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Publication Date: 2001
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Bloem & Carpenter: Population suppression by irradiated Lepidoptera


EVALUATION OF POPULATION SUPPRESSION
BY IRRADIATED LEPIDOPTERA AND THEIR PROGENY

STEPHANIE BLOEM1 AND JAMES E. CARPENTER2
1USDA-APHIS-PPQ-CPHST-NBCI, at University of Florida, NFREC, Monticello, FL 32344

2USDA-ARS-Crop Protection and Management Research Unit, Tifton, GA 31793


Lepidopteran species are among the most im-
portant pests of major annual and perennial
crops, forests, and stored products throughout the
world. More than 25% of the species that appear
on a list of the 300 most important exotic insects
that threaten the United States are in the order
Lepidoptera (ESA 2001). In a supplement to that
list, where the 30 most serious threats to Agricul-
ture are named, 50% of the species are lepidopter-
ans (ESA 2001). Unfortunately, control of lepi-
dopteran pests worldwide is achieved almost en-
tirely through the use of synthetic insecticides.
This dependence on insecticides has contributed
to the development of insecticide resistance in
many of the most serious pests. Relevant exam-
ples include the codling moth, Cydia pomonella
(Lepidoptera: Tortricidae) (Varela et al. 1993) and
the diamondback moth, Plutella xylostella (Lepi-
doptera: Plutellidae) (Shelton et al. 1993), where
resistance has developed even against the micro-
bial insecticide Bacillus thuringiensis (Tabash-
nick et al. 1990). Heavy reliance and frequent
indiscriminant use of pesticides also has had a
significant negative impact on the environment.
Of particular importance to agriculture is the de-
struction of crop pollinators and natural enemies
that keep secondary pests in check (Edwards
2000). Development of alternative tactics to the
unilateral use of insecticides is a major emphasis
of most local, national and international research
organizations concerned with pest control.

The Sterile Insect Technique and Lepidoptera

Genetic pest suppression is unique among bio-
logical methods in that it involves the release of
genetically modified insects to control the same
species (LaChance 1985). Sterile Insect Tech-
nique (SIT) programs have been quite successful
against a number of pest Diptera (including the
screwworm fly, Cochliomyia hominivorax, and the
Mediterranean fruit fly, Ceratitis capitata), and
numerous mass rearing facilities have been con-
structed worldwide to support these programs.
However, compared to dipterans, lepidopterans
generally are more expensive to rear and have a
propensity to fly greater distances. Additionally,
lepidopterans are more resistant to the effects of
ionizing radiation than dipterans. As a conse-
quence, the greater amount of radiation required
to completely sterilize lepidopterans negatively
impacts their competitiveness and performance


in the field. Nevertheless, two SIT programs are
currently operating against pest Lepidoptera,
namely the pink bollworm, Pectinophora gossyp-
iella (Saunders), program in the USA (Staten et
al. 1993), and the codling moth program in Can-
ada (Dyck et al. 1993; Bloem & Bloem 2000), and
both of these programs have been very successful.
One approach to reduce the negative effects of
radio-resistance in Lepidoptera has been the use
of inherited or F, sterility. Proverbs & Newton
(1962) first documented F, sterility during the
course of their studies on the codling moth. Sub-
sequently, investigators have reported F, sterility
in many lepidopteran species of economic impor-
tance (LaChance 1985). Like SIT, F, sterility in-
volves the mass rearing and release of genetically
altered insects to insure that when matings occur
in the field, a significant proportion of matings in-
volve a treated, released insect. However, F, ste-
rility takes advantage of two unique genetic
phenomena in Lepidoptera. First, lepidopteran
females generally are much more sensitive to ra-
diation than are males of the same species. This
may allow the dose of radiation to be adjusted so
that treated females are completely sterile and
males are partially sterile. Second, when these
partially sterile males mate with fertile females
the radiation-induced deleterious effects are in-
herited by the F, generation. As a result, egg
hatch is reduced and the resulting (F1) offspring
are both highly sterile and predominately male.
The lower dose of radiation used in F, sterility in-
creases the quality and competitiveness of the re-
leased insects (North 1975). In addition, because
F1 sterile progeny are produced in the field, the re-
lease of partially sterile insects offers greater sup-
pressive potential than the release of fully sterile
insects (LaChance 1985) and is more compatible
with other pest control mechanisms or strategies
(Carpenter 1993).
Knipling (1970) explored the theoretical appli-
cation of F, sterility for control of lepidopteran
pests. Using mathematical models, he suggested
that when releasing partially sterile insects, the
sterile-to-wild overflooding ratio could be as low
as 14 of what is normally required for fully sterile
insects. Population models developed by other re-
searchers using data collected from several pest
species (Carpenter 1993; Anisimov 1998) corrobo-
rate Knipling's findings.
Field releases of partially sterile insects have
demonstrated the potential of using F, sterility to







Florida Entomologist 84(2)


control many lepidopterans, including the cab-
bage looper, Trichoplusia ni (North & Holt 1969),
the corn earworm, Helicoverpa zea (Carpenter et
al. 1987; Carpenter & Gross 1993), the gypsy
moth, Lymantria dispar (Mastro 1993) and the
codling moth, Cydia pomonella (Proverbs et al.
1978; Bloem et al. 1999b; Bloem et al. 2001). In
addition, many studies have shown that F, steril-
ity can be effectively combined with other biolog-
ical controls such as pheromone disruption
(Bloem et al. 2001), entomopathogens (Hamm &
Carpenter 1997), host plant resistance (Carpen-
ter & Wiseman 1992a, b) and natural enemies
(Carpenter et al. 1996; Greany & Carpenter 1999).
As a result of these many studies, F, sterility is
regarded as the most favorable genetic method
for most applications against Lepidoptera (for a
review, see Carpenter & Bartlett 1999).

The FAO/IAEA Sponsored
Coordinated Research Programs (CRP's)

The Joint Division of Nuclear Techniques in
Food and Agriculture of the Food and Agriculture
Organization (FAO) and the International Atomic
Energy Agency (IAEA) promotes agricultural de-
velopment through the peaceful use of atomic en-
ergy. This mission is accomplished through their
Technical Cooperation Projects, Coordinated Re-
search Projects, publications, and meetings and
training courses. In response to the recommenda-
tions of a group of consultants that met at the
IAEA in Vienna in 1984, the Insect and Pest Con-
trol sub-program of the Joint FAO/IAEA Division
designed and initiated the first five-year (1987-
1991) Coordinated Research Program (CRP) on
"Radiation Induced F, Sterility in Lepidoptera for
Area-Wide Control." Research by CRP participat-
ing scientists focused largely on modeling the ef-
fects of releasing partially sterile moths on the
field dynamics of feral populations, conducting
laboratory studies to evaluate the relationship
between radiation dose and sterility and conduct-
ing selected field-cage evaluations. Scientists
from ten countries participated in this CRP, and
the research results were published by the IAEA
in 1993 (Anonymous 1993). As a result of the re-
search progress during the CRP, the participants
recommended to the FAO/IAEA that a second Co-
ordinated Research Program should be consid-
ered which would emphasize field applications of
inherited or F, sterility for lepidopteran pests.
A second CRP entitled "Evaluation of Popula-
tion Suppression by Irradiated Lepidoptera and
Their Progeny" was therefore initiated in 1995
with the objective of assessing the potential for
controlling populations of pest Lepidoptera by re-
leasing irradiated moths and/or their progeny in
combination with other biological control meth-
ods. This CRP concluded in 1998. During this
time period, three Research Coordination Meet-


ings (RCM) were held to allow participants to dis-
cuss initial results and share ideas. The first RCM
was held in Jakarta, Indonesia (24-28 April,
1995), the second meeting was held in Vienna,
Austria (2-6 September, 1996), and the final
meeting was held in Penang, Malaysia (28 May-2
June, 1998) in conjunction with the FAO/IAEA
International Conference on "Area-Wide Control
of Insect Pests Integrating The Sterile Insect and
Related Nuclear and Other Techniques."
Twenty-five scientific teams from twenty-two
different countries (Bangladesh, China, Pakistan,
Myanmar, Syria, India, Java, Philippines, Mauri-
tius, Vietnam, Tunisia, Bulgaria, Romania, Czech
Republic, Russia, Ukraine, Iran, Austria, United
States, Brazil, Cuba and Canada) participated in
this second CRP. Participants conducted research
on important pests of annual and perennial crops
and stored-product pests (see Tables 1 and 2). The
research findings have been published in three
separate venues: as refereed publications in scien-
tific journals, as part of a Technical Document or
meeting proceedings published by the Interna-
tional Atomic Energy Agency, and as a block of four
manuscripts following this introductory article.
The manuscripts in this volume report important
research findings from four different countries and
on four different species. Ocampo (2001) describes
the effects of a substerilizing dose of gamma radia-
tion (100 Gy) on the mating competitiveness and
mating propensity of the Old World cotton boll-
worm, Helicoverpa armigera, in the Philippines.
Seth & Sharma (2001b) report on the effects of dif-
ferent doses of radiation on the common cutworm,
Spodoptera litura reared on two different diets in
India. Koudelova & Cook (2001) examine the com-
petitiveness of mutant and irradiated males of the
Mediterranean flour moth, Ephestia kuehniella, in
the laboratory by counting eupyrene (fertile) and
apyrene (non-fertile) sperm transferred to the fe-
male during copulation. Finally, Nguyen Thi &
Nguyen Thanh (2001) report on the potential of
combining F, sterility and the parasitoid Cotesia
plutellae in a system to manage the diamondback
moth, Plutella xylostella, in Vietnam.

Major Findings and Impact of the Research
Conducted During the F, Sterility CRP

The research conducted during this CRP re-
vealed principles that were common to all species
studied. These can be summarized into two major
points: (1) F, sterility is an effective and environ-
mentally safe tactic for lepidopteran pest sup-
pression that is useful under a variety of
environments and crop production strategies. (2)
F1 sterility is compatible with all pest control tac-
tics. The combination of F, sterility with phero-
mones, natural enemies, host plant resistance,
entomopathogens and insecticides results in syn-
ergistic pest population suppression.


June 2001







Bloem & Carpenter: Population suppression by irradiated Lepidoptera


TABLE 1. LISTING OF THE SPECIES OF LEPIDOPTERA INVESTIGATED BY PARTICIPANTS OF THE FAO/IAEA RESEARCH
COORDINATED PROGRAM ON EVALUATION OF POPULATION SUPPRESSION BY IRRADIATED LEPIDOPTERA AND
THEIR PROGENY.


