 2007, 360 plants were examined and 11% of
them had groups of nymphs.
Although Rotundicerus sp. nymphs occur
mainly in groups, some individuals were found
alone. In 2006 a mean of 2.65 groups per plant
was found (sd = 2.35; min = 1, max = 9) and each
group had a mean of 19.4 nymphs (sd = 18.47; min
= 1, max = 80). The groups are formed by nymphs
of different instars and eventually some images.
In some cases we observed the presence of an
adult female together with the young nymphs,
however no egg laying or egg masses were found.


TABLE 1. FREQUENCY OF ANT SPECIES ASSOCIATED WITH
GROUPS OF ROTUNDICERUS SP. NYMPHS COL-
LECTED IN 2006 AND 2007 DURING WEEKLY
SAMPLING TRIPS IN A CERRADO AREA OF THE
FEDERAL DISTRICT, BRAZIL.

2006 2007

Species (n = 29) (n = 38)

MYRMICINAE
Cephalotes pusillus Klug 5 4
Cephalotes depressus Klug 1 -
Cephalotes grandinosus Smith 2
Cephalotes betoi de Andrade 1 -
Cephalotes liepini de Andrade 3
Crematogaster stollii Forel 2
Crematogaster victim Smith 1
Pheidole capillata Emery 4
Solenopsis sp. 1 -
FORMICINAE
Camponotus crassus Mayr 10 13
Camponotus arboreus Smith 2 -
Camponotus (Myrmaphaenus) sp. 2 5
Camponotus (Myrmaphaenus) sp. 1 2 3
Brachymyrmex sp. 3
DOLICHODERINAE
Azteca instabilis Smith 2 5


December 2009







Maravalhas & Morais: Ants and Leafhopper Association in Brazil


^sss&^'^'^%L


Fig. 2. A Camponotus crassus worker foraging honeydew from Rotundicerus sp. nymph (Cicadellidae, Idioceri-
nae) in a cerrado area of the Federal District, Brazil.


It is possible, as described for R. minutus (Di-
etrich & McKamey 1990), that eggs may be in-
serted almost completely into the stem or the leaf
vein of the host plant, but we did not observe this.
The occurrence of Rotundicerus sp. was con-
centrated in Oct when the host plants have the
peak of leaf production (Fig. 1). Along with the
monitoring made between Apr and Oct 2007, we
observed a few groups of nymphs in other months,
always on R. montana individuals that had young
leaves. In the borders of a gallery forest groups of
nymphs were observed between Sep 2007 and
May 2008, always on young leaves. The observed
exceptions were recorded in Jun in a nearby cer-
rado area, and in Apr and May in the border of a
gallery forest where nymphs were feeding on in-
florescences of R. montana. These observations
suggest that Rotundicerus sp. have a defined re-
productive season between Aug and Nov, together
with the production of new leaves by the host
plant, but can reproduce opportunistically when-
ever there is available food for the offspring.
Adults of Rotundicerus sp. were not found on R.
montana during the rest of the year in the study
site, suggesting that after the development of the
nymphs onR. montana's young leaves the adults ex-
ploit another unknown host plant species.
Of a total of 66 plants examined in 2006 and
2007 with groups of the leafhopper, 83% had tend-
ing ants. We found 15 ant species associated with
Rotundicerus sp. in the diurnal observations of


2006 and 2007 (Table 1). Camponotus crassus was
the most frequent ant species found with the
nymph (Fig. 2). A group of nymphs could be at-
tended by more than one ant species at the same
time, and this was observed twice, with Cephal-
otes liepini attending a Crematogaster victim
dominated group, but without any agonistic inter-
action.
During the 24-h monitoring samples made in
2006 we recorded 9 ant species (Table 2 A). Only 1
group had ants in all the 12 periods. The other 6
groups had from 4 to 7 periods without ant pres-
ence. Another group had attendance of C. crassus
on 6 consecutive periods, during the day. A similar
result was obtained in 2007 (Table 2 B). With the
exception of the groups attended byAzteca insta-
bilis and Pheidole capillata, all the others had at
least 4 periods of ant absence. Azteca instabilis
species monopolizes the resource and exploit it
continuously.
There is a clear ant species turnover during a
day, so a single nymph group can be attended by 2
or more ant species in a 24-h period (Table 2).
Camponotus atriceps, Camponotus (Tanae-
myrmex) sp. and Crematogaster evallans were the
most frequent during the night and Camponotus
crassus, Camponotus arboreus, Cephalotes pusil-
lus, and Cephalotes depressus the most frequent
during the day.
Altogether, we found 21 ant species associated
with groups of nymphs of Rotundicerus sp., and









TABLE 2. OCCURRENCE OF ANT SPECIES ASSOCIATED WITH SEVEN GROUPS OF ROTUNDICERUS SP. IN A 24-H OBSERVATION, IN NOV 2006 (A) AND 2007 (B) IN A CERRADO AREA OF
THE FEDERAL DISTRICT, BRAZIL. PERIODS 18:30 TO 04:30 IN (A) AND 19:00 TO 05:00 IN (B) ARE NIGHT PERIODS. DURING PERIOD 01:00 AND 05:00 IN (B) NO COLLECTIONS
WERE MADE DUE TO HEAVY RAIN.


Nymph Group Ant species 16:30 18:30 20:30 22:30 00:30 02:30 04:30 06:30 08:30 10:30 12:30 14:30

(A)
Camponotus crassus Mayr x x
Camponotus melanoticus Emery x
Crematogaster evallans Forel x x x x x
2 Camponotus crassus Mayr x x x x x
Camponotus (Tanaemyrmex) sp. x x x
3 Cephalotes depressus Klug x x x x
Camponotus crassus Mayr x x
4 Camponotus crassus Mayr x x x
Brachymyrmex sp. x x x x
5 Azteca instabilis Smith x x x x x x x X x x x x
6 Camponotus (Myrmaphaenus) sp.2 x x
Camponotus (Tanaemyrmex) sp. x x x
Azteca instabilis Smith x
Camponotus (Myrmaphaenus) sp.l x
7 Azteca instabilis Smith x x x x x x
Camponotus (Tanaemyrmex) sp. x

Nymph Group Species 11:00 13:00 15:00 17:00 19:00 21:00 23:00 01:00 03:00 05:00 07:00 09:00

(B)
1 Azteca instabilis Smith x x x x x x x x x x
Cephalotes liepini de Andrade x
2 Camponotus arboreus Smith x x x x
Nesomyrmex spininodis (Mayr) x
Camponotus (Tanaemyrmex) sp. x
3 Pheidole capillata Emery x x x x x x x x x x
Camponotus (Myrmaphaenus) sp.2 x
Pseudomyrmex tenuis (Fabricius) x
4 Camponotus crassus Mayr x x x x
Cephalotes pusillus Klug x x x x
Camponotus (Tanaemyrmex) sp. x x








Maravalhas & Morais: Ants and Leafhopper Association in Brazil


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the species composition, with predominance of
Camponotus spp. and Cephalotes spp., is similar
to that found in Membracidae and extrafloral nec-
taries ant associations in the cerrado (e.g., 01-
iveira et al. 1995; DelClaro & Oliveira 1999; Mor-
eira & DelClaro 2005).
The Idiocerinae are phloem feeders and the
nymphs of Rotundicerus sp. are gregarious and
non-jumping. These characteristics help the asso-
ciation with ants (Delabie 2001), and it seems to
be a characteristic of the genus (Dietrich & McK-
amey 1990).
Ant attendance to the nymphs most of the time
is not continuous, and there is a temporal varia-
tion of the ant species associated to Rotundicerus
sp. groups. This suggests that the ants explore
Rotundicerus sp. nymph's honeydew opportunis-
tically, contrasting with other ant-hemipteran as-
sociations. This characteristic makes this associa-
tion interesting to compare costs and benefits to
the involved species.

ACKNOWLEDGMENTS

Thanks to Dr. Jacques Delabie for identification of
ant species and to Dr. Gabriel Mejdalani for identifica-
tion of the leafhopper; to Pedro A. S. Cirotto for informa-
tion about nymphs on a forest edge; to the Postgraduate
Ecology Program and technician Mard6nio Timo (De-
partment of Ecology) for support in field transportation;
to the Universidade de Brasilia for infrastructure in the
campus and in the Fazenda Agua Limpa, to PIC-UnB-
CNPq for the scientific initiation scholarship granted to
J. Maravalhas; and to FINATEC for financial support.

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Florida Entomologist 92(4)







Auad et al: Biology and Life Table ofRhopalosiphum padi


THE IMPACT OF TEMPERATURE ON BIOLOGICAL ASPECTS
AND LIFE TABLE OF RHOPALOSIPHUM PADI
(HEMIPTERA: APHIDIDAE) FED WITH SIGNAL GRASS

A. M. AUAD, S. 0. ALVES, C. A. CARVALHO, D. M. SILVA, T. T. RESENDE AND B. A. VERISSIMO
Embrapa Gado de Leite, Caixa Postal, Juiz de Fora-MG, 36038-330, Brazil


ABSTRACT

The impact of temperature was evaluated on Rhopalosiphum padi (L.) (Hemiptera: Aphid-
idae). Nymphs, 12-h-old, were placed individually in cylindrical plastic dishes (2.5 x 2.5 cm),
with a layer of 1% agar in which leaf disks of signal grass had been placed. The nymphs were
reared at 12, 16, 20, 24, 28, and 32C 1 C, RH of 70 + 10% and 12-h photophase. We eval-
uated number of instars, duration of each instar and the nymphal period, survival of instars,
duration of the reproductive period, daily and total production of nymphs, and longevity of
the nymphal and adult phases. To construct the life expectancy table and fecundity, daily ob-
servations were made of 70 nymphs at each temperature, from birth to death. Development
of R. padi was faster with increased temperature, but they did not complete the last
nymphal instar at 32 C. The same pattern occurred for the pre-reproductive, reproductive,
and post-reproductive periods. The highest fecundity rates were between 16 C and 24 C.
The highest fertility (4 nymphs/female/day) was recorded at 12 C and 20 C. The highest net
reproduction rates were at 24 C and 28 C, and the time interval between each generation
(T) and the population doubling time (DT) diminished as temperature increased. The finite
rate of increase (X = 1.9 nymphs/female/day) and the intrinsic rate of increase (rm = 0.64)
were greatest at 24 C and 28 C, respectively. There was a negative impact on the biology and
life table ofR. padi at 32 C, but the range of 12 C to 28 C, despite some fluctuations, was fa-
vorable for survival and reproduction.

Key Words: ecology, aphid, signal grass, pest


RESUMO

Objetivou-se avaliar o impact da temperature nos aspects biol6gicos e na tabela de
vida de Rhopalosiphum padi (L.) (Hemiptera: Aphididae). Ninfas com ate 12 horas de
idade, foram individualizadas em places cilindricas de plastico (2,5 x 2,5 cm). No interior
destas, foi depositada uma camada de agar sobre a qual foram dispostos discos foliares
de braquidria que receberam as ninfas, as quais foram submetidas a 12, 16, 20, 24, 28,
e 32C + 1 C, UR 70 10% e fotofase de 12 horas. Avaliou-se o numero de instares, a du-
racao de cada instar e do period ninfal, a sobrevivencia nos diferentes instares, a du-
racao do period reprodutivo, capacidade didria e total de produgao de ninfas,
longevidade da fase ninfal e adulta. Para a tabela de esperanca de vida e de fecundidade
efetuaram-se observagoes didrias de 70 ninfas, para cada temperature, do nascimento
ate a more. Verificou-se que a velocidade de desenvolvimento de R. padi aumentou a me-
dida que a condicgo t6rmica era mais elevada; no entanto, nao completaram os dois ulti-
mos estadios ninfais a 32 C. A mesma tendencia foi constatada para os periods pre-
reprodutivo, reprodutivo e p6s-reprodutivo. As maiores taxas de fecundidade estiveram
entire 16 e 24 C. A maior fertilidade especifica foi registrada a 12 e 20 C, sendo de 4 nin-
fas/femea/dia. As maiores taxas liquidas de reproducgo, foram a 24 e 28C e o intervalo
de tempo entire cada geracgo (T) e o tempo necessario para a populagao duplicar em nu-
mero de individuos (TD) diminuiram com o aumento da temperature. A razao finita de
aumento (X = 1,9 ninfas/femea/dia) e a capacidade inata de aumentar em numero (rm =
0,64) foram superiores a 24 e 28 C, respectivamente. Constatou-se impact negative na
a biologia e tabela de vida de R. padi a 32 C, por6m, na faixa de 12 a 28 C, apesar das
oscilacoes, constata-se que o inseto encontra condigoes favoraveis para sobrevivencia e
reproducgo.

Translation by the authors.


The quality of forage plants is one of the most grown in pastures in Brazil (Botrel et al. 1987).
important factors for reducing milk production Various aphids in Brazil, including Rhopalosi-
costs. Signal grass (Brachiaria sp.), a perennial phum padi (L.) vector viruses to grasses (Hutch-
with large production of leaf mass, is widely inson & Bale 1994), and although R. padi is con-







Florida Entomologist 92(4)


sidered only an occasional pests on signal grass,
at high population levels its can considerably re-
duce the quantity and quality of forage. Aphids in
the genus Rhopalosiphum Koch, are widely dis-
tributed in tropical and subtropical regions
(Blackman & Eastop 2000) and affect various
crops of economic importance, such as sorghum,
corn, sugarcane, oats, rye and barley (Robison
1992; Jauset et al. 1998). Hence, strategies to
manage R. padi should be defined.
The effect of temperature on the biological
aspects of R. padi on various host plants has
been studied by Leather & Dixon (1984), Elliott
& Kieckhefer (1989), De Barro et al. (1992),
Hutchinson & Bale (1994), and Asin & Pons
(2001), but there are no published studies ofR.
padi on signal grass. Knowledge in this respect
will make it possible to predict the intensity of
occurrence, to serve as a base for developing
control strategies, and consequent improve-
ment in management programs. In the present
study we examined the effect of temperature on
biology and development of R. padi and con-
structed a life table for R. padi fed with signal
grass.

MATERIALS AND METHODS

Biological Aspects of R. padi on Signal Grass

Adults of R. padi were collected on signal
grass, Brachiaria ruziziensis (Germain & Ever-
ard), grown in greenhouses at the Embrapa Dairy
Cattle Research Center in Juiz de Fora, Minas
Gerais, Brazil. The aphids were multiplied in the
laboratory in Petri dishes kept in climate con-
trolled chambers at a temperature of 24 1C, RH
of 70 10% and 12-h photophase. Nymphs up to
12-h age were collected with tweezers and placed
individually in cylindrical plastic dishes (2.5 cm
diameter and 2.5 cm height). Each dish was first
filled with a solution of 1% agar in water to a
height of 1.0 cm, in which leaf disks of signal
grass were placed in order to keep the leaves tur-
gid. The dishes were covered with fabric and se-
cured with rubber bands.
The aphids were reared at temperatures of 12,
16, 20, 24, 28, and 32C 1C, RH of 70 10% and
photophase of 12 h. Each treatment contained
150 nymphs in a fully randomized setup. The fol-
lowing parameters were evaluated: number ofin-
stars; duration of each instar and nymphal pe-
riod; survival of the different instars; duration of
the reproductive period; daily and total capacity
to produce nymphs; and longevity of the nymphal
and adult phases.
The data were submitted to variance analysis
and the means were compared by the Scott Knott
(1974) test at 5% significance. Data were submit-
ted to regression analysis and the lower threshold
temperatures (LTT) and development time in de-


gree-days (DD) were estimated based on a hyper-
bolic relation, according to the method proposed
by Bean (1961).

Life Table of R. padi on B. ruziziensis

Newly emerged R. padi nymphs were main-
tained individually in plastic dishes containing a
layer of agar solution and leaf disk of signal grass,
as described above. Nymphs were kept at temper-
atures of 12, 16, 20, 24, 28, and 32C, with 70 in-
sects at each temperature. The insects were ob-
served daily from birth to death to record the via-
bility throughout their life cycle and to enable cal-
culating the life tables at each temperature.
The life expectancy tables were based on the
number of survivors at the start of age x (1l), num-
ber of individuals that died during interval x (d),
age structure (E.), corresponding to the number of
live individuals between one day and the next, life
expectancy for individuals of age x (e.) and proba-
bility of death at age x (100q.), which indicates the
probability of dying before the time established in
e.. The following equations were used, as pro-
posed by Silveira Neto et al. (1976): E,=[L,+(L ,1)]/
2; e,=TJL, and 100 q,=(dJl1).100.
For the fertility tables, the reproduction rates
(Ro), time interval between generations (T), in-
trinsic rate of increase (rm), finite rate of increase
(X), time necessary for the population to double
(DT), means of the age interval (x), specific fertil-
ity (my) and probability of survival (1) were calcu-
lated, according to the following equations: Ro =
1(mJ,); T = 1(m,.l,.x)/ 1(mJ.); rm= log, Ro/T = In
Ro/T; X = e" and DT = In(2)/rm.

RESULTS AND DISCUSSION

Temperature influenced the duration of the in-
stars and the nymphal cycle of R. padi, with in-
creased rate of development as the temperature
increased (Table 1, Fig. 1). These results agree
with those reported by Elliott & Kieckhefer (1989)
and Asin & Pons (2001) for the same aphid spe-
cies. The last 2 instars at the highest tempera-
ture, 32C, did not survive (Table 1). Asin & Pons
(2001) found average survival of 55% of imma-
tures of R. padi fed on corn at 30C. According to
Campbell et al. (1974), higher temperatures lead
to greater mortality due to the denaturing of pro-
teins or metabolic disturbances from the accumu-
lation of toxic products, and these harmful effects
mainly occur if the temperature is held constant.
The period in which R. padi remained in the
immature phase was shortest at 28C. This
agrees with the findings of Asin & Pons (2001).
However, Elliot & Kieckhefer (1989) and Dean
(1974), using barley as food forR. padi, found that
the maximum development rate occurred at 25C.
At 16 and 20C, the insects remained in the
nymphal stage longer than found by Segonca et


December 2009










TABLE 1. MEAN DURATION IN DAYS, FOR 1ST, 2ND, 3TH AND 4TH INSTAR TO DEVELOP, PRE-REPRODUCTIVE, REPRODUCTIVE AND POST-REPRODUCTIVE PERIODS, DAILY AND
TOTAL FERTILITY, AND LONGEVITY OF NYMPHS AND ADULTS OF RHOPALOSIPHUM PADI AS A FUNCTION OF TEMPERATURE.

Nymphal stage

Temperatures (C)

12 16 20 24 28 32

l instar 3.42 0.16 e 2.87 0.06 d 2.01 0.06 c 1.73 0.04 b 1.64 0.07 b 1.33 0.07 a
(n = 112) (n = 150) (n = 141) (n = 150) (n = 75) (n = 54)


2.93 0.16 c 3.10 0.08 c 2.01 0.06 b 1.79 0.04 b 1.47 0.08 a 1.88 0.21 b
(n = 84) (n = 135) (n = 126) (n = 150) (n = 63) (n = 18)


4.35 0.25 d 3.40 0.07 c
(n = 57) (n =99)


2.22 0.04 b 1.71+ 0.05 a
(n = 113) (n = 136)


1.48 0.07 a
(n = 52)


6.84 0.49 d 3.83 0.11 c 2.33 0.09 b 1.94 0.06 a 1.64 0.09 a
(n = 43) (n = 69) (n = 57) (n = 95) (n = 44)


17.3 0.53 e 12.93 0.17 d
(n = 43) (n = 69)


Nymphal stage




Pre-reproductive


Reproductive


Post-reproductive


8.37 0.13 c 7.13 0.11 b 6.24 + 0.15 a
(n = 57) (n = 95) (n = 44)


Adult stage

3.00 0.43 c 2.42 0.26 b 1.88 + 0.12 a 1.86 0.08 a 1.92 0.09 a
(n = 12) (n = 19) (n = 35) (n = 77) (n = 36)
7.91 1.48 b 11.31 1.67 c 4.63 0.45 a 6.75 0.44 b 4.08 0.42 a
(n = 12) (n = 19) (n = 35) (n = 77) (n = 36)
2.67 0.48 c 1.84 0.33 b 1.22 0.08 a 1.40 0.08 a 1.30 0.09 a
(n = 12) (n = 19) (n = 35) (n = 77) (n = 36)
13.58 1.59 c 15.58 1.77 c 7.74 0.45 a 10.00 0.40 b 7.30 0.44 a
(n = 12) (n = 19) (n = 35) (n = 77) (n = 36)
Fertility (Number of nymphs produced)

1.54 + 0.14 a 1.81+ 0.14 a 2.68 0.18 b 2.73 0.14 b 1.83 0.09 a
(n = 12) (n = 19) (n = 35) (n = 77) (n = 36)


Longevity


Total 13.00 3.18 a
(n = 12)


21.21 + 3.98 b
(n = 19)


12.88 1.55 a
(n = 35)


19.97 + 1.83 b
(n = 77)


7.75 0.94 a
(n = 36)


Means followed by the same letter in the rows did not differ by Scott Knott (1974) at 5%.A dash, -, indicates absence of data due mortality.


2nd instar


3rd instar


4th instar


Daily


h instar


Daily







Florida Entomologist 92(4)


y = 0.0048x2 0.3128x + 6.5238
= 0.979


5
4
3
2
1
0
8


8 12 16 20 24 28 32 36


y = 0.0094x' 0.5614x + 9.8054
8 = 0.9914








8 12 16 20 24 28 32


y = 0.0459X2 2.5334x + 41.233
F2 = 0.9927








8 12 16 20 24 28 32


y = 0.0047x2 0.282x + 5.8938
F= 0.8018


12 16 20 24 28 32


y = 0.0291x2- 1.4711x + 20.167
F = 0.9887


8 12 16 20 24 28 32


y = 0,0031x 0,5785x + 21,06
F = 0,6236


0 -
8 12 16


20 24 28 32


Temperatures (C)

Fig. 1. Fitted regression curves for the duration of the first (A), second (B), third (C), fourth (D) instars, nymphal
(E), and adult (F) phases of Rhopalosiphum padi, as a function of temperature.


al. (1994) at the same temperatures. Elliott &
Kieckhefer (1989) also found a shorter nymphal
cycle at similar temperatures to those studied
here. These results suggest that the type of host
plant offered as well as the geographic origin of
the aphid can affect its development, even when
kept under similar heat conditions. This is in line
with the observations of Smith (1922).
The nymphal period of R. padi was on average 3
times longer at the lowest temperature (12'C) com-
pared with the upper limit for survival (28C). The
effects of low temperature on the duration ofR. padi
were described by Hutchinson & Bale (1994), show-


ing that sublethal stress affects the development
process and longevity, which can exercise a domi-
nant influence on the aphid's population dynamic.
Asin & Pons (2001) also found that development in-
creased with rising temperature. They reported
complete development of the immature phase at
30'C, with a period significantly equal to that of the
insects maintained at 27.5C. These results also
agree with the findings of Campbell & Mackauer
(1975) that the average development time of aphids
declines as temperature increases.
The lower threshold temperatures (LTT) for
nymphs of R. padi are shown in Table 2. The in-


II)

"0



0

5


December 2009







Auad et al: Biology and Life Table ofRhopalosiphum padi


TABLE 2. REGRESSION EQUATIONS, DETERMINATION COEFFICIENTS (R2),THE LOWER THRESHOLD TEMPERATURES (LTT),
AND DEVELOPMENT TIME IN DEGREE-DAYS (DD) OF IMMATURES OF RHOPALOSIPHUM PADI FED WITH SIGNAL
GRASS.

Nymphal stage Equation R2 LTT ( C) DD

l' instar -0.0229 + 0.0251x 0.97 0.91 C 39.84
2" instar -0.1069 + 0.0282x 0.97 3.79 C 35.46
3"d instar -0.1439 + 0.0295x 0.98 4.87 C 33.89
4th instar -0.1996 + 0.0296x 0.99 6.74 C 33.78
Nymphal stage -0.0227 + 0.0067x 0.98 3.38 C 149.2


sects were tolerant up to the limit of 28C and the
LTT increased as the insects reached the adult
phase. Higher values were found by Elliott &
Kieckhefer (1989) for the different instars (5.73 to
6.10 d) and nymphal phase (5.76 d), except for the
fourth instar, when the temperature was the
same as that found here. The numbers of degree-
days (DD) required for the development of the dif-
ferent instars and the nymphal cycle were higher
than those reported by Elliott & Kieckhefer
(1989) for R. padi kept at temperatures from 11 to
29C. With 3.38 as the lower temperature limit for
development of the nymphal phase, 149.2 degree-
days are required for this aphid species to reach
the adult phase. These results are important to
predict the number of annual generations of this
insect.
The longest pre-reproductive and post-repro-
ductive periods for R. padi maintained on B. ruz-
iziensis were at the lowest temperature (12'C)
and no significant differences were found in the
range from 20'C to 28C (Table 1). In this interval
the adults began laying nymphs more quickly
than at the other temperatures (Table 1 and
Fig. 2). Elliott and Kieckhefer (1989) reported a
shorter pre-reproductive period in the range from
15 to 26C in relation to the extreme conditions of
11 and 29C. Segonca et al. (1994) did not find any
significant differences in the pre-reproductive pe-
riod of R. padi in the range from 4 to 20'C. The
values found by these authors were lower than
those found in the present study.
The aphid's reproductive period was longest at
16'C, and there was no reduction in this biological
parameter with increasing temperature (Table 1).
Likewise, Segonca et al. (1994) did not find signif-
icant reductions in the duration of the reproduc-
tive period of R. padi submitted to temperatures
from 8 to 20'C.
The highest fecundity rates occurred at 16 and
24oC, with 21.2 and 20.0 nymphs/female, respec-
tively. Higher rates were found by Asin & Pons
(2001) in the range from 18 to 27.5C, varying
from 35.2 to 45.0 nymphs/female, and a similar
number of nymphs/female was found by Dean
(1974) at 25C. At 16 and 20'C, the average num-
ber of nymphs produced was 21.2 and 12.9, re-
spectively. In contrast, Segonca et al. (1994) found


twice as many nymphs produced when the aphids
were reared at the same temperatures, indicating
that the same aphid species, under the same cli-
mate conditions, can have different reproductive
potentials, which are possibly influenced by other
biotic or abiotic factors.
Hesler et al. (2005a) found production of 7.5 to
11.4 R. padi nymphs in a 24-h period when 3
winged females were reared at temperature of
20'C on transgenic wheat plants. The effect of the
host plant on nymph production was also reported
by Hesler (2005b), in which the same aphid spe-
cies, during 7 d of observation, produced between
23.6 and 43.3 nymphs/female, according to the
type of host grass. Nutritional factors are also re-
sponsible for variation in fecundity, as mentioned
in the study by Adams (2007), in which an in-
crease in nitrogen content was responsible for
higher fecundity of R. padi.
The daily and total nymph production was
lower at the extreme temperatures (12 and 28C),
as shown in Table 1. Asin & Pons (2001) also
found a low reproductive capacity of R. padi when
the females were reared at 30'C. According to
Tang et al. (1999), aphids respond in different
ways to adapt to different geographic regions and
climate conditions. Nava-Camberos et al. (2001)
added that besides temperature, the host plant
also influences the aphid's physiological response.
Therefore, the number of nymphs found in other
studies possibly was higher than our findings be-
cause the R. padi aphids originated from other
populations and were provided with host plants
that permitted better development. However, Lo-
eck et al. (2006) did not find significant differ-
ences in the reproduction of R. padi when testing
different oat cultivars.
The survival of the various instars was above
70%, from 12 to 28C, except at 20'C for the
fourth instar (51%), indicating that in this tem-
perature range the nymphs reach the adult
phase. Third and fourth instars did not survive at
the highest temperature (32C), corroborating the
results of Dean (1974), according to which 30'C
was lethal to R. padi nymphs. However, Asin &
Pons (2001) reported survival of 55% of this aphid
species at this temperature. Tolerance of this
aphid species to high temperatures was reported







Florida Entomologist 92(4)


y = 0.008x2 0.3894x + 6.5331
P = 0.9875


12
-10





4
0


8 12 16 20 24 28 32
Temperature (C)


y = 0.0101x' 0.4831x + 6.9889
P = 0.9557


53








0
I'O


y = -0.0149x2 + 0.2909x + 7.5589
* FP= 0.4683


8 12 16 20 24 28 32

Temperature (C)


y= -0.141x2+0.6018x- 3.8234
R = 0.7804


8 12 16 20 24 28 32
Temperature (OC)


2
R 20
-15



Z 5

0


8 12 16 20 24 28 32
Temperature (C)


y = -0.1136X2 + 4.2494x 20.962
R = 0.4827


8 12 16 20 24 28 32
Temperature (OC)

Fig. 2. Fitted regression curves for the duration of pre-reproductive (A), reproductive (B), post-reproductive (C),
and daily (D) and total (E) fertility of Rhopalosiphum padi, as a function of temperature.


by De Barro et al. (1992), who mentioned that the
initial mortality of this aphid was 320C, and that
above 390C mortality was greater than 90%.