Family


Species


Common name


Crop


Reference


Noctuidae Spodoptera litura

Spodoptera exigua
Spodoptera frugiperda


Helicoverpa armigera





Helicoverpa zea


Common cutworm or
Tobacco caterpillar
Beet armyworm
Fall armyworm


Corn earworm or
Cotton bollworm
(Old World)



Corn enrworm


Gelechiidae Pectinophora gossypiella Pink bollworm
Diatraea saccharalis Sugarcane borer


Tortricidae Cydia pomonella





Cydia molesta
Pyralidae Ectomyelois ceratoniae

Ephestia kuehniella


Crocidolomia binotalis
Chilo suppressalis
Crambidae Ostrinia furnacalis
Ostrinia nubilalis

Plutellidae Plutella xylostella







Arctiidae Spilosoma obliqua


Codling moth





Oriental fruit moth
Carob or date moth

Mediterranean flour
moth

Cabbage webworm
Asian rice stem borer
Asian corn borer
European corn borer

Diamondback moth


Soybeans, sorghum,
corn, vegetables
Cotton, vegetables
Forage grass, corn


Corn, cotton, tomato





Corn, cotton, tomato


Cotton
Sugarcane


Pome fruit, walnut





Stone & pome fruit
Date, carob, pome-
granate
Stored grain


Crucifer vegetables
Rice
Corn
Corn

Crucifer vegetables


Hairy jute caterpillar Jute


Seth et al. 1997
Seth & Sharma 2001a, b
Carpenter et al. 1996
Carpenter et al. 1997
Proshold et al. 1998
Arthur et al. 2001
Pham Van et al. 1996
Ocampo et al. 2000
Rimas et al. 2000
Ocampo 2001
Lu et al. 2001
Ocampo & deLeon 2001
Mannion et al. 1995
Hamm & Carpenter 1997
Proshold et al. 1998
Ahmad et al. 2001a, b
Garcia & Garcia 1996
Arthur et al. 2001
Anisimov & Shvedov
1996
Chernyi et al. 1996
Bloem et al. 1999a, b,
2001
Mansour 2001
Genchev 1996, 2001
Dhouibi & Abderahmane
2001
Matolin & Marec 1998
Marec et al. 1999
Koudelova & Cook 2001
Sutrisno Apu 2001
Esmaili et al. 1996
Wang et al. 2001
Chernyi et al. 1996
Rosca & Barbulescu 1996
Dunhawoor 1996
Okine et al. 1998
Mitchell et al. 1999
Nguyen Thi & Nguyen
Thanh 2001
Sutrisno Apu 2001
Yang et al. 2001
Maung 2001
Rahman et al. 2001


This CRP also highlighted several areas that
would benefit from further research and develop-
ment to increase the economic viability of F, ste-
rility programs. Development of diets using
locally available ingredients would reduce rear-
ing costs, especially in locations with developing
economies. Improvements in mass rearing are
needed to take advantage of the economy of scale
as evidenced in dipteran SIT programs. Develop-
ment of genetic sexing techniques, especially


those that would eliminate females at the egg or
early larval stage, would reduce rearing costs,
would increase the efficiency of rearing, irradia-
tion and release by 100% and would eliminate as-
sortative mating of released moths in the field.
The FAO/IAEA sponsored CPR's have had ma-
jor impacts in the direction of future research for
the control of lepidopteran pests. For example, ex-
panded research and implementation programs
on F1 sterility in combination with natural ene-







Florida Entomologist 84(2)


June 2001


TABLE 2. LISTING OF THE TYPE OF STUDIES CONDUCTED AND SPECIES INVESTIGATED BY PARTICIPANTS DURING THE
FAO/IAEA RESEARCH COORDINATED PROGRAM ON EVALUATION OF POPULATION SUPPRESSION BY IRRADI-
ATED LEPIDOPTERA AND THEIR PROGENY.


Species and Reference


Diet Development and Insect Rearing






Radiation Biology










Genetics and Genetic Sexing

Effects of Radiation on Sperm Development and
Sperm Competitiveness

Mating Competitiveness of Irradiated Insects
and Their Progeny






Compatibility of F, Sterility with Other Control
Strategies








Use of F, Sterility to Enhance Parasitoids


Field Releases for Suppression





Population Models


Chilo suppressalis (Esmaili et al. 1996)
Cydia molesta (Genchev 2001)
Helicoverpa armigera (Lu et al. 2001; Ocampo et al. 2000,
Pham Van 1996)
Pectinophora gossypiella (Ahmad et al. 200 1b)
Spilosoma obliqua (Rahman et al. 2001)
Spodoptera litura (Seth & Sharma 2001a)
Cydia pomonella (Chernyi et al. 1996; Bloem et al. 1999a;
Mansour 2001)
Diatraea saccharalis (Arthur et al. 2001)
Ectomyelois ceratoniae (Dhouibi & Abderahmane 2001)
Helicoverpa armigera (Lu et al. 2001; Ocampo & de Leon 2001)
Ostrinia nubilalis (Chernyi et al. 1996)
Pectinophora gossypiella (Ahmad et al. 200 1b)
Plutella xylostella (Nguyen Thi & Nguyen Thanh 2001)
Spilosoma obliqua (Rahman et al. 2001)
Spodoptera frugiperda (Arthur et al. 2001)
Spodoptera litura (Seth et al. 1997; Seth & Sharma 2001b)
Ephestia kuehniella (Koudelova & Cook 2001; Marec 1998;
Marec et al. 1999; Matolin & Marec 1998)
Helicoverpa armigera (Rimas et al. 2000)
Ostrinia furnacalis (Wang et al. 2001)
Spodoptera frugiperda (Carpenter et al. 1997)
Cydia pomonella (Bloem et al. 1999b, Mansour 2001)
Cydia molesta (Genchev 1996)
Crocidolomia binotalis (Sutrisno Apu 2001)
Diatraea saccharalis (Garcia & Garcia 1996)
Helicoverpa armigera (Ocampo 2001)
Ostrinia furnacalis (Wang et al. 2001)
Plutella xylostella (Nguyen Thi & Nguyen Thanh 2001;
Sutrisno Apu 2001; Yang et al. 2001)
Cydia pomonella (Bloem et al. 2001)
Helicoverpa zea (Hamm & Carpenter 1997; Mannion et al.
1995)
Pectinophora gossypiella (Ahmad et al. 2001a, b)
Plutella xylostella (Sutrisno Apu 2001; Maung 2001;
Okine et al. 1998; Dunhawoor 1996)
Ostrinia furnacalis (Wang et al. 2001)
Ostrinia nubilalis (Rosca & Barbulescu 1996)
Spodoptera exigua (Carpenter et al. 1996)
Spodoptera frugiperda (Hamm & Carpenter 1997)
Helicoverpa zea (Proshold et al. 1998)
Plutella xylostella (Mitchell et al. 1999)
Spodoptera frugiperda (Proshold et al. 1998)
Cydia molesta (Genchev 2001)
Cydia pomonella (Bloem et al. 2001; Chernyi et al. 1996)
Ostrinia furnacalis (Wang et al. 2001)
Ostrinia nubilalis (Rosca & Barbulescu 1996)
Plutella xylostella (Nguyen Thi & Nguyen Thanh 2001;
Sutrisno Apu 2001; Yang et al. 2001; Maung 2001)
Cydia pomonella (Anisimov & Shvedov 1996; Anisimov 1998)
Plutella xylostella (Carpenter 2000)


mies are underway in Tunisia for suppression of
the carob moth, Ectomyelois ceratoniae, and on
the island of Mauritius for control of the diamond-
back moth, Plutella xylostella. F, sterility pro-


grams for other lepidopteran pest species also are
being considered. Furthermore, the research from
the F, sterility CRP's contributed to the develop-
ment of a new Coordinated Research Program on


Type of Study







Bloem & Carpenter: Population suppression by irradiated Lepidoptera


"Evaluating the Use of Nuclear Techniques for
the Colonization and Production of Natural Ene-
mies". This new CRP was initiated in October
1999, with scientific teams from fourteen coun-
tries. As one of the research objectives in this new
CRP, F, sterility is being developed as a tactic to
study possible lepidopteran biocontrol agents for
invasive noxious weeds (Carpenter et al. 2001).

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Florida Entomologist 84(2)


June 2001


EFFECT OF GAMMA RADIATION AND SEX-LINKED RECESSIVE LETHAL
MUTATIONS ON SPERM TRANSFER IN EPHESTIA KUEHNIELLA
(LEPIDOPTERA: PYRALIDAE)

J. KOUDELOVA1 AND P. A. COOK2
1Department of Genetics, Institute of Entomology, Branigovska 31, CZ-370 05 Cesk6 Budejovice, Czech Republic

2School of Health, Liverpool John Moores University, 79 Tithebarn Street, Liverpool L2 2ER, UK

ABSTRACT
Sperm quality and sperm competition play important roles in determining the efficiency of
genetic methods for suppression oflepidopteran populations. Herein we have examined com-
petitiveness of mutant and irradiated males of Ephestia kuehniella by counting eupyrene
(fertile) and apyrene (non-fertile) sperm transferred to the female during copulation. Mu-
tant BL-2 males, trans-heterozygous for two sex-linked recessive lethal mutations sl-2 and
sl-15, produced 50% fewer of both types of sperm as compared to with WT-C (wild-type)
males. However, the ratio of apyrene to eupyrene sperm remained the same in both male
types (9.5:1). Irradiation of mature male pupae, heterozygous for either sl-2 or sl-15 muta-
tions, with doses between 150 and 350 Gy showed dose-dependent effects on the amount of
sperm transferred and on the total length of mating times. As the treatment dose increased
the volume of sperm transferred by the male decreased and the mating times got longer. In
the F, descendants of the treated males, males were found to transfer either a relatively nor-
mal or a very small volume of sperm, which could reflect changes in gamete segregation and
in chromosomal aberrations that are inherited. The dose of 175 Gy is suggested as optimal
for irradiation of Ephestia kuehniella.

Key Words: irradiation, sperm transfer, eupyrene, apyrene, sperm ratio, sperm competition,
Ephestia kuehniella

RESUME
La calidad y la competitividad del esperma es de suma importancia en la determination de
la eficacia de los metodos geneticos destinados a la supresion de poblaciones de Lepidoptera.
En este articulo hemos examinado la competitividad de machos de una sepa mutante asi
como la competitividad de machos irradiados de la especie Ephestia kuehniella utilizando
como indicador el numero total de esperma nucleado (eupyreno) y ancleado (apyreno) que es-
tos machos transfieren durante la copula. Los machos de la sepa mutante (BL-2) que son
trans-heterocigotos para dos mutaciones letales recesivas ligadas al sexo (sl-2 y sl-15), pro-
dujeron 50% menos cantidad de los dos tipos de esperma que los machos de tipo salvaje (WT-
C). Sin embargo, la relacion entire la cantidad de esperma apyreno y eupyreno se mantuvo
constant en ambos tipos de macho (9.5:1). La irradiacion de pupas maduras, heterocigotas
para las mutaciones sl-2 o sl-15 con dosis entire 150 y 350 Gy mostro efectos dependientes de
la dosis en cuanto a la cantidad de esperma transferido y el tiempo que los machos perma-
necen en copula. A media que la dosis de irradiacion se incrementa, el volume de esperma
transferido decrece y el tiempo que los machos permanecen en copula aumenta. Asimismo,
se encontro que los descendientes de machos irradiados (F,) transfieren ya sea una cantidad
normal de esperma o una cantidad muy reducida de esperma lo cual indica que los cambios
en la segregacion de gametos en cromosomas irradiados son heredados. La dosis de 175 Gy
parece ser optima para la irradiacion de Ephestia kuehniella.


Substerilizing doses of radiation induce inher-
ited sterility in Lepidoptera (Anisimov et al. 1989;
Carpenter 1991), and the impact of gamma rays
at the chromosomal level is well known (Astaurov
& Frolova 1935; LaChance 1967; Carpenter 1991).
The effectiveness of radiation-induced inherited
sterility in the Mediterranean flour moth (Ephes-
tia kuehniella), an extremely radio-resistant spe-
cies, is enhanced by the use of a genetic sexing
technique (Marec & Mirchi 1990; Marec et al.
1999). However, when evaluating the potential of
inherited sterility for population suppression it is


important to quantify the competitiveness of the
released males.
In Lepidoptera, the amount of sperm trans-
ferred during mating may affect reproductive suc-
cess (Cook et al., in press), and as such has
important consequences in the success of a sterile
male release program. Females that receive in-
sufficient amounts of sperm during mating may
remate in order to secure enough sperm to fertil-
ize their complement of eggs. Even if females
have received enough sperm to fertilize the eggs,
they might be able to perceive that insufficient







Koudelova & Cook: Effect of radiation on sperm transfer in Ephestia kuehniella 173


sperm quantities were transferred during mat-
ing. In the Indian meal moth (Plodia interpunc-
tella), females that received smaller amounts of
sperm did not exhibit reduced fertility, even at the
end of their life (Cook 1999). Nonetheless, these
females were more likely to remate than females
receiving adequate sperm numbers (Cook & Gage
1995). In experiments with Plodia interpunctella,
where females were mated twice to males that
differed in the amount of sperm transferred Cook
et al. (1997) found that males that transferred
more sperm were able to fertilize a higher propor-
tion of the females eggs. Similar results were re-
ported by Wedell & Cook (1998) for the pierid
butterfly Pieris rapae. In accordance with the
sperm competition theory (Parker 1970), male
Lepidoptera have the capacity to increase sperm
numbers when sperm competition risk is high
(Cook & Gage 1995; Cook & Wedell 1996; Wedell
& Cook 1999a). This phenomenon has been re-
ported in other invertebrates (Gage 1991; Gage &
Baker 1991; Simmons et al. 1993) and verte-
brates (Baker & Bellis 1989; Bellis et al. 1990).
Several other factors affect sperm numbers.
For example, in P. interpunctella, males transfer
fewer sperm in their second and third matings
(Gage & Cook 1994), whereas males of P. rapae
transfer more sperm in the second when com-
pared to the first mating (Cook & Wedell 1996;
Wedell & Cook 1999b). Gage & Cook (1994) exam-
ined the effect of high versus low protein larval di-
ets on sperm number and size inP. interpunctella.
They found that the number but not the size of
sperm was reduced when male larvae suffered re-
source restrictions. However, in a study of suble-
thal viral larval infection in the same species,
Sait et al. (1998) found that sub-lethally infected
males, despite having lower fertility, did not have
significantly fewer or smaller sperm.
Lepidopterans produce two types of sperm
cells, nucleated eupyrene (Figs. 1, 2) and smaller,
anucleated apyrene sperm (Fig. 3). The two sperm
types result from two distinct modes of spermato-
genesis (Friedlander 1997), with apyrene out-
numbering eupyrene sperm by about ten to one
(Gage & Cook 1994; Cook & Wedell 1996, 1999;
Marec et al. 1996). Both types are transferred to
the female during copulation via the spermato-
phore and both reach the site of sperm storage,
the spermatheca. Apyrene sperm bundles dissoci-
ate and become motile prior to male ejaculation
while eupyrene sperm remain in bundles (Fig. 1).
Until recently, the function of apyrene sperm was
unknown although several hypotheses have been
postulated (reviewed by Silberglied et al. 1984).
Osanai et al. (1987, 1989) suggested that apyrene
sperm aid in the motility of eupyrene sperm bun-
dles inside the female. They characterized apy-
rene function as that of promoting dissociation of
eupyrene bundles and separation of individual
eupyrene sperm both mechanically and by bio-


r


Fig. 1. Eupyrene sperm bundle of Ephestia kueh-
niella taken from the spermatophore and seen in
Nomarski interference contrast using Aristoplan micro-
scope. Scale bar = 100 mm.


chemical degradation. It has also been suggested
that apyrenes play a role in sperm competition
(Silberglied et al. 1984; Cook & Gage 1995). Also,
there is good evidence that they protect a males
reproductive investment by delaying female re-
mating (Cook & Wedell 1999). Thus, apyrene
sperm, as well as eupyrene sperm, must be con-
sidered when investigating male reproductive
success.
In this study we examined the effects of muta-
tion and doses of gamma radiation on the number
of eupyrene and apyrene sperm transferred by
males ofE. kuehniella in the P and F, generations
in an effort to assess the sperm competitiveness of
the released males used in an F, sterility manage-
ment program for Ephestia kuehniella.

MATERIALS AND METHODS

Insects

Insects were reared and experiments carried
out in rooms held at constant temperature (25 +
1C), 12L:12D photoperiod and ambient relative


/







Florida Entomologist 84(2)


A'4' ' '- F ""




-J
I. %


Fig. 2. Eupyrene sperm of Ephestia kuehniella after
dissociation from the sperm bundle and seen in Nomar-
ski interference contrast using Aristoplan microscope.
Scale bar = 100 mm.



humidity. The Mediterranean flour moth, Ephes-
tia kuehniella Zeller (Lepidoptera: Pyralidae) has
been in laboratory culture since 1984 and the
strain is considered to be wild-type (WT-C) (for
details see Marec 1990). A balanced lethal strain,
BL-2, was constructed in 1990 (Marec 1991) and
has been reared since 1990 in single pair cultures.
BL-2 males are trans-heterozygous for two non-
allelic sex-linked recessive lethal mutations, sl-2
and sl-15. The genetic structure of the BL-2 strain
is described in Marec et al. (1999). The number
and ratio of eupyrene and apyrene sperm (see be-
low) and the total time in copula for BL-2 and WT-
C males mated to WT-C females was recorded in
the laboratory.
BL-2 males were individually mated to virgin
WT-C females one generation before irradiation.
Heterozygous males of two genotypes (sl-2 or sl-
15) were produced (both male types are desig-
nated here as sl/+), whereas female zygotes died
during embryogenesis (since they were hemizy-
gous for the lethal sl-2 or sl-15 mutations). Six to
eight day-old pupae were removed from their co-
coons and irradiated.


Fig. 3. Apyrene sperm of Ephestia kuehniella taken
from the spermatophore and seen in Nomarski interfer-
ence contrast using Aristoplan microscope. Scale bar =
100 mm.



Irradiation

Irradiation was performed at the Entomology
Unit of the FAO/IAEA Laboratories in Seibers-
dorf, Austria, using a Co60 Gammacell 220 (AE of
Canada Ltd.) with dose rates ranging between 50-
60 Gy/min. The sl/+ male pupae were placed in
plastic petri-dishes (5 cm diam.) In the first ex-
periment we examined the effect of dose on the P
generation. Pupae were treated with 150, 175,
200, 250 or 350 Gy of gamma radiation. In the
second experiment we examined the effect on the
F1 generation. A sixth dose was added in this ex-
periment (300 Gy). As a control for the irradia-
tion procedure, untreated sl/+ and WT-C males
were subjected to the same handling as irradiated
males. Pupae were placed in containers at the
conditions mentioned above and adults were col-
lected as they emerged.

Sperm Counts

Males were allowed to mate with WT-C virgin
females. For each mating, the total time in copula


June 2001



'+







Koudelova & Cook: Effect of radiation on sperm transfer in Ephestia kuehniella 175


and the number of eupyrene and apyrene sperm
transferred was recorded. Sperm were counted us-
ing the methods outlined in Gage & Cook (1994)
and Cook & Wedell (1996). Immediately after mat-
ing, the female was dissected and the spermato-
phore removed from the bursa copulatrix and
placed in a drop of modified Barth saline solution
on a glass slide (Gurdon 1991). Using a dissecting
microscope (40x magnification), the spermato-
phore was ruptured to release the sperm. The eu-
pyrene sperm bundles were counted and the
number of eupyrene sperm was obtained by mul-
tiplying the number of bundles by 256 (which is
the number of sperm per bundle present after
both mitotic and meiotic divisions are completed).
To count the apyrene sperm, the sample was
washed off from the glass slide with more Barth
saline and diluted with distilled water. Six 10 gl
sub samples were placed on slides and allowed to
dry. Each was examined using dark-field phase
contrast microscopy (100x magnification) and the
number of apyrene sperm in each dried sample
was counted. The sperm ratio was expressed as a
quotient of apyrene to eupyrene sperm.

Statistical Analysis

Data was analyzed using Instat and GraphPad
Prism software. The number and ratio of eu-
pyrene and apyrene sperm and the total time in
copula for the BL-2 and WT-C males was analyzed
with one way analysis of variance (ANOVA). Data
collected for the F, generation appeared to deviate
from normality. As such, Kruskal-Wallis one-way
analysis of variance by ranks was used to decide if
samples came from populations with the same
median (Siegel & Castellan 1988). This non-para-
metric method has the advantage of not requiring
the assumption of normally distributed error. To-
tal time in copula for the F, generation was com-
pared with one-way ANOVA.

RESULTS

Data on the number of eupyrene and apyrene
sperm, sperm ratio and the total time in copula


for BL-2 and WT-C males is presented in Table 1.
The number of both eupyrene and apyrene sperm
were significantly lower in males from the BL-2
strain. However, the ratio of apyrene to eupyrene
sperm was the same in both male groups (9.5:1).
The total time in copula was slightly (although
not significantly; P = 0.0774) longer for the mu-
tant male strain (BL-2) than for the wild type
(WT-C) males.
Radiation dose significantly affected both eu-
pyrene (Fig. 4-P < 0.05, F = 3.844) and apyrene
(Fig. 5-P < 0.05, F = 2.953) sperm numbers in the P
generation as males were treated with increasing
doses of gamma radiation. When males were
treated with 175 Gy the number of eupyrene (Fig.
4) and apyrene (Fig. 5) sperm was higher than for
both controls (sl/+ and WT-C). At doses above 175
Gy the amount of sperm decreased significantly
to reach a 25% reduction at 350 Gy for both sperm
types (F = 3.85, P = 0.013 and F = 2.95, P = 0.009
for eupyrene and apyrene sperm, respectively).
There were no significant differences in the
apyrene to eupyrene sperm ratios among treat-
ments (Fig. 6) (F = 1.25, P = 0.28). The total time
in copula for control and treated P generation
males is shown in Figure 7. There was a signifi-
cant treatment effect, with males treated at 350
Gy remaining in copula for the longest period of
time (F = 5.55, P = 0.0001).
No adult progeny developed from crosses in-
volving P males treated with 300 and 350 Gy. As
such, no data is included for the F, generation at
these doses. In the remaining F, male groups
(150-250 Gy), no dose-response was evident when
the mean numbers of sperm in the spermatophore
were compared. However the numbers of eu-
pyrene to apyrene sperm suggested a bimodal dis-
tribution but could not be tested for normality
because of insufficient sample size. Therefore, a
Kruskall-Wallis test was used to compare medi-
ans (Table 2). The number of sperm transferred
by F, generation males was highly variable. Fig-
ures 8 and 9 illustrate the distribution of num-
bers of both sperm types at various doses. These
distributions clearly show the difference between
WT-C control and untreated sl/+ control in the


TABLE 1. NUMBER OF EUPYRENE AND APYRENE SPERM AND COPULA DURATION IN THE BL-2 AND WT-C STRAINS OF
EPHESTIA KUEHNIELLA.

Total time in copula
Eupyrene sperm (no.) Apyrene sperm (no.) Sperm ratio' (min.)

Strain n Mean + SD n Mean + SD n Mean + SD n Mean + SD

bl-2 21 6644+ 4146** 23 60868 + 28793** 16 9.5 + 3.3 21 121.9 + 32.5*
WT-C 22 14604 + 7028 26 112182 + 75046 19 9.5 + 2.9 25 106.2 + 26.4

'The difference between strains was not significant at P = 0.05 (compared using ANOVA).
*Marginally significant at P = 0.0774.
** P< 0.001.