Table of Life Expectancy and Fecundity of R. padi on
Signal Grass

The survival (lx) started to diminish after the
first day at a temperature of 120C, as of 3.5 d at
160C and from 1.5 d at the other temperatures
(Fig. 3). These figures to not agree with those re-


ported in the literature for other aphid species,
probably because the insect's life expectancy is as-
sociated with the particular species (Tamaki et al.
1982) or host plant (Wale et al. 2000) being stud-
ied.
The highest life expectancies (ex) were 14.9,
14.3, 9.7, 10.3, 7.5 and 2.5 d in the nymph phase
at 12, 16, 20, 24, 28, and 320C, respectively In the
adult phase, the longest life expectancies were
6.7, 10.8, 5.2, 6.2, and 4.7 days at 12, 16, 20, 24,
and 280C, respectively. At the highest tempera-


4-




0

1-


3


02





0-


December 2009







Auad et al: Biology and Life Table ofRhopalosiphum padi


12'C


0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32


0 2 4 6 8 10 12 14 16 18 20 22 24


28'C


0 I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16


0 I 2 3 4 5 6 7 8


Time (Days)

Fig. 3. Survivorship (Lx) and life expectancy (ex) of Rhopalosiphum padi as a function of temperature.


ture (32C), life expectancy of nymphs was lowest,
and the individuals died before reaching the adult
phase.
The probability of death before the period es-
tablished (100 )q occurred during the entire
nymph and adult phase, with the majority of nil
values for this parameter occurring at 16C. The
highest mortality rates (d.) were 10, 19, 9, 14, 13,
and 23 dead individuals, and the probability of
death (100q.) on these occasions was 71.4, 29.7,
45.0, 28.6, 18.6, and 40.4% at 12, 16, 20, 24, 28,
and 32C, respectively.
The highest specific fecundity (m. = 4
nymphs/female/day) was recorded at 12 and


20C. The aphids did not reach the adult phase
at 32C, which prevented calculating the fe-
cundity table figures at this temperature. Like-
wise, Asin & Pons (2001) found a significant de-
cline in the reproductive capacity of R. padi
maintained at 30C. The highest net reproduc-
tion rates (Ro) occurred at 24 and 28C (Table
3). At 16 and 20C, the Ro values were 9.92 and
9.85, respectively. Segonca et al. (1994), study-
ing the same temperature conditions, found re-
spective values of 46.6 and 38.0, indicating that
the number of times R. padi increases in popu-
lation from 1 generation to the next was nearly
5 times higher.


0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32


24C


. 2 4 6 8 1 12 14 16 18 2. .
0 2 4 6 8 10 12 14 16 ie 20







Florida Entomologist 92(4)


TABLE 3. FERTILITY TABLE FOR RHOPALOSIPHUM PADI
AS A FUNCTION OF TEMPERATURE

Parameter
Temperatures
(C) T R, rm X DT

12 21.90 6.59 0.086 1.09 8.06
16 17.80 9.92 0.128 1.14 5.42
20 11.32 9.85 0.202 1.22 3.43
24 9.43 13.42 0.280 1.32 2.47
28 4.08 13.88 0.640 1.89 1.08

T = time interval between each generation; Ro= Net repro-
ductive rate; rm= intrinsic rate of population increase; = finite
rate of increase and DT = time necessary for the population to
double (days).


The time interval between each generation (T)
diminished with increasing temperature (Table
3). A significant reduction in this interval was
also found by Segonca et al. (1994) studying R.
padi kept at 8 to 20C.
The intrinsic rate of increase (r.) was higher at
28C (0.64), indicating that at this temperature
the population increases faster, while aphids kept
at 12C had a lower rm (0.09). These results cor-
roborate those of Asin & Pons (2001) for R. padi,
according to which the highest rate occurred at
27C, considered to be the most suitable tempera-
ture for the species to develop. Segonca et al.
(1994) found a gradual increase in r. from 8 to
20C, and when compared to the results of the
present study (16 and 20C), the authors observed
higher rm values. The same results occurred when
R. padi individuals were exposed to temperatures
fluctuating between 12.8 to 26.6C or 20 to
33.40C.
We found that rm was 4 times higher when the
temperature increased from 16 to 280C. Asin &
Pons (2001) found that in the range of 18 to 270C,
rm of this aphid doubled at the highest tempera-
ture, suggesting the ability of this species to
adapt to higher temperatures. The finite rate of
increase was higher at 280C, at 1.9 nymphs/fe-
male/day. The population doubling times (DT)
were 8.06 and 1.08 days at 12 and 280C, respec-
tively.
The results show that with higher tempera-
tures, up to a limit of 280C, although the life ex-
pectancy of R. padi fed with signal grass is lower,
the aphid's fecundity is higher when compared to
lower temperatures, explaining population spikes
at certain times of the year. These findings can as-
sist in planning control measures. In conclusion,
in the range of 12 to 28 C, nymphs reached the
adult phase, and 16 to 240C was the most favor-
able temperature range for reproduction of R.
padi on B. ruziziensis. The life expectancy of R.
padi decreased with increasing temperature, but
fecundity was best at 280C.


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Florida Entomologist 92(4)


December 2009


TEMPERATURE-DEPENDENT DEVELOPMENT OF THE CYCAD AULACASPIS
SCALE, AULACASPIS YASUMATSUI (HEMIPTERA: DIASPIDIDAE)

RONALD D. CAVE, CORA SCIACCHETANO AND RODRIGO DIAZ
Indian River Research and Education Center, University of Florida, 2199 S. Rock Road,
Ft. Pierce, FL 34945-3138, U.S.A.
E-mail: rdcave@ufl.edu


ABSTRACT

Egg duration period, immature development time, and pre-oviposition period of the cycad
aulacaspis scale,Aulacaspis yasumatsui Takagi, were measured at 9 constant temperatures
in the laboratory. Egg duration period ranged from 15 d at 20C to 7 d at 30C. First instar
development time was 30 d at 18C but only 4 d at 35C. No first instars completed develop-
ment below 18C or above 35C. Development time of second instar females ranged from 19
d at 18 and 20C to 9 d at 30C. Development time of male second instar + pupa ranged from
15 d at 20C to 9-10 d at 25-32C. Pre-oviposition period averaged 14 d at 20C to 8 d at 25-
32C; no females laid eggs at 18 and 35C. The lowest temperature threshold for all stages
ranged from 8 to 12oC and 538 degree-days were required for female immature development
in a linear model. Development rates of the scale are compared to those of 3 of its natural
enemies, Cybocephalus nipponicus Endr6dy-Younga, Rhyzobius lophanthae (Blaisdell), and
Coccobius fuluus (Compere and Annecke).

Key Words: armored scale, Cycas revoluta, development time, degree-days, natural enemies

RESUME

Se midieron el period de duraci6n del huevo, el tiempo de desarrollo de los inmaduros, y el
period de pre-oviposici6n de la escama aulacaspis de las cicadas, Aulacaspis yasumatsui
Takagi, a nueve temperatures constantes en el laboratorio. El period de duraci6n del huevo
vari6 de 15 d a 20oC hasta 7 d a 30C. El tiempo de desarrollo del primer estadio fue 30 d a
18C pero solamente 4 d a 35C. Ninguna ninfa en el primer estadio cumpli6 su desarrollo
abajo de 18C ni arriba de 35C. El tiempo de desarrollo de hembras en el segundo estadio
vari6 de 19 d a 18 y 20oC hasta 9 d a 30C. El tiempo de desarrollo de machos en el segundo
estadio + pupa vari6 de 15 d a 20oC hasta 9-10 d a 25-32oC. El period de pre-oviposici6n
tuvo un promedio de 14 d a 20oC hasta 8 d a 25-32oC; ninguna hembra deposit huevos a 18
ni 35C. Se estim6 que el umbral tnrmico mas bajo para todas las etapas vari6 de 8 a 12oC y
se necesitaron 538 grados-dias para el desarrollo inmaduro de las hembras en un modelo li-
neal. Se compare la tasa de desarrollo de la escama con las de tres de sus enemigos natura-
les, Cybocephalus nipponicus Endr6dy-Younga, Rhyzobius lophanthae (Blaisdell), y
Coccobius fulvus (Compere y Annecke).


Translation provided by the authors.


The cycad aulacaspis scale, Aulacaspis ya-
sumatsui Takagi, is a native of Southeast Asia,
and an invasive pest in Florida, Texas, Hawaii,
West Indies, Costa Rica, New Zealand, and Ivory
Coast (Germain & Hodges 2007) and in Guam
where it is killing large numbers of the native Cy-
cas micronesica K. D. Hill (Terry & Marler 2005).
It was first detected in south Florida in 1998 and
quickly spread throughout the state. The scale in-
fests several species of cycads (Howard et al.
1999), but the king sago, Cycas revoluta Thun-
berg, a popular landscape plant due to its attrac-
tive form and minimal maintenance require-
ments, appears to be especially susceptible. Since
1998, large numbers of king sagos in south Flor-
ida have been destroyed by the cycad aulacaspis
scale. A number of natural enemies are being


studied in a biological control program to manage
the pest. In order to better understand host-natu-
ral enemy relationships, the development time of
the cycad aulacaspis scale was examined at 9 con-
stant temperatures and compared to the develop-
ment time of 3 natural enemies occurring in Flor-
ida, the parasitic wasp Coccobius fulvus (Comp-
ere and Annecke) (Hymenoptera: Aphelinidae)
and the predatory beetles Rhyzobius lophanthae
(Blaisdell) (Coleoptera: Coccinellidae) and Cyb-
ocephalus nipponicus Endr6dy-Younga (Co-
leoptera: Cybocephalidae).

MATERIALS AND METHODS

Eggs were collected from females on the day
they were deposited. From 12-20 eggs were gently







Cave et al.: Development ofAulacaspis yasumatsui


transferred to a glass vial (9 mm x 50 mm) by us-
ing a fine camel hair brush and the vial was
plugged with sterile cotton that was moistened
daily with a drop of distilled water. Vials were
placed in a Percival environmental chamber set
with 50-60% RH, 14:10 h light:dark photoperiod,
and one of the following constant temperatures:
18, 20, 25, 30, 32, or 35C. Vials were examined
daily for presence of crawlers, which were
counted and killed, until all eggs hatched or be-
came collapsed.
Cycas revoluta plants in 4.4-L pots were in-
fested with crawlers by placing them next to
plants with female scales that began ovipositing 6
d previous. The leaves were intermixed to allow
movement of crawlers from infested to uninfested
plants. One day after crawlers moved onto unin-
fested plants, the pots were placed individually in
a Percival environmental chamber set with a con-
stant temperature, 50-60% RH, and 14:10 h
light:dark photoperiod. Experimental tempera-
tures were 11, 18, 20, 25, 30, 32, 35, 38, and 42C
for females and 18, 20, 25, 30, and 32C for males.
Each settled crawler was assigned a number, the
settling date was recorded, and its position on the
leaflet was mapped. Each nymph was examined
daily under a dissecting microscope. Nymphs
were sexed when they molted to second instar. Fe-
males are recognized by the production of a round
armor covering the nymph. Males are recognized
by an initially 3-pronged scale cover that eventu-
ally becomes elongate and tricarinate. For males,


dates of molting to second instar and emergence
of adult from the scale cover were recorded. For
females, dates of molting to second instar and to
adult, or date of death prior to adulthood, were re-
corded. Starting 5 d after molting to adult, the ar-
mor of the female was gently lifted daily and the
date of first egg produced was noted.
Since active crawlers require a day to settle,
1 d was added to the time period spent as a settled
crawler to quantify the development time of the
first instar. Means were statistically compared by
analysis of variance and Student-Newman-Keuls
test with a = 0.05.
For the female nymphal stages, the linear por-
tion of the developmental rate curve [R(T) = a + bT]
was modeled by least squares linear regression
(PROC GLM, SAS Institute 1999), where T was
temperature, and a and b were estimates of the in-
tercept and slope, respectively. Development rates
that were not part of the linear portion of the curve
were not included in the regression analysis. The
base temperature threshold was estimated by the
intersection of the regression line at R(T) = 0, To =
-a/b. Degree-day requirements for each stage were
calculated from the inverse slope of the fitted lin-
ear regression line (Campbell et al. 1974).

RESULTS

The effect of temperature on egg hatching is
shown in Table 1. No eggs hatched at 18C (n =
25). Significant differences were detected among


TABLE 1. MEAN DEVELOPMENT TIME IN DAYS SEM OF IMMATURE STAGES, TOTAL NYMPHAL DEVELOPMENT TIME,
AND PRE-OVIPOSITION PERIOD OFA. YASUMATSUI AT 6 CONSTANT TEMPERATURES FOR FEMALES AND 5 CON-
STANT TEMPERATURES FOR MALES. SAMPLE SIZE FOR EACH MEAN IS INDICATED IN PARENTHESES.

Females

Pre-oviposition Egg
Temp (C) Egg 1t instar 2"d instar Total nymph period to adult

18 30.8 1.0 a (18) 19.0 2.0 a (2) 46.5 1.5 a no eggs -
20 15.5 + 0.5 a (2) 17.8 0.8 b (17) 19.3 0.3 a (15) 36.8 0.9 b 13.6 0.5 a (14) 52.6
25 9.6 0.2 b (27) 11.0 0.2 c (72) 11.9 0.2 bc (72) 22.9 0.3 c 8.1 + 0.3 b (52) 32.5
30 6.9 0.1 c (63) 10.4 0.2 c (140) 9.3 0.2 c (110) 19.7 0.2 c 7.9 0.2 b (54) 26.6
32 7.4 0.2 c (18) 8.9 + 0.1 d (30) 12.7 0.3 bc (30) 21.6 0.4 c 7.7 0.5 b (15) 29.0
35 7.9 0.1 c (51) -no eggs -
Males

Egg
Temp (C) Egg 1" instar 2nd instar + pupa Total nymph to adult

18 29.6 0.5 a (45) -
20 16.4 0.5 b (20) 14.6 1.2 a (5) 29.6 1.8 a 46.5
25 10.8 0.2 c (31) 9.7 0.3 b (27) 20.4 0.4 b 30.1
30 10.3 0.3 c (50) 9.7 0.7 b (12) 19.9 0.6 b 26.9
32 8.6 0.2 d (59) 9.4 0.2 b (59) 18.0 0.2 c 25.4

Means within a column for each sex followed by the same letter are not significantly different (P > 0.05; Student-Newman-Keuls test).







Florida Entomologist 92(4)


the egg duration periods at the other 5 tempera-
tures tested (F = 78.93, df = 4, P < 0.001). As ex-
pected, increasing temperature decreased hatch-
ing time up to 30'C, but 32' and 35C did not de-
crease hatching time. Percentage egg hatch at
30'C (84.0%, n = 75) was greater than percentage
egg hatch at 35C (62.2%, n = 82), more than dou-
ble that at 25C (40.9%, n = 66), and nearly 3
times the percentage at 32C (29.5%, n = 61).
No development of the first instar occurred at
11, 38, and 42C. Significant differences were de-
tected among the development times of female
first and second instars and total development
times of female nymphs at the other 6 tempera-
tures (F = 402.3, 83.8, and 148.2, respectively, df =
5, P < 0.001) (Table 1). First instars developed
slowest at 18'C and fastest at 35C. There was no
significant difference between first instar devel-
opment times at 25 and 30'C, both of which were
significantly different from development times at
20 and 32C. Female second instars required sig-
nificantly more days to reach adulthood at 18 and
20'C than at temperatures >25C. Many female
nymphs produced deformed armors during the
molt to second instar at 18 and 35C, and only 3
individuals at these temperatures reached adult-
hood. Total female nymphal development times at
25C and higher were not significantly different
but were significantly different from the develop-
ment time at 20'C. Female nymphs required more
than twice as many days to complete development
at 18'C than at >250C.
Females that reached adulthood at 18 and
350C died without producing eggs. Pre-oviposition
period was significantly longer at 200C (F = 35.1,
df = 3, P < 0.001), but there were no significant
differences among the pre-oviposition periods at
the other 3 temperatures (Table 1).
Significant differences were detected among
the development times of male first instar (F =
645.7, df = 4, P < 0.001), second instar + pupa (F
= 14.4, df = 3, P < 0.001), and total development
times of male nymphs (F = 59.3, df = 5, P < 0.001)
(Table 1). First instar males developed slowest at
180C and fastest at 320C. There was no significant
difference between first instar development times
at 25 and 300C, both of which were significantly
different from development time at 200C. Second


instar + pupal development time was signifi-
cantly longer at 200C than at the 3 higher temper-
atures, which were not significantly different
among themselves; no males completed develop-
ment after attaining the second instar at 180C.
Total male immature development time was slow-
est at 200C and fastest at 320C, with no difference
in times between 25 and 300C.
The linear model provided a good means to de-
scribe the relationship between developmental
rate (1/D) and temperature (T). Table 2 shows the
lower threshold temperature and total degree-
days required to complete development of each
immature stage. The linear model estimated that
the lower temperature threshold for all stages
ranged from 8 to 120C and 538 degree-days were
required for female immature development. The
number of day-degree requirements for first and
second instars were very similar.

DISCUSSION

Cycad aulacaspis scales had great difficulty
developing beyond the first instar below 200C. Av-
erage minimum temperatures are below this level
in south Florida during the months Nov to Apr, so
scales settled on the plant leaves would suffer in-
creased mortality or develop very slowly, if at all.
Scales under megasporophylls or on the roots,
however, may be protected from the lower ambi-
ent temperatures. The optimal temperature for
female development is 300C. Average maximum
temperatures approximate or slightly exceed this
level in south Florida during the months May to
Oct, thus supporting greater population growth
and plant infestation in this period (unpublished
data). Egg incubation period at this temperature
is 7 d. Therefore, the time interval from egg to egg
at 300C is on average 34.5 d for this insect. The
linear model underestimated the lower tempera-
ture threshold because laboratory results con-
firmed that eggs and nymphs did not complete de-
velopment below 180C.
In Florida and elsewhere, the 3 principal
natural enemies of the cycad aulacaspis scale
are C. fulvus, R. lophanthae and C. nipponicus.
The development time of female C. fulvus para-
sitizing the arrowhead scale, Unaspis yanonen-


TABLE 2. PARAMETER ESTIMATES DESCRIBING THE RELATIONSHIP BETWEEN TEMPERATURES AND DEVELOPMENTAL
RATES (1/D) OF A. YASUMATSUI FEMALES.

Stage Intercept Slope R2 Threshold 'C Degree-days'

Egg -0.096 0.0080 0.99 12.0 124.3
1 instar -0.050 0.0051 0.90 9.9 194.9
2" instar -0.041 0.0050 0.96 8.2 202.4
Egg to adult -0.017 0.00186 0.95 9.3 537.6

Total degree-days to complete development.


December 2009







Cave et al.: Development ofAulacaspis yasumatsui


sis Kuwana, is 52, 27, and 26 d at 19, 25, and
30C, respectively (Ogata 1987). Rhyzobius lo-
phanthae development from egg to egg is 44, 32,
and 24 d when preying on Aspidiotus nerii
Bouche (Stathas 2000) and 48, 34, and 27 d
when feeding on Ci.' ....u.. 4.... ....aonidum (L.)
(Stathas et al. 2002) at 20, 25, and 30C, respec-
tively. In comparison, the life cycle times from
egg to egg for A. yasumatsui at the same 3 tem-
peratures are 50, 31, and 28 d, respectively.
These data indicate that C. fulvus and R. lo-
phanthae develop at similar rates or slightly
more quickly than the cycad aulacaspis scale
and may produce as many or a few more gener-
ations than their host within a defined period of
time, which is a favorable characteristic of bio-
logical control agents. The time interval for de-
velopment from egg to egg ofC. nipponicus feed-
ing on the euonymus scale, Unaspis euonymi
(Comstock), is 48 d at 22C (Alvarez & van Dri-
esche 1998), whereas the time interval is 44 d
for the predator feeding on A. yasumatsui at
25C (Smith & Cave 2006). The development
rate of A. yasumatsui at this temperature is
more rapid (31 d). Therefore,A. yasumatsui can
build up population numbers faster than C. nip-
ponicus, which, coupled with the fact that C.
nipponicus pupae are parasitized by Aphanog-
mus albicoxalis Evans and Dessart (Evans et
al. 2005), may limit the effectiveness of this
predator as a biological control agent of the
scale. The fecundity ofA. yasumatsui is yet un-
studied, but the prolific reproduction of this
pest may be one reason why its natural enemies
are not providing adequate control on king sa-
gos in Florida.

ACKNOWLEDGMENTS

Celia Branch, Michael Burton', Julieta Castillo, Veron-
ica Manrique, and Greta Thorson are gratefully acknowl-
edged for assistance in data collection. This research was
supported by grants from the Florida Department ofAgri-
culture and Consumer Services and the Department of In-
terior Cooperative Conservation Initiative.


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Florida Entomologist 92(4)


December 2009


IMPACT OF URBANIZATION ON TRI-TROPHIC
INTERACTIONS IN AN ENDEMIC SCRUB COMMUNITY

S. E. SUMOSKI1, A. J. JOHNCOX2, D. M. ALTHOFF3 AND K. A. SEGRAVES3
'State University of New York College of Environmental Science and Forestry, 1 Forestry Drive,
Syracuse, NY 13210

2Department of Biological Sciences, Le Moyne College, 1419 Salt Springs Road, Syracuse, NY 13214

'Department of Biology, Syracuse University, 107 College Place, Syracuse, NY 13244


ABSTRACT

Human-mediated disturbances have altered every ecosystem on the planet and these
changes may have important consequences for biodiversity and community structure. We
tested how the degree of urbanization impacts a tri-trophic interaction among the Florida
scrub endemic plant Palafoxia feayi, a gallmaking midge, and the associated parasitoid
wasps. A combination of field surveys and laboratory hearings were used to determine
whether habitat disturbance associated with housing development (e.g., land clearing, fire
suppression) was correlated with changes in plant architecture, gallmaker abundance, or
parasitoid diversity. We found significant differences in the number of side branches of
plants at urban sites, and that the number of galls per plant increased with both the number
of side branches and plant height. More parasitoids were found in galls collected from urban
sites, but parasitoid diversity was unchanged by urbanization. We conclude that although
urbanization influenced plant architecture, there was only a minor impact on gallmaker
abundance and parasitoid diversity.

Key Words: Palafoxia, Asphondylia, parasitoid, tri-trophic interactions, urbanization, con-
servation

RESUME

La urbanizaci6n ha alterado cada ecosistema en el planet y estos cambios pueden tener
consecuencias significantes para la biodiversidad y estructura de la comunidad. Nosotros
probamos c6mo la urbanizaci6n afecta una interacci6n tri6-tr6fica entire el arbusto end6mico
Floridano de la plant Palafoxia feayi, unjej6n que produce agallas, y las asociadas avispas
parasitoides. Una combinaci6n de examenes y crianzas en el laboratorio fueron utilizadas
para determinar si los disturbios con la urbanizaci6n (por ejemplo: claro de tierra, supresi6n
de fuego) eran correlacionados con la arquitectura de la plant, la abundancia del jej6n que
crea las agallas, y la diversidad del parasitoide. Encontramos diferencias significativas en el
numero de ramas laterales de plants en sitios urbanos, y que el numero de irrita en una
plant fue correlacionado con el numero de ramas laterales y la altura de una plant. Mas
parasitoides fueron encontrados adentro agallas recogidos en sitios urbanos, pero la diver-
sidad de los parasitoides no fue cambiado por la urbanizaci6n. Concluimos que aunque la ur-
banizaci6n influenciara la arquitectura de la plant, tenia un minimo impact en la
abundancia del jej6n que produce agallas y la diversidad del parasitoide.


Translation provided by the authors.


Tri-trophic interactions among plants, herbiv-
orous insects, and their natural enemies are a
major component of most terrestrial ecosystems.
These 3 groups comprise a grand majority of the
Earth's described species (Strong et al. 1984). As a
consequence, human disturbance of terrestrial ec-
osystems has a strong potential to produce a cas-
cading impact on biodiversity and community
structure across trophic levels. For example, hu-
man-mediated disturbance has been shown to
have significant consequences for insect commu-
nities by influencing species diversity, insect


abundance, and community structure (Hamer et
al. 1997; Denys & Schmidt 1998; Rambo & Faeth
1999; Botes et al. 2006; Jones & Paine 2006; Co-
mor et al. 2008; Tscharntke et al. 2008). Moreover,
disturbance induced changes in plant communi-
ties and plant architecture can have cascading ef-
fects on plant-feeding insects and their predators
(Rambo & Faeth 1999; Scheirs & De Bryun 2002;
Thies et al. 2003; Moreau et al. 2006; Tylianakis
et al. 2007).
Urbanization is an obvious form of human-me-
diated disturbance that has been shown to have







Sumoski et al.: Urbanization and Tri-trophic Interactions


both rapid and extreme impacts on arthropod
abundance, distribution, and community compo-
sition (reviewed by McIntyre 2000). For instance,
Rango (2005) showed that community composi-
tion of arthropods on creosote bush differed sub-
stantially between urban and fringe deserts, and
that one-third of the species found in fringe
deserts were absent from urban sites. Although a
number of studies have examined how urbaniza-
tion affects insect communities, relatively few
have addressed how urbanization impacts tri-
trophic interactions. Examining how urbaniza-
tion affects interactions among plants, phytopha-
gous insects, and natural enemies is essential for
understanding the additive and non-additive ef-
fects that these disturbances have on biodiversity.
Florida scrub is a rare ecosystem that has been
heavily impacted by urbanization (Peroni & Abra-
hamson 1986). Fire suppression, habitat frag-
mentation, and land clearing are the major
threats of concern (Myers 1990). Florida scrub
has a high level of endemism, especially of plants,
and covers areas along the Lake Wales Ridge of
central Florida (Estill & Cruzan 2001). Primarily
due to habitat loss, this ecosystem is home to ap-
proximately 22 federally listed threatened and
endangered plant species. Currently, Florida
scrub is highly fragmented and consists of iso-
lated habitat islands encompassing both pro-
tected and non-protected areas along the Lake
Wales Ridge. In non-protected areas, agriculture
and housing developments have created a patch-
work mosaic of scrub habitat. Thus, urbanization
may have significant effects on species diversity
and abundance due to reduction in population
sizes through habitat loss and reduction in dis-
persal among isolated patches.
The purpose of this study was to examine how
urbanization influences tri-trophic interactions
among the endemic Florida scrub plant Palafoxia
feayi A. Gray (Asteraceae), a gallmaking midge,
Asphondylia sp. (Diptera: Cecidomyiidae), and
the associated parasitoid guild. We compared
plant architecture, number of galls, and parasi-
toid species diversity between pristine and urban
sites situated in and surrounding the Archbold
Biological Station in Lake Placid, Florida. In par-
ticular, we addressed the following questions: (1)
Do plants growing in urban vs. pristine sites dif-
fer in architecture? (2) If so, does this difference
correspond to a change in the incidence of gall for-
mation? (3) Is parasitoid species diversity and
abundance increased or decreased in urban sites?

Study System

The plant Palafoxia feayi (Asteraceae) is found
in scrub and pinelands of central and southern
Florida. The genus Palafoxia contains a total of 9
species, and the center of diversity for the genus is
in Texas (USDA, NRCS 2009). Members of this


genus occur across the southern half of the United
States from coast to coast (USDA, NRCS 2009).
Palafoxia feayi is the only perennial in the genus.
These plants are host to an undescribed dipteran
gallmaker in the genus Asphondylia that pro-
duces galls mainly in the floral tissue (Gagne
1989; McIntyre unpublished data). Midge larvae
are attacked by 4 undescribed hymenopteran par-
asitoid species in 3 different families: Torymus
(Torymidae), Galeopsomyia (Eulophidae), Tenui-
petiolus (Eurytomidae), and Rileya (Eurytomi-
dae) (McIntyre unpublished data).