Florida Entomologist 84(2)


J 20000-

E
15000-
CL

10000-
S5000-
5000-
u-


sl/+ WT-C 150 175 200 250 350
Dose of irradiation [Gy]

Fig. 4. Eupyrene sperm numbers transferred in P
generation of irradiated sl/+ males of Ephestia kueh-
niella. Untreated controls: sl/+, WT-C. Bars indicate
standard deviation.


numbers of both sperm types transferred. Apy-
rene to eupyrene sperm ratios in the F, genera-
tion were lower in the controls (sl/+ and WT-C)
than in the treatments (Table 2). Some of the
males from each treated group exhibited very
high apyrene to eupyrene ratios as shown in Fig.
10. The total time in copula in F, males did not fol-
low a dose-dependent pattern (Fig. 11-F = 4.605,
P = 0.0009). When compared with one-way
ANOVA, significant differences were found
among controls and treated groups. Males treated
with 175 Gy remained in copula for the longest
time (the mean was 160 minutes).
In addition, some F, males exhibited various
abnormalities in spermatophore production, in-
cluding spermatophore bulb formation, sperm
transfer to the spermatophore and accessory
gland fluid transfer to the bursa copulatrix. We
recognized three types of copulatory defects in F,
males: (1) no accessory gland fluid present in the
bursa copulatrix; (2) no spermatophore or acces-


sl/+ WT-C 150 175 200 250 350
Dose of irradiation [Gy]

Fig. 5. Apyrene sperm numbers transferred in P gen-
eration of irradiated sl/+ males of Ephestia kuehniella.
Untreated controls: sl/+, WT-C. Bars indicate standard
deviation.


0o

10 -
CL

5-


0
sl/+ WT-C 150 175 200 250 350
Dose of irradiation [Gy]

Fig. 6. Apyrene to eupyrene sperm ratio in the P gen-
eration for irradiated sl/+ males of Ephestia kuehniella.
Untreated controls: sl/+, WT-C. Bars indicate standard
deviation.


sory gland secretion present, only apyrene sperm
present in bursa copulatrix, and (3) no spermato-
phore or sperm present. These defects were ob-
served in 7.3% of the total (110 males) studied
and occurred only in treated males.

DISCUSSION

In order to predict the amount of sperm trans-
ferred by Ephestia kuehniella males, we analyzed
sperm numbers in parents of irradiated sl/+
males, heterozygous for either the sl-2 or sl-15
mutations, and in BL-2 males which carried both
mutations. We discovered that mutations had an
effect on the amount of sperm in the spermato-
phore but not on the apyrene to eupyrene sperm
ratio (Table 1). A 9.5:1 apyrene to eupyrene ratio
was found in the control (WT-C) males and in the
BL-2 mutant males, and this ratio is similar to
that reported for other Lepidoptera. For example,


200-
S180. P generation
E 160-
T 140-




10-



sIl/+ WT-C 150 175 200 250 350
Dose of irradiation [Gy]

Fig. 7. Total time in copula in the P generation for ir-
radiated sl/+ males of Ephestia kuehniella. Untreated
controls: sl/+, WT-C; bars indicate standard deviation.


June 2001







Koudelova & Cook: Effect of radiation on sperm transfer in Ephestia kuehniella 177


TABLE 2. EUPYRENE AND APYRENE SPERM NUMBERS AND APYRENE: EUPYRENE SPERM RATIO IN GENERATION OF IR-
RADIATED MALES AND UNTREATED CONTROLS IN EPHESTIA KUEHNIELLA.

Eupyrene sperm (no.) Apyrene sperm (no.) Sperm ratio

Dose (Gy) n Median Mean + SD Median Mean + SD Median Mean + SD

0 (WT-C) 10 20860 19120 + 4666 106800 176200 + 54290 9.90 9.3 + 2.4
0 (sl/+) 26 9728 10670 + 5082 108300 113000 + 36450 11.25 11.6 + 3.6
150 22 6528 9169 + 7752 203500 132200 + 64470 16.10 24.3 + 21.6
175 10 10240 11420 + 7290 142900 179000 + 94860 15.35 26.4 + 38.2
200 18 3712 6585 + 6009 121800 164500 + 80370 33.30 47.4 + 37.8
250 9 6912 8107 + 6015 182100 130400 + 45210 25.40 20.7 + 8.1
Kruskal-Wallis statistics' 23.43** 12.58* 27.16***

'Medians vary significantly at: *, P < 0.05; **, P < 0.001; ***, P < 0.0001.


a 9:1 ratio was reported for Plodia interpunctella
by Gage & Cook (1994); a 5:1 ratio for Spodoptera
litura by Etman & Hooper (1979) and a 9.6:1 ratio
for Manduca sexta by J. G. Shepherd, unpub-
lished data in Silberglied et al. (1984).
Several authors have suggested that anucle-
ated apyrene sperm play a role in sperm competi-
tion (Silberglied et al. 1984; Osanai et al. 1987;
Cook & Gage 1995). Cook & Weddel (1999) found
that female Pieris napi that had more apyrene
sperm in their storage organ were more likely to
delay remating than females with less apyrene
sperm. Marec et al. (1999) examined the effect of
irradiation on mating success and fecundity ofsl/
+ E. kuehniella and found that radiation dose did
not produce any major physiological changes that
would decrease reproductive capacity in treated
males. Holt & North (1970) studied the effect of
gamma radiation on sperm distribution and cop-
ulation in Trichoplusia ni. They reported that
there was no difference in sperm volume ejacu-
lated but found differences in the number of
sperm present in the spermatophore and con-
cluded that irradiation affects the complex pro-
cess of copulation. In the present study, gamma
radiation reduced the number of sperm trans-
ferred in treatedE. kuehniella males of the P gen-
eration at all doses except for 175 Gy. At higher
doses, the number of eupyrene and apyrene
sperm were reduced and the total time in copula
for these males was increased. We suggest that to-
tal time in copula was dose and sperm volume-de-
pendent (i.e., the higher the treatment dose and
the lower the sperm number, the longer the total
time in copula). Holt & North (1970) also found
that irradiated males copulated for longer times
than did untreated controls.
We suggested that the impact of irradiation
would be greater in the progeny (F1) of treated sl/
+ Ephestia kuehniella males than in the parental
(P) generation. North (1975) suggested that F,
males originating from a partially sterile male
parent in Pectinophora gossypiellla or from apho-


late treated males of Bombyx mori had problems
in the production and transfer of sperm. F, males
of B. mori produced normal numbers of apyrene
but fewer numbers of eupyrene sperm. Basically,
inherited (or F1) sterility is caused by radiation-
induced chromosomal aberrations that affect the
regular segregation of chromosomes, thus gener-
ating unbalanced gametes (Anisimov et al. 1989)
which can lead to changes during copulation. In
our experiments, F, males showed great variabil-
ity in the numbers of sperm transferred to fe-
males (Table 2). In the range of doses tested (150-
250 Gy) some males showed sperm numbers that
were similar to those found in the untreated con-
trols. However, at the same doses, some males
transferred reduced eupyrene and increased apy-
rene sperm when compared to controls. These re-
sults agree with those of a cytogenetic study
(Tothova & Marec, subm.) which showed variabil-
ity in the number and type of chromosome aberra-
tions inherited from irradiated male parents in F,
males of E. kuehniella. This variability was mag-
nified by abnormal pairing of chromosomes dur-
ing meiosis. This abnormal pairing most probably
influenced chromosome segregation in meiosis I,
finally resulting in either genetically unbalanced
or balanced gametes. However, the level of steril-
ity predicted according to the observed frequency
of chromosome aberrations was much higher than
the level of inherited sterility found by Marec et
al. (1999). Thus, it has been suggested that there
is a regulation mechanism which enables the
moths to correct the predicted unbalanced state
towards balanced segregation of chromosomes
during meiosis I (Tothova & Marec, subm.).
In the F, males we observed no clear dose-
dependence between sperm transfer and the dose
of gamma radiation. In general, the mean num-
ber of eupyrene sperm transferred decreased,
whereas the mean number of apyrene sperm
transferred increased. This resulted in a high ra-
tio of apyrene to eupyrene sperm, which fluctu-
ated between values similar to those of controls






Florida Entomologist 84(2)


20


c 15-

E
0 10-
J3
E
z 5-


0-



20


a 15-

E
o 10-

E
z 5-


<10000 <20000 <30000
Eupyrene sperm [no.]


<10000 <20000 <30000
Eupyrene sperm [no.]


F1 males: sl/+ (control)











<10000 <20000 <30000
Eupyrene sperm [no.]


<10000 <20000 <30000
Eupyrene sperm [no.]


F1 males: 250 Gy

15-


10-


5-


0 I-
<10000 <20000 <30000
Eupyrene sperm [no.]


Fig. 8. Frequency distribution of eupyrene sperm numbers transferred by control (sl/+, WT-C) and treated males
of F, generation Ephestia kuehniella.


WT-C males (control)











<10000 <20000 <30000
Eupyrene sperm [no.]


F1 males: 150 Gy


L


I -


June 2001






Koudelova & Cook: Effect of radiation on sperm transfer in Ephestia kuehniella 179


20


cu 15-

E
'4-
0 10-

E
Z 5-


n-


<100 <200 <300
Apyrene sperm [no. x 103]


<100 <200 <300
Apyrene sperm [no. x 103]


(I) Iy
0a)
E
o 10
-0
E
z


<100 <200 <300 <400
Apyrene sperm [no. x 103]


II I


co 15-

E
0 10-






20
E






z 5-
0-










20-
m 15-











0 10-
E
Z 5-


0-



20-


u) 15-
a)


0 10-

E
z 5


0-


SF, males: sl/+ (control)


Fig. 9. Frequency distribution of apyrene sperm numbers transferred by control (sl/+, WT-C) and treated males
of F, generation Ephestia kuehniella.


WT-C males (control)













<100 <200 <300
Apyrene sperm [no. x 103]


F1 males: 175 Gy













<100 <200 <300 <400
Apyrene sperm [no. x 103]


F1 males: 250 Gy













<100 <200 <300
Apyrene sperm [no. x 103]







Florida Entomologist 84(2)


20
F1 males: sl/+ (control)
15- WT-C males (control)


10-


5-


0 -
10 20 30 40 50 60 70 80 90 100 120 140
Apyrene to epyrene sperm ratio

Is---------------


0
10 10
E0
W
z 5-


U-
10 20 30 40 50 60 70 80 90 100 120 140
Apyrene to epyrene sperm ratio

n .


I 5.
--
E


10 20 30 40 50 60 70 80 90 100 120 140
Apyrene to epyrene sperm ratio

Fig. 10. Frequency distribution of apyrene to eu-
pyrene sperm ratios in control (sl/+, WT-C) and treated
males of F, generation Ephestia kuehniella.



and those which could be clustered into four
groups: (1) higher than 10:1, (2) higher than 20:1,
(3) higher than 30:1 and (4) the ratios that ex-
ceeded the value 100:1. At 200 and 250 Gy the
most frequent ratios fluctuated around the value
30:1. Several authors have reported reduced eu-
pyrene to apyrene ratios in the F, progeny of lep-
idopteran species. Proshold et al. (1993) reported


" 180- F, generation
160-
.5 140-








sl/+ WT-C 150 175 200 250
Dose of irradiation [Gy]

Fig. 11. Total time in copula in the F, generation of
Ephestia kuehniella (sl/+ male parents were irradiated).
Untreated controls: sl/+, WT-C.


reduced eupyrene sperm transfer in Lymantria
dispar. Twenty-five percent of the females that
mated with F, males from 100 Gy treated male
parents contained no eupyrene sperm in their
spermatheca. Similarly, Cheng & North (1972)
found that nearly half of normal P. gossypiella fe-
males that mated with F, males from 150 and 200
Gy treated male parents had only apyrene sperm
in their spermathecae. LaChance et al. (1977)
suggested that females with no eupyrene sperm
in their spermathecae behaved as virgins and re-
mated. However, apyrene sperm can influence
sperm competition as reported for Pieris napi by
Cook & Wedell (1999). They found that more apy-
rene sperm is stored in females that do not re-
mate. As such, males that are able to transfer
more apyrene sperm might reduce the possibility
of sperm competition. We observed that some
treated F, males were able to transfer large
amounts of apyrene sperm. Matings with these F,
males may suppress female remating.
An interesting result was obtained when
Ephestia kuehniella males were treated with 175
Gy. In both the P and the F, generation, this dose
caused an increase in the number of eupyrene
sperm transferred compared to the sl/+ control
(but not WT-C control in the P generation). In F,
males, the number of apyrene sperm was higher
than in both controls and this number had a
broader frequency distribution. Therefore we sug-
gest that 175 Gy may be the best dose to treat
Ephestia kuehniella for F, sterility induction.
We conclude that males inheriting many chro-
mosomal aberrations may change either the
mechanisms regulating spermatogenesis or those
underlying copulation and sperm transfer to the
spermatophore. In this study we found that apy-
rene sperm are transferred in higher numbers in
irradiated males and in very high numbers in
their progeny. Given that apyrene sperm num-
bers are related to female remating in untreated
bers are related to female remating in untreated


F-- F, males: 150 Gy
= F, males: 175 Gy








; B I a 0 n n


S F, males: 200 Gy
F, males: 250 Gy


June 2001


1







Koudelova & Cook: Effect of radiation on sperm transfer in Ephestia kuehniella 181


Lepidoptera, the effect of receiving such high apy-
rene sperm numbers on female behavior merits
further study.

ACKNOWLEDGMENTS

We thank Ms. Ivana Kollarova for technical
support and Dr. Rostislav Zemek for help in the
statistical analysis. We are grateful to Dr. Fran-
tisek Marec for discussion of genetics questions.
The research was supported by the research con-
tract no. 7161/RB of the International Atomic En-
ergy Agency (Vienna) and by grant no. A6007609
of the Czech Academy of Sciences (Prague).

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June 2001







Seth & Sharma: Inherited sterility in Spodoptera litura


INHERITED STERILITY BY SUBSTERILIZING RADIATION
IN SPODOPTERA LITURA (LEPIDOPTERA: NOCTUIDAE):
BIOEFFICACY AND POTENTIAL FOR PEST SUPPRESSION

R. K. SETH AND V. P. SHARMA
Department of Zoology, University of Delhi, Delhi 110 007, India

ABSTRACT
Spodoptera litura reared on host plants and on synthetic diet were irradiated with two sub-
sterilizing doses of gamma radiation, 100 Gy and 130 Gy, and examined for inherited steril-
ity. Irradiation affected mating success in the parental (P) and F, generations. F, sterility was
higher than P sterility, and F, males inherited more sterility than did F, females. F, progeny
developed at a slower rate compared with controls. F, survival to adulthood decreased with
increasing dose of radiation. Sex ratio in F, moths was skewed towards male. Life tables were
constructed for S. litura reared on host plant and synthetic diets, and the impact of radiation
on population characteristics was ascertained. Reproductive rate (R0) was significantly de-
creased as a consequence of irradiation, and the effect was more severe in F, crosses than in
P crosses. There was a negative correlation between the dose of radiation and the percent em-
bryo formation in P crosses. Whereas in F, crosses, radiation dose (given to male parents) was
positively correlated with the percent unhatched embryonated eggs. Early mortality of eggs
prevailed in unhatched eggs derived from P crosses, and late embryonic lethality was the ma-
jor cause ofF, sterility. Effects of irradiation are discussed with an emphasis on assessing the
potential of the inherited sterility principle for pest control.
Key Words: common cutworm, irradiation, F, sterility, synthetic diet, embryonic lethality,
population dynamics

RESUME
Insectos de la especie Spodoptera litura provenientes de colonies mantenidas en plants
huesped o en dieta artificial se irradiaron a dos dosis subesterilizantes de radiacion gamma,
100 Gy y 130 Gy, y fueron examinados para detectar la presencia de esterilidad hereditaria.
La irradiacion afecto la habilidad de copula en las generaciones P y F,. Se encontro que la es-
terilidad en la generation F, es mas alta que en la generation P y los machos F, heredaron
mas esterilidad que las hembras de la misma generation. Asimismo, se encontro que la ve-
locidad de desarrollo de la generation F, es mas lenta que en el grupo control (no irradiado),
y la supervivencia al estado adulto se redujo a media que los insects fueron expuestos a do-
sis de radiacion mas altas. Finalmente, la tasa sexual se vio favoracida hacia el sexo macho
en la generation F,. Se construyeron tablas de vida para ambas colonies y se investigo el
efecto de la radiacion sobre various parametros de estas poblaciones. La tasa reproductive (R.)
se redujo significativamente y el efecto fue mas severo en parejas de insects de la genera-
cion F,. Asimismo, se encontro una correlacion negative entire la dosis de radiacion y la for-
macion de hevecillos embrionados en la generation P. Esta corralacion fue positive en la
generation F, cuando la radiacion se aplico solamente a los machos. Se detecto mortalidad
temprana dentro de los huevecillos no eclosionados provenientes de cruces en la generation
P y se detecto muerte tardia de los huevecillos embrionados en cruces en la generation F,.
En este articulo discutimos estos efectos con enfasis a la aplicacion de la esterilidad F, como
metodo de control para esta especie.


Spodoptera litura (Fabricius) (Lepidoptera:
Noctuidae), the common cutworm, is an economi-
cally serious and polyphagous pest in India. This
pest is reported to attack a wide range of food
plants (112 cultivated plants belonging to 44 fam-
ilies worldwide and 60 plants in India) (Lefroy
1908; Moussa et al. 1960; Thobbi 1961; Chari &
Patel 1983). A multifaceted approach is required
for the control of this pest because it has devel-
oped resistance against a range of insecticides
and because other control measures are inade-
quate when applied alone (Ramakrishnan et al.
1984; Armes et al. 1997). The sterile insect tech-
nique (SIT) has been used for Lepidoptera but


insects in this order are radio-resistant, presum-
ably due to their holokinetic chromosomal config-
uration (Bauer 1967). Therefore, lepidopterans
require large doses of radiation for sterilization,
leading to somatic damage and reduced competi-
tiveness in the irradiated insect.
A favored alternative to using fully sterile
moths in SIT is the use of F, sterility. F, survivor
progeny of sub-sterile parental (P) male moths
result when sub-sterilizing doses of radiation are
applied to the P males. The resulting F, progeny
are more sterile than the irradiated parent, and
the irradiated moths are more competitive as a re-
sult of receiving a lower dose of radiation. Inherited







Florida Entomologist 84(2)


sterility in the progeny of treated males has been
shown to have potential in suppressing popula-
tions of lepidopteran pests (North & Holt 1969;
Knipling 1970; North 1975; LaChance 1985).
Previous studies of substerilizing gamma-radia-
tion doses on the growth, bioenergetics and repro-
ductive behavior of S. litura in the F, progeny of
treated moths indicated the potential of managing
this pest by using inherited sterility (Seth & Sehgal
1993). In this study we evaluated the reproductive
performance and mating behavior ofS. litura in re-
sponse to two substerilizing doses (100 Gy and 130
Gy) when reared on two different diets.

MATERIALS AND METHODS

Insect Rearing

Spodoptera litura was reared on two diets, the
natural food, castor leaves (Ricinus communis)
and a meridic diet containing chickpea seeds and
sinigrin as a phagostimulant (Table 1). Insects
were held at ambient environmental conditions,
26.8 1C temperature, 75 5% R.H. and a pho-
toperiod of 12L:12D in the insectary. Larvae de-
veloping on the castor leaves were allowed to
pupate in moist, loose soil. Larvae developing on
the chickpea diet pupated in the diet container.

Irradiation of Insects

Irradiation of 0-24-h old adult males was con-
ducted in the Genetics Division, Indian Agricul-


TABLE 1. CONSTITUENTS OF THE SEMI-SYNTHETIC DIET PRO-
POSED FOR REARING OF SPODOPTERA LITURA.

Ingredients Amount

Agar 25 g
Deionized water 750 ml
Casein 44 g
Ground chickpea seeds 93.50 g
Wessons salts 12.50 g
Cholesterol 1.25 g
Yeast (dried, brewer's) 19 g
Methyl-p-hydroxybenzoate 1.25 g
Sugar 39 g
Sorbic acid 2 g
Deionized water 400 ml
4 M KOH 6.25 ml
Corn oil 2.50 ml
Linseed oil 2.50 ml
Formaldehyde (10%) 5.50 ml
Sinigrin (1%) 3.53 ml
Antibiotic and vitamin mixture' 7.50 g
Choline chloride 1.25 g

'Composition: chloramphenicol (2 g), streptomycin (4 g), tetracycline
(36 g), ascorbic acid (80 g), Evion (vitamin E; 0.2 g; Merck Co.), vitamin
mixture (2 g; Roche Co.).


tural Research Institute, New Delhi, using a Co60
source at the dose rate of about 13.5 Gy/min. On
the basis of our initial studies (Seth & Sehgal,
1993), two gamma-radiation doses, 100 Gy and
130 Gy, were selected for this study.

Reproductive Performance and Viability
of Irradiated Moths and Their Progeny

Various reproductive parameters were as-
sessed by pairing treated insects (irradiated P
males and F, moths derived from the treated P
males crossed with normal females) with their
normal (N) counterparts from the stock culture.
Eggs from single-pair matings were counted daily
and the number hatching was monitored in 10
replicated samples of 80-100 eggs each (up to first
3 days of egg laying). Corrected sterility and con-
trol of reproduction of insect population due to ir-
radiation were determined according to the
methods described by Abbott (1925) and Seth &
Reynolds (1993).
Experiments on mating success were con-
ducted in laboratory cages (each cage having 10-
15 pairs, comprising one replicate). The mating
success of moths was assessed by dissection of fe-
males immediately after death. The presence of a
spermatophore in the bursa copulatrix indicated
that the female had mated; the number of sper-
matophores indicated the mating frequency.
The viability of treated moths, and the sur-
vival and developmental pattern of F, progeny
were determined. Diurnal observations on the in-
sect behavior and the growth index for each treat-
ment was calculated.

Population Characteristics

Various features of population dynamics were
studied by constructing life tables for S. litura on
castor leaves and chickpea diet. The female life ta-
bles of P and F, generation were constructed as de-
scribed by Birch (1948), and elaborated by Howe
(1953), Morris & Miller (1954), and Atwal & Bains
(1989). For ascertaining the life table characteris-
tics, a defined size of population was established
in field-cages for a particular cross. Then seven
batches of 200-250 eggs collected from each cross
were reared to determine age specific mortality in
different life stages and successful adult emer-
gence. The life tables gave the probability at birth
of a female being alive at age x, designated as L (1,
= 1). The age schedule for female births denoted
the mean number of female offspring produced per
unit time by a female of age x, designated as mi.
The net reproductive rate (Ro) was calculated from
1. and mx. Mean length of generation time (T), in-
nate capacity for increase in number (rm) and fi-
nite rate of increase (X) were calculated from the
data generated in the life tables.