MATERIALS AND METHODS

Surveys for plants, galls, and insects were con-
ducted during May and Jun 2008 in 2 areas near
Lake Placid, Florida-the Archbold Biological
Station (ABS), 27011' 20" N, 81 20' 26"W, and va-
cant lots in the Placid Lakes housing subdivision
(PL), 27015' 22" N, 81022' 57"W. These areas differ
in degree of urbanization. The Archbold Biological
Station contains some of the last remaining con-
tiguous tracts of Florida scrub. Sites at ABS are
pristine and are only disturbed by frequent natu-
ral fires and prescribed burns. In contrast, Flor-
ida scrub in the Placid Lakes subdivision is sub-
ject to land clearing associated with housing de-
velopment. Many of the urban sites used in this
study were vacant lots that had been cleared for
prospective development, although some of the
sites were relatively undisturbed patches of scrub
that were frequently subdivided by paved roads.
As a result, urban scrub sites are severely frag-
mented by roads and development of surrounding
lots.
At each of these locations we surveyed 10 m by
10 m plots separated by a minimum of 100 m.
Plots were haphazardly selected and only plots
containing P feayi were included in the surveys.
Initially, plants were surveyed to assess whether
the presence of galls was correlated with plant ar-
chitecture. Ten plots at ABS and 20 plots at PL
were surveyed. Each plant with a minimum stem
length of 30 cm was measured for a number of ar-
chitectural features: main stem length to the
nearest cm, the number of side branches, and the
number of galls. Plants of this size were used be-
cause individuals were more easily located in the
scrub and were more likely to have flowered (As-
phondylia primarily forms galls in floral tissue).
This survey provided enough plants to test for a
correlation between plant architecture and gall
presence. Subsequently, 20 plots at ABS and 15
plots at PL were surveyed for the number of
plants and the number of galls per plant. This sec-
ond survey allowed us to test whether there was a
difference in the average number of galls per plot
in pristine versus urban sites.
We examined parasitoid diversity by collecting
100 galls from plants at both ABS and PL. The







Florida Entomologist 92(4)


galls were individually placed into 88.7-mL plas-
tic cups and covered with Glad Press'N Seal@
wrap. Galls were checked daily and emergence of
insects was recorded. Insects were placed in 1.5-
mL microcentrifuge tubes containing 70% ethanol
and stored in a freezer at -20C for later identifi-
cation. If a gall appeared infected with fungus or
became severely desiccated, it was dissected im-
mediately to salvage as many collectable, identifi-
able insects as possible. After a period of 30 d all
galls were dissected to determine the abundance
of the remaining insects. Parasitoids were identi-
fied to genus with keys in Goulet & Huber (1993)
and Gibson et al. (1997).
We examined how plant architecture influ-
enced the presence of galls by using linear regres-
sion to examine how plant height and the number
of side branches related to the number of galls per
plant. We also examined whether plant architec-
ture differed between sites by using a t-test to
compare the length of the main stem of plants at
ABS and PL and a Mann-Whitney U-test to com-
pare the number of side branches per plant at
these sites. For comparisons of urban and pristine
plots, we used t-tests to determine whether the
average number of plants per plot, the average
number of galls per plot, and the average gall den-
sity per plot differed between sites. Finally, we
compared parasitoid species diversity between
ABS and PL via the Shannon-Wiener index (a
joint measure of species richness and evenness). A
chi-square test was used to determine if parasi-
toid abundance differed with degree of urbaniza-
tion. All statistical analyses were performed with
JMP 5.01 (SAS Institute).

RESULTS

A total of 1473 plants was surveyed- 587 (104
from ABS and 483 from PL) plants were exam-
ined for plant morphology and number of galls,
and an additional 886 plants for surveys of gall
occurrence only (423 from ABS and 463 from PL).
Plots from PL had more plants and more galls,
but the average gall density per plot did not differ
with urbanization (Table 1). Overall, both plant
height and the number of side branches were pos-
itively correlated with the number of galls per
plant; however, neither characteristic explained a
large proportion of the variation associated with


the presence of galls (Fig. 1). Plants from ABS and
PL did not differ in height (ABS = 105.91 cm
4.22 (SE), PL = 100.02 cm 1.96; t = 1.267, df =
585, P = 0.21), but did differ in the number of side
branches per plant (Mann-Whitney U-test, U =
24448, P < 0.0001). PL plants had slightly more
and a greater range of side branches (range 1 to
34, median = 3.00) than ABS plants (range 1 to 9,
median = 2.00). As a result, the average number
of galls per individual plant was slightly greater
for PL than ABS (PL = 0.57 0.04 (SE), ABS =
0.42 0.06; t = 2.105, df = 1471, P = 0.0354).
Wasps obtained from P feayi galls were from 4
genera-Galeopsomyia (Eulophidae), Rileya (Eu-
rytomidae), Tenuipetiolus (Eurytomidae), and To-
rymus (Torymidae)-and all genera were repre-
sented at both sites (Table 2). Of the 100 galls col-
lected from each site, parasitoids were more fre-
quently found in galls from PL (38 galls from PL
and 24 galls from ABS, x2 = 4.58, df= 1, P = 0.032).
The hearings and dissections produced 161 hy-
menopterans, 53 from ABS and 108 from PL. Al-
though more parasitoids were reared from galls
at PL, the average rate of successful parasitism
per gall was not different between sites (t = 0.988,
df= 49, P = 0.328). Similarly, for plots that yielded
parasitoids, Shannon-Wiener diversity indices
were similar between ABS and PL (ABS = 0.665
0.16 (SE), PL = 0.566 + 0.232; t = 0.360, df = 9, P
= 0.72).

DISCUSSIoN

The Florida scrub ecosystem is considered to
be one of the most endangered ecosystems in Flor-
ida and North America (Scott 2003). Urbanization
and conversion to agricultural lands have frag-
mented Florida scrub into a patchwork consisting
of a few large, pristine sections and a number of
small areas interspersed among urbanized
patches. Understanding how the native scrub
subsisting in this mosaic has been impacted by
urbanization is an important first step to make
informed decisions in scrub conservation. Here
we compared how urbanization may influence tri-
trophic interactions among an endemic scrub
plant, a gallmaking midge, and the associated
parasitoids.
Comparisons of plants in urban versus pristine
scrub indicated substantial differences in plant


TABLE 1. SURVEY RESULTS FOR ASPHONDYLIA GALLS ON PALAFOXIA FEAYI IN PRISTINE AND URBAN PLOTS (MEAN
SE). STATISTICAL SIGNIFICANCE BASED ON T-TESTS.

Site Pristine (ABS) Urban (PL) Significance

No. Plots 30 35
Average no. plants/plot 17.57 2.62 27.03 2.42 P < 0.007
Average no. galls/plot 7.33 1.65 15.37 3.39 P < 0.038
Average gall density/plot 0.41 0.11 0.54 0.09 P < 0.349


December 2009







Sumoski et al.: Urbanization and Tri-trophic Interactions


P<0001
R'=007



*




~- - ----------


0* 0Mee s*000040 0 e

40 60 8b 100 120 140 160 180 200 220
Height (cm)


P< 00001
SR2=014


* *


" 0 6




*


* S__ O *
06S6 beg h


Fig. 1. Linear regression of the number of galls produced by the gall midge Asphondylia in comparison to plant
height and the number of side branches for Palafoxia feayi. Only plants taller than 30 cm that had galls were in-
cluded (n = 145 plants).


architecture. Plants growing in urban sites did
not differ in overall height from plants growing in
pristine scrub, but urban plants did have signifi-
cantly more and a greater range of side branches.
Unlike other Palafoxia species, P feayi is a peren-
nial and continues growing by initiating side
branches on already existing stems that have fin-
ished flowering (McIntyre unpublished data).
One potential reason for the difference in the
number of side branches is that plants growing in
housing subdivisions are less likely to experience
fire. Although P feayi can resprout after fire, all
aboveground stems of the plant are destroyed.
Plots located at the Archbold Biological Station
are regularly subjected to natural and prescribed
burns to maintain the Florida scrub ecosystem
(Ostertag & Menges 1994). Hence, plants have to
re-initiate above ground growth more often at
ABS. The higher density of P feayi at urban sites
suggests that the plants may potentially avoid
mortality associated with fire.
Although there were clear differences in the ar-
chitecture of plants with the degree of urbaniza-


tion, this did not transcend into differences at
higher trophic levels. At the individual plant level,
we detected a significant correlation between gall
density and plant height and gall density and
number of side branches; however, this pattern
only explained a small proportion of the variation.
Plants at urban sites had more side branches and
a higher density of galls per plant than plants in
pristine scrub. At the plot level, however, gall den-
sity did not differ with degree of urbanization. Ur-
ban plots had more plants with a greater number
of side branches, but the average density of galls
per plot was not significantly different from pris-
tine sites. This was surprising given that differ-
ences in the number of plants and the number of
side branches has direct implications for the num-
ber of galls that are possible at a plot. Because each
side branch terminates in a flower head that is the
primary site ofAsphondylia gall formation, this re-
sult suggests thatAsphondylia has not responded
to the increase in availability of potential oviposi-
tion sites or that other factors may limit midge
populations at these sites.


TABLE 2. DISTRIBUTION OF HYMENOPTERANS REARED OR DISSECTED FROM EQUAL NUMBERS (100) OF GALLS ON
PALAFOXIA FEAYI FROM PRISTINE AND URBAN FLORIDA SCRUB. (MEAN S.E.; N.S. = NOT SIGNIFICANT).

Parasitoids Pristine (ABS) Urban (PL) Significance

Number of Galeopsomyia 28 54 -
Number of Rileya 1 8 -
Number of Tenuipetiolus 16 2 -
Number of Torymus 8 44 -
Percent galls parasitized 24% 38% P < 0.05
Average parasitism per gall 2.51 0.84 3.6 0.70 n.s.
Average Shannon-Wiener index/plot 0.665 + 0.16 0.566 0.232 n.s.


0 2 4 6 8 10 12 14 16 18 20 22
No. side branches







Florida Entomologist 92(4)


One possible factor that may limit midge pop-
ulations in urban areas is suppression by parasi-
toids. Indeed, more parasitoids were reared from
galls collected in disturbed sites and these sites
also exhibited a higher proportion of parasitized
galls (38% parasitized versus 24% in pristine
sites). These results, however, should be inter-
preted with caution as studies have shown year to
year variation and among site variation in parasi-
toid attack rate (e.g., Heard et al. 2006). The ques-
tion remains whether these modest differences in
parasitism translate into consistently higher
mortality rates of the gallmakers across years.
Differences in the degree of urbanization did
not appear to influence species richness of parasi-
toids attacking Asphondylia. The parasitoid com-
munities in urban and pristine sites were similar.
All 4 species were found at both sites and Shan-
non-Wiener indices of diversity were similar.
Whether these parasitoid species are specialists
on Asphondylia utilizing P feayi is unclear. Spe-
cies from the same 4 genera are found on As-
phondylia borrichiae that uses sea oxeye daisy
Borrichia fructescens along Florida's Gulf Coast
(Stiling et al. 1992; Rossi et al. 2006). If parasi-
toids that attack Asphondylia are recruited from
other gallmakers in the local community, then
there may be a stable species pool of parasitoids
in scrub habitats that can use galling larvae on P.
feayi. If this were the case, changes caused by ur-
banization would need to influence the entire
host-parasitoid insect community in order to po-
tentially change parasitoid species diversity on
urban P. feayi.
Urbanization has direct impacts on plant com-
munity structure and plant architecture of the
Florida endemic P feayi. These impacts, however,
do not appear to significantly influence utilization
patterns by the galling midgeAsphondylia and its
associated parasitoid community.

ACKNOWLEDGMENTS

We thank H. Swain and M. Deyrup for valuable ad-
vice on the study system, and P. McIntyre for sharing
his unpublished study. Mylenne Salinas graciously
translated the abstract. The Archbold Biological Station
kindly provided facilities and access to field sites. This
project was supported by a grant to K. A. S. and D. M. A.
from the National Science Foundation (DEB 0743101).

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Florida Entomologist 92(4)


December 2009


PARASITOIDS ATTACKING THE EMERALD ASH BORER
(COLEOPTERA: BUPRESTIDAE) IN WESTERN PENNSYLVANIA

J. J. DUAN', R. W. FUESTER', J. WILDONGER', P. B. TAYLOR', S. BARTH1 AND S. E. SPICHIGER2
'USDA-ARS, Beneficial Insects Introduction Research Unit, Newark, DE 19713

'Pennsylvania Department of Agriculture, Bureau of Plant Industry, Harrisburg, PA 17110
E-mail: Jian.Duan@ars.usda.gov

ABSTRACT

Field surveys of the emerald ash borer (EAB), Agrilus planipennis Fairmaire, and associ-
ated parasitoids were conducted in Cranberry Township, PA from 11 Mar to 23 Oct 2008.
Several species of parasitic Hymenoptera were collected from EAB-infested green ash trees
or reared from late-instar EAB larvae, prepupae, and/or pupae. These included Balcha in-
dica (Mani & Kaul), Eupelmus pini Taylor (Eupelmidae), Dolichomitus uitticrus Townes
(Ichneumonidae), and 2 additional unidentified ichneumonids, Orthizema sp. Townes and
Cubocephalus sp. Townes. Together, these parasitoids caused about 3.6% parasitism of EAB
in the field. The most abundant species was B. indica accounting for 82% of all parasitoids
recovered during our survey. Subsequent laboratory assays confirmed that B. indica and E.
pini are solitary ectoparasitoids of EAB larvae, prepupae, and/or pupae. In addition, both B.
indica and E. pini reproduce through thelytokous parthenogenesis; i.e., virgin females pro-
duce daughters. These parasitoids may be complementary to current classical biological con-
trol programs against EAB in North America, which have been focusing primarily on the
introduction of exotic larval and egg parasitoids from China.

Key Words:Agrilus planipennis, parasitoid, Balcha indica, Eupelmus pini, biological control

RESUME

Se realizaron sondeos de campo para el barrenador esmeralda de fresno (BEF),Agrilus pla-
nipennis Fairmaire, y los parasitoides asociados en el pueblo de Cranberry, PA desde el 11
de marzo hasta el 23 de octubre de 2008. Varias species de parasitoides del orden Hyme-
noptera fueron recolectadas de arboles de fresno verde infestados de BEF o criadas de los 1l-
timos estadios de las larvas, prepupas y/o pupas de BEF. Estos incluyen Balcha indica (Mani
& Kaul), Eupelmus pini Taylor (Eupelmidae), Dolichomitus vitticrus Townes (Ichneumoni-
dae) y 2 ichneum6nidos no identificados adicionales, Orthizema sp. Townes y Cubocephalus
sp. Townes. Juntos, estos parasitoides causaron aproximadamente 3.6% del parasitismo de
BEF en el campo. La especie mas abundante fue B. indica que represent 82% de todos los
parasitoides recuperados durante el sondeo. Ensayos de laboratorio subsiguientes confir-
man que el B. indica y E. pini, son ectoparasitoides solitarios de larvas, prepupas y/o pupas
de BEF. Ademas, ambos B. indica y E. pini se reproducen por medio de la partenog6nesis de
thelytokia, donde hembras virgenes produce hijas. Estos parasitoides pueden complemen-
tar los programs de control biol6gico clasico actuales contra el BEF en Norteamerica, los
cuales han sido enfocados principalmente en la introducci6n de parasitoides ex6ticos de
China sobre larvas y huevos.


The emerald ash borer (EAB), Agrilus pla-
nipennis Fairmaire, is a serious invasive pest that
has killed millions of ash trees in North America
since its discovery in 2002 in Michigan and On-
tario (Haack et al. 2002; Poland & McCullough
2006). Regulatory efforts to contain the pest's
spread via early detection, quarantine, and re-
moval of infested ash trees, have had limited ef-
fects on containing this pest (Cappaert et al.
2005), and chemical control cannot be used to pro-
tect native ashes in forest ecosystems because of
prohibitive cost and general impracticality (Po-
land & McCullough 2006). Currently, EAB is es-
tablished in Michigan, Indiana, Illinois, Ohio,
Pennsylvania, Maryland, West Virginia, Wiscon-


sin in the US, and Ontario and Quebec in Canada.
An isolated infestation was detected in southeast-
ern Missouri as recently in summer of 2008
(NAPPO Phytosanitary Alert System 2008).
Following the discovery of EAB in Michigan,
classical biological control programs were initi-
ated against this invasive pest. The programs
have primarily focused on the introduction and
release of exotic parasitoids from China, the prob-
able origin of the pest (USDA APHIS 2007; Liu et
al. 2007; Bauer et al. 2008). Recent field surveys
in Michigan and Ontario, however, indicate that
some indigenous parasitoids have already become
associated with EAB and may play a role in sup-
pressing the local populations of EAB (Bauer et







Duan et al.: Parasitoids Attacking the Emerald Ash Borer


al. 2004; Cappaert & McCullough 2008; Lyons
2008). In the present study, we investigated ex-
tant parasitoid guilds associated with emerald
ash borers in western Pennsylvania, where the
pest was first discovered in 2007. We also con-
ducted laboratory tests with the recovered parasi-
toid species against the larvae, prepupae, and/or
pupae of EAB to further confirm their associa-
tions.

METHODS

Field Survey

A total of 41 green ash (Fraxinus pennsylvan-
ica Marshall) trees with an average diameter
breast height (DBH) of 21.5 cm (range 10-45 cm)
with symptoms of EAB infestation such as wood-
pecker feeding, thin canopy, epicormic shoots, and
bark splits (Cappaert et al. 2005) were searched
for and located in Cranberry Township, PA (Fig. 1)
from 11 Mar to 23 Oct 2008. On each sampling oc-
casion in the months of Mar, Apr, Jun, Sep, and
Oct, we removed the bark from the boles of 3 to 12
trees from the ground to the height of 2 m using
draw knives. To avoid damaging immature EAB
larvae, prepupae and/or pupae under the bark, we
first cut the bark using the draw knife to reach
the cambium and the surface of the wood tissue,
and then peeled down the bark to expose imma-
ture EAB and parasitoids. Exposed immature
EAB and parasitoids were collected with soft alu-
minum forceps, placed in cells of plastic culture
plates (12 or 24 cells per plate, each lined with wet
filter paper) or plastic tubes with ventilated caps,
and returned to the USDA ARS Beneficial Insects
Introduction Research quarantine facility (New-
ark, DE) to rear the parasitoids to the adult stage.
Most of the parasitoids collected by debark sam-
pling were immature stages (larvae and/or pu-
pae), but a few were emerging adults. Adult para-
sitoids either directly collected from the field or
recovered later from rearing in the laboratory
were sent to the USDA ARS Systematic Entomol-
ogy Laboratory for identification to species, and
voucher specimens were deposited there.

Laboratory Exposure Assay

To evaluate parasitoid-host associations, we
presented adult parasitoids to EAB late-instars,
prepupae, or pupae that were inserted into ash
sticks (about 10 cm long x 1 cm diameter) freshly-
cut from green ash trees by methods modified
from Liu & Bauer (2007). Our modified method
involved cutting a thin flap (about 5 cm long x 0.5
cm wide) of the outer bark from the top of the stick
with a utility knife, leaving the end of the flap at-
tached in the middle of the stick. We then cut a
narrow groove (3-4 mm deep) in the exposed wood
of the stick underneath the flap with a wood-carv-


ing V-shaped chisel. An EAB larva, prepupa or
pupa was then placed in the groove, covered with
the flap, and secured with thin bands of parafilm
at the top and bottom of the flap. The sticks were
placed, with the insertion-end up, into cells of a
12-well tissue culture plate, which was placed in-
side a ventilated plastic box (17.6 x 12.6 x 10 cm).
To maintain moisture in the ash sticks, water was
added to the cells of the culture plate so that the
base of each stick was submerged in about 1/4 cm
of water. Parafilm was used to cover the surface of
the plate to prevent spillage of water.
The adult parasitoids used in the laboratory
assay originated from the field-collected imma-
ture stages of EAB or ash bark infested with EAB.
At least 7 d after emergence from the immature
(cocoon or pupa) stages, 1 to 6 adult parasitoids
were released into the plastic box containing
EAB-infested ash sticks at parasitoid to host ra-
tios ranging from 1:4 to 1:6. The exposure dura-
tion was 3 to 7 d, depending upon the longevity of
the test parasitoids. At the end of each trial, ex-
posed ash sticks containing immature EAB were
incubated in a growth chamber at 20-26C; 55% -
65% RH, and 16:8 h (L:D) photoperiod for recov-
ery of F, parasitoid progeny. When no adult para-
sitoids emerged from the exposed ash sticks after
4 weeks of incubation, we dissected them and
transferred parasitoids or hosts to wells of 12-well
tissue cell plates lined with moist filter paper;
these were incubated for 8 to 12 weeks of incuba-
tion or until adult parasitoids or EAB emerged.
Approximately equal proportions of the EAB late
instars (3rd to 4th), prepupae, and pupae were
tested across different trials for each species of
the parasitoid tested. Percentage parasitism for
each trial was calculated as proportion of imma-
ture EAB successfully attacked, as evidenced
with the presence of parasitoid progeny produced
by the test parasitoid.

RESULTS AND DISCUSSION

Five species of hymenopteran parasitoids were
recovered from 1,091 EAB larvae, prepupae, and/
or pupae collected in the field survey, including
Balcha indica Mani & Kaul (Eupelmidae), Eupel-
mus pini Taylor (Eupelmidae), Dolichomitus vitti-
crus Townes (Ichneumonidae), and 2 unidentified
ichneumonids, Orthizema sp. Townes and Cuboce-
phalus sp. Townes. These parasitoids together
parasitized 3.6% of the sampled EAB hosts
(Table 1). Balcha indica was the most abundant
species of parasitoid recovered from the field sur-
vey, accounting for 82% of the parasitoids recov-
ered. While all the B. indica adults (n = 32) recov-
ered from the field survey were females, both
sexes ofD. vitticrus (n = 2), and Cubocephalus sp.
(n = 3) were recovered. Only 1 individual female
was recovered from the field survey for both E.
pini and Orthizema sp.







Florida Entomologist 92(4)


Fig 1. Locations where the first EAB were discovered and field survey samples were taken in Cranberry
Township, PA.


Larval stages ofB. indica were observed in as-
sociation with remains of emerald ash borer lar-
vae, prepupae, and/or pupae in the field. However,
the association of E. pini, D. vitticrus, Cuboceph-
alus sp., and Orthizema sp. with the emerald ash
borer could not be positively confirmed from field
observations as stages of these parasitoids had al-
ready progressed to pupae and/or pharate adults
and their host remains were not recognizable.


Thus, all the recovered female adult parasitoids
were further tested in the laboratory assay
against EAB late-instars, prepupae, and pupae.
Results from our laboratory assays demon-
strated that both field-recovered eupelmids, B. in-
dica and E. pini, successfully attacked and devel-
oped from EAB late-instars, pre-pupae, and/or
pupae inserted into the ash sticks, while the other
parasitoids did not (Table 2). All the progeny pro-


December 2009








Duan et al.: Parasitoids Attacking the Emerald Ash Borer


TABLE 1. PARASITOIDS RECOVERED/COLLECTED FROM EAB-INFESTED ASH TREES, THEIR RELATIVE ABUNDANCE AND
PARASITISM (11 MAR-24 OCT). A TOTAL OF 1091 EAB LARVAE, PREPUPAE/PUPAE WERE COLLECTED FROM
THE SURVEY.

No. Individuals Relative Abundance %
Parasitoid Taxon Recovered (%) Parasitism of EAB'

Balcha indica 32 82.0 2.9
Eupelmus pini 1 2.6 0.1
Dolichomitus vitticrus 2 5.1 0.2
Orthizema sp. 1 2.6 0.1
Cubocephalus sp. 3 7.7 0.3

Parasitism calculated as percent of the total parasitoids recovered relative to the total number of the EAB (1091) and parasi-
toids (39) throughout the entire survey.


duced by both virgin B. indica and E. pini were fe-
males, indicating that both species had repro-
duced through thelytokous parthenogenesis.
Although EAB was just recently discovered in
western PA, the extent of the damage to ash trees
in the area and the size of the infestation suggest
EAB has been present at this site for several
years (Pennsylvania State University 2009). Dur-
ing that time, 2 locally extant parasitoids, B. in-
dica and E. pini, became associated with EAB, ex-
erting about 3% parasitism (Table 1). However,
the association of the 3 ichneumonids, D. vitti-
crus, Cubocephalus sp. and Orthizema sp., with
EAB could not be confirmed from our field survey
and laboratory experiments. It is possible that
these ichneumonid parasitoids collected from our
field samples were from insects other than EAB
infesting ash tree bark.
Gibson (2005) reported the first collection ofB.
indica in Virginia in 1994 and suggests this para-
sitoid was probably introduced to the U.S. from
Southeast Asia in the 1960s. Balcha indica was
next found in 2003 in Michigan parasitizing EAB
(Bauer et al. 2004, 2005) and in Maryland from
wood-boring beetles in a cherry tree (Prunus sp.)
(Gibson 2005). Only females have been collected,
and Gibson (2005) proposed that B. indica was
likely parthenogenetic. Data from our laboratory
assay indicates that B. indica reproduces via the-


lytokous parthenogenesis, and successfully para-
sitizes larvae, prepupae, and/or pupae of EAB
(Table 2).
We collected a single female of E. pini associ-
ated with EAB-infested ash trees at our field
sites, and it was our least abundant parasitoid
species (Table 1). It was also recovered from EAB-
infested ash in southeast Michigan from 2002 to
2004 (Bauer et al. 2004). Using laboratory assays,
we have now confirmed that this species repro-
duces via a thelytokous parthenogenesis on EAB
larvae, prepupae, and/or pupae (Table 2). Surpris-
ingly, E. pini previously was only recorded para-
sitizing late-instars of the white pine weevil, Pis-
sodes strobi (Peck), in eastern US (Taylor 1929;
Harman & Kulman 1967) and Quebec (Williams
& Langor 2002). Although there are no records of
E. pini attacking buprestids, species from Eu-
pelminae are more niche-specific than host-spe-
cific, and Eupelmus spp. generally parasitize a
wide range of insect larvae or pupae in concealed
locations (G. Gibson, Agriculture and Agri-Food
Canada, personal communication).
These eupelmid species may be complemen-
tary to the ongoing EAB biological control efforts
in the U.S., which include 1 egg and 2 larval par-
asitoids that attack EAB in China (USDA APHIS
2007; Liu et al. 2007). Another native ectoparasi-
toid found to attack EAB larvae,Atanycolus hico-


TABLE 2. RESULTS OF EXPOSURE ASSAYS OF PARASITOIDS AGAINST IMMATURE EAB (LARVAE/PRE-PUPAE/PUPAE) CON-
TAINED IN GREEN ASH TWIGS IN THE LABORATORY.

No. Parasitoid: Host Total No. % Parasitoid
Parasitoid Taxon Trials ratio Host Exposed' Parasitism2 Progeny produced3

Balcha indica 15 1:4 130 16.9 22
Eupelmus pini 58 1:4 58 13.8 8
Dolichomitus vitticrusz 3 1:5 15 0 0
Orthizema sp. 4 1:6 24 0 0
Cubocephalus sp. 8 1:4 42 0 0

1Parasitized host stages (EAB larvae, prepupae and pupae) were pooled for calculation of percent parasitism.
'Approximately equal proportions of late instars (3" to 4'), prepupae, and pupae were presented in different trials.
'All progeny produced by virgin B. indica (Fo) and E. pini (Fo to F ) are females.











riae (Braconidae) (Bauer et al. 2004, 2005), is be-
ing evaluated for possible use as a biocontrol
agent of EAB (Cappaert & McCullough 2008).
Studies of B. indica and E. pini are ongoing in our
laboratory, where we are focusing on reproductive
and developmental biology, host finding and selec-
tion behavior, and the eventual development of
mass rearing methods for use in augmentative bi-
ological control agents against EAB in the U.S.