June 2001







Seth & Sharma: Inherited sterility in Spodoptera litura


Embryonic Development

The embryonic development in F, and F2 eggs
was studied to understand the stage at which ir-
radiation induced lethality was manifested. The
developmental state of embryos was assessed in
dechorionated eggs. The eggs were treated with 3-
5% NaC10 for 30-40 min, fixed with 10% formalin
for 8-12 h, and stained with lactoacetic-orcein or
Feulgen stain. The stained embryos were exam-
ined under the microscope. The embryonic devel-
opment was classified as stage I (cleavage/early
development), stage II (germ band stage), and
stage III (embryo stage). Some embryos were re-
corded as abnormal.

Statistical Analysis

The effect of radiation on various parameters
in the two diets was subjected to analysis of vari-
ance (ANOVA). Data were usually obtained in
replicates often, unless otherwise specified in the
text. Percentage data were transformed using


arcsine Kx before ANOVA. Means were separated
at the 5% significance level by least significant
difference (LSD) test (Snedecor & Cochran 1989).


RESULTS

Reproductive Performance and Viability
of Irradiated Moths and Their Progeny

For the untreated controls, the pre-oviposition
period ranged from 1.65 to 1.72 d on the castor
leaf diet and 1.69 to 1.70 d on the chickpea. The
dose of radiation did not significantly affect pre-
oviposition period for the P and F, crosses in ei-
ther diet. The oviposition period was not affected
in P crosses, but was significantly reduced in most
F1 crosses (Table 2). Also, the radiation treat-
ments caused a significant reduction in fecundity
of the mated female during P crosses and F,
crosses, with the greatest reduction in F, crosses.
Even normal females mated with treated males
(P or F1) showed a reduction in fecundity. For ex-


TABLE 2. EFFECT OF SUBSTERILIZING GAMMA-RADIATION DOSES AND DIET ON THE OVIPOSITIONAL BEHAVIOR AND LON-
GEVITY OF SPODOPTERA LITURA PARENTS AND F, PROGENY.

Preoviposition Oviposition Eggs per Life span (days)
Dose Cross period period female
Diet (Gy) type' (days) (days) (no.) Male Female

P crosses
Castor leaf 0 Nm x Nf 1.65 + 0.07 a 7.23 + 0.32 a 1893 + 59 a 10.0 + 0.4 a 9.4 + 0.3 a
100 Pm x Nf 1.68 + 0.17 a 7.25 + 0.17 a 1847 + 68 b 9.4 + 0.7 a 9.4 + 0.5 a
130 Pm x Nf 1.89 + 0.13 a 7.21 + 0.25 a 1795 + 93 b 9.4 + 0.4 a 9.1 + 0.5 a
F, crosses
0 NmxNf 1.72 + 0.08 a 7.62 + 0.31 a 1990 + 49 a 10.3 + 0.4 a 9.3 + 0.3 a
100 Flmx Nf 1.62 + 0.14 a 6.71 + 0.11 b 1659 + 78 b 9.4 + 0.6 a 8.1 + 0.5 b
Nm x Ff 1.72 + 0.19 a 7.01 + 0.09 a 1693 + 80 b 9.8 + 0.2 a 9.3 + 0.3 a
Flmx Ff 1.60 + 0.24 a 6.22 + 0.49 b 1520 + 56 b 9.6 + 0.4 a 8.3 + 0.4 b
130 Flm x Nf 1.78 + 0.16 a 6.22 + 0.25 b 1464 + 85 b 9.1 + 0.4 b 8.9 + 0.4 ab
Nm x Ff 1.73 + 0.11 a 6.29 + 0.42 b 1499 + 69 b 8.8 + 0.3 b 8.7 + 0.9 ab
Fmx Ff 1.85 + 0.24 a 6.22 + 0.59 b 1219 + 30 c 8.8 + 0.3 b 8.4+ 0.1b
P crosses
Chickpea diet 0 Nm x Nf 1.70 + 0.09 a 7.40 + 0.23 a 2011 + 70 a 10.1 + 0.4 a 9.5 + 0.4 a
100 PmxNf 1.80 + 0.10 a 7.16+ 0.11 a 1886 + 84 b 9.3+ 0.1 a 8.7 + 0.2 a
130 Pm x Nf 1.88 + 0.11 a 6.66 + 0.42 a 1727 + 69 b 8.9 + 0.3 a 8.6 + 0.2 a
F, crosses
0 Nm x Nf 1.69 + 0.09 a 7.33 + 0.24 a 2155 + 74 a 9.8 + 0.5 a 9.5 + 0.4 a
100 Fmx Nf 1.63 + 0.17 a 7.09 + 0.08 a 1649 + 70 b 8.8 + 0.4 a 8.5 + 0.4 b
NmxFFf 1.62 +0.16 a 6.90 + 0.37 ab 1592 + 84 b 8.6 + 0.4 a 8.5 + 0.4 b
Flmx Ff 1.69 + 0.13 a 6.34 + 0.10 bc 1492 + 59 bc 7.2 + 0.2 c 7.8 + 0.1 c
130 F1m x Nf 1.89 + 0.17 a 6.40 + 0.13 bc 1304 + 58 d 8.6 + 0.3 b 8.0 + 0.3 bc
Nm x Ff 1.75 + 0.17 a 5.95 + 0.26 bc 1363 + 75 cd 8.5 + 0.4 b 7.9 + 0.2 bc
Flmx Ff 1.84 + 0.20 a 5.83 + 0.22 c 1099 + 53 e 7.0 + 0.3 c 7.1 + 0.3 c

'N, normal; P, treated parent; F progeny of treated males; m, male, f, female. Means + SE followed by the same letter in a column in each generation
in case of each diet are not significantly different at P < 0.05 (ANOVA followed by LSD posttest); n = 10.







Florida Entomologist 84(2)


ample, the reduction in oviposition in P crosses,
with respect to untreated crosses, was 6.2% at
100 Gy and 14.1% at 130 Gy on the chickpea diet.
This reduction in oviposition was further in-
creased in the F, crosses (23-30% at 100 Gy, and
39-49% at 130 Gy on chickpea diet). Both doses
significantly reduced F, male and female longev-
ity when the chickpea diet was used. Although
the same trend was evident when moths were
reared on castor leaves, the reduced longevity for
males at the 100 Gy dose was not significant.
A significant, dose-dependent effect was ob-
served on the mating success of P and F, moths
reared on both diets. The treated males were less
successful at mating than untreated males (Table
3). For example, at 130 Gy on chickpea diet, the
mating success of P males and F, males was
76.4% and 72.7%, respectively, as compared with
89.6% in the control. The mating percentage was
more adversely affected in F, female crosses and
F1 self crosses. However, the radiation did not sig-
nificantly affect the mating frequency of the


moths from either diet. Radiation-induced sup-
pression effects were examined in terms of cor-
rected sterility and control of reproduction (Table
3). At 100 Gy on chickpea diet, the F, male x N fe-
male cross gave 76.5% of the total eggs laid by
controls (see Table 2) and resulted in 28.3% egg
hatch as compared with 78.6% hatch in controls.
Therefore, this cross exhibited 63.9% sterility,
and 72.3% control of reproduction. For both the
diets tested, the control of reproduction in F,
males (mated with normal females) was more
than 71% at 100 Gy and about 83-87% at 130 Gy.

Growth and Survival of F, Progeny

The developmental time of F, eggs, larvae, pu-
pae and adults reared on the two diets were signif-
icantly affected by the radiation treatment given
to the male parent (crossed with a normal female).
Their developmental rate was slower than that of
the control progeny (Table 4); the total develop-
mental time between eggs and adults was in-


TABLE 3. EFFECT OF SUBSTERILIZING GAMMA-RADIATION DOSES AND DIET ON MATING BEHAVIOR, FERTILITY AND RE-
PRODUCTIVE SUPPRESSION OF SPODOPTERA LITURA PARENTS AND THEIR PROGENY.

Mating Mating Corrected Control of
Dose Cross frequency success2 Fertility2 Sterility Reproduction
Diet (Gy) type' (no. SE) (%) (%) (%) (%)

P crosses
Castor leaf 0 Nm x Nf 1.7+ 0.1 a 92.8+ 2.2 a 89.7+ 2.1a 0 0
100 PmxNf 1.9+ 0.3 a 84.0 + 3.1b 52.3+ 2.0b 41.1+ 1.6 48.0+ 1.9
130 Pm x Nf 1.9 + 0.3 a 72.6 + 3.8 c 44.8 + 2.5 c 49.5 + 2.8 56.6+ 3.2
F, crosses
0 NmxNf 1.7+ 0.1 a 94.1+ 1.9 a 88.9+ 1.4 a 0 0
100 Fm x Nf 1.9 + 0.2 a 80.9 + 3.1 bc 31.6 + 2.1 cd 64.4 4.3 71.7 + 4.8
Nm x Flf 1.8 + 0.2 a 73.2 + 2.2 cd 44.5 + 2.9 b 49.9 + 3.3 59.4 + 3.9
Flm x Flf 1.7 + 0.3 a 71.4 + 2.9 de 28.3 + 3.1 d 68.2 + 7.4 76.8 + 8.4
130 Fim x Nf 2.0 + 0.2 a 75.6 + 3.5 bc 21.8 + 1.8 de 75.5 + 6.2 82.8 +6.8
Nm x Fif 1.7 + 0.3 a 66.0 + 2.4 ef 35.9 + 2.2 c 59.6 + 3.7 71.0 + 4.4
Fim x Fif 1.9 + 0.5 a 59.0 + 4.0 f 20.2 + 1.1 e 77.3 + 4.2 86.7 + 4.7
P crosses
Chickpea diet 0 Nm x Nf 1.7 + 0.1 a 89.2 + 2.2 a 84.4 + 2.9 a 0 0
100 PmxNf 1.8+ 0.3 a 81.5+ 2.2 b 46.2 + 4.1b 41.2+3.7 48.6+ 4.3
130 PmxNf 1.8+ 0.2 a 76.4+ 3.2 b 41.6 + 2.1b 47.1+ 2.4 57.6+ 2.9
F, crosses
0 Nm x Nf 1.7 + 0.2 a 89.6 + 2.4 a 78.6 + 3.0 a 0 0
100 Flm x Nf 1.5 + 0.3 a 84.0 + 3.1 ab 28.3 + 2.3 c 63.9 +5.4 72.4+ 6.1
Nm x Flf 1.6 + 0.1 a 75.4 + 1.5 bc 43.2 + 2.9 b 45.0 + 3.0 59.4 + 4.0
Fm x Flf 1.5 + 0.1 a 65.9 + 2.8 de 22.3 + 2.0 cd 71.6 + 6.4 80.4 + 7.1
130 Flm x Nf 2.1 + 0.1 a 72.7 + 3.1 cd 17.5 + 1.8 de 77.8 + 7.9 86.5 + 8.8
Nm x Flf 2.4 + 0.1 a 60.9 + 1.3 ef 26.7 + 2.2 c 66.1 + 5.4 78.5 + 6.5
Fm x Flf 2.0 + 0.2 a 56.1+ 3.1 f 14.6 + 2.1 e 81.4 8.7 90.6 +8.1

'N, normal; P, treated parent; F, progeny of treated males; m, male, f, female.
For statistical analysis by ANOVA, the percentage data were transformed using arcsine x. Means + SE followed by the same letter in a column in
each generation in case of each diet are not significantly different at P < 0.05 (ANOVA followed by LSD posttest); n = 10.


June 2001

























TABLE 4. DEVELOPMENTAL PROFILE AND SURVIVAL OF F, PROGENY OF IRRADIATED SPODOPTERA LITURA REARED ON TWO DIETS.