ACKNOWLEDGMENT

We thank Mitchell Dykstra (APHIS PPQ Cranberry,
PA), Donald Eggen (PA Bureau of Forestry, Middletown,
PA), Shalah Werner (Sierra Club, Madison, WI) and Du-
ane McKee (Cranberry Towhship, PA) for providing in-
formation concerning the infestation of EAB in West PA,
and Greg Sahene (Mine Safety Appliance Company, PA)
for allowing us to sample green ash trees on the com-
pany's property. We are grateful to Michael Gates and
Robert Kula (USDA ARS, Systematic Entomology Lab-
oratory, Beltsville, MD) for identification of B. indica
and Ichneumonidae, and Gary Gibson (Canadian Na-
tional Collection of Insects, Agriculture and Agri-Food
Canada, Ontario Canada) for identifying E. pini. We
thank Leah Bauer (USDA FS), Juli Gould (USDA
APHS, PPQ), Roy Van Driesche (University of Massa-
chusetts), and Douglas Luster (USDA ARS) for criti-
cally reviewing the manuscript prior to submission to
the journal for publication.

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AND PETRICE, T. R. 2004. Natural enemies of emer-
ald ash borer in southeastern Michigan, pp. 33-34 In
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MILLER, D. L., AND PETRICE, T. R. 2005. Emerald
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letter of the Michigan Entomological Society 47: 1-5.
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don, and G. Parra [Compilers], Proc. Emerald Ash
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SIEGERT, N. W. 2005. Emerald ash borer in North


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(Hymenoptera: Chalcidoidea: Eupelmidae), Parasi-
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lia Press, Aukland, New Zealand. 62 pp.
HARMAN, D. M., AND KULMAN, H. M. 1967. An Annotat-
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land, Natural Resources Institute, College Park,
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HAACK, R. A., JENDEK, E., LIU, H-P., MERCHANT, K. R.,
PETRICE, T. R., POLAND, T. M., AND YE, H. 2002. The
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ica. Newsletter of the Michigan Entomol. Soc. 47: 1-
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LIU, H-P., BAUER, L. S., MILLER, D. L, ZHAO, T., GAO, R.,
SONG, L., LUAN, Q, JIN, R, AND GAO, C. 2007. Season-
al abundance of Agrilus planipennis (Coleoptera:
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nipennisi (Hymenoptera: Eulophidae), a gregarious
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na, pp. 61-62 In V. Mastro, D. Lance, R. Reardon,
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LYONS, B. 2008. Emerald ash borer: it's here to stay,
let's learn how to manage it. Forest Health and
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http://www.pestalert.org/oprDetail.cfm?oprID=335.
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Florida Entomologist 92(4)







Gagn6 et al: Description ofLophodiplosis trifida Larva


DESCRIPTION OF THE LARVA OF LOPHODIPLOSIS TRIFIDA, AN
AUSTRALIAN GALL MIDGE (DIPTERA: CECIDOMYIIDAE) AND
BIOCONTROL AGENT OF PAPERBARK IN FLORIDA, USA

RAYMOND J. GAGNE', SUSAN A. WRIGHT2, MATTHEW F. PURCELL3, BRADLEY T. BROWN3, PAUL D. PRATT4
AND TED D. CENTER4
'Systematic Entomology Laboratory, PSI, Agricultural Research Service, U.S. Department of Agriculture,
c/o Smithsonian Institution MRC-168, P.O. Box 37012, Washington, DC 20013-7012, USA
E-mail: raymond.gagne@ars.usda.gov
2Invasive Plant Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture,
P.O. Box 147100, Gainesville, FL 32614-7100, USA
E-mail: susan.wright@ars.usda.gov
3Australian Biological Control Laboratory, Agricultural Research Service, U.S. Department of Agriculture,
CSIRO Entomology, 120 Meiers Road, Indooroopilly, Queensland, Australia 4068
E-mail: matthew.purcell@csiro.au; bradley.brown@csiro.au
4Invasive Plant Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture,
Ft. Lauderdale, FL, 33314, USA
E-mail: paul.pratt@ars.usda.gov; ted.center@ars.usda.gov

ABSTRACT
Lophodiplosis trifida Gagn6, an Australian gall midge on paperbark, Melaleuca quinque-
nervia (Myrtaceae), is a recent release in southern Florida for the biological control of that
host. The larval stage is described for the first time and compared to that of other Lopho-
diplosis species. Photos of galls and illustrations of larvae are provided. Second and third in-
stars of L. trifida are unusual among Cecidomyiidae for the lack of setae on most papillae.

Key Words: Melaleuca, biological control, bud gall, cecidomyiid

RESUME
Lophodiplosis trifida Gagn6, una mosquita de agalla sobre Melaleuca quinquenervia (Myr-
taceae) de origen Australiano, fue reci6n liberada en el sur del estado de Florida para el con-
trol biol6gico de este hospedero. Se describe el estadio larval por primera vez y se compare
contra larvas de otras species de Lophodiplosis. Se provee fotos de las agallas e ilustracio-
nes de las larvas. El segundo y tercer estadio de L. trifida no son comunes entire los Cecido-
myiidae por la falta de setas sobre la mayoria de las papilae.


Lophodiplosis trifida Gagne has been success-
fully introduced into southern Florida to aid in the
control of paperbark, Melaleuca quinqueneruia
(Myrtaceae) (P. D. Pratt, unpublished). This insect
was initially described as one of 5 Lophodiplosis
species reared from Melaleuca spp. in the vicinity of
Townsville, Qld., Australia (Gagne et al. 1997). No
further species has been added to Lophodiplosis
and, except for L. trifida in Florida, the genus is still
known only from eastern Australia. The other 4 spe-
cies, Lophodiplosis bidentata Gagne, Lophodiplosis
cornuata Gagne, Lophodiplosis indentata Gagne,
and Lophodiplosis denticulata Gagne, were taken
from separate and distinctive galls of paperbark.
Lophodiplosis trifida was initially thought to be an
inquiline in galls of 3 of the other species because it
was reared in association with them but not associ-
ated with any particular distinctive gall of its own
(Gagne et al. 1997). Subsequent observations in the
vicinity of Brisbane, Qld., showed that L. trifida
forms separate, often very prominent galls (Figs. 1-


4) in young shoots (Purcell et al. 2007). In the earlier
situation in Townsville, stem galls ofL. trifida galls
were evidently inconspicuous, perhaps containing
only 1 or 2 larvae, and had presumably been
masked by galls of the other Lophodiplosis species
and associated plant tissue in rearing cages. The
larval stage ofL. trifida was not described initially,
unlike those of 3 of the other species of Lophodiplo-
sis (that of L. denticulata remains undescribed).
Now that L. trifida has taken on biological control
importance, we take this opportunity to describe the
instars for future identification.

MATERIALS AND METHODS

Larvae of known L. trifida, as determined from
adults and pupae reared in quarantine in Ft. Lau-
derdale, FL, were removed from paperbark shoot
galls in various stages of growth. Specimens were
the progeny, many generations removed, of material
originally sent from Indooroopilly, Qld., Australia.







Florida Entomologist 92(4)


Figs. 1-4. Shoots of paperbark galled by Lophodiplosis trifida. 1, Galled young, soft shoot. 2, Longitudinal section
of same largely filled with spherical larval cells containing first instars. 3, Older, woody gall with exit holes, the ar-
rows indicating pupal exuviae still attached. 4, Same, longitudinal section, the topmost cell with pupa still inside.


December 2009







Gagn6 et al: Description of Lophodiplosis trifida Larva


Specimens were placed on slides for viewing in ac-
cordance with the method outlined in Gagne (1989).
Other specimens were critical-point dried and
placed on SEM stubs. Terminology for larval mor-
phology follows that in Gagne (1989).


Description of larva of L. trifida
Third Instar (Figs. 5-12)

Length, 1.3-2.0 mm (n = 10). Body cylindrical,
spindleform. Head capsule hemispherical, with


Figs. 5-9. Lophodiplosis trifida, third instar. 5, Entire larva (dorsolateral). 6, Anterior segments (anteroventral).
7, First thoracic segment with details of spatula and papillae (ventral). 8, Posterior segments showing anus at cen-
ter of terminal segment (lateroventral). 9, Detail of eighth and terminal segments (laterodorsal). Abbreviations: Ip
= triplets of lateral papillae; spat = spatula; spir = spiracle; tp = terminal papillae.







Florida Entomologist 92(4)


apodemes as long as capsule; antennae short, as
long as wide. Spiracles present on prothorax and
on first through eighth abdominal segments. In-
tegument with horizontal rows of minute spicules
ventrally on anterior third of 2nd and 3rd thoracic
and all abdominal segments, remainder smooth if
not weakly and irregularly lined. Spatula (Figs. 6,
11) widest posteriorly when fully sclerotized, with
long shaft, anteriorly with 2 large, triangular
teeth and a shorter tooth between. Full comple-
ment of papillae present except only 2 pairs on
terminal segment, 1 corniform, the other seti-


form. Setae present on 2 of 3 of each of the 4
groups of lateral papillae and on 1 of the pairs of
terminal papillae.

Second Instar (Figs. 13-14)

Length, 0.6-0.9 mm (n = 10). Body shape, head,
spiracles, integument and papillae as for third in-
star. Spatula present, short, the acutely triangu-
lar anterior tooth up to half length of shaft. As
with third instar, full complement of papillae is
present except for 2 pairs on terminal segment


.14


.4


S12
Figs. 10-15.Lophodiplosis trifida. 10-12, Third instar: 10, entire larva (dorsal), showing position of sternal spat-
ula on venter; 11, detail of anterior segments with inset showing 2x detail of lateral papillae (ventral); 12, detail of
posterior segments (dorsal). 13-14, Second instar: 13, entire larva (dorsal view), showing position of sternal spatula
on venter; 14, sternal spatula (ventral). 15, First instar, eighth and terminal segments (laterodorsal).


December 2009


er 2009







Gagn6 et al: Description ofLophodiplosis trifida Larva


and setae present on only 2 of 3 of each of the 4
groups of lateral papillae and on 1 pair of termi-
nal papillae.

First Instar (Fig. 15)

Length, 0.3-0.5 mm (n = 10). Body shape, head,
and integument as for second and third instars.
Spiracles present only on prothorax and eighth
abdominal segment. Spatula absent. Presence of
papillae as for second and third instars except
that pleural and dorsal papillae and the non-set-
ose pair of terminal papillae of second and third
instars all with minute seta.

Comments on Larval Stage of L. trifida

The first and second instars of this species are
unusual among Cecidomyiidae for the almost
complete absence of papillar setae. Setae occur on
only 2 papillae in each of the 4 sets of lateral trip-
lets and on 2 terminal papillae. The first instar, on
the other hand, has tiny setae, no longer than the
diameter of their papillae, on all pleural and dor-
sal papillae, in addition to the setae already men-
tioned for the third and second instars. The only
other example known of the lack of papillar setae
generally is the genus Caryomyia, a North Amer-
ican genus, in which most of species completely
lack papillar setae, even those on the lateral and
terminal papillae (Gagn6 2008). The integument
is remarkably smooth in L. trifida, with the an-
teroventral horizontal bands of spicules the only
sculpturing.

DISCUSSION

The 5 species originally placed in Lophodiplo-
sis come from simple to complex galls of Mela-
leuca spp. (Gagn6 et al. 1997). These species are
an eclectic mix that, besides having a host genus
in common, share the distinguishing pupal char-
acter of vertexal extensions that are evidently
used in cutting through gall tissue prior to adult
emergence. Many gall midges have variously
shaped prominences on the head for that purpose,
but they are almost always extensions of the an-
tennal bases, not of the vertex. Larvae of Lopho-
diplosis are diverse, but this is not unusual in
genera with a variety of gall shapes (Gagn6 2008).


Larvae of L. trifida are unique in Lophodiplosis
for the lack of setae on most papillae, their long,
narrow, tridentate spatula in the third instar, and
mostly smooth integument. The other species of
Lophodiplosis for which larvae are known, L. bi-
dentata Gagn6, L. cornuata Gagn6, and L. inden-
tata Gagn6, have short setae on most papillae, a
short, wide, bidentate spatula, and a rugose in-
tegument. The type species, L. indentata, and L.
trifida both have only 4 papillae on the terminal
segment, but all 4 papillae have setae onL. inden-
tata while only 2 have setae on L. trifida. Both
species have a spatula in the second instar. In gall
midges, a second instar spatula occurs only in
some gall-making genera where it seems to have
arisen de novo in each of the genera where it ap-
pears (Gagn6 2008).

ACKNOWLEDGMENTS

We thank P. Malikul for making the slide prepara-
tions, Scott D. Whittaker, SEM Laboratory Manager,
Smithsonian Institution for assistance with the scan-
ning electron microscope, Diana Marquez for electroni-
cally arranging the photos and drawings onto plates,
and James A. Lollis and Elizabeth D. Mattison for assis-
tance in collecting galls and larvae, and the South Flor-
ida Water Management District for financial support to
this project. We are grateful also to Keith M. Harris,
Woking, Surrey, United Kingdom, David A. Nickle and
Allen L. Norrbom, Systematic Entomology Laboratory,
and 2 anonymous reviewers for critical comments on
the manuscript.

REFERENCES CITED

GAGNE, R. J. 1989. The Plant-Feeding Gall Midges of
North America. Cornell University Press, Ithaca,
New York. xiii & 355 pp. and 4 pls.
GAGNE, R. J. 2008. The gall midges (Diptera: Cecidomyi-
idae) of hickories (Juglandaceae: Carya). Mem.
American Entomol. Soc. 48: 1-147.
GAGNE, R. J., BALCIUNAS, J. K., AND BURROWS, D. W.
1997. Six new species of gall midges (Diptera: Cecid-
omyiidae) from Melaleuca (Myrtaceae) in Australia.
Proc. Entomol. Soc. Washington 99: 312-334.
PURCELL, M. F., WINEWRITER (SIC! FOR WINERITER),
AND S. A., BROWN, B. T. 2007. Lophodiplosis trifida
Gagn6 (Diptera: Cecidomyiidae), a stem-galling
midge with potential as a biological control agent of
Melaleuca quinquenervia (Myrtaceae). Australian
Entomol. 34: 123-125.







Florida Entomologist 92(4)


THE PARASITOID FLY ORMIA OCHRACEA
(DIPTERA: TACHINIDAE) CAN USE JUVENILE CRICKETS AS HOSTS

CRYSTAL M. VINCENT' AND SUSAN M. BERTRAM
Carleton University, 1125 Colonel By Drive, Nesbitt Building, Ottawa, Ontario, Canada
'Corresponding author

ABSTRACT

The parasitoid fly Ormia ochracea uses the calling song of its host Gryllus spp. to locate an
area inhabited by potential hosts. Once a calling male has been located, 0. ochracea deposits
live larvae on the host, and on substrates surrounding the host to enable the larvae to attach
to and enter individuals in the vicinity of the calling male. In Texas, where 0. ochracea par-
asitizes the Texas field cricket Gryllus texensis, we observed juvenile crickets in the mating
aggregations that form around calling males. Juvenile G. texensis crickets are, therefore, po-
tentially susceptible to parasitism by 0. ochracea. Here we investigated whether laboratory
reared juvenile field crickets could successfully host 0. ochracea larvae. We found that juve-
nile crickets were appropriate hosts for 0. ochracea.

Key Words: parasitoid, cricket, host, parasite, juvenile

RESUME

La mosca parasitoide Ormia ochracea usa el canto de cortejo del hospedero Gryllus spp. para
ubicar las areas habitadas por hospederos potenciales. Una vez que el canto del macho ha
sido localizado, 0. ochracea deposit larvas vivas sobre el hospedero y sobre los sustratos al-
rededor del hospedero para que larvas pueden atar y entrar los individuos en la vecindad del
canto del macho. En Texas, donde 0. ochracea parasita el grillo de campo Tejano, Gryllus
texensis, observamos grillos juveniles en las agregaciones de apareamiento que se forma al-
rededor de los machos cantando. Los grills juveniles de G. texensis entonces son potencial-
mente susceptibles al parasitismo por 0. ochracea. Aqui, investigamos si los juveniles de los
grills de campo criados en el laboratorio pueden exitosamente ser hospederos de las larvas
de 0. ochracea. Encontramos que los juveniles de los grills fueron hospederos apropiados
para 0. ochracea.


Parasites and parasitoids are often limited in
host range because of 4 factors: (1) parasites and
potential hosts may not overlap in their ranges;
(2) they may not come into direct contact for a va-
riety of behavioral or ecological reasons; (3) poten-
tial hosts fail to support the essential spatial or
metabolic demands of the parasite or parasitoid;
and (4) potential hosts may employ a variety of
defence mechanisms to avoid being parasitized
(Euzet & Combes 1980, referenced in Combes
2001). These limits on host availability may ex-
plain why parasites often utilize a specific life
stage of their hosts as it is not often that larval
and adult, or juvenile and adult life stages of
hosts coalesce with respect to all 4 of these limit-
ing factors. However, if a species changes its dis-
tribution or behavior, it may put itself at risk of
becoming host to a previously absent parasite.
How species traverse these boundaries and
whether parasites are capable of exploiting these
boundary breaches is an exciting area of investi-
gation for parasitologists. Here we investigate
whether crickets and their parasitoid fly demon-
strate such a violation of host specificity.
Singing males of the Texas field cricket, Gryl-
lus texensis (Orthoptera: Gryllidae), are acousti-


cally stalked and parasitized by gravid females of
the parasitoid fly Ormia ochracea (Diptera: Ta-
chinidae) (Cade 1975). Ormia ochracea are larvip-
arous; once they locate their host, the gravid fe-
male deposits her planidia (1t instar Ormia) on
and around the cricket (Cade 1975). Planidia laid
around the cricket wave their anterior ends in the
air in an attempt to come in to contact with a host.
Once contact is established, the planidia burrow
into the host's body. Inside the host, the larvae
feed on the cricket's fat body, and abdominal and
thoracic muscles (Adamo et al. 1995). Parasitized
crickets initiate an encapsulation response to kill
the planidia before they have a chance to estab-
lish themselves (Vinson 1990). However, encapsu-
lation responses are often co-opted by the parasi-
toid larvae and used to construct a trachea that
they attach to the cricket's abdominal wall (Vin-
son 1990). After 7 to 10 d, the larvae emerge from
the host (Adamo et al. 1995). Larval emergence
invariably results in the death of the host cricket.
The observation that gravid flies deposit lar-
vae around calling males (Cade 1975) as well as
observations made during behavioral trials con-
ducted in our laboratory, suggests that all crickets
in the vicinity of a calling male are in danger of


December 2009







Vincent & Bertram: Juvenile Crickets as Tachinid Hosts


parasitization by 0. ochracea. In fact, several au-
thors have observed parasitized females in the
wild even though female crickets do not acousti-
cally signal (Walker & Wineriter 1991; Zuk et al.
1993; Adamo et al. 1995). To our knowledge, par-
asitized juvenile crickets have yet to be observed,
although juvenile crickets have been observed in
the vicinity of calling males. While collecting G.
texensis in the field in Texas during Sep 2007,
more than 30% of the individuals that we col-
lected from the area immediately around calling
male crickets were penultimate and last instar ju-
veniles. Thus, these juvenile G. texensis were in
danger of becoming parasitized. Using 3rd genera-
tion lab reared G. texensis, we compared the
progress of an 0. ochracea infestation in penulti-
mate juveniles to that of adults to determine
whether juvenile G. texensis are suitable hosts for
0. ochracea larvae.

MATERIALS AND METHODS

Crickets and flies were collected nightly from
several locations between Austin and Smithville,
Texas, in 2007. Flies were trapped with a sound
trap modelled after Walker's (1989) slit trap de-
sign. A Durabrand CD-566 compact disc player
(Lennox Electronics Corporation, 35 Brunswick
Avenue, Edison, NJ 08817) was connected to an
Amplified Speaker AMX 18 (RadioShack Corpora-
tion, Fort Worth, Texas 76102) and then placed
underneath the sound trap. This speaker broad-
casted a natural call of a G. texensis male at a
decibel level of 61db from 30 cm. The broadcasted
call was recorded from a laboratory-reared male
G. texensis and had a dominant frequency of 4.6
kHz. Gravid female 0. ochracea were attracted to
the broadcast and entered the trap via the slit, be-
coming ensnared. Captured flies were housed in a
terrarium and provided with ad libitum hum-
mingbird feed. Crickets and flies were brought
back to establish laboratory colonies of each at
Carleton University, Canada.
We used a manual parasitization method (Vin-
cent & Bertram in press) to parasitize the crick-
ets. Briefly, a gravid 0. ochracea female was
chilled and her abdomen removed. The abdomen
was gently teased apart, the reproductive tract
opened, and the planidia exposed. Once planidia
were exposed to the air, they 'stood' on their pos-
terior ends and moved back and forth in a 'wav-
ing' motion. These planidia were gently probed
with a blunt dissecting tool until they latched on
to the tool. They were then transferred to a
cricket. Two actively waving planidia were gently
placed on the articular sclerites (located at the
anterior end of the thorax where the wings at-
tach) of each host cricket. In total, 74 penultimate
juveniles (37 males/37 females) and 57 adults (28
males/29 females) were parasitized in this man-
ner.


Crickets were housed individually and pro-
vided ad libitum food and water until larval para-
sitoid emergence. After mature parasitoid larvae
emerged from the host and pupated, the pupae
were housed separately in an incubator so that the
success (eclosion) of each cricket's larvae could be
determined. The dates of parasitization and larval
emergence were recorded as well as the number of
days after larval emergence the cricket survived.
All crickets that did not succumb to parasitization
were frozen on the 14th d following manual parasit-
ization. Crickets were later thawed and dissected
to ascertain whether there was any visible evi-
dence of parasitism (e.g., encapsulated parasitoid
larvae or parasitoid trachea).
Comparisons of parasitoid success in juvenile
and adult cricket hosts were based on the follow-
ing measurements: number of days from manual
parasitization to first mature larval emergence;
the total number of larvae that emerged from
each cricket (0, 1, or 2); the number of days that
the cricket survived following larval emergence;
success of parasitoid pupae (proportion of pupae
that closed: none-0, half-1, all-2); the number of
larvae that established, as determined by count-
ing the number of parasitoid trachea found in
cricket's abdomen during dissections; the propor-
tion of crickets with larval emergence; and the
proportion of larvae that emerged versus became
established in the cricket host.

RESULTS

Ormia ochracea planidia were able to establish
themselves in juvenile crickets. We found no sig-
nificant difference between adult and juvenile
crickets in the number of planidia that success-
fully established, the number of larvae that suc-
cessfully emerged, or in the success of emerged
larvae (Table 1). Juvenile crickets lived signifi-
cantly longer than adult crickets post larval
emergence (juveniles = 1.03 days, adults = 0.61
days, P = 0.015; Table 1).

DISCUSSION

The observance of penultimate juvenile crick-
ets in mating aggregations led us to postulate
that juvenile G. texensis may be parasitized by 0.
ochracea in the wild, and indeed in laboratory
studies, juvenile crickets can serve as hosts for 0.
ochracea. Penultimate stage juvenile G. texensis
appear to be ideal hosts for 0. ochracea. The num-
ber of parasitoid larvae that established within a
cricket, the number of larvae that emerged, the
proportion of established versus emerged larvae,
and the time it took for the larval parasitoids to
emerge from the cricket did not differ between ju-
venile and adult cricket treatments. Our results
suggest that 0. ochracea could successfully use
penultimate juvenile crickets as hosts in nature.







Florida Entomologist 92(4)


December 2009


TABLE 1. ORMIA OCHRACEA SUCCESS WITHIN JUVENILE AND ADULT CRICKETS.


Mean No. Adults Eclosing from Pupae (0-2)



Mean No. Larvae Established (0-2)



Proportion of Crickets with Larval Emergence



Mean No. Larvae Emerged (0-2)



Mean No. Days Until Larval Emergence



Mean Proportion of Emerged / Established


ADULT JUVENILE

1.71 0.1405 1.92 0.0576



1.35 0.104 1.30 0.102


0.634


0.763


1.18 0.0969 1.31 0.0778


X"= 3.23
n = 45
P = 0.2004
X"= 0.934
n = 107
P= 0.82
X"= 3.75
n = 107
P= 0.053
"= 4.19
n = 86
P = 0.19


6.71 3.45 7.44 2.98 ANOVA: n = 86
F = 1.99
P = 0.16


0.7804


Mean No. Days Cricket Survived Following Larval Emergence


0.844


0.614 0.839 1.03 1.02


X- = 0.579
n = 86
P= 0.45
ANOVA: n = 86
F = 6.13
P = 0.015


'dffor all analyses was 1.


Our results show that 0. ochracea has a simi-
lar life history in hosts of different stages of devel-
opment in a laboratory population of crickets.
However, it remains to be demonstrated whether
juvenile crickets become parasitized in the wild.
If juvenile crickets are regularly utilized by 0.
ochracea as hosts in nature, this might impact
populations of G. texensis.

ACKNOWLEDGMENTS

We are grateful to I. Thompson, E. Whattam, and V
Rook for field assistance and to M. Forbes and H. Rundle
for helpful comments on this manuscript. We thank the
University of Texas at Austin and the staff at Bracken-
ridge and Stengl field stations. This work was funded by a
grant from Carleton University, a grant from the Canada
Foundation for Innovation Leaders Opportunity Fund, a
grant from the Ministry of Research and Innovation On-
tario Research Fund, and a Natural Science and Engi-
neering Research of Canada Discovery Grant to S.M.B.

REFERENCES CITED

ADAMO, S. A. 1999. Evidence for adaptive changes in egg
laying in crickets exposed to bacteria and parasites.
Anim. Behav. 57: 117-124.
ADAMO, S. A., ROBERT, D., AND HOY, R. 1995. Effects of
a tachinid parasitoid, Ormia ochracea, on the behav-


ior and reproduction of its male and female field
cricket hosts (Gryllus spp.). J. Insect Physiol. 41:
269-277.
COMBES, C. 2001. Parasitism: The Ecology and Evolu-
tion of Intimate Interactions. The University of Chi-
cago Press, Ltd. London.
GULLAN, P. J., AND CRANSTON, P. S. 2004. The Insects:
An Outline Of Entomology. Blackwell Publishing
Ltd. Oxford, England.
KOLLURU, G. R., ZUK, M., AND CHAPPELL, M. A. 2002.
Reduced reproductive effort in male field crickets in-
fested with parasitoid fly larvae. Behav. Ecol. 13:
607-614.
LEHMANN, G. U. C., AND LEHMANN, A. W. 2000. Sper-
matophore characteristics in bushcrickets vary with
parasitism and remating interval. Behav. Ecol. So-
ciobiol. 47: 393-399.
OROzCO, S. X., AND BERTRAM, S. M. 2004. Parasitized
male field crickets exhibit reduced trilling bout rates
and durations. Ethology 110: 909-917.
VINCENT, C. M., AND BERTRAM, S. M. in press. Collect-
ing and laboratory culture of Ormia ochracea
(Diptera: Tachinidae). J. Entomol. Sci.
WALKER, T. J. 1989. A live trap for monitoring Eu-
phasiopteryx and tests with E. ochracea (Diptera:
Tachinidae). Florida Entomol. 72: 314-319.
WALKER, T. J., AND WINERITER, S. A. 1991. Hosts of a
phonotactic parasitoid and levels of parasitism
(Diptera: Tachinidae: Ormia ochracea). Florida En-
tomol. 74: 554-559.







Weiberlahl & Liburd: Epizootic ofA. vaccinii Caused by H. thompsonii



EPIZOOTIC OF ACALITUS VACCINII (ACARI: ERIOPHYIDEA)
CAUSED BY HIRSUTELLA THOMPSONII ON SOUTHERN HIGHBUSH
BLUEBERRY IN NORTH-CENTRAL FLORIDA

ELKE WEIBELZAHL AND OSCAR E. LIBURD
Entomology and Nematology Department, University of Florida/IFAS, 970 Natural Area Drive,
Gainesville, FL 32611-0620

ABSTRACT

The blueberry bud mite (BBM),Acalitus vaccinii (Keifer), is gaining importance as a pest of
southern highbush blueberries. During a BBM population development study in a north-
central Florida location, an epizootic was observed, and the mesothermic acarine myco-
pathogen, Hirsutella thompsonii (Fisher), was identified as the causal organism. In order to
better understand the progression of an epizootic resulting from H. thompsonii, the area was
extensively sampled from Mar 2007 until Apr 2008. Terminal buds of the following develop-
mental stages were collected, as follows: (1) tightly closed buds, (2) symptomatically swollen
and reddened buds, and (3) separating or opened buds. A red food coloring staining tech-
nique commonly used to stain phyto-parasitic nematodes in or on roots was used to improve
the visibility of the microscopic mite. Within 1 year, the population declined from 50% of the
flower buds infected (up to 2,000 BBM per bud) to less than 5% of the flower buds infected
(about 20 BBM per bud). During the summer, fall, and winter months, the preferred flower
buds are scarce, causing low numbers of BBM to colonize less favorable leaf buds. At this
time, infection by H. thompsonii remained above 50%. In months with average temperatures
below 25 C (Dec through Mar), the frequency of the disease was reduced to 50% and less, al-
lowing the BBM population to recover slightly.

Key Words:Acalitus vaccinii, blueberry bud mite, epizootic, Hirsutella thompsonii, southern
highbush blueberry

RESUME

El acaro del brote de arandano, (ABA), Acalitus vaccinii (Keifer), esta aumentando en im-
portancia como una plaga del arandano de clase alto sureio. Durante un studio del desa-
rrollo de la poblaci6n de ABA en un sitio en el norte central de la Florida, una epizo6tica fue
observado, y el micopat6geno mesot6rmico de acaros, Hirsutella thompsonii (Fisher), fue
identificado como el organismo causante. Para entender mejor la progresi6n de una epizo6-
tica como resultado de H. thompsonii, la area fue muestreada extensivamente desde marzo
del 2007 hasta abril del 2008. Los brotes terminales de los siguientes estadios de desarrollo
fueron recolectados, de la siguiente manera: (1) brotes fuertemente cerrados, (2) brotes sin-
tomaticamente hinchados y enrojecidos y (3) brotes separandose o abiertos. Una t6cnica de
utilizar colorante rojo para comida que se usa para colorear nematodos fito-parasiticos en o
sobre las races fue usada para mejorar la visibilidad del acaro microsc6pico. Dentro de 1
aio, la poblaci6n disminuyo desde 50% de los brotes de las flores infectados (hasta 2000 ABA
por brote) a menos del 5% de los brotes de flores infectados (aproximadamente 20 ABA por
brote). Durante el verano, el otoio y meses de invierno, los brotes de flores preferidos son es-
casos, causando numerous bajos de ABA para colonizar los brotes de hojas menos favorables.
En este tiempo, la infecci6n por H. thompsonii se mantuvo mas de 50%. En los meses con un
promedio de la temperature menor a 25 C (diciembre a marzo), la frecuencia de la enferme-
dad fue reducida un 50% o menos, permitiendo que la poblaci6n de ABA se recuperara.


The blueberry bud mite (BBM),Acalitus vacci-
nii (Keifer), is an important pest of cultivated
blueberries with greatest activity in areas with
mild winter climates (Isaacs et al. 2004). It is ca-
pable of causing severe yield losses through vari-
ous degrees of flower bud damage (Cromroy &
Kuitert 2001), ranging from a reduction in the
number of fruits per cluster to a total desiccation
of developing flower buds (Baker & Neunzig
1970). Persistent feeding by large numbers of
mites results in reddening and swelling of the


base of the bud scales. Buds appear rosetted, are
delayed in their development, and often fail to
open. The symptoms are easily confused with
winter damage. Populations of BBM remain fre-
quently undetected by the growers (Isaacs et al.
2004). From several highbush blueberry groves in
north-central Florida, blueberry bud mites have
been isolated and identified by Dr. Marjorie Hoy.
In this region, a rapid expansion of the blueberry
acreage has occurred for more than a decade (Wil-
liamson & Lyrene 2004). In selected locations, up







Florida Entomologist 92(4)


to 50% of the developing flower buds were symp-
tomatic. Recommendations for control are cur-
rently limited to selective pruning of infected
plants, application of horticultural oil, and post-
harvest application of the organochlorine insecti-
cide, endosulfan (Thiodan) (Krewer et al. 2008).
The chemical cannot be used safely in the vast
majority of cases for which it is currently ap-
proved, and may be banned by 2011 (Anonymous
2009a).
Hirsutella thompsonii (Fisher) is a mesother-
mic mycopathogen of various invertebrates in-
cluding insects, mites, and nematodes (Boucias et
al. 2007). The fungus requires an optimal temper-
ature of 25 to 30C and a RH of 98% for growth
and sporulation (Boucias et al. 2007; Gerson et al.
1979; Kenneth et al. 1979; McCoy 1996). As a par-
asite it has been reported to cause epizootics of
several eriophyoid mites including Calacarus he-
ueae on rubber trees (Tanzini et al. 2000), Phyllo-
coptruta oleivora, citrus rust mite (Fisher et al.
1949), andAceria guerreronis on coconut (Gopal &
Gupta 2001).
In Apr 2007, the presence ofH. thompsonii be-
came apparent during a population study con-


ducted in a commercial southern highbush blue-
berry planting in Windsor, FL (Fig. 1). Hirsutella
thompsonii, has previously been found in associa-
tion with the BBM in North Carolina (Baker &
Neunzig 1968, 1970) but an epizootic has not been
observed in Florida.
The objective of this study was to determine
the seasonal development of a well- established
BBM population and its mycopathogen, H. th-
ompsonii, in a commercial planting of southern
highbush blueberries under north-central Florida
conditions.

MATERIALS AND METHODS

The selected study site was a 1.2-ha commer-
cial southern highbush blueberry (Vaccinium
corymbosum L. X V. darrowi Camp.) planting, in-
fested with high populations of BBM. Bushes
were 1.6-2 m high (approximately 10 years old)
and consisted of a mixed planting of Windsor and
Star cultivars. Blueberry bushes were spaced
about 1 m apart. No insecticide or acaricide was
applied to the field prior to field survey. However,
foliar fungal pathogens were controlled with Ca-


Fig. 1. Scanning electron microscopy images of blueberry bud mites, Acalitus vaccinii (Keifer), healthy (upper
left) and infected by the micopathogen, Hirsutella thompsonii (upper right), isolated from blueberry flower buds of
commercially grown southern highbush blueberry. The fungus forms single conidia (lower left), and may also colo-
nize mite eggs (lower right).


December 2009







Weibelzahl & Liburd: Epizootic ofA. vaccinii Caused by H. thompsonii


brio, Switch, Pristine, Bravo, Indar, and
Elevated. The fungicides were applied according
to the recommended calendar spray technique
(Krewer et al. 2008). With the first appearance of
the fungal infection, distorted BBM were plated
on sterilized water agar, incubated at 27C (Bou-
cias et al. 2007), and sporulating fungal struc-
tures were submitted for expert identification.
The fungus was identified by Dr. Drion Boucias in
the insect pathology laboratory at the University
of Florida.
In order to determine the population dynam-
ics of BBM, 10 terminal flower buds were col-
lected randomly from the cultivar 'Windsor' in
the following developmental stages: (1) tightly
closed buds, (2) symptomatically swollen and
reddened buds, and (3) separating or opened
buds. Buds were collected bi-weekly from two,
10-m sections of the planting from Mar until Jun
2007. After Jun, when BBM populations are
known to decline to low numbers (Barker & Ne-
unzig 1970), samples were collected every 8
weeks from the same two 10-m sections. At each
sampling date, the percent of symptomatic buds
was estimated visually by examining bushes for
at least 1 min, and counting and recording the
number of buds that appeared reddened and ro-
setted, as well as the number of buds with
healthy appearance. The percentages of 10


40

I-
35 -

S30

& 25 A

I 20
m0
15

S10 --

5

0 -
-


^s
^ .


plants were averaged for each section at each
sampling date.
Buds were placed in a cooler, brought to the
Small Fruit and Vegetable IPM Laboratory at the
University of Florida, and stained with red food
coloring (McCormick & Co., Hunt Valley, MD) by a
slightly modified technique commonly used to
stain phyto-parasitic nematodes in or on roots
(Thies et al. 2002). The number of mites per bud
was determined by dissecting the stained buds
and counting individual mites of all life stages
(excluding the eggs) on the inside of the bud
scales with the aid of a dissecting microscope at
X63. Based on their appearance, blueberry bud
mites were differentiated into healthy, oblong to
cigar-shaped individuals of reddish color, and
other distorted and grayish individuals.
A sub-sample of symptomatic buds was pre-
served in 1% glutharaldehyde and prepared for
scanning via electron microscopy (SEM) applying
the critical point drying technique commonly
used for soft bodied specimen. Samples were ob-
served on a Hitachi S-400 FE-SEM (Hitachi High
Tech America). Average relative humidity and
temperature data were recorded by the Florida
Automated Weather Network station in Citra,
FL, with a CSI 107 temperature probe and a CSI
CS215 temperature and humidity probe (Anony-
mous 2009b).


a
ted j-70 I

60

A 50

-40

30 '

. 20

10o


0 5


4', I


Dates


Fig. 2. Mean population density and health of blueberry bud mites (BBM) in tightly closed flower buds of south-
ern highbush blueberry cv. Windsor, Florida.


i 56







Florida Entomologist 92(4)


The total population density was determined
by pooling the data from healthy and distorted
BBM. The population health (healthy versus dis-
torted) was determined by subtracting the num-
ber of distorted BBM from the number of healthy
BBM. We used paired t-tests to compare symp-
tomatic and opened buds for the period of 5 Mar-
5 May 2007 to determine statistical differences
(SAS Institute 2003).

RESULTS

Blueberry bud mites and its fungal parasite,
Hirsutella thompsonii, were present in all 3 devel-
opmental stages of the blueberry buds (Figs. 1-4).
From the first sampling (5 Mar 2007) to the end of
the observation period (3 Apr 2008), the BBM
population declined drastically. The percentage of
symptomatic buds recorded visually was reduced
from over 50% to less than 5% (data not shown).
Infection by H. thompsonii remained undetected
until mid Apr 2007. By mid May 2007 and there-
after, the infection rate by H. thompsonii ex-
ceeded 50% in months with an average tempera-
ture higher than 25C (Figs. 2-5). Voucher speci-
mens ofAcalitus uaccinii and H. thompsonii were


saved and deposited in the Entomology and Nem-
atology collection at the University of Florida.

Population Density

With a population peaking at about 2,000 BBM
per bud in Mar 2007, the population was signifi-
cantly higher in symptomatic buds than in any
other buds at any time during the spring 2007 ob-
servation period (t value = 8.51; P < 0.0001).
Symptomatic buds opened or desiccated due to
mite feeding by the end of May. In the newly
formed closed buds, BBM numbers remained very
low throughout the late summer, fall, and winter
months only to increase slightly during spring
2008 (Figs. 2-4). Symptomatic buds were difficult
to detect in Apr 2008, and were not present there-
after.

Population Health

In early Mar 2007, the numbers of healthy
BBM in symptomatic buds were significantly
higher than numbers of distorted BBM (t value =
2.81; P < 0.0053). In mid Apr, infection rates in
opened buds increased rapidly to exceed 50% un-


-U- distorted
- -- Total
A % infected A


-

S I
/
i



I
i
- i


- I

* I
i
r (


A



A A

A



'

A


A
/. \



jQA


S100

.90

.80

S70



S500

.40

-30

.20

*10

0


Date
Fig. 3. Mean population density and health of blueberry bud mites (BBM) in opened flower buds of southern
highbush blueberry cv. Windsor, Florida.


100

901

801


70 -


ISO-

50.

40-

I 30.

20
S0-

10

0


---'


Ll LI


December 2009







Weibelzahl & Liburd: Epizootic ofA. vaccinii Caused by H. thompsonii


til the winter months (Fig. 3). In symptomatic
buds the same trend was observed with a two-
week delay (Fig. 4). In all 3 bud types (closed,
opened, and symptomatic), the BBM population
and the mycopathogen were nearly undetectable
during the months of Sep 2007 through Jan 2008.
In 2008, the number of distorted BBM in symp-
tomatic and opened buds started to increase at
the end of Jan to exceed 50% in the beginning of
Apr, and BBM numbers were suppressed to about
20 per bud.

DISCUSSION

During 2007/08 season, favorable temperature
and humidity conditions supported an epizootic of
BBM by H. thompsonii at our study site in north-
central Florida. Although the average daily rela-
tive humidity (RH) did not exceed 75%, overhead
irrigation contributed to the higher humidity
ideal for fungal growth. However, from mid-Nov
through mid-Mar the average temperature
dropped below the optimum of 25C, causing the
frequency of the disease to decrease. During this
time, the BBM population recovered only to be
suppressed to below an average number of less
than 20 BBM per bud in spring of 2008. The lack
of symptomatic buds at this time suggests that


450

400

350

300

1250-
1 260

S200

! 160


It


the BBM population decreased to non-significant
levels.
By the end of May, as opened flower buds des-
iccated due to mite feeding, BBM appeared to
have moved to tightly closed buds, which are
newly formed on early summer growth. This ob-
servation agrees with Baker & Neunzig (1970),
who reported that in North Carolina BBM moved
to secondary growth starting in mid Jun.
Throughout the observation period, the infection
rate of BBM remained high in closed buds. McCoy
& Lye (1995) demonstrated that copper sprays ap-
plied for foliar disease control in citrus reduced
the magnitude of the epizootic ofH. thompsonii on
citrus rust mites, Phyllocoptruta oleivora. It is not
clear whether the frequent application of fungi-
cides had an effect on the population densities of
either organism in blueberries.
The cryptic habits of BBM within the blue-
berry bud make them difficult to control with
miticides, but they can be devastating if allowed
to develop in the field. Our research is in agree-
ment with McCoy (1981), who considered H. th-
ompsonii a key biological control agent regulating
mites. The fungus can play an important role in
regulating populations of BBM in southern high-
bush blueberries in Florida. Although the full ex-
tent of the damage caused by BBM and the fre-


A distorted
- Total
A % infected


A A


A




-. -. .9


100

-90

- 80
.6O



so





.30

-O20

- 10

- 0


Fig. 4. Mean population density and health of blueberry bud mites (BBM) in symptomatic flower buds of south-
ern highbush blueberry cv. Windsor, Florida.


,m u


.


*


m


~

r.


100

0 I







Florida Entomologist 92(4)


- RH in %

- Averagetemp in C


- --Lowtemp. in C

-----High temp. in C


70

80

I 5080

40



20 wo

10







Dare

Fig. 5. Climatic data for Windsor, Florida.


quency of epizootics are still unknown, BBM man-
agement programs should consider possible ef-
fects on H. thompsonii when selecting fungicides
for foliar disease control. With an increasing num-
ber of Florida blueberry farms installing mtcro-jet
irrigation systems to improve quality and quan-
tity of the fruit (Williamson, pers. comm.), it re-
mains to be seen if changing cultural practices
will affect the frequency and population density of
BBM.

ACKNOWLEDGMENTS
We thank the Florida Blueberry Growers Associa-
tion for the financial support, Alto Straughn and his
team for access to the blueberry farm and for technical
assistance, and Dr. Drion Boucias and Marjorie Hoy for
guidance in working with the fungus and mite. The au-
thors acknowledge the help of the staff of the Small
Fruit and Vegetable IPM lab.

REFERENCES CITED
ANONYMOUS 2009a. http://www.panna.org/campaigns/
endosulfan
ANONYMOUS 2009b. http://www.wunderground.com/
weatherstation/WXDailyHistory. asp?ID=MAP573
BAKER, J. R., AND NEUNZIG H. H. 1970. Biology of blue-
berry bud mite. J. Econ. Entomol. 63: 74-79.
BAKER, J. R., AND NEUNZIG, H. H. 1968. Hirsutella th-
ompsonii as a fungal parasite of the blueberry bud
mite. J. Econ. Entomol. 61: 1117-1118.


BOUCIAS, D. G., MEYER, J. M., POPOONSAK, S., AND
BREAUX, E. 2007. The genus Hirsutella: A polyphyl-
etic group of fungal pathogens infecting mites and
insects, pp. 1-34 In S. Ekesi and N. K. Maniania
[eds.], Use of Entomopathogenic Fungi in Biological
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phia, PA.
CROMROY, H. L., AND KUITERT, L. C. 2001. Blueberry
bud mite,Acalitus vaccinii (Keifer) Arachnida: Acari
Eriophyidae. EDIS-Publication #EENY-186. Uni-
versity of Florida Gainesville, Florida.
FISHER, J. T., GRIFFITHS, J. R., AND. THOMPSON, W. L.
1949. An epizootic ofPhyllocoptruta oleivora (Ashm.)
on citrus in Florida. Phytopath. 39: 510-512.
GERSON, U, KENNETH, R., AND MUTTATH, T. I. 1979. Hir-
sutella thompsonii, a fungal pathogen of mites. II.
Host-pathogen interactions. Ann. Appl. Biol. 91: 29-
40.
GOPAL, M., AND GUPTA, A. 2001. Has Hirsutella thomp-
sonii the wherewithal to counter coconut eriophyid
mite scourge? Current Science, 80: 831-836.
ISAACS, R., MORRONE, V., AND GAJEK, D. 2004. Potential
acaricides for management of blueberry bud mite in
Michigan blueberries. Hort. Technology 14: 18-21.
KENNETH, R., MUTTATH, T. I., AND GERSON, U. 1979.
Hirsutella thompsonii, a fungal pathogen of mites. I.
Biology of the fungus in vitro. Ann. Appl. Biol. 91: 21-
28.
KREWER, G., BRANNEN, P., CLINE, B., HALE, F., AND
HORTON, D. 2008. South Eastern Regional Blueber-
ry Integrated Management Guide. http://
www.smallfruits.org/SmallFruitsRegGuide/Guides/
2008/BlueberrySprayGuide2 1008.pdf


100

90

80


*1


- 100

- 90

-8 60













-O10
40







S0


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McCoY, C. W. 1996. Pathogens of eriophyoid mites, pp.
481-490 In E. E. Lindquist, M. W. Sabelis, and J.
Bruin [eds.], Eriophyoid Mites: Their Biology, Natu-
ral Enemies and Control. Elsevier, Amsterdam, The
Netherlands.
McCoY, C. W. 1981. Pest control by the fungus Hirsutel-
la thompsonii. pp. 499-513 In H. D. Burges [ed.], Mi-
crobial Control of Insects and Mites. Vol. 2.Academic
Press, London, England.
McCoY, C. W., AND LYE, B. H. 1995. Effect of copper
sprays on the population dynamics of the citrus rust
mite, Phyllocoptruta oleivora (Acari: Eriophyidae)
and its fungal pathogen, Hirsutella thompsonii.
Proc. Florida State Hort. Soc. 108: 126-129.
SAS INSTITUTE 2003. The SAS System 9.1 for Windows.
SAS Institute, Cary, NC.


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J., AND FERLA, N. J. 2000. An epizootic of Calacerus
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ompsonii on rubber trees. Exp. Appl. Acarol. 24: 141-
144.
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Red food coloring stain: New, safer procedures for
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Nematol. 34: 179-181.
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commercial blueberry industry. #HS742 EDIS -
Publication University of Florida Gainesville,
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Florida Entomologist 92(4)


December 2009


DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) INFECTION AND
DISSEMINATION OF THE ENTOMOPATHOGENIC FUNGUS ISARIA
FUMOSOROSEA (HYPOCREALES: CORDYCIPITACEAE) UNDER
LABORATORY CONDITIONS

PASCO B. AVERY1, WAYNE B. HUNTER2, DAVID G. HALL2, MARK A. JACKSON3, CHARLES A. POWELL'
AND MICHAEL E. ROGERS4
'University of Florida, Institute of Food and Agricultural Sciences, Indian River Research and Education Center,
2199 South Rock Road, Fort Pierce, FL 34945, USA

2USDA, ARS, U.S. Horticultural Research Laboratory, Subtropical Insect Research Unit,
2001 South Rock Road, Ft. Pierce, FL 34945, USA

3USDA, ARS, National Center for Agricultural Utilization Research, Crop Bioprotection Research Unit,
1815 North University Street, Peoria, IL 61604, USA

4University of Florida, Citrus Research and Education Center, 700 Experimental Station Road,
Lake Alfred, FL 33850, USA

ABSTRACT
The infectivity and horizontal transfer of Isaria fumosorosea Wize among Diaphorina citri Ku-
wayama was measured using a detached leaf bioassay in which blastospores were sprayed on
citrus leaf sections or yellow plastic tags (artificial attractant surface). Four leaf sections or three
leaf sections and one yellow tag were placed together in a Petri dish chamber. One to four of the
leaf sections or the yellow tag was sprayed with I. fumosorosea (1.2 1.7 x 103 blastospores/mm2).
After treatments dried, a single adult psyllid was released into each chamber. Mortality due to
I. fumosorosea for the adult psyllid was observed 4.9 0.21- 6.1 0.37 d following exposure to
the pathogen. The rate of colonization by I. fumosorosea on adults in chambers with untreated
leaf sections and one treated yellow tag was as effective in inducing mortality as in chambers
with one treated leaf section at 8 days post application. Under high humidity, I. fumosorosea
blastospores readily produced hyphae on the surface of leaves, which was useful for determining
if adults were responsible for transmission of the fungus. In chambers with a single treated leaf
section, adults came into contact with blastospores and moved these around to the non-treated
leaves. The same phenomenon, of psyllid infection and subsequent spreading of the fungus to
non-treated leaves, was observed when psyllids were placed into chambers with a treated yellow
tag. The use of I. fumosorosea inoculated yellow tags has potential as a psyllid dissemination
technique for managing pest populations.
Key Words: autodissemination; blastospores; Diaphorina citri; fungal development index;
Isaria fumosorosea; Huanglongbing

RESUME
La infectividad y transferencia horizontal de Isaria fumosorosea Wize entire Diaphorina citri
Kuwayama fue media usando un bioensayo de una hoja despegada en el cual blastosporas
fueron rociadas sobre secciones de hojas de citricos o sobre etiquetas plasticas de color ama-
rillo (superficie de atrayente artificial). Cuatro secciones de hojas o tres secciones de hojas y
una etiqueta amarilla fueron puestas juntas en una camara de plato Petri. Una de las cuatro
secciones de hojas o la etiqueta amarilla fueron rociados con I. fumosorosea (1.2 1.7 x 103
blastosporas/mm2). Despu6s de que se seco los tratamientos, un solo adulto de psilido fue li-
berado dentro de cada camara. La mortalidad debido a I. fumosorosea para el adulto del psi-
lido fue 4.9 0.21- 6.1 0.37 dias despu6s de exponerlo al pat6geno. La tasa de colonizaci6n
de I. fumosorosea sobre los adults en las cameras con secciones de hojas no tratadas y con
una etiqueta amarilla tratada fue tan efectiva en inducir mortalidad como en las camaras
con una secci6n de hoja tratada a los 8 dias despu6s de la aplicaci6n. Bajo condiciones de alta
humedad, las blastosporas de I.fumosorosea facilmente producieron hifae sobre la superficie
de las hojas, que fue util para determinar si los adults psilidos fueron responsables para la
transmisi6n del hongo. En las camaras con una sola secci6n de una hoja tratada, los adults
se pusieron en contact con las blastosporas y los removieron a las hojas no tratadas. El
mismo fenomeno, de la infecci6n del psilido y el espacir el hongo a hojas no tratadas fue ob-
servado cuando los psilidos fueron puestos en camaras con una etiqueta amarilla tratada. El
uso de etiquetas amarillas inoculadas con I. fumosorosea tiene un potential como una t6c-
nica para diseminar los psilidos para el manejo de poblaciones plagas.







Avery et al.: Infectivity and Dissemination of I. fumosorosea


The Asian citrus psyllid, Diaphorina citri Ku-
wayama (Hemiptera: Psyllidae), was first discov-
ered in Florida in 1998 and has since dispersed
rapidly throughout the state (Halbert & Manju-
nath 2004). The insect has a narrow host range
consisting of plants in the family Rutaceae, in-
cluding citrus and citrus relatives such as orange
jasmine, Murraya paniculata (L.) Jack (Tsai et al.
2000). Diaphornia citri is a vector of the phloem-
limited bacterium Candidatus Liberibacter asiat-
icus, which is always associated with citrus- hua-
nglongbing (HLB), commonly referred to as 'cit-
rus greening disease' (Hung et al. 2004; Manju-
nath et al. 2007). HLB is one of the most serious
plant diseases in citrus on a worldwide scale
(Bove 2006) and has been reported in Florida
(Tsai et al. 2000; FDACS 2009).
Direct feeding by D. citri nymphs is primarily
on new citrus growth or "flush" (Hall & Albrigo
2007) which can result in distorted, reduced
growth of new leaf tissue. Probing by the adult
psyllid while searching for the best feeding area
on a leaf can transmit HLB. Infected citrus trees
may only live 5-8 years and produce irregular
shaped, bitter, unmarketable fruit (Halbert &
Manjunath 2004; Bove 2006). Considering the se-
riousness of the disease and its -vector, control-
ling psyllid populations by the use of chemical in-
secticides, removing confirmed diseased trees and
planting disease-free nursery stock are recom-
mended as management strategies for this patho-
system (Childers & Rogers 2005; Brlansky et al.
2006; Rogers et al. 2006). The present paradigm of
an intensive insecticidal control program is eco-
nomically unsustainable for the grower and will
likely interfere with biological control programs
in Florida citrus (Michaud & Grant 2003;
Michaud & Olsen 2004; Hoy 2005; Stansly &
Qureshi 2008). Thus, an integrated pest manage-
ment (IPM) strategy is needed to minimize the
use of chemical insecticides and to develop sus-
tainable alternatives for managing psyllid popu-
lations.
The entomopathogenic fungus, Isaria fumoso-
rosea (Ifr) Wize (= Paecilomyces fumosoroseus)
(Hypocreales: Cordycipitaceae), was recently iso-
lated from mycosed D. citri collected from the un-
derside of foliage on orange trees in Polk County,
Florida (28o06'295" N, 8142'895"W) (Meyer et al.
2008). Presently, 2 Ifr strains are available for re-
search as blastospore formulations in the U.S.A.,
PFR 97 20% WDG@ (Certis, Columbia, MD, USA)
and Ifr 3581 from the USDA/ARS, NCAUR, Peo-
ria, IL, USA (Jackson et al. 1997). Ifr has several
characteristics that favor its further evaluation
for controlling D. citri; it is native to Florida, can
infect a wide range of citrus pests, and is compat-
ible with non-target arthropods (Sterk et al.
1995a, b; Avery 2002; Avery et al. 2008).
Growing concerns about the negative effects of
chemical insecticides on workers, food supply, and


the environment make microbial control of ar-
thropod pests of tree fruit crops an attractive al-
ternative (Puterka 1999; Subandiyah et al. 2000;
Slininger et al. 2003; Dolinski & Lacey 2007;
Lacey & Shapiro-Ilan 2008). The most common
fungal pathogen application technique, spraying
trees with conidial suspensions, can become cost
prohibitive for multiple treatments of groves.
Therefore, development of a low-cost autodissem-
ination technique for entomopathogenic fungi
where the insect can spread the fungus via hori-
zontal transmission to conspecifics (e.g., during
mating) is warranted. Similar autodissemination
techniques for controlling pests have been evalu-
ated in other systems (Maniania 2002; Dowd &
Vega 2003; Tsutsumi et al. 2003; Scholte et al.
2004; Maniania et al. 2006).
Adult psyllids are attracted to yellow sticky
cards in the field (Hall & Albrigo 2007; Hall et
al. 2007, 2008; Hall 2009); therefore, it was hy-
pothesized that yellow tags (non-sticky artificial
attractant) sprayed with Ifr blastospores could
potentially be used to horizontally spread the
fungus by acquisition and dissemination to
other leaves and psyllids in the field. The objec-
tives were (1) to compare the efficacy of yellow
tags and leaves sprayed with Ifr blastospores for
infecting and colonizing the psyllid and (2) to as-
sess the horizontal transfer of blastospores by
the movement of the adult psyllids under labo-
ratory conditions. A Fungal Development Index
(FDI), similar to that of Avery et al. (2004), was
designed to assess the effect and development of
Ifr dosages on the post-lethal period of infected
adult psyllids.

MATERIALS AND METHODS

Source of Insects

The USDA-ARS laboratory colony of D. citri
was established during early 2000 at the U.S.
Horticultural Research Laboratory, Fort Pierce,
FL. Originally collected from citrus, the psyllids
have been continuously reared on orange jasmine,
Murraya paniculata (L.) Jack housed in Plexiglas
(0.6 x 0.6 x 0.6 m) or BugDorm-2 cages (MegaView
Science Education Services Co., Ltd., Taichung,
Taiwan). Original colony has not had field col-
lected psyllids added since establishment.

Citrus Leaves

Duncan grapefruit (Citrus paradise Macf)
seedlings were grown in Premier Pro-mix General
Purpose Growing Medium from seed in size C10
"Cone-tainers"TM (Stuewe & Sons, Inc., Corvalis,
OR) for approximately 6 months. Detached leaves
of similar age and size were washed with water
and placed in a fume hood to air dry.