Developmental period (days)
Dose Adult eclosion' Sex ratio
Diet (Gy) Egg Larva Pupa Total % pupation' % Growth index2 (M:F)

Castor leaf 0 3.6 + 0.1 a 15.8 + 0.3 a 7.9 + 0.2 a 27.2 + 0.4 a 89.3 + 4.6 a 82.3 + 2.5 a 3.0 + 0.1 a 1:1.03 a
100 3.9 + 0.1 b 16.7 + 0.5 ab 8.5 + 0.2 ab 29.2 + 0.6 ab 71.2 + 2.7 b 64.0 + 2.8 b 2.2 + 0.1 b 1:0.69 ab
130 4.2 0.1 b 17.3 + 0.4 b 8.6 + 0.2 b 30.1 0.8 b 62.8 + 2.9 c 56.3 + 3.4 b 1.8 + 0.1 1:0.64 b

Chickpea diet 0 3.6 + 0.1 a 16.2 + 0.4 a 8.4 + 0.2 a 28.3 + 0.7 a 90.2 + 3.3 a 79.2 + 2.8 a 2.8 + 0.1 a 1:0.99 a
100 3.9 + 0.1 b 17.9 + 0.4 b 8.5 + 0.3 ab 30.7 + 1.0 ab 68.4 + 4.5 b 60.9 + 3.1 b 2.0 + 0.1 b 1:0.71 b
130 4.0 0.1 b 18.7 + 0.3 b 9.1 0.3 b 31.5 + 0.3 b 58.8 + 2.1b 51.7 + 2.2 c 1.6 0.1 c 1:0.65 c

Observed in group of 25 individuals comprising each replicate, n = 7. For statistical analysis by ANOVA, the percentage data were transformed using arcsine x.
Growth index = % adult eclosion/total developmental period.
Means SE followed by the same letter in a column within each diet are not significantly different at P < 0.05 (ANOVA followed by LSD posttest).
Ct+







Florida Entomologist 84(2)


creased by about 7-9% at 100 Gy and 10-13% at
130 Gy. The F, insects experienced significantly
more larval and pupal mortality than the controls,
and also exhibited a higher rate of pupal and adult
malformation than the controls. The proportion of
F1 insects surviving to adults decreased with the
increasing dose on both diets tested. About 61-
64% of the F, progeny of 100 Gy treated male par-
ents emerged as adults, whereas 79-82% adults
emerged in the controls. The F, growth index was
significantly decreased according to the dose of ra-
diation administered to the male parent. The sex
ratio of the F, generation was significantly skewed
towards males as a result of the radiation treat-
ments. For example, F, females constituted about
41% of the population at 100 Gy and about 50% of
the population in the control.

Population Characteristics

Increase in the total developmental period of
F1 progeny delayed the commencement of oviposi-
tion with further debilitating effects on adult sur-
vival, life expectancy, oviposition rate and female
births with respect to the pivotal age group. The
reproductive rate (Ro) was significantly decreased
as a consequence of radiation treatment, with the
effect being more pronounced in F, crosses than in
P crosses (Table 5). For instance, Ro for untreated
insects was 785.2 on castor and 740.2 on chickpea
diet. On chickpea diet, Rowas decreased by 16.4%
at 100 Gy and 36.6% at 130 Gy in P generation. Ro
was further reduced by about 70% at 100 Gy and
about 80% at 130 Gy in F, crosses, with respect to
the control.
The mean generation time (To) was 31.3 days
on castor and 32.3 days on chickpea diet for un-
treated insects. In F, crosses, To was significantly
higher, indicating a protraction of 2-4 days as
compared with the control. The To delay showed a
positive correlation with doses of radiation (Table
5). The intrinsic rate of increase (rm) in untreated
insects was 0.21 on castor and 0.20 on chickpea
diet. The effect of irradiation on the intrinsic rate
of increase was not apparent in P crosses, but was
significant in F, crosses, where there was a reduc-
tion in the rm value (23-25% at 100 Gy and 31-
36% at 130 Gy). Irradiation also affected the finite
rate of increase of this insect population. For ex-
ample, on chickpea diet at 130 Gy, it was reduced
to 1.15 in F, male crosses from 1.23 in the control.
A similar pattern was observed on castor leaves.

Embryonic Development of F, and F2 Zygotes

In F, and F2 generations, the embryonic devel-
opment was disrupted at different stages that re-
flected a specific pattern of radiation-mediated
lethality in P and F, moths (Table 6). The reduced
egg hatch from P and F, males crossed with normal
females may have been caused by infertility of eggs


or embryonic death. Certain fertilized eggs exhib-
ited embryonic development but were incapable of
hatching. The development of such zygotes was ar-
rested at different levels of embryogenesis showing
partial to complete embryonation. Some of these
eggs could reach the "black head" stage wherein,
blackish brown colored head cuticle of the pharate
1st instar larva was visible through translucent
chorion of the egg. This type of egg was categorized
as an unhatched embryonated egg. At 130 Gy on
the chickpea diet, early mortality (EM) of F, eggs
(derived from P crosses) was 36.9% with no detect-
able embryonic development. Late embryonic le-
thality (LEL) was 22.4%, mainly due to embryonic
stages blocked at cleavage or early development
stage. In F2 eggs (from F, crosses) at the same dose,
EM constituted 16.8% and LEL constituted 65.8%.
Notably, more than 75% of LEL was restricted to
germ band and embryo stages (Table 6, Fig. 1). The
formation of embryos was significantly reduced in
F, eggs (100 Gy and 130 Gy), but was not statisti-
cally different from the controls in F2 eggs at both
doses. However, the percentage zygotic viability in
P and F, crosses was significantly reduced by the
dose of radiation (Table 6). EM appeared predomi-
nantly in F, eggs, whereas F2 eggs showed sterility
mainly due to LEL.

DISCUSSION

The phenomenon of F, sterility in S. litura was
examined over a range of substerilizing doses
(Seth & Sehgal 1993). As a result of the reproduc-
tive performance and somatic damage caused by
the radiation, we selected two doses (100 Gy and
130 Gy) for a more in-depth study. The reduced re-
productive performance of the treated moths re-
sulted from the combined effects of reduced
longevity, fecundity, mating success, and fertility.
The mating percentage was more adversely af-
fected in F, females crossed with normal males
and in F, self crosses as compared with the F,
males paired with normal females. A greater re-
duction in reproductive performance was ob-
served at the 130 Gy dose than the 100 Gy dose.
The effects of radiation dose on reproductive per-
formance were not significantly influenced by
diet. Similar debilitating effects of irradiation on
the reproduction of moths have been reported by
several workers (North & Holt 1968; Proshold &
Bartell 1970; Cheng & North 1972; North 1975;
LaChance et al. 1976; Carpenter et al. 1983;
LaChance 1985; Sallam & Ibrahim 1993; Omar &
Mansor 1993; Ismail 1994; Carpenter et al. 1987;
Makee & Saour 1997). Poor reproductive perfor-
mance and low fertility in treated S. litura could
be due to one or more of the following reasons: (i)
poor ability to mate (El-Sayed & Graves 1969), (ii)
failure to produce and transfer as many sper-
matophores as normal males (Rule et al. 1965;
Flint & Kressin 1969), (iii) transfer spermato-


June 2001

















TABLE 5. POPULATION CHARACTERISTICS OF IRRADIATED SPODOPTERA LITURA AND THEIR F, PROGENY REARED ON TWO DIETS.

Mean generation Innate capacity Finite rate
Dose Net reproductive rate2 length (days) for increase of increase
Diet (Gy) Cross type' Ro = lknm, Exlxm, To = Exxm/R0o rm = logeR/T, (X) = antilogerm

Castor leaf 0 Nm x Nf 785.2 + 27.4 a 24583.0 + 683.3 a 31.30 a 0.2129 a 1.237 a
100 Pm x Nf 602.4 + 25.4 b 18643.5 + 774.2 b 30.94 a 0.2068 a 1.229 a
F,m x Nf 227.4 + 10.2 c 7489.2 + 292.0 c 32.92 b 0.1648 bc 1.179 bc
Nm x Flf 212.8 + 10.3 c 7262.3 + 355.1 c 34.12 c 0.1571 bc 1.170 bc
Fm x Flf 206.5 + 6.1 c 6975.2 + 180.5 c 33.76 bc 0.1578 bc 1.170 bc
130 Pm x Nf 591.4 + 27.0 b 18279.8 + 835.0 b 30.90 a 0.2065 a 1.229 a
F,m x Nf 167.7 + 7.6 d 5805.4 + 150.2 d 34.62 c 0.1479 cd 1.159 cd
Nm x Ff 161.8 + 7.3 d 5626.6 + 153.1 d 34.76 c 0.1463 cd 1.157 cd
Fm x Ff 111.1 + 12.1 e 3819.7 + 110.7 e 34.36 c 0.1371 d 1.146 d
Chickpea diet 0 Nm x Nf 740.2 + 38.7 a 23913.1 + 738.8 a 32.30 a 0.2045 a 1.226 a
100 Pm x Nf 618.1 + 28.5 b 19898.6 + 693.4 b 32.19 a 0.1996 ab 1.220 a
F,m x Nf 235.6 + 21.4 d 8207.3 + 220.7 d 34.82 bc 0.1568 b 1.169 bc
Nm x Flf 220.5 + 19.3 d 7669.1 + 131.7 de 34.76 bc 0.1552 b 1.167 bc
Fm x Flf 209.1+13.3 d 7321.7 + 161.0 e 35.01 c 0.1526 b 1.164 bc
130 Pm x Nf 469.0 + 36.7 c 14941.7 + 328.7 c 31.85 a 0.1931 ab 1.213 a
F,m x Nf 152.0 + 13.1 e 5461.3 + 120.1 f 35.92 c 0.1399 c 1.150 c
Nm x Ff 145.0 + 11.1 e 5226.3 + 101.9 f 35.97 c 0.1384 c 1.148 c
Fm x Ff 101.9 + 8.9 f 3602.7 + 76.4 g 35.34 c 0.1308 c 1.139 d

'N, normal; P, treated parent; F progeny of treated males; m, male, f, female.
'I, female survival; m., female offspring produced per unit time; x, pivotal age.
Means + SE followed by the same letter in a column within each diet are not significantly different at P < 0.05 (ANOVA followed by LSD posttest); n= 7.













TABLE 6. EMBRYONIC DEVELOPMENT AND ZYGOTE VIABILITY IN EGGS OVIPOSITED BY P AND F, MOTHS OF SPODOPTERA LITURA.