Florida Entomologist 92(4)


Fungal Blastospore Preparation, Deposition, and Viability

A fungal, dessication-tolerant, blastospore-di-
atomaceous earth formulation of Ifr ARSEF
strain 3581, supplied as a powder in vacuum
packed 10-g bags was produced and stabilized as
previously described (Jackson et al. 2003) and
stored at 4C. The blastospore suspension was
prepared by mixing 2 g of the powder in 100 mL of
sterile distilled water, stirring the suspension
with a magnetic bar for 30 min and then allowing
the diatomaceous earth to settle from the suspen-
sion for an additional 30 min. The suspension (50
mL) was then pipetted to a Nalgene aerosol
sprayer (Nalge Nunc International, Rochester,
NY). Two aliquots were taken prior to spraying
from the suspension and the concentration of Ifr
blastospores/mL was determined with a hemacy-
tometer.
To determine the deposition oflfr blastospores/
mm2, 12 plastic microscope cover slips (Fisher-
brand@ 22 x 22 mm, Fisher Scientific, Pittsburgh,
PA) were placed randomly on paper among the
leaf sections and yellow plastic tags and sprayed
simultaneously and in an identical fashion. The
cover slips were allowed to dry for 30 min in a
fume hood, then placed upside down on a glass
microscope slide in a 50-pL drop of acid fuschin
stain. Blastospore density was assessed with a
compound light microscope (400X) and a 10 mm
reticle grid (Hunt Optic and Imaging, Pittsburg,
PA).
The viability of blastospores were assessed
with 2 potato dextrose agar plates sprayed at a
rate of 6.0 x 10 blastospores/mL. After the plates
had been incubated for 12 h at 25 1.0C, 100%
RH, the percent viability was determined by
viewing a total of 200 blastospores. Blastospores
were considered to have germinated if a germ
tube had formed. This procedure was repeated for
each repetition of the experiment, and the mean
percent viability was 85 8.3%.

Bioassay Petri Dish Chambers

Petri dishes (100 mm x 15 mm) were lined with
filter paper and moistened with 800 pL sterile dis-
tilled water. To prepare the leaf sections, each leaf
was cut 2.5 cm from the tip across the midrib. The
adaxial side of leaves of similar size and top side
of yellow plastic tags (Xpress Tags, Brooklyn,
NY), were cut to mimic the shape and surface
area (range: 101-125 mm2) of the leaf section.
These sections were sprayed until runoff with a
Nalgene aerosol sprayer held at approximately
a 450 angle. The spray was either sterile distilled
water (DW) or an Ifr blastospore suspension (6.0 x
107 blastospores/mL) in sterile water. Sprayed
leaves and yellow tags were air dried for 30 min.
Bioassay leaf section treatments inside the
Petri dish consisted of 4 sections total, with 1, 2, 3


or 4 leaf sections) sprayed with Ifr. The yellow tag
treatments consisted of 3 leaf sections (sprayed
with water) and 1 yellow tag (sprayed with either
Ifr or water) placed on moistened filter paper.
Leaf section treatment combinations were ar-
ranged in the following ratios of fungus (blas-
tospores) to distilled water (Ifr to DW): 0:4 (con-
trol), 1:3, 2:2, 3:1, 4:0, and yellow tag treatment
combinations 1:3 and 0:4 (control). Treatment
combinations were oriented in a cross pattern
with the leaf section or yellow tag tip pointed to-
ward the center of the dish prior to introducing an
adult psyllid inside the Petri dish.
A single (<1 week old) adult psyllid (sex not
identified) was allowed to walk on the inside of
the Petri dish lid. The lid was then turned over,
placed over the bottom of the dish and the adult
psyllid was allowed free movement. Each dish
chamber was sealed with Parafilm@ and trans-
ferred to a Precision 818 low temperature fluo-
rescent illuminated incubator (Precision, Win-
chester, VA, USA). All treatments were main-
tained at 25 1.0C under a photoperiod of 16:8
(L:D) at approximately 100% RH for 14 d and ob-
served on a daily basis. There were 8 replicate
dish chambers for each treatment and the experi-
ment was repeated 4 times.

Determining Ifr Acquisition and Horizontal Transfer by
the Psyllid

Leaf sections and yellow tags were treated and
arranged inside the dish chambers as described
above for all treatments. Two groups of treatments
(8 replicates/treatment) were compared, one with
the psyllid present, the other without the psyllid
present. The group without the presence of an
adult psyllid served as a control for assessing
spread of the blastospores within dish chambers in
the absence of a psyllid. Leaf section treatment
combinations were arranged in the following ratios
of fungus (blastospores) to distilled water (Ifr to
DW): 0:4 and 1:3. The yellow tag treatments were
conducted as previously described.
After a pilot study, fungal hyphae from spores
transferred by the psyllid were first observed to
grow on the leaf surface under high relative hu-
midity conditions (Avery, unpublished data; Fig.
1). Therefore, this new finding was used to evalu-
ate the transfer of fungal spores among leaves in
the dish chamber. Untreated leaf sections inside
the dish chambers were monitored for the pres-
ence of Ifr hyphae growing on the whole leaf with
a dissecting binocular microscope (40X). Data ob-
tained from replicated experiments after 14 d
were used as criteria for determining acquisition
and horizontal transfer by the adult psyllid. In
cases where the insect died and mycosed on an
untreated leaf section, the leaf was recorded as
contaminated by Ifr and horizontally transferred
by the adult psyllid.


December 2009







Avery et al.: Infectivity and Dissemination of I. fumosorosea


Fungal Development Index (FDI) Assessment

The degree of fungal development of Ifr on
psyllid adults was assessed by a Fungal Develop-
ment Index (FDI; see Table 3 for summary) modi-
fied from Avery et al. (2004). The FDI was used as
a measure for estimating establishment speed or
infection rate of hosts in each treatment. All as-
says were rated daily until sporulation of Ifr was
observed (FDI value 3.0) on the insect host. Each
adult was assessed under a dissecting binocular
microscope (40X), and the FDI value for the stage
of fungal development observed was recorded.
The FDI was used to assess the fungal growth of
blastospores after infection of the adults until col-
onization at 25 1.0oC and 100% RH.
The FDI values of 0.0-0.5, which represented
the beginning of the growth phase and initial ger-
mination of the blastospore, were not assessed.
An FDI value of 1.0 was assumed once the insect
died; however, this value was not recorded until
confirmation of Ifr fungal hyphae was first no-
ticed extending from any part of the body or
wings. Once the fungus protruded through the ex-
oskeleton of the host insect (FDI values 1.5-2.0),
the insect would not recover from the infection.
Conidiogenesis was represented by FDI values
2.5-3.0. Each adult was scored for 8 d according to
the FDI as a replicate and results were expressed
as a daily mean value for all adult psyllids in each
treatment.

Statistical Analysis

The mean number of days of adult psyllids sur-
vival post Ifr leaf section treatment compared
with a yellow tag treatment were assessed by
ANOVA (a = 0.05) with mean separation by a
Tukey's HSD test. In order to determine the per-
cent transfer of Ifr blastospores to untreated leaf
sections by adult psyllid movement compared to
no psyllid present, data were arcsine-transformed
and analyzed by ANOVA (a = 0.05) with mean
separation by a Tukey's HSD test. A Ryan-Einot-
Gabriel-Welsh Multiple Range Test was used to
analyze the effect of increasing the number of
treated leaf sections on the development of Ifr on
the adult psyllid (after initial mycosis until colo-
nization; FDI value 3.0) and between the single
treated leaf section compared to the yellow tag
treatment using the FDI values. A regression
analysis was used to determine if the infection
rate of 1 treated yellow tag was as high or higher
compared to a treated single leaf section against
the psyllid over time. If results and trends per
treatment were not significantly different be-
tween repetitions of the experiment based on an
ANOVA (a = 0.05), then the data were pooled and
analyzed. All statistical tests were conducted by
PROC GLM procedures of SAS (SAS Institute,
Cary, NC, USA).


RESULTS


Efficacy of Treatments

All Ifr treatments were effective in inducing
mortality in adult psyllids under the laboratory
conditions tested. No significant differences in
treatment results were observed (F = 0.01; df= 3,
15; P = 0.100) between repetitions of the experi-
ment; therefore, the data over all repetitions were
pooled and analyzed. The mean number of Ifr vi-
able blastospores/mm2 deposited on the leaf sec-
tions was 1,344 + 149.7.
The number of days for the fungus to infect
and induce mortality in an adult psyllid ranged
from 4.9 to 6.1, and no mortality was observed in
the control treatment (Table 1). Mortality rates of
adults in chambers with an Ifr- treated yellow tag
were not significantly different (P > 0.05) than
mortality rates of adults in chambers with Ifr-
treated leaves. The number of days adult psyllids
survived in chambers with 3 Ifr- treated leaf sec-
tions was significantly shorter (F = 5.60; df = 4,
155; P < 0.001) compared to those treatments
with fewer leaf sections treated. The days the
psyllid survived in treatments with 3 or 4 leaf sec-
tions sprayed were similar (P > 0.05), 4.9 0.21
and 5.2 + 0.30, respectively.

Ifr Acquisition and Horizontal Transfer by the Psyllid

The acquisition and percent horizontal trans-
fer of blastospores to untreated leaf surfaces
(edge or center) is presented in Table 2. Psyllid
movement within chambers did not affect the per-
cent horizontal transfer of the blastospores to the
edge of the untreated leaf sections in either the
leaf section or yellow tag treatments (F = 1.12; df
= 2, 95; P = 0.348). However, in both the leaf sec-
tion and yellow tag treatments, the presence and


TABLE 1. MEAN TIME TO DEATH IN DAYS ( SEM) FOR
ADULT PSYLLIDS AFTER RELEASE INTO PETRI
DISH CHAMBERSa CONTAINING CITRUS LEAF
SECTION (S) OR YELLOW TAGS SPRAYED WITH
ISARIA FUMOSOROSEA (IFR).

Treatment Time to death (days)

1 Yellow tag sprayed 5.7 0.23 ab
1 Leaf section sprayed 6.1 0.37 b
2 Leaf sections sprayed 5.9 0.30 b
3 Leaf sections sprayed 4.9 0.21 a
4 Leaf sections sprayed 5.2 0.30 ab

*Total number of leaf sections per Petri dish chamber was 4.
The leaf sections and yellow tags were sprayed with blas-
tospores of Ifr and allowed to dry before introducing a psyllid.
The yellow tag replaced 1 leaf section. No mortality was ob-
served for the untreated controls (n = 32/treatment).
'Mean survival values followed by different letters in a col-
umn are significantly different (Tukey's HSD test, P < 0.001).








Florida Entomologist 92(4)


TABLE 2. PERCENT HORIZONTAL TRANSFER ( SEM) OF BLASTOSPORES OF ISARIA FUMOSOROSEA (IFR) FROM TREATED
TO THE UNTREATED LEAF SECTION EDGE OR CENTER BY ADULT PSYLLID MOVEMENT IN PETRI DISH CHAM-
BERS' HELD AT 25C UNDER A 16-H PHOTOPHASE AFTER 14 D.

% Horizontal transfer SEMb

Treatment Psyllid Leaf edge Leaf center

1 Leaf section sprayed Absent 81.9 + 6.4 a' 23.5 5.8 a
1 Leaf section sprayed Present 90.3 4.3 a 84.8 4.9 b
1 Yellow tag sprayed Absent 73.6 7.0 a 13.8 4.0 a
1 Yellow tag sprayed Present 90.4 3.1 a 90.3 3.7 b

Total number of leaf sections per Petri dish chamber was 4. The leaf sections and yellow tags were sprayed with blastospores of
Ifr and allowed to dry before introducing a psyllid. The yellow tag replaced 1 leaf section. No mortality for the control was observed
(n = 24/treatment).
bMean percent horizontal transfer values were arcsine transformed before being analyzed. Untransformed values followed by
different letters in a column are significantly different (Tukey's HSD test, P < 0.001).
'Mechanical transfer of blastospores from a treated leaf section or yellow tag to an untreated leaf edge occurred in all treatments.


movement of psyllids enhanced and had a signifi-
cant positive effect on the acquisition and spread
of the fungus to the central part of untreated leaf
sections (F = 6.67; df = 2, 95; P < 0.001).

FDI Assessment of Ifr

Adult psyllids began succumbing to the fungus
2 d post release in all Ifr treatments. A 100-per-
cent mortality of the adult psyllids occurred (FDI
value 1.0) and all psyllids in treatments with 3-4
leaves sprayed had mycosed (FDI value 1.5) 5 d
post release (Table 3). The fungi on the leaf sec-
tion effectively infected and colonized the adult
psyllid, as compared with the controls for the du-
ration of the experiment under these growing con-
ditions. The yellow tag treatment had a similar ef-
fect on the Ifr development as compared to the
single leaf section treatment. Ifr developed on the
adult psyllids exposed to the tag treatment at a
similar rate compared with the sprayed leaf sec-
tion treatments, except 5 d post application where
3-4 leaf section treatments showed a higher rate
(F = 1.92; df = 3, 127; P = 0.0009) compared with
the 1-2 leaf section treatments. The total percent-
age psyllid adults colonized (FDI value of 3.0: cov-
ered with mycelium and conidia) for all experi-
ments after 8 d post release was 63 8.7, 55 + 9.1,
77 + 7.6, 75 7.8% for 1, 2, 3, 4 leaf sections)
treated and 73 8.2% for the yellow tag treat-
ments. The final percent mortality was 91 5.2,
97 + 3.2, 97 + 3.2, 97 + 3.1% and 100 + 0.0%, re-
spectively. Regression analyses between FDI
value (Y) and days of exposure to Ifr treatment (X)
were similar between the yellow tag treatment (Y
= -0.9 + 0.44X; F = 347.0, Pr > F = < 0.0001, r2 =
0.59, slope SEM = 0.023, 239 df) and single leaf
section treatment (Y = -0.8 + 0.39X; F = 276.2, Pr
> F = < 0.0001, r2 = 0.52, slope SEM = 0.023, 239
df). These analyses indicated that Ifr blastospores
sprayed on either a leaf or card, infected and de-
veloped on the adult psyllid at a similar rate over


time. No natural mortality of the adult psyllids
(controls) occurred until 8 d post release for either
the 4 leaf sections (3.2 3.2%) or 1 yellow tag plus
3 leaf sections (25.8 8.0%) treated with water.

DISCUSSION

Assessment of Ifr Treatments

All Ifr-sprayed leaf section treatments resulted
in a mortality of >95% of the adult psyllids under
laboratory conditions after 8 d with 100% mortal-
ity on the yellow tag treatments during the same
period. In addition, fungal development of Ifr on
psyllids in the yellow tag treatment was similar to
the single leaf section treatment, and comparable
to the other leaf section treatments. This indi-
cates that psyllids were attracted to the artificial
yellow tag and then able to acquire and dissemi-
nate the blastospores to the surface of other un-
treated leaves. Some of the untreated leaf section
edges may have become contaminated with the
blastospores by mechanical transfer while in the
Petri dishes.
Under these optimum growing conditions in
the dish chambers, fungal hyphae were observed
to grow on both the leaf (edge and center) and
plastic tag surface. Lopez-Llorca et al. (1999) ob-
served that I. farinosa first grew on the edges of
the leaves and then colonized the palm leaf sur-
face. This is the first report of Ifr producing hy-
phal extensions on either a leaf or an artificial
surface (yellow tag) directly from Ifr blastospores
(Fig. 1).
Moribund psyllids that were attached by myce-
lium to the filter paper, Petri dish or leaf section
had succumbed to the fungal infection after they
had walked around and contaminated the un-
treated leaf surfaces. Under high humidity (RH >
80%) some insects would mycose and form a
sporulating cadaver cemented in a feeding posi-
tion to any surface by hyphae growing from their


December 2009





















TABLE 3. FUNGAL DEVELOPMENT INDEX (FDI) VALUES OF MYCOSIS OBSERVED DAILY ON ADULT PSYLLIDS INFECTED WITH ISARIA FUMOSOROSEA (IFR) AFTER EXPOSURE TO
SPRAYED CITRUS LEAF SECTIONS) OR A YELLOW TAG IN PETRI DISH CHAMBERS' HELD AT 25C UNDER A 16-H PHOTOPHASE.

FDI valuesc days post release

Treatment 2 3 4 5 6 7 8

1 Yellow tag sprayed 0.1 + 0.01 a 0.1 + 0.07 a 0.3 0.14 ab 1.0 0.23 b 1.7 0.23 ab 2.4 + 0.15 a 2.8 0.08 a
1 Leaf section sprayed 0.1 + 0.07 a 0.1 + 0.07 a 0.3 + 0.11 ab 1.0 + 0.21 b 1.6 + 0.20 b 2.2 0.19 a 2.4 0.17 a
2 Leaf sections sprayed 0.1 + 0.01 a 0.1 + 0.08 a 0.4 0.14 ab 1.1 + 0.21 b 1.7 + 0.21 ab 2.0 + 0.21 a 2.5 0.13 a
3 Leaf sections sprayed 0.1 + 0.03 a 0.1 + 0.04 a 0.6 + 0.13 a 1.7 + 0.19 a 2.2 + 0.16 a 2.5 + 0.17 a 2.7 + 0.12 a
4 Leaf sections sprayed 0.0 0.00 a 0.1 + 0.08 a 0.6 + 0.15 a 1.7 + 0.19 a 2.2 + 0.18 a 2.5 + 0.16 a 2.7 + 0.11 a
4 Leaf sections untreated 0.0 + 0.00 a 0.0 + 0.00 a 0.0 + 0.00 b 0.0 + 0.00 c 0.0 + 0.00 c 0.0 + 0.00 b 0.1 + 0.05 bd
1 Yellow tag untreated 0.0 0.00 a 0.0 0.00 a 0.0 0.00 b 0.0 0.00 c 0.0 0.00 c 0.0 0.00 b 0.1 + 0.03 bd

*Total number of leaf sections per Petri dish chamber was 4. The leaf sections and yellow tags were sprayed with blastospores ofIfr and allowed to dry before introducing a psyllid. The yellow
tag replaced 1 leaf section (n = 32/treatment).
FDI values are as follows: 1.0 = insect is dead (whether infected with Ifr fungus or due to natural mortality); 1.5 = appearance of Ifr fungal hyphae protruded through the exoskeleton of
the psyllid body; 2.0 = Ifr fungal hyphae protruded through head, thorax, wings of the psyllid body; 2.5 = Ifr fungal hyphae protruded through the exoskeleton as in 2.0, plus conidia are first
formed anywhere on the psyllid body; 3.0 = Ifr fungus has colonized and formed conidia on all sections of the psyllid body
'FDI values in a column followed by the same letter are not significantly different (REGW multiple range test, P < 0.001).
Natural mortality (FDI = 1.0) 8 d post release for leaf section and yellow tag treatments were 3.2 + 3.2% and 25.8 + 8.0%. No mortality was observed due to Ifr infection.








Florida Entomologist 92(4)


Fig. 1. Isaria fumosorosea hyphal growth on a citrus leaf surface exposed to high humidity conditions (40X).


tarsi. Similarly, Meyer et al. (2008) observed that
moribund adult psyllids were lightly fastened to
the leaf or to the side of a centrifuge tube by white
mycelium of IfrAsCP emerging from the tarsi. In
addition, Ifr hyphae were observed to spread out-
wards from the cadavers and contaminate the
surrounding leaf surface. On plant leaves, Ifr has
been observed to colonize several millimeters
across the leaf surface and infect aleyrodids
(Wraight et al. 1998). Avery (2002) noted that the
Ifr hyphae grew 21 mm across a simulated leaf
surface to colonize other susceptible greenhouse
whitefly pharate adults.

FDI Assessment

In all treatments, 83% of the adult psyllids
were colonized and sporulating (FDI value: 2.5-
3.0) by d 8. Infected insects (FDI value: 1.5) were
alive and had fungal hyphae protruding from
their leg joints immediately prior to mortality,
similar to effects observed by Meyer et al. (2008).
After psyllids succumbed to fungal infections,
fungal development of Ifr progressed to an FDI
value of 2.0 or higher the following day under con-
tinuous high (RH > 80%) humidity conditions.
Ifr infection rate on the adult psyllids was com-
parable to that recorded for the greenhouse
whitefly maintained under similar laboratory
conditions (Avery et al. 2004). All whitefly
pharate adults were completely colonized (FDI
value of 3.0) in 8 d following topical application
and infection with Ifr blastospores under a 16 hr
photophase and high relative humidity. Simi-


larly, in sprayed leaf section treatments, over
97% of the adult psyllids were colonized in 8 d,
while 100% of the psyllids were colonized in the
yellow tag treatments. Overall, our data sup-
ported that a yellow card impregnated with blas-
tospores is as effective in contaminating and kill-
ing the adult psyllid as spraying several leaf sec-
tions under laboratory conditions. However, the
efficacy of Ifr for managing the psyllids by either
spraying trees or by using yellow cards contami-
nated with fungal spores requires evaluation un-
der field conditions. In addition, evaluation of the
most suitable material for retaining the blas-
tospores on cards in the field also warrants fur-
ther investigation.
In autodissemination strategies, the ability of
insects to acquire and horizontally transfer viable
spores is vital to the effectiveness and ultimate
success of a fungal biocontrol pest management
program (Roy et al. 2001; Dowd & Vega 2003; Tsut-
sumi et al. 2003; Scholte et al. 2004; Maniania et
al. 2006). The increase in the amount of viable in-
oculum on the leaf surface appears to positively
correlate with the rate of acquisition and concomi-
tant increase in mortality of the psyllid. For in-
stance in Table 3 on d 5, as the concentration of Ifr
inoculum increased among leaf sections per dish
chamber from 1 leaf section to 3 leaf sections, the
host infection and fungal development rate also in-
creased 7 times. Bailey et al. (2007) found that
when the Microsphaeropsis ochracea was in-
creased in concentration per leaf surface, the host
infection rate also increased. In contrast, Ugine et
al. (2005) noted an inverse relationship between


December 2009







Avery et al.: Infectivity and Dissemination of I. fumosorosea


acquisition rate conidiaa acquired/total conidia ap-
plied) and residue concentration of Beauueria
bassiana by western flower thrips. This size ratio
concept is very important when designing an auto-
dissemination system with entomopathogenic
fungi and warrants further research.
A high incidence of Ifr hyphae being observed
along the edges of some non-treated leaves with-
out psyllids present during the experiments was
noted (Table 2), which could be attributed at least
partially to the edges of these non-treated leaves
coming into accidental contact with the edge of a
treated leaf. This scenario could be avoided in fu-
ture studies by fixing the leaf sections to the filter
paper. However, the significant increases in Ifr
hyphae growing within the center of leaves and
yellow tags was attributed to active dissemina-
tion by the adult psyllids. Regardless of whether
the sprayed surface was an authentic leaf or an
artificial attractant tag, psyllid movement caused
significant contamination of unsprayed leaf sec-
tion centers. Meyling et al. (2006) found that in-
sects living in nettle plants could help spread and
disperse B. bassiana from one site to another. In
the field, the transfer of Ifr to leaves or flush
where psyllids congregate could potentially lead
to secondary infection. In a preliminary bottle
cage experiment, it was observed that an entire
psyllid population living on a citrus seedling be-
came infected after several days exposure to a yel-
low tag sprayed with Ifr blastospores (Avery, un-
published data). Also, in a pilot field trial (Avery
et al. 2009), 33-50% of psyllid eggs and 29-50%
nymphs on citrus flush were found infected with
Ifr 10-21 d post-spray, respectively. In addition,
100% (3/3) of the adult psyllids caught per yellow
card were contaminated and infected with Ifr 28 d
post-spray (Avery et al. 2009).
The autodissemination system using a yellow
tag contaminated with Ifr blastospores has poten-
tial; however, there are many parameters that
need to be investigated further in order to deter-
mine the efficacy of this strategy for managing
psyllid populations in a citrus grove. The efficacy
of a yellow tag contaminated with Ifr blastospores
as a source for D. citri to spread the fungus to
young citrus plants and other psyllids is presently
being tested in cages (Moran et al. 2009); if re-
sults are promising then this autodissemination
strategy will be evaluated in Texas door-yard cit-
rus. However, persistence and viability of the Ifr
blastospores on the yellow tag or leaf surface over
time under field conditions will help determine
the cost effectiveness of such a pest management
strategy. To increase the persistence, viability and
efficacy of the fungal blastospores, perhaps an ad-
juvant could be added. Dunlap et al. (2007) indi-
cated that the speed of the Ifr blastospore germi-
nation was improved by adding keratin hydroly-
sate and the number of infective propagules was
increased as well.


The yellow tag may only attract a few psyllids
for dissemination of Ifr into the grove and timing
of application will be crucial. In a field study
where D. citri populations were monitored with
yellow sticky traps, the mean number of adult D.
citri per trap decreased significantly during peri-
ods of abundant new flush compared to trap cap-
tures immediately before and after new flush was
present (Rogers, unpublished data). Therefore,
the yellow tags will need to be hung prior to the
emergence of the new preferred flush depending
on the climatic conditions and phenology (usually
before Mar and just prior to Aug) by the psyllids
to be most effective. However, this autodissemina-
tion strategy could be augmented by the addition
of an attractant in the future (El-Sayed et al.
2006; Suckling et al. 2007). Recently, Wenninger
et al. (2008) provided behavioral evidence for a fe-
male-produced volatile sex pheromone for the
adult psyllid. Perhaps this pheromone, once iden-
tified and synthesized could be added to the yel-
low tag to increase the effectiveness of attracting
other adult psyllids and increase the dissemina-
tion of the Ifr into the grove, irrespective of the
presence of flush.
In the field, the transfer of Ifr to leaves or flush
where psyllids congregate could potentially lead to
secondary infection producing sporulating cadav-
ers and eventually under high humidity conditions
an epizootic effect. In a preliminary caged labora-
tory experiment, it was observed that an entire
psyllid population living on a citrus seedling be-
came infected after 7 d of exposure to a yellow tag
sprayed with Ifr blastospores (Avery, unpublished
data) Also, in qualitative assays, Meyer (2007) re-
corded 100% mortality of adult D. citri that were
exposed to Pfr AsCP on sporulating psyllid cadav-
ers. The extent of the epizootic effect is dependent
upon the density of the insects in the area where
the sporulating cadavers are located (Furlong and
Pell 2001; Avery 2002; Klinger et al. 2006).
Entomopathogenic fungi, which are efficient in
killing soft bodied, sucking-piercing insects, are
being investigated in different parts of the world
as biocontrol agents for controlling the Asian cit-
rus psyllid (Subandiyah et al. 2000; Pell 2008).
However, currently there are no Ifr biopesticides
registered for spraying and controlling the psyllid
on fruit crops in the USA. Presently, Certis@ in
Maryland, USA, produces a blastospore formula-
tion of Ifr (Pfr-97 20% WDG@ that should become
registered for use by citrus growers in 2010 (Di-
mock, personal communication).
Blastospores of different entomopathogenic
fungi, including Ifr have been used extensively in
pest management programs on a worldwide scale
(Avery 2002). Ifr blastospores easily can be mass
produced in a shake-flask liquid culture medium
(Jackson et al. 1997, 2003; Lozano-Contreras et
al. 2007) and only require 6-8 h to germinate
(Vega et al. 1999). Considering that southern and








Florida Entomologist 92(4)


central Florida experiences high humidity, it
seems that the environmental conditions are con-
ducive for the use of this fungal biopesticide as
part of an IPM program in managing all stages of
the psyllid population.
Biopesticides can be used as an alternative in a
spray program to break the cycle of harder chem-
icals and prevent the development of resistance
(Moore 2008a). The use of Ifr is an environmen-
tally friendly alternative that will have minimal
effect on non-target beneficial arthropods present
in the grove (Sterk et al. 1995a, b) and can be used
with other strategies for sustainable pest man-
agement (Shah & Pell 2003). For instance, Eti-
enne et al. (2001) reported Tamarixia radiata, a
parasitoid of the Asian citrus psyllid established
in the Guadeloupe Islands, has provided excellent
control of the psyllid even in the presence of an
entomopathogenic fungus of the psyllid, Hirsu-
tella citriformis. Both H. citriformis and Ifr are
types of native entomopathogenic fungi found in
the Florida groves (Meyer et al. 2007, 2008), and
should be compatible with T radiata previously
released for the control of the psyllid pest. How-
ever, the compatibility of Ifr with T radiata for
controlling the psyllids needs to be tested under
field conditions. Lastly, biopesticides, such as Ifr,
may be used effectively either alone or in rotation
with traditional pesticides for added genetic re-
sistance prevention (Er & Gd6ke 2004; Kantz
2007; Moore 2008b). However, which chemicals
sprayed in the field are compatible with Ifr war-
rants further investigation.
These autodissemination laboratory studies are
the first to evaluate the potential for using Ifr
against the Asian citrus psyllid, whether sprayed on
trees or on an artificial attractant surface. Based on
the results, the use of Ifr for managing the citrus
psyllid has demonstrated potential and warrants
further testing under field conditions. Lastly, be-
cause the psyllid is attracted to the yellow color
(Hall & Albrigo 2007; Hall et al. 2007; Hall et al.
2008; Hall 2009), the use of yellow cards impreg-
nated with Ifr blastospores as part of an IPM strat-
egy has potential for providing citrus growers with a
cost-effective method for managing psyllids.

ACKNOWLEDGMENTS

We thank Matthew Hentz and Kathy Moulton for
technical assistance in rearing and providing D. citri
and Anna Sara Hill at the USDA, ARS, US Horticul-
tural Research Laboratory for growing the citrus seed-
lings for this research project. Thanks to Phyllis
Rundell and Eliza Duane at IRREC for assistance in the
preparation of the spray trials and evaluation of the
treatments. Statistical analysis assistance and sugges-
tions by Dr. P. Stoffella at UF-IFAS-IRREC in Ft.
Pierce, Florida, were greatly appreciated. Reviews by
Drs. A. Arevalo, S. Arthurs, P. Stansly, L. Stelinski, E.
Wenninger, and V. Wekesa provided constructive criti-
cism for improving the manuscript. This project was


funded by the following grant: The Direct Grower Assis-
tance: Development and Evaluation of Citrus Grower
Psyllid Management Programs 2008 awarded by the
Florida Citrus Advanced Technology Program
(FCATP08: Control of Citrus Greening, Canker and
Emerging Diseases of Citrus).

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Florida Entomologist 92(4)







Watts et al.: Blood Meal ID from FL Mosquitoes


BLOOD MEAL IDENTIFICATION FROM FLORIDA MOSQUITOES
(DIPTERA: CULICIDAE)


STACEY L. WATTS, DANIEL M. FITZPATRICK AND JAMES E. MARUNIAK
Department of Entomology and Nematology, University of Florida, P.O. Box 110620, Gainesville FL, 32611-0620

ABSTRACT

Mosquitoes from different species endemic to North Florida were collected over a 12-month
period, and blood meal analyses were conducted on engorged females to determine the range
of vertebrate hosts each species fed on. Thirty-one mosquito blood meals from 6 mosquito
species were identified, and blood meal hosts identified included horse, cow, armadillo, deer,
raccoon, rabbit, and owl. Several mosquitoes captured in the study should be considered po-
tential bridge vectors for encephalitis viruses considering their ubiquity, their preference for
mammalian blood meals, and their competence as viral vectors.

Key Words: host identification, host preference, Alachua County, Florida, polymerase chain
reaction, viral vectors

RESUME

Varias species end6micas de mosquitos de la region norte de Florida fueron colectadas du-
rante un period de 12 meses y la comida de sangre de hembras fue analizada para deter-
minar los hospederos de los cuales los mosquitos se habian alimentado. Un total de treinta
y una comidas de sangre obtenidas de seis diferentes species de mosquitos identificaron a
los siguientes hospederos: caballo, vaca, armadillo, ciervos, mapache, conejo, y buho. Various
de los mosquitos capturados en este studio pueden considerarse vectores potenciales de vi-
rus de encefalitis debido a su omnipresencia, su preferencia por sangre de mamiferos, y por
ser vectores virales competentes.


Translation provided by the authors.


Mosquitoes vector a variety of pathogens of
medical and veterinary importance. Growing con-
cern over the spread of West Nile virus (WNV)
and related encephalitis viruses has prompted ex-
tensive investigation into the host-feeding pat-
terns and preferences, as well as vectoral capaci-
ties, of pests now viewed mainly as cosmopolitan
nuisances (Sardelis et al. 2001; Turell et al. 2005;
Rodrigues & Maruniak 2006; Molaei et al. 2008).
Host-feeding patterns and preferences vary ac-
cording to a number of innate, seasonal, and envi-
ronmental tendencies, including host availability
and abundance, flight behavior and feeding peri-
odicity of mosquitoes (Molaei et al. 2008) as well
as by region (Turell et al. 2005). The current study
sought to collect an array of mosquito species na-
tive to North Florida over a 12-month period and
identify the range of vertebrate hosts that those
mosquitoes fed on through blood meal analysis.

MATERIALS AND METHODS

Mosquito Collection and Identification

Mosquitoes were collected in CO,-baited Cen-
ter for Disease Control (CDC) light traps (John W.
Hock, Gainesville, FL) from May 2006 through
Apr 2007 from 11 sites in Alachua County, Flor-


ida. The habitats chosen were sampled due to
proximity either to aquatic environments or to
habitats of suspected vertebrate hosts. Traps were
placed on site in late afternoon and collected the
next morning, approximately 18 h later. Captured
mosquitoes were transferred to the University of
Florida while still alive in mesh collection bags
from the trap, placed in a -70'C freezer to eutha-
nize them, and stored in the freezer until subse-
quent identification and processing. Mosquitoes
were sorted by species as described in Darsie &
Morris (2003) on a -20'C chill-table, and those
without apparent blood meals were pooled in
groups of up to 25 in sterile microcentrifuge tubes,
while female mosquito abdomens with apparent
blood meals were homogenized individually.

PCR Amplification and Sequencing of Mosquito Blood
Meals

DNA was extracted with the DNAzol@ Direct
Extraction Kit (MRC, Cincinnati, OH). Poly-
merase chain reactions (PCR) were conducted on
extracted DNA with vertebrate-specific primer
sets and cycling conditions described in Cupp et
al. (2004) that preferentially amplified a 290-bp
region from the cytochrome b gene within the mi-
tochondrial DNA of vertebrates. Briefly, the PCR








Florida Entomologist 92(4)


reactions were conducted in 50 pL reactions with
the following reagents: 5.0 pL of 10X reaction
buffer (600 mM Tris-HCl pH 8.5, 150 mM
(NH ),SO,, 35 mM MgCl,), 0.2 mM dNTPs, 25
pmol of each primer, 2.5 units of Taq Polymerase
(Invitrogen, Carlsbad, CA) and 2.5 pL of DNA
template. If 290 bp amplicons were not produced
or were not able to be identified, a second PCR
was conducted with mammalian-specific primer
sets and cycling conditions described in Ngo &
Kramer (2003) to preferentially amplify a 772-bp
region from the cytochrome b gene. Briefly, the
PCR reactions were conducted in 25 pL reactions
with the following reagents: 2.5 pL of 10X reac-
tion buffer (200 mM Tris-HC1 pH 8.4, 500 mM
KC1), 3.0 mM MgCl,, 0.5 mM dNTPs, 5.0 pmol of
each primer, 1.25 units of Taq Polymerase, and
2.5 pL of blood meal DNA template. Agarose gel
electrophoresis was used to separate amplicons,
and DNA was purified from excised bands with
the expected size with QIAquick columns
(QIAGEN, Valencia, CA). Fragments were se-
quenced with BigDye Terminator Sequencing
Ready Reaction Kits (Applied Biosystems, Foster
City, CA) at the Interdisciplinary Center for Bio-
technology Research at the University of Florida.
Sequences were analyzed and edited with Se-
quencherTM software (Gene Codes Co., Ann Arbor,
MI), and were compared with the GenBank data-
base entries by the BLAST program from NCBI
(http://blast.ncbi.nlm.nih.gov) to identify the ver-
tebrate host on which each mosquito had fed.


RESULTS

Sixty-one of the 45,692 mosquitoes collected
showed apparent blood meals. Upon amplifica-
tion, 57 of those 61 samples (93%) showed ampli-
cons of the expected size. Of those, vertebrate
hosts were identified in 33 individuals (58%) (Ta-
ble 1). Blood meals were most commonly identi-
fied from hosts fed on by Coquillettidia pertur-
bans Walker, a particularly opportunistic feeder
(Molaei et al. 2008). Coquillettidia perturbans
blood meals represented 5 mammalian species
(horse, armadillo, deer, rabbit, and raccoon) and 1
avian species (owl). Blood meals were identified
from 6 horses and 4 cows in the 10 Mansonia tit-
illans Walker, from 2 horses and 1 cow in 3Aedes
uexans Meigen, from 1 cow and 1 deer in 2 Culex
salinarius Coquillett, and from 1 rabbit in Culex
erraticus Dyer & Knab, while all 3Anopheles cru-
cians Wiedemann fed upon horses. The identity of
the blood meal match with GenBank sequences
ranged from 89-100%, and the best match was
shown in Table 1.


DISCUSSION

Of the 6 species of blood-fed mosquitoes cap-
tured in the study, 4 species fed from horses,


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Watts et al.: Blood Meal ID from FL Mosquitoes


which are particularly susceptible to diseases
caused by encephalitis viruses. Preferential feed-
ing on horses byAe. vexans is a major concern, as
Ae. vexans is considered a competent to highly-ef-
ficient vector for Saint Louis encephalitis (SLE),
western equine encephalomyelitis (WEE), east-
ern equine encephalomyelitis (EEE), and WNV
(Goddard et al. 2002; Turell et al. 2005).
Coquillettidia perturbans is considered a mod-
erately competent vector of EEE (Vaidyanathan
et al. 1997) and an inefficient but competent vec-
tor for WNV (Sardelis et al. 2001) in lab settings.
EEE and WNV have been isolated from mosqui-
toes in nature throughout the Eastern and South-
eastern United States (Srihongse et al. 1980;
Crans & Schulze 1986; Edman et al. 1993; Godsey
et al. 2005; Lukacik et al. 2006). Anopheles cru-
cians and Ma. titillans showed a considerable af-
finity for horse hosts in a previous study by Cupp
et al. (1986) as well, and have been positive for
WNV isolations in the field (CDC 2007; Cupp et
al. 2007). The paucity of research onAn. crucians
and Ma. titillans vectoral capacities for encepha-
litis viruses indicates that neither have histori-
cally been considered important disease vectors.
However, considering their pervasiveness and
their preference for mammalian blood meals in
our study, as well as their moderate competence
as viral vectors (Molaei et al. 2008), the potential
for Cq. perturbans and Ma. titillans to be bridge
vectors in the transfer of EEE and WNV from
viremic primary hosts to mammals should still be
considered.
CO2-baited light traps, used in a series of stud-
ies conducted on the mosquitoes captured, are not
considered particularly attractive to engorged
mosquitoes, thus explaining the low percentage
(61 of 45,692, or > 0.002%) of blood-fed mosquitoes
captured. Future studies in mosquito blood meal
analysis will supplement CO2-baited light traps
with mosquito resting boxes.
Of particular interest is the low yield of posi-
tive blood meal identifications in An. crucians (3
positive identifications in 12 blood meals). Town-
zen et al. (2008) had similar complications, re-
porting that co-amplification of Anopheles genes
occurred often with cyt b primer sets, which con-
sequently prevented positive host identification.

ACKNOWLEDGMENTS

We thank Dr. Alejandra Garcia-Maruniak for invalu-
able contributions and for help with editing the manu-
script. This research was funded by USDA-CSREES, T-
STAR grant # 00057913.

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Gillett-Kaufman & Kimbrough: Beauveria Infection Sites on Diaprepes Adults 623



A MODIFIED METHOD TO VISUALIZE INFECTION SITES OF SPORES OF
THE ENTOMOPATHOGEN BEAUVERIA BASSIANA (DEUTEROMYCOTINA:
HYPHOMYCETES) ON THE EXOSKELETON OF CITRUS ROOT WEEVIL
DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONIDAE) ADULTS


JENNIFER L. GILLETT-KAUFMAN1AND JAMES W. KIMBROUGH2
1Department ofEntomology and Nematology, P.O. Box 110620, Gainesville, FL 32611-0620

2Department of Plant Pathology, P.O. Box 110680, University of Florida, Gainesville, FL 32611


ABSTRACT

Beauveria bassiana is a widespread entomopathogen which is infectious to a great variety
of insects. A commercial preparation of this fungus was used to study its potential as a bio-
control agent of the citrus root weevil, Diaprepes abbreviatus. Laboratory reared adult wee-
vils were placed in clean plastic bags with 0.05 g of powdered inoculum per bag, shaken for
thirty seconds, and placed in holding cages. In order to determine the spore concentration,
germination, and position of ingress, inoculated weevils were dipped in a collodion solution
after 30 min, 6 h, 12 h, 18 h, and 30 h. Collodion peels from various areas of the exoskeleton
were removed, stained with lactophenol cotton blue, and observed microscopically. At 12 h
post-inoculation, spores began to swell, and at 18 h close to 25% germinated on all body parts
except on the elytra. After 30 h, between 45% and 75% of the spores germinated, depending
on the body part. Most active spore germination occurred around the eyes (75%), followed by
the ventral abdomen (65%), the dorsal pronotum (60%), scales (45%), and dorsal surface of
the elytra (7%). Spores were washed from the surface of inoculated weevils with distilled wa-
ter and plating onto potato dextrose agar. Spore density on the weevils' surface was deter-
mined to be 15,815 479 spores initially after inoculation, and was reduced to 5,782 136
spores after 3 h.

Key Words: biocontrol, conidia, entomopathogenic fungus, infection sites, spore germination

RESUME

Beauveria bassiana es un entomopat6geno cosmopolita que es infeccioso para un gran varie-
dad de insects. Una preparaci6n commercial de este hongo fue usada para estudiar su poten-
cial como un agent de control biol6gico para el picudo de la raiz de citricos, Diaprepes
abbreviatus. Adultos del picudo criados en el laboratorio fueron puestos en bolsas plasticas
y limpias con 0.05 g de inoculo en polvo en cada bolsa, agitado por treinta segundos, y pues-
tos enjaulas de espera. Para determinar la concentraci6n de esporas, germinaci6n y la posi-
ci6n de su entrada, picudos inoculados fueron metidos en una soluci6n adhesive despu6s de
30 minutes, 6 h (horas), 18 h y 30 h. Las cascaras de colodio de varias areas del exoesqueleto
fueron quitadas, tintadas con el algod6n azul de lactofenol, y observadas bajo el microscopio.
A los 12 h despu6s del inoculo, las esporas empezaron a hincharse, y a los 18 h aproximada-
mente 25% germinaron sobre todas las parties del cuerpo menos los elitros. Despu6s de 30 h,
entire 45% y 75% de las esporas germinaron, segun la parte del cuerpo. La mayor parte de la
germinaci6n de las esporas sucedi6 alrededor de los ojos (75%), seguida por la parte ventral
del abdomen (65%), el dorso de pronoto (60%), escamas (45%) y la superficie dorsal de los eli-
tras (7%). Las esporas fueron lavadas de la superficie de los picudos inoculados con agua des-
tilada y puestas en places de agar de dextrosa de papa. La densidad de las esporas sobre la
superficie de los picudos fue determinada como 15,815 479 esporas inicialmente despu6s
del inoculo, y fue reducida a 5,782 + 136 esporas despu6s de 3 horas.


Beauveria bassiana (Balsamo) Vuillemin (Deu- habitats (Ferron 1981). The fungus regulates pop-
teromycotina: Hyphomycetes) is a disease-caus- ulations of coleopterous insects of the genera Di-
ing agent in insects (Steinhaus 1949). De Hoog abrotica, Colaspis, and Maecolaspis in soybeans
(1972) has done the most definitive work on the grown in Argentina and Brazil. In Brazil, the fun-
genus Beauveria in which he was able to delimi- gus achieves high prevalence in populations of
tate Beauveria from the genera Isaria, Trititach- Aracanthus, an important pest of beans (Sosa-Go-
ium, andAcrodontium. Scrutinized as a biological mez et al. 1994), and in France, England, and Mo-
control agent of hypogeous insects, B. bassiana rocco it can be found on Sitonia weevils, a major
has been found around the world and in many pest of cultivated Fabacae (Poprawski et al. 1988).







Florida Entomologist 92(4)


Isolates from the United States, Canada, and
China have been collected and used in immu-
nochemical characterization to identify a quality
control program to ensure virulence of a strain
(Tan & Ekramoddoullah 1990). Although most B.
bassiana isolates are morphologically indistin-
guishable, they vary in virulence (Safavi et al.
2007). Biochemical characterization and large-
scale production of these virulent isolates are im-
portant for successful biological control (Zhang &
Tan 1987). In 2003 B. bassiana (strain GHA) pro-
duced by the MycoTech Corporation (USA, My-
cotrol ES, Emerald BioAgriculture, Butte, MT,
59702) was in commercial production as a my-
coinsecticide and this product was used in all of
the experiments reported herein.
The Diaprepes root weevil (Diaprepes abbrevia-
tus L.) (Coleoptera: Curculionidae) is a pest in
Florida on citrus and other crops. It was first re-
ported in the United States in a Florida nursery
(Woodruff 1964); now the weevil infests more than
50,000 ha of the 300,000 ha of citrus in production
(Duncan & McCoy 2001). The weevil larvae feed
on roots of trees creating an entry for fungal and
other plant pathogens. It has been shown that spe-
cies of Phytophthora can gain ingress into citrus
root systems where root integrity has been com-
promised by weevil damage (Rogers et al. 1996;
Graham et al. 1997). Graham et al. (1997) found
that in some soil types Phytophthora palmiuora
caused more damage to structural roots than the
weevil larvae. Current control of the Diaprepes
root weevil relies on timed applications of chemi-
cal pesticides, but these control measures have yet
to suppress weevil populations (Bullock et al.
1988). With growing concerns in the use of chemi-
cal pesticides, costs, and deleterious effects to the
environment, it is important that we explore the
potential of enhancing biological control.
This study was initiated to examine the pro-
pensity of a formulated strain of B. bassiana to
germinate on the exoskeleton of adult D. abbreuia-
tus. It is known that B. bassiana will infect adult
and larval D. abbreuiatus in nature (Futch & Mc-
Coy 1992). However, fungal modes of ingress and
germination rates on different areas of adult Di-
aprepes exoskeleton have yet to be established.
Humidity and temperature are necessary factors
for the germination of B. bassiana (Thomas &
Blanford 2003); however, neither humidity nor
water alone is enough to stimulate germination of
conidia (Hunt et al. 1984). The insect exoskeleton
is known to have chemical compounds that affect
the germination of B. bassiana (Woods & Grula
1984). A lack of nutrients on sclerotized beetle cu-
ticle can be a limiting factor in fungal growth and
development, including conidial germination
(Hunt et al. 1984). Amino acids and glucosamine
have been found on the larval cuticle of Heliothis
zea. These fluctuate in levels during larval devel-
opment but are always sufficient to trigger the


germination of B. bassiana (Woods & Grula 1984).
Amines and peptides on the larval exoskeleton of
H. zea do not inhibit the germination of B. bassi-
ana (Woods & Grula 1984). Information gathered
on the most vulnerable areas of the insect exoskel-
eton will be considered in the future production of
a spore dissemination method for the use of Beau-
ueria in an integrated pest management system to
control adult population levels ofD. abbreviatus.

MATERIALS AND METHODS

Conidial Delivery

Cages (Sho-Bowl, 64 oz. tub, ribbed dome lid,
Ultra Pac, Rogers, Mn. 55374) with pin holes in the
lid for ventilation were prepared that contained
~100g of dry play sand. The sand and cages were
sterilized in a UV hood for 1 h. Work surfaces, in-
cluding the balance used, were cleaned with a 10%
Clorox@ solution before and after treatment to
avoid contamination of control insects.
Diaprepes abbreviatus were reared at the Flor-
ida Department of Agriculture facility in Gaines-
ville, Florida, by Beavers' (1982) methods for rear-
ing on an artificial diet. A dry commercially pro-
duced wettable powder formulation of B. bassiana
conidia produced by the MycoTech Corporation
(strain GHA, Mycotrol ES, Emerald BioAgriculture,
Butte, MT, 59702) was used as an inoculum. This
strain was previously used by Furlong & Groden
(2003) in their study of larvae of the Colorado potato
beetle, Leptinotarsa decemlineata (Say). Citrus root
weevils were separated by gender as described by
Harari & Landolt (1997) and placed in batches into
clean plastic sandwich bags containing 0.05 g of
powder per insect. Bags with insects and powder
were shaken for 30 s. This method of conidial deliv-
ery was similar to that used by Hedlund & Pass
(1968) on the alfalfa weevil. Insects were trans-
ferred to holding cages, based on gender, with 2 or-
ganically grown baby carrots and a cotton ball satu-
rated with sterilized water. Control groups were ag-
itated in a clean plastic bag to simulate the possibil-
ity of mechanical damage that the insects might
incur during treatment. Insects were maintained in
an incubator with a 14 h:10 h light:dark cycle, a rel-
ative humidity of 85-90%, and a temperature range
between 22C and 240C.

Modified Collodion Method

Thirty min after treatment, 5 insects of each
gender were removed from the holding containers
and the live insects were pinned through the right
elytra with a number 2 nylon headed insect pin.
Insects were dipped into a collodion solution (Col-
lodion Flexible USP, Alcohol 22%, Ether 67%, Py-
roxylin 4.75%, Camphor 2% and Castor Oil 3%,
HUMCO, Texarkana, TX 75501), and allowed to
stand at room temperature until the layer was so-


December 2009







Gillett-Kaufman & Kimbrough: Beauveria Infection Sites on Diaprepes Adults 625


lidified but not hardened (approximately 45 min)
(Delp 1954; Kimbrough 1963). The solidified collo-
dion film was removed from the insect cuticle with
jeweler's forceps. This process removes loose and
attached conidia and some insect cuticular com-
ponents. Peels were made from the ventral ab-
dominal sterna and related inter-segmental mem-
branes, and from the dorsal elytra to the beak in-
cluding the dorsal pronotum, eyes, and antennae.
Each peel was placed with the exoskeleton con-
tact side facing up to reduce interference from the
collodion when visualizing the peel with the com-
pound light microscope. A drop of lactophenol cotton
blue stain was placed on the peels of collodion on
glass slides (Shurtleff & Averre 1997). After 5 min
excess stain was wicked off of the peels with absor-
bent tissue. The slides were sealed with a few drops
of CytosealTM 60 (Stephens Scientific, Division of Ri-
chard-Allan Scientific, Kalamazoo, MI 49007-3538)
around the edges of a 50 x 24-mm cover glass. To en-
sure thin slides and remove air bubbles, slides were
placed on a slide warmer for 6 h at 50C with a 15-g
lead weight on the cover glass. Slides were removed
from the slide warmer and allowed to cool for 6 h be-
fore the weight was removed from the cover glass.
Collodion dipping and the preparation of epidermal
peels were repeated every 6 h with 5 fresh insects of
each gender from the treatment cohort until 30 h
past the initial inoculations. Conidial density, ger-
mination rate, and growth including the formation
of germ tubes and points of fungal ingress were ob-
served and photographed with a Nikon 990 digital
camera attached to a light microscope. Dorsal and
ventral photographs of weevils were labeled for
rapid identification of areas.

Conidial Viability and Weevil Contact

A simple procedure was used to determine the
number of viable conidia on each treated insect.
Twenty insects were treated with 0.05 g of dry com-
mercially produced wettable powder ofB. bassiana
per insect using the previously described plastic bag
procedure. The weevils were then placed in a hold-
ing container for 3 h. The weevils were then re-
moved from the holding container and placed indi-
vidually in 100-mL glass vials. One milliliter of de-
ionized water was added to the vial containing the
weevil. The vial was shaken vigorously for 1 min.
The water from each weevil rinse was poured onto
potato dextrose agar (PDA) (Difco Laboratories, De-
troit, MI, 48232-7058) and the Petri dishes were
sealed with Parafilm M Lab Wrapping Film and
placed inverted in an incubator at room tempera-
ture for 48 h. Twenty control insects were treated
with the powder and immediately washed as de-
scribed above. After incubation, the plates were ex-
amined for signs of fungal growth. Colonies forming
on the plates were counted on a grid system under a
dissecting microscope at 40x. Data were analyzed
with Excel (Microsoft Corp 1997).


RESULTS

Spores on the surface of the weevil were not
evenly distributed at 30 min post inoculation. Total
spore density on weevil surfaces could not be deter-
mined with the collodion method. It could be deter-
mined, however, by washing spores from the sur-
face of the weevils with distilled water and plating
onto PDA. Spore density on the weevils' surface
was determined to be 15,815 + 479 spores initially
after inoculation. Spore load on the surface of the
weevils was reduced to 5,782 + 136 spores after 3 h.
During this 3 h time frame, weevil activities such
as contact with inanimate objects and other wee-
vils or self-grooming were observed in the holding
cage. Thirty min post inoculation, conidia were
found in clusters around ommatidia on the eyes, on
setae, in depressions in the integument, on the
ventral pronotum, and in the grooves of scales lo-
cated along the ventral surfaces. At 6 h, weevils
were checked for signs of spore enlargement or ger-
mination. No evidence was found to suggest spore
growth at that time. At 12 h, peels were similar to
those taken at 30 min and 6 h post inoculation.
Spores were found on plumose setae, around the
base of setae, and in clusters around ommatidia.
Spores were abundant in pits of the pronotum and
in the grooves between the ommatidia of both eyes.
Debris from insect rearing medium collected on
some of the peels could account for spores cluster-
ing in particular areas. Clusters of spores associ-
ated with rearing medium were not tabulated with
the results.
Initially, spores measured 1.3 pm in length,
and began to enlarge at 12 h to 2.9 pm. Measure-
ments were taken for spore length not width. Ger-
mination observed at 18 h was 24 3% on abdom-
inal sections, 10 + 3% on the pronotum and beak,
35 3% on the eyes, and 9 + 2% on scales on ven-
tral abdominal sections (Fig. 1). Enlarged conidia
were present on the elytra but had not begun to
germinate at 18 h. Germination observed at 30 h



mis hour
s o- = MUM





0 "






Fig. 1. Percentage of Beauveria bassiana conidia ger-
minating on different areas of adult Diaprepes abbre-
uiatus exoskeleton at 18 and 30 h post inoculation.







Florida Entomologist 92(4)


December 2009


Figs. 2-12. Beauveria bassiana conidia on adult Diaprepes abbreviatus exoskeleton 30 h post inoculation. 2.
Conidia germinating on ovipositor. 3. Conidia germinating in cluster on edge of sterna. 4. Conidia germinating on
edge of sterna. 5. Conidia germinating on ventral metathorax. 6. Conidia in clusters germinating among scales of
sterna. 7. Conidia germinating on pronotum. 8. Conidia germinating around hair on sterna. 9. Conidia germinating
on ommatidia of left eye. 10. Conidia germinating on beak near eye. 11. Conidia germinating on ommatidia of right
eye. 12. Conidia germinating on edge of eye. Scale bars: 5 lm.







Gillett-Kaufman & Kimbrough: Beauveria Infection Sites on Diaprepes Adults 627


was 58 3% on the pronotum, 74 3% on the
eyes, 65 3% on abdominal sections, and 44 + 3%
on scales on abdominal sections (Fig. 1). At 30 h
post inoculation conidia were germinating on the
ovipositor (Fig. 2), in clusters on the edge of ster-
nal segments (Fig. 3-4), on the ventral metaster-
num (Fig. 5), and among scales of sterna (Fig. 6).
At 30 h, germination was observed on the dorsal
surface of both of the elytra at a rate of 7 3%.
Conidia were also found germinating on the dor-
sal surface of the pronotum (Fig. 7), around hairs
on the sterna (Fig. 8), on the ommatidia of the left
eye (Fig. 9), on the beak near the left eye (Fig. 10),
on the ommatidia of the right eye (Fig. 11), and on
the edge of the right eye (Fig. 12).

DISCUSSION

Diaprepes weevils were infected with a dry,
commercially produced wettable powder of B.
bassiana conidia, strain GHA, a strain previously
used in studies of Colorado potato beetle larvae by
Furlong & Groden (2003). Conidial lengths of B.
bassiana isolates tested by Sosa-Gomez et al.
(1994) showed lengths varying among isolates
from 0.95 to 3.41 pm. These dimensions agree
with those measured in the present study at the
time of inoculation. The development of B. bassi-
ana on adults and larvae ofD. abbreviatus is sim-
ilar to other entomopathogenic deuteromycetes,
including Nomuraea (Boucias & Pendland 1982).
Essentially nothing was known as to how
spores ofB. bassiana would attach to and aggre-
gate on the surface of Diaprepes root weevil
adults after inoculation. Our results demon-
strated that spore concentrations on the weevils
surface was reduced approximately 3-fold 3 h post
inoculation. This reduction may be explained by
weevil activity, contact, or self grooming observed
during this time. This research showed that
conidia tend to condense in intersegmental areas
and around hairs and the base of appendages.
Natural grooves in scales also filled with spores.
There has been little definitive data as to the tim-
ing of spore germination once spores make con-
tact with adult D. abbreviatus integument. We
have shown that spores germinate at different
rates on different areas of the weevils' exoskele-
ton. Several authors have studied why B. bassi-
ana spores germinate when they come in contact
with insect cuticle. Factors that may inhibit or en-
hance germination and penetration include cuti-
cle density or compounds on insect integument
(Woods & Grula 1984; Soza-Gomez et al. 1997). In
addition, the lack of nutrients on sclerotized bee-
tle cuticle is a limiting factor in fungal growth and
development (Hunt et al. 1984). Results obtained
from this study indicate that germination is lim-
ited on the heavily sclerotized elytra. By observ-
ing germination rates on various regions we could
document (1) where the largest numbers of spores


will germinate most readily on the Diaprepes ex-
oskeleton, and (2) the viability of conidia in a dry
commercially available wettable powder. Data
generated in this phase of research will enable
one to project the likely source of direct fungal en-
try and provide researchers with the information
they need to produce an effective conidial delivery
system for Diaprepes root weevil control.

ACKNOWLEDGMENTS

The senior author thanks Drs. K. L. Buhr, J. W. Kim-
brough, C. W. McCoy, J. M. Sivinski, and F. W. Zettler for
service on her Ph.D. Supervisory Committee. We greatly
appreciate J. M. Sivinski and C. J. Stuhl for reviewing
the manuscript in preparation for publication.

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Florida Entomologist 92(4)








Larsen & Nuessly: Reduced Insecticide Rates for Wireworms


EFFECTIVENESS OF REDUCED RATES OF INSECTICIDES FOR THE CONTROL
OF MELANOTUS COMMUNIS (COLEOPTERA: ELATERIDAE) IN SUGARCANE


NICHOLAS A. LARSEN AND GREGG S. NUESSLY
Everglades Research and Education Center, University of Florida, Institute of Food and Agricultural Sciences,
3200 Palm Beach Road, Belle Glade, FL 33430


ABSTRACT

Wireworms (larval Elateridae) are perennial pests of newly planted sugarcane causing
stand loss directly by damaging growing points and indirectly by introducing disease. Two
organophosphate insecticides, phorate and ethoprop, are currently labeled for controlling
wireworms in sugar cane. In the first experiment, 4 rates of phorate (100, 87.5, 75, and
62.5% of the current maximum field rate) were used in 2 different types of soil: Dania Muck
and Immokalee Fine Sand. In the second experiment, 4 rates of phorate and ethoprop (100,
75, 50, and 25% of current maximum field rates) were used in a Lauderhill Muck soil. A no-
treatment and a wireworm-free treatment were used as controls in both experiments. The
effect of the insecticide was evaluated in simulated field experiments conducted in 18.9-L (5-
gallon) buckets filled with soil and artificially infested with 10 wireworms (Melanotus com-
munis (Gyllenhall), Coleoptera: Elateridae) per bucket. After 60 d, the contents of the buck-
ets were emptied to evaluate damage to the plant and count the surviving wireworms.
Insecticides resulted in fewer live wireworms and reduced damage to sugarcane shoots,
roots, and seed pieces compared to the no-treatment control. In the first experiment, phorate
proved to be very effective at controlling wireworms even at the 62.5% rate. Phorate was
more effective in the Immokalee Fine Sand than in the Dania Muck. In the second experi-
ment, phorate was found to be more effective at reducing stand loss and wireworm numbers
than ethoprop.

Key Words: Elateridae, wireworms, phorate, ethoprop, histosol, spodosol

RESUME

Gusanos de alambre son continues plagas insectiles en nuevamente sembrado cana de azucar
que reduce el numero de plants directamente por dahar el punto de crecimiento y indirec-
tamente por introducir agents pat6genos. Dos insecticides organofosforados, phorate y etho-
prop, son registrados para controlar gusanos de alambre en cana de azucar. En el primer
experiment, se usan cuatro tazas de phorate (100, 87.5, 75, y 62.5%) de la taza maxima ac-
tual en dos suelos de tipo diferente: Dania Muck y Immokalee Fine Sand. En el segundo ex-
perimento, se usan cuatro tazas de phorate y ethoprop (100, 75, 50, y 25%) de la taza maxima
actual en un suelo: Lauderhill Muck. Se usan una prueba sin insecticide y una prueba sin gu-
sanos alambres como pruebas de control en cada experiment. Se evalu6 el efecto de la insec-
ticida en experiments de campo simulados en baldes de 18.9 litros (5 galones), llenados con
suelo, e infestados artificialmente con 10 gusanos de alambre (Melanotus communis (Gyllen-
hall), Coleoptera: Elateridae). A partir de 60 dias, se vaciaron los contenidos de los baldes
pare evaluar el daho a la plant y para contar los gusanos de alambre sobrevivientes. El uso
de insecticides reduce el numero de gusanos de alambre sobrevivientes y reduce el daho al ta-
1lo, a la raiz, y la semilla en comparaci6n a la prueba de control. En el primer experiment,
phorate fue efectivo hasta la taza de 62.5%. Phorate fue mas efectivo en el Immokalee Fine
Sand que en el Dania Muck. En el segundo experiment, phorate fue mas efectivo en reducir
p6rdida de plants y numerous de gusanos de alambre que ethoprop.

Translation by the authors.


Wireworms (Melanotus communis (Gyllen- and a large percentage of the land that gradually
hall), Coleoptera: Elateridae) have been a con- would go into sugarcane production was devoted
stant threat to sugarcane since the beginning of to pasture (Kidder 1979). Elevated populations of
its production in the 1920s and 1930s in the Ever- wireworms often are found in pasture or other-
glades Agricultural Area (EAA) (Gifford 1964). wise grassy fields (Fox 1961; Parker & Seeney
Prior to the advent of chemical insecticides some 1997).
growers had to replant several times to get ac- The wireworm is a hardy insect with a long life
ceptable stands (Wilson 1940). Prior to the 1960s, cycle of 1 tolO years depending on the climate of
most of the EAA was devoted to cattle production the area (Capinera 2001). Fields of sugarcane







Florida Entomologist 92(4)


have been reported to have upwards of 100,000
larvae per hectare in histosols (Wilson 1946). Re-
moving the food source by clean fallowing will
starve out most of the worms and may prevent
more oviposition in that field (Wilson 1946); how-
ever, it will not provide complete control (Cherry
& Stansly 2007). Fallow flooding has proven to be
very effective, but even flooding requires that the
water be above a certain temperature (22C) and
must last for 6 weeks (Hall & Cherry 1993).
Planting time and varietal selection can also af-
fect the amount of damage that is incurred. Plant-
ing soon after oviposition may reduce damage be-
cause small wireworms are less likely to damage
germinating sugarcane and this time period cor-
responds with a warmer time of the year which
greatly speeds germination and emergence (In-
gram et al. 1950). Adult M. communis are most
numerous during Apr to Aug with peak oviposi-
tion in May to Jun (Cherry & Hall 1986). Applica-
tion of insecticide soon after peak oviposition may
be more effective because young wireworms may
be more susceptible to insecticides (Genung
1972). However, this tactic is of little use in most
of the sugarcane growing region as it is too wet to
plant until Oct. Certain varieties avoid damage
by germinating quickly (Hall 2001). Stand losses
are greatest when wireworms eat the buds before
germination (Hall 1985).
Only 2 insecticides, ethoprop and phorate, are
currently registered for wireworm control in Flor-
ida sugarcane. Current chemicals will not kill ev-
ery wireworm (Nuessly et al. 2007). Stand was re-
ported to decline by 7% per wireworm per 1.5 m of
row and yield was reported to decline by 3.8% per
wireworm per 1.5 m of row (Hall 1990). While
Hall (2001) reported that the threshold for treat-
ment might be as low as 2610 wireworms per
hectare, current studies indicate that this level of
infestation may be well below what is needed to
cause economical damage (N.A.L., unpublished
data). Even in studies by Hall (1990), 17,600 to
35,200 wireworms per hectare were required to
produce statistically significant stand and yield
loss. Sugarcane has the ability to compensate for
stand loss by tillering, with certain varieties
tillering more profusely than others; therefore,
sugarcane can withstand some degree of early
stand loss.


Harris (1972) outlines how pesticide biological
activity in the soil is affected by a number of
chemical and environmental factors. Chemical
factors that affect the biological activity of an in-
secticide include pesticide toxicity to the target
organism, insecticide volatility, insecticide half-
life, solubility of the insecticide, and adsorption
characteristics of the insecticide. Soil factors that
affect pesticide effectiveness are texture, struc-
ture, organic matter, moisture, pH, and tempera-
ture. A soil-dwelling insect must come into con-
tact with a toxic dose of insecticide to be killed.
Most (75%) sugarcane in Florida is grown on
histosols (referred to as muck soils), while the re-
mainder is grown in a mixture of spodosols,
alfisols, and entisols (referred to as sandy soils)
that are typical of the flatwoods of central and
southern Florida. Despite the obvious differences
between mucks and sandy soils, such as organic
matter content and water holding capacity, there
are no differences in recommended insecticide
rates for use on either soil. For example, the
Thimet 20G (phorate, AMVAC, Los Angeles, CA)
label recommends the use of 16.4 to 21.9 kg/hect-
are (14.6 to 19.5 lb/acre) and the Mocap 20G
(ethoprop, Bayer CropScience, Research Triangle
Park, NC) label recommends 16.8 to 22.4 kg/hect-
are (15 to 20 lb/acre), regardless of soil type.
Cherry & Raid (1999) found that it took 7 times as
much chemical to kill wireworms in a muck soil
versus a sandy soil. The only other guidance given
for insecticide use is that the applicator should
use the lower rate for lighter wireworm infesta-
tions. Many growers do not treat sugarcane grow-
ing on sandy soil due to the belief that wireworms
are not problematic on sandy soils; however,
Cherry & Stansly (2007) reported that while
there were fewer wireworms in sandy soils, they
did occasionally reach populations high enough to
warrant treatment.
Based on the characteristics of the insecticides
in Table 1, phorate is more tightly bound to soil,
has a longer half-life, and is less soluble than
ethoprop. Phorate should be less available to the
insect and less prone to leaching, while ethoprop
should be more available to the insect and more
prone to leaching. Given their similar toxicities,
one would expect ethoprop to be a more effective
insecticide for wireworm control. However,


TABLE 1. CHARACTERISTICS OF PESTICIDES USED FOR WIREWORM CONTROL IN FLORIDA SUGARCANE.

Name and Active Half-life' Solubility' Toxicity (LD50)2
formulation ingredient (days) Koc'(mg/kg) (mg/L) [ig/wireworm)

Thimet 20G phorate 60 1000 22 0.64-1.03
Mocap 20G ethoprop 25 70 750 0.44-2.00

Vogue et al. 1994.
'Cherry & Hall 1985.


December 2009







Larsen & Nuessly: Reduced Insecticide Rates for Wireworms


Cherry & Raid (1999) determined that phorate
was the more effective insecticide. The purpose of
this research was to evaluate the effectiveness of
reduced rates of ethoprop and phorate in killing
wireworms and reducing damage to sugarcane in
soils typical for Florida sugarcane production.

MATERIALS AND METHODS

Simulated field experiments were used to eval-
uate insecticides for wireworm control and sugar-
cane protection in different soil types, because
uniform natural infestations in commercial or ex-
perimental fields could not be relied upon. Artifi-
cially infesting a natural field with 3.33 wire-
worms per linear meter of row did not cause
enough damage to be detected (N.A.L., unpub-
lished data). It is possible that a greater level of
infestation may have caused recognizable dam-
age; however, the sheer quantity of wireworms re-
quired for such an experiment (3000-5000 wire-
worms) could not be procured. A simulated field
experiment done in a greenhouse allowed for reg-
ulation of several otherwise uncontrollable vari-
ables such as temperature, the number of stalks
and buds, soil moisture, and the number of wire-
worms. Experimental units for the tests were
18.9-L (5-gal) buckets filled with the specific soil
type and planted with sugarcane variety CP89-
2143 seed pieces. Sugarcane stalks were har-
vested at the EREC 3 d prior to setting up each
experiment. Seed pieces with live, undamaged
buds (eyes) were taken from the center third of
each stalk. Buckets were infested with M. com-
munis larvae collected from sugarcane fields
within the Everglades Agricultural Area by over-
turning stools between Oct and Jan. Collected lar-
vae were maintained in buckets of muck soil on a
carrot diet within an insectory room at 27C and
14:10 L:D h photoperiod until used.

Soil and Rate Interaction Experiment

The experiment was conducted within a fan
and pad cooled greenhouse with temperature
loosely maintained between 20 and 30C from 12
Feb 2007 to 13 Apr 2007 in a histosol and spodo-
sol. The histosol used was Dania Muck (Euic, hy-
perthermic, shallow Lithic Haplosaprists, pH 7.4,
65% organic matter) and the spodosol used was
Immokalee Fine Sand (Sandy, siliceous, hyper-
thermic Arenic Alaquods, pH 7.8, 1.9% organic
matter). Phorate (Thimet 20G) at 62% to 100% of
the maximum label rate: 13.6, 16.4, 19.2, and 21.9
kg/hectare (12.1, 14.6, 17.1, and 19.5 lb/acre), a
no-chemical control and a no-wireworm control
were tested. To set up the experimental units, soil
was first added to each bucket and compacted to
field density. This was accomplished by adding
7.25 kg of Dania Muck or 13.36 kg of Immokalee
Fine Sand to the bucket and then tamping and


packing the soil to 17.8 cm below the rim of each
bucket to achieve field bulk densities of 0.76 g/cm3
and 1.55 g/cm3, respectively. Ten late-instar M.
communis larvae (>1.75 cm) were then added to
the soil in each bucket 2 d prior to planting the
sugarcane seed pieces. Seed pieces were im-
mersed in hot water (40C) for 30 min immedi-
ately before planting to treat for pineapple dis-
ease (Ceratocystis paradoxa) and red rot disease
(Glomerella tucumanensis). Three seed pieces 24-
29 cm long, with 2 to 3 nodes per seed piece (total
of 7 to 8 nodes per bucket) were placed in the
buckets and then the granular insecticide was ap-
plied. Additional soil was added over the seed
pieces and compacted to field bulk density 2.5 cm
from the lip of the bucket so that the planting
depth was 15.2 cm. Ten buckets were set up for
each of the treatments.
Shoot counts were conducted weekly after
shoot emergence to keep track of dead hearts,
damaged shoots, and healthy shoots. After 60 d,
plant height was measured from the soil surface
to the top visible dewlap of the tallest shoot in
each bucket. The buckets were then upended and
the soil in each bucket extensively searched for
wireworm larvae, pupae, and adults. Plants were
examined for wireworm-damaged seed pieces,
eyes, and shoots. An eye, shoot, or tiller was
counted as damaged if there was evidence of wire-
worm feeding. Seed pieces were each rated on the
following rating scale: 0 = no damage, 1 = surface
feeding only, 2 = 1 hole in the seed piece, 3 = 2
holes in the seed piece, and 4 = 3 or more holes in
the seed piece. After examination, plants were
dried and dry weights were measured for roots,
shoots, and seed pieces.

Chemical and Rate Interaction Experiment

Wireworm control and sugarcane damage was
compared between 24 Jan 2008 and 24 Mar, 2008
at 25, 50, 75, and 100% of the maximum labeled
rates for phorate (Thimet 20G) and ethoprop
(Mocap 20G) in a Lauderhill Muck (euic, hyper-
thermic shallow Lithic Haplosaprist, pH 6.2, 60%
organic matter). Ten treatments were evaluated,
including phorate at 5.5, 10.9, 16.4, and 21.9 kg/
ha (4.9, 9.8, 14.6, and 19.5 lb/acre), ethoprop at
5.6, 11.2, 16.8, and 22.4 kg/ha (5, 10, 15, 20 lb/
acre), a no-chemical control, and a no-wireworm
control. Experimental units were set up as before,
except that seed pieces were treated immediately
prior to planting in an attempt to reduce infection
by pineapple disease and red rot disease by soak-
ing for 1 h in a 0.59% solution of propiconozole
(Tilt, Syngenta, Greensboro, NC). Ten buckets
were set up for each treatment.
In the soil and rate interaction experiment,
soil type, chemical rate, and the interaction of
these factors were modeled to evaluate their ef-
fects on seed piece damage rating, wireworm







Florida Entomologist 92(4)


survival, and percentage stand loss. Analysis of
variance was conducted and least squared
means were generated with JMP 6 (SAS Insti-
tute 2005). Treatment rate of each chemical was
modeled by regression analysis to evaluate its ef-
fect on seed piece damage rating, wireworm sur-
vival, and percentage stand loss in the chemical
and rate interaction experiment. An LSD test
was used for means comparisons where ANOVA
determined that a factor was a significant source
of model variation. The no-wireworm control
data were removed from all statistical analyses
evaluating wireworm control since there was no
variation.

RESULTS AND DISCUSSION

Soil and Rate Interaction

Numbers of surviving M. communis were not
significantly affected by soil type (df = 1, 8; F =
3.4021;P = 0.0684). While all treatments reduced
M. communis numbers significantly, none were
able to completely eliminate M. communis (Table
2). There was not an apparent rate response in
the Dania Muck as the lowest rate was statisti-
cally equivalent to the highest rate. While M.
communis numbers did decline with increased
rates of insecticide, the decline was not significant
(F = 2.9477; P = 0.0941; r2 = 0.0720). The soil and
chemical interaction term was not significant (df
= 4, 8; F = 0.8093; P = 0.5224).


Seed piece damage ratings (Table 3) were lower
in the Immokalee Fine Sand than in the Dania
Muck (df= 1, 8; F = 12.36; P = 0.0007). While there
was separation among the various rates of phorate
in the Dania Muck, there was no clear rate re-
sponse as the lowest rate was statistically equiva-
lent to the highest rate (F = 2.4503; P = 0.1258; r2
= 0.06). The soil and insecticide interaction was not
significant (df = 4, 8; F = 1.3049; P = 0.2742).
Percentage stand loss (Table 4) was similar in
the Dania Muck and the Immokalee Fine Sand (df
= 1, 8; F = 3.194; P = 0.0774). The addition of phor-
ate reduced percentage stand loss in both soils. In
both the Dania Muck (F = 8.8596; P = 0.0051; r2 =
0.1890) and the Immokalee Fine Sand (F = 6.8608;
P = 0.0126; r2 = 0.15), a rate response to increasing
rates of phorate was apparent with decreasing
rates of phorate resulting in increasing percentages
of percentage stand loss. Despite a significant re-
gression model for both soils, the model only ex-
plained a small fraction of the variability. In the
Dania Muck the mean decrease in stand loss per
2.8 kg/ha increase in rate was 3.5%. In the
Immokalee Fine Sand, the rate response was
dampened and leveled off. The increase from 13.6
kg/ha to 16.4 kg/ha in the Immokalee Fine Sand de-
creased percentage stand loss by 52%, the increase
from 16.4 kg/ha to 19.2 kg/ha decreased percentage
stand loss 57%. The final increase from 19.2 kg/ha
to 21.9 kg/ha only resulted in a further 38% reduc-
tion. The soil and insecticide interaction was not
significant (df = 4, 8; F = 0.2919; P = 0.8826).


TABLE 2. MEAN ( SEM) NUMBER OF SURVIVING WIREWORMS PER BUCKET 60 D AFTER PLANTING.

Treatment Rate (kg/ha) Dania Muck Immokalee Fine Sand

no chemicals 0 9.2 + 0.2 A 9.3 + 0.5 A
phorate 13.6 4.4 0.4 B 5.0 0.5 B
phorate 16.4 3.1 0.4 C 4.2 0.5 B
phorate 19.2 2.9 0.6 C 4.1 0.6 B
phorate 21.9 3.9 0.4 BC 3.7 0.5 B
df 4, 45; F = 36.43; P< 0.0001 df 4,45;F = 19.21; P < 0.0001

Means within a column followed by the same letter are not significantly different (LSD, P < 0.05).



TABLE 3. MEAN ( SEM) SEED PIECE DAMAGE RATING PER BUCKET 60 D AFTER PLANTING.

Treatment Rate (kg/ha) Dania Muck Immokalee Fine Sand

no chemicals 0 2.0 + 0.2 A 1.6 + 0.3 A
phorate 13.6 0.5 0.3 C 0.1 0.1 B
phorate 16.4 1.0 0.2 B 0.2 0.1 B
phorate 19.2 0.6 0.2 BC 0.1 0.1 B
phorate 21.9 0.1 0.1 C 0.1 0.1 B
df 4, 45; F = 13.55; P< 0.0001 df 4, 45; F = 17.37; P < 0.0001

Means within a column followed by the same letter are not significantly different (LSD, P < 0.05).


December 2009









Larsen & Nuessly: Reduced Insecticide Rates for Wireworms


TABLE 4. MEAN ( SEM) PERCENTAGE STAND LOSS PER BUCKET 60 D AFTER PLANTING.

Treatment Rate (kg/ha) Dania Muck Immokalee Fine Sand

no chemicals 0 55.7 6.5 A 47.6 6.6 A
phorate 13.6 12.0 4.2 B 7.7 3.4 B
phorate 16.4 8.0 2.2 B 3.7 1.4 B
phorate 19.2 4.8 1.8 B 1.6 1.1 B
phorate 21.9 1.5 1.5 B 1.0 0.7 B
df 4, 45; F= 35.42; P< 0.0001 df 4, 45; F = 32.44; P < 0.0001

Means within a column followed by the same letter are not significantly different (LSD, P < 0.05).


Chemical and Rate Interaction

All ethoprop and phorate treatments had sig-
nificantly fewer surviving wireworms than the
untreated check (Table 5). Only the high rate of
ethoprop performed as well as the phorate treat-
ments. Ethoprop did show a significant rate re-
sponse with progressively higher rates resulting
in fewer surviving M. communis (F = 26.2909; P <
0.0001; r2 = 0.4089). There was a 20-40% drop in
wireworm mortality with each 5.5 kg/ha reduc-
tion in ethoprop rate. There was no rate response
with phorate, and all rates dropped the surviving
M. communis counts to around one per bucket (F
= 0.0383; P = 0.8459; r2 = 0.0010).
Phorate application resulted in a mean seed
piece damage rating of 0.16, which was signifi-
cantly lower (df = 1, 78; F = 27.068; P < 0.0001)
than ethoprop which had an average seed piece
rating of 0.95. The 22.4 kg/ha rate of ethoprop did
as well as all rates of phorate (Table 5). Ethoprop
showed a distinct rate response with decreased
rates leading to increased seed piece damage (F =
47.1636; P = < 0.0001; r2 = 0.5538). There was a
233% increase in the seed piece damage rating be-


tween the 11.2 kg/ha and 5.6 kg/ha ethoprop
rates. Phorate did not show a rate response for
seed piece damage at the rates tested (F = 1.5024;
P= 0.2278; r2 = 0.0380).
Percentage stand loss is a measure of the per-
centage of damaged eyes, shoots, and tillers.
Phorate had less stand loss than ethoprop (Table
5). There was a rate response for ethoprop with
increases in stand loss equal to or greater than
50% for each 5.5 kg/ha reduction below the 16.8
kg/ha rate (F = 24.4167; P< 0.0001; r2 = 0.3912). A
similar rate response was not detected for phorate
(F = 3.0363; P =0.0895; r2 = 0.0740).

DISCUSSION

Soil type, chemical, and rate interact to deter-
mine the efficacy of the insecticide. The toxic
doses of both phorate and ethoprop to M. commu-
nis were determined previously by Cherry & Hall
(1985); however, little research has been done to
investigate how the soil interacts with phorate
and ethoprop to eventually deliver a toxic dose.
The results of the soil-rate interaction experiment


TABLE 5. MEAN ( SEM) DATA FOR SURVIVING M. COMMUNIS, SEED PIECE DAMAGE RATING, AND PERCENTAGE STAND
LOSS PER BUCKET 60 D AFTER PLANTING IN LAUDERHILL MUCK.

Rate Surviving Seed piece Percentage
Treatment (kg/ha) M. communis damage rating' stand loss

no chemicals 0.0 8.4 + 0.2 A 2.8 + 0.1 A 60.0 + 5.5 A
ethoprop 5.6 5.8 0.3 B 2.1 0.3 B 40.5 4.5 B
ethoprop 11.2 3.5 0.4 C 0.9 0.2 C 24.1 6.3 C
ethoprop 16.8 2.8 0.3 CD 0.6 + 0.1 CD 14.9 2.8 CD
ethoprop 22.4 1.9 0.3 DE 0.3 0.1 DE 11.8 2.3 DE
phorate 5.5 1.0 + 0.1 E 0.3 0.1 DE 4.9 1.5 EF
phorate 10.9 1.1 + 0.2 E 0.0 0.0 E 4.2 1.8 EF
phorate 16.4 1.1 + 0.3 E 0.3 0.2 DE 1.6 0.8 F
phorate 21.9 0.9 0.2 E 0.0 0.0 E 2.2 1.2 EF
df8, 81;F = 34.30; df8, 81; F = 44.10; df8, 81; F = 32.59;
P<0.0001 P<0.0001 P<0.0001

Means within a column followed by the same letter are significantly different (LSD, P < 0.05).
'Seed piece damage rating scale: 0 = no damage, 1 = surface feeding only, 2 = 1 hole in the seed piece, 3 = 2 holes in the seed piece,
and 4 = 3 or more holes in the seed piece.







Florida Entomologist 92(4)


suggest that a reduced rate of phorate is effective
at delivering a toxic dose of insecticide in both
muck and sand soils. In the model, the soil and in-
secticide rate interaction term was never signifi-
cant for any of the response variables. There were
similar levels of mortality in both soils even though
the seed piece damage rating and percentage stand
loss in the Immokalee Fine Sand were numerically
lower. This may suggest that activity was more
rapid in the Immokalee Fine Sand or perhaps the
chemical diffused through the sandy soil causing
mortality before damage could be done.
In the chemical-rate interaction experiment, a
wider range of rates were used to better detect a
rate response. All rates of phorate caused mortal-
ity of nearly 90%, which was much higher than
the observed mortality in the soil-rate interaction
experiment where mean mortality was only
around 60% across all soil types and treatments.
In the soil-rate experiment, many M. communis
larvae pupated and finished development. In the
soil-rate experiment more pupae and adults were
found than in the chemical and rate experiment,
so it is possible that some larvae were not ever ex-
posed to the insecticide. The soil pH in the chem-
ical and insecticide rate interaction experiment
was lower (Lauderhill Muck, 6.2) than both of the
soils in the soil and insecticide rate interaction ex-
periment (Dania Muck 7.8; Immokalee Fine
Sand, 7.2). Harris (1972) indicated that some soil
insecticides may have reduced activity in a high
pH environment due to alkaline hydrolysis.
None of the rates ofethoprop caused 90% mortal-
ity and decreasing rates caused increased survivor-
ship. Despite similar levels of toxicity, ethoprop was
less able to kill M. communis. The chemical charac-
teristics indicate that phorate will be bound to the
soil and inactivated more effectively than ethoprop;
however, mortality numbers indicate that perhaps
something more than binding characteristics were
at play. Phorate does have a longer half-life than
ethoprop, so it is possible that a lethal dose was
available for a longer time. These experiments do
suggest that the label rates for phorate may be su-
praoptimal for controlling wireworms in sugarcane.

ACKNOWLEDGMENT
We thank Bijeita Thapa, Rosa Innocent, Kimberly
Notarian, Oleides Mendez, Hardev Sandhu, and
Gaurav Goyal for help in collecting wireworms, cutting
and transporting sugarcane stalks, and experiment set
up. The Immokalee Fine Sand used in the experiment
was provided and transported to EREC from Clewiston
by the United States Sugar Corporation. This work was
made possible by financial support fromAMVAC Chem-
ical Corporation (Los Angeles, CA) and the Sugar Cane
Growers Cooperative of Florida.

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December 2009




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