Eggs showing3 (%)
Unhatched
Eggs per Embryonic stage of unhatched eggs Embryo embryonated
Dose Cross female2 No formation4 eggs4
Diet (Gy) type' (no.) development Stage I Stage II Stage III Abnormal (%) (%)

Castor leaf 0 Nm x Nf 2088 6.5 + 0.2 2.8 +0.1 0.9 + 0.0 0.7 + 0.0 0 93.3 + 3.0 a 4.7 + 0.2 a
(88.9%)
100 Pm x Nf 1847 26.3 + 1.7 13.4 +0.5 2.5 + 0.1 2.0 + 0.1 3.3+ 0.1 73.5 + 4.7 bc 28.8 + 1.4 b
(52.3%) (55.5) (28.0) (5.2) (4.1) (6.9)
Fim x Nf 1755 11.5 + 0.6 8.4 + 0.3 7.6 + 0.4 37.0 + 2.1 4.0 + 0.3 88.6 + 4.2 a 64.3 + 3.1 c
(31.6%) (16.6) (12.2) (11.1) (54.0) (5.8)
130 Pm xNf 1795 33.1+ 1.6 9.4 + 0.2 6.4 + 0.4 3.2 + 0.2 2.3+ 0.1 66.4 + 3.2 c 32.0 + 1.9 b
(44.8%) (60.8) (17.0) (11.5) (5.7) (4.1)
Flm x Nf 1464 13.2 + 0.7 7.5 + 0.2 10.3 + 0.6 39.4 +2.2 7.1+ 0.3 86.1 + 4.1 ab 74.6 + 4.6 c
(21.8%) (17.9) (9.6) (13.2) (50.2) (9.1)
Chickpea diet 0 Nm x Nf 2155 11.2 + 0.7 5.0 + 0.2 1.6 +0.1 2.2 + 0.1 0.8 + 0.03 88.2a + 4.4 10.8 + 0.7 a
(78.6%) C2
100 Pm x Nf 1886 28.9 + 1.5 14.1 +0.7 4.2 + 0.2 2.8 +0.1 4.2 + 0.2 71.5 + 3.7 bc 35.8 + 2.2 b
(46.2%) (52.9) (26.2) (7.8) (5.2) (7.8) 00

Flm x Nf 1750 11.6 + 0.6 7.7 + 0.4 9.6 + 0.4 36.5 + 1.8 3.8 + 0.2 88.5 + 4.5 a 65.3 + 3.1 c I-
(30.7%) (16.5) (11.1) (13.8) (52.6) (5.4)
130 Pm x Nf 1727 36.9 + 1.4 14.3 + 0.8 3.1 +0.1 1.5 + 0.1 3.1+ 0.2 63.6 + 2.4 c 34.5 + 1.9 b
(41.6%) (62.3) (24.4) (5.3) (2.5) (5.3)
Flm x Nf 1394 16.8 + 1.0 5.5 + 0.3 6.5 + 0.2 47.0 + 2.3 6.8 + 0.4 83.5 + 4.9 ab 78.8 + 4.4 d
(17.5%) (20.1) (6.7) (7.9) (57.0) (8.3)

N, normal moth; P, treated parent; F progeny of treated males; m, male; f, female.
Ovipositional data computed out of mated females; data in parentheses represent % egg hatch.
Data represent % inviable eggs in the particular stage out of total eggs laid; data in parentheses represent % inviable eggs in the stage out of total unhatched eggs; Stage I, cleavage or early developmental phase; Stage
II, germ band phase; Stage III, embryo phase.
'Percentage data were transformed using arcsine x before ANOVA, but data shown are back transformations ; means SE followed by the same letter in a column in case of each diet are not significantly different at P
< 0.05 (ANOVA followed by LSD post-test); n= 7; each replicate comprising of 80-100 eggs. C-4


bC







Seth & Sharma: Inherited sterility in Spodoptera litura


75- Castor
**
EM **
LEL
50-



25 **



0 x
NmxNf PmxNf F~mxNf PmxNf F~mxNf


Control 100 Gy
Crosses


130 Gy


NmxNf PmxNf FlmxNf Pm x Nf FrmxNf
Control 100 Gy 130 Gy
Crosses
Fig. 1. Effect of substerilizing gamma-radiation
doses on the development of eggs derived from the
crosses of treated Spodoptera litura males and their F,
progeny, reared on castor leaves and semi-synthetic
diet. Bars show mean SE; EM, early mortality in eggs;
LEL, late embryonic lethality, standard error of the
mean (SE). N, normal moth; P, treated parent; F,, prog-
eny of treated males; m, male; f, female. ns, non-signifi-
cant at P < 0.05; *, significant at P < 0.05; **, significant
at P < 0.01 (t-test conducted between and LEL in each
cross).


phores that contain little or no sperm; (iv) abnor-
mal sperm structure, which fails to fertilize the
eggs (Ashrafi & Roppel 1973), or (v) inheritance of
special chromosome rearrangements (LaChance
1985; Anisimov et al. 1989).
The developmental rate of F, larvae originat-
ing from the crosses between treated males and
normal females of S. litura was slower than that
of controls, and this delay in development was
greater when males were treated with 130 Gy. Be-
cause insect development and differentiation are
controlled by hormones (Gilbert 1964), the pro-
tracted development of F, larvae might be due to
alteration in hormonal or enzymatic production
caused by chromosomal rearrangements, as indi-
cated by Proshold & Bartell (1970, 1972). The F,


growth index showed a decrease as a consequence
of irradiation of male parents. The sex ratio in F,
generation was skewed towards male as com-
pared with a 1:1 ratio in the control group of in-
sects reared on castor leaves as well as chickpea
diet. Sex distortion appears to be general phenom-
enon occurring in the progeny of irradiated male
lepidopterans (Proverbs 1962; Carpenter et al.
1986), probably resulting from the expression of
recessive lethal mutations on the single Z chromo-
some in females but not in ZZ males (Marec 1990).
The effect of irradiation on population charac-
teristics was not observed in the P generation, es-
pecially in case of mean generation time, survival
and finite rate of increase, because the treatment
was given in the adult stage and it could manifest
its impact only in the first filial generation. How-
ever, irradiation significantly affected population
characteristics in F, and F2 generations, particu-
larly the production of females. The production of
more males than females, and the reduction in
female longevity due to sublethal radiation ulti-
mately reduced the net reproductive rate and the
potential rate of increase.
Our data suggest that both inability of irradi-
ated males to fertilize eggs and dominant lethal
mutations (DLM) induced in sperm were respon-
sible for reduced egg viability in P crosses. This is
because both unhatched embryonated eggs (LEL)
and eggs showing no development (EM) were
induced by irradiation. The eggs marked in the
category of early mortality (EM) could be unfertil-
ized, indicating physiological damage in irradi-
ated males that reduced the ability to transfer
sperm. Alternatively, these eggs could have
ceased their development at an early stage due to
induced DLM (see discussion in Marec et al.
1999). Whereas in F, crosses, sterility was largely
a result of induced chromosomal aberrations.
Therefore, eggs from these crosses showed embry-
onic development but were unable to hatch, indi-
cating late egg lethality (LEL) as the main cause
of egg inviability. Similar findings were made by
Proshold & Bartell (1970) in the tobacco bud-
worm, Heliothis virescens, and by Bughio (1988)
in Chilo partellus. LEL was clearly expressed by
F1 males, whereas EM was expressed less, unlike
in P males of the codling moths (Anisimov et al.
1989). Similarly, Seth & Reynolds (1993) reported
that the main cause of sterility in F, generation of
Manduca sexta was the induction by radiation of
lethal mutations that arrested the development
in late embryonic life. Also, Marec et al. (1999)
observed sterile eggs (eggs with early embryonic
lethality as well as unfertilized eggs) and inviable
eggs (in which embryos died during different
stages of embryogenesis) while studying the
gamma radiation induced sterility combined with
genetic sexing in Ephestia kuehniella.
In view of the overall reproductive perfor-
mance of P and F, moths, and developmental be-







Florida Entomologist 84(2)


havior of F, insects, our findings suggest the use
of 100 Gy as an effective dose for the suppression
of S. litura populations by the release of partially
sterile males. This dose gives better viability of F1
insects among normal population and thus, a
higher inherited sterility effect. Although the
higher dose (130 Gy) could be more effective in P
crosses, it would less effective in the F, crosses. It
is worth noting that degree of irradiation impact
on the net reproductive rate of P and F, crosses
observed in their life tables was almost similar to
the control of reproductive potential calculated.
Proper sperm competition is required for the
treated insects to induce reproductive suppres-
sion of the feral pest population by using F, steril-
ity. Studies on sperm formation, transfer and
their competitiveness are in progress (RKS).

ACKNOWLEDGMENTS

The author (RKS) is grateful to the Interna-
tional Atomic Energy Agency for supporting this
research work under Research Contract No.
IAEA/IND-7162/RB in a coordinated research
programme. Thanks are due to technical assis-
tance offered by D. K. Rao and J. J. Kaur.

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Florida Entomologist 84(2)


June 2001


EFFECT OF A SUBSTERILIZING DOSE OF RADIATION ON THE MATING
COMPETITIVENESS OF MALE AND ON THE MATING PROPENSITY OF
FEMALE HELICOVERPA ARMIGERA (LEPIDOPTERA: NOCTUIDAE)

VIRGINIA R. OCAMPO
Department of Entomology, University of the Philippines Los Banos, College, Laguna 4031, Philippines

ABSTRACT
In this article we report the results of experiments conducted with Helicoverpa armigera
(Htibner) to determine the effects of a substerilizing dose of gamma radiation (100 Gy) on the
mating competitiveness of treated males and the effect on the mating propensity of females
with which they mate. Mating competitiveness of treated and untreated male moths was
measured at two different release ratios inside field-cages in a cabbage field. A 15:1 and a
4 5:19 ratio were used while keeping a constant density of moths per cage. The mean num-
ber of matings recorded was not significantly different at either ratio, suggesting that
treated males of this species are equally as competitive as their untreated counterparts. In
the mating propensity studies, virgin female H. armigera were first mated to treated or un-
treated males and then re-exposed to untreated males 24 hours later. No statistical differ-
ences were found in the number of females that re-mated from either group. Thirty point
eight percent of the females first mated with treated males and 29.17% of the females first
mated to untreated males re-mated in this study. When both types of females were re-ex-
posed to untreated males in the same field-cage, a higher percentage (38.3%) of females that
had initially mated with a treated male re-mated than those initially mated with a un-
treated male (31.7%), although the differences were not significant.

Key Words: F, sterility, partial sterility, remating, corn earworm

RESUME
En este articulo se reportan los resultados de experiments con la especie Helicoverpa armi-
gera. El objetivo de estos experiments era determinar el efecto de una dosis sub-esterili-
zante de radiacion gamma (100 Gy) en la competitividad de copula de machos irradiados y
el efecto indirecto sobre la propensidad de re-copula de las hembras con las cuales copularon
estos machos. La competitividad de machos fertiles e irradiados se evaluo a dos diferentes
tasas de liberation dentro dejaulas de campo colocadas en una plantacion de repollo. Las ta-
sas de liberation utilizadas fueron 1 macho:1 hembra y 4 machos:1 hembra mientras se man-
tuvo el numero de insects constant dentro de las jaulas. El numero promedio de copulas
que se recobraron en cadajaula no fue significativamente distinto, lo que sugiere que los ma-
chos irradiados pueden competir efectivamente con los machos fertiles. En studios sobre
propensidad de re-copula, hembras virgenes fueron apareadas con machos fertiles o irradia-
dos y luego fueron re-expuestas a machos fertiles despues de 24 horas. No se encontro una
diferencia estadistica en el porcentaje de re-copulas entire los grupos de hembras. Treinta
punto ocho porciento de las hembras que primero se aparearon con machos irradiados re-co-
pularon despues de 24 horas mientras que 29.17% de las hembras primero apareadas con
machos fertiles re-copularon despues de 24 horas. Cuando se expusieron estas hembras (ini-
cialmente apareadas con distintos tipos de machos) simultaneamente a machos fertiles des-
pues de 24 horas, un porcentaje mas alto (38.3%) de hembras inicialmente apareadas con
machos irradiados se re-aparearon que las hembras apareadas con machos feritles (31.7%),
pero las diferencias no fueron estadisticamente significativas.


The corn earworm, Helicoverpa armigera
(Hubner), is one of the most destructive pests of
agricultural crops in the Philippines. It is a highly
polyphagous insect, feeding on 84 different plant
species (Gabriel 1997). One environmentally ac-
ceptable control strategy currently being explored
for the control of this insect is the release of irra-
diated partially sterile males capable of trans-
ferring sterility to the next generation. The
advantages of using partial, inherited or F, steril-
ity over classical sterile insect release methods is
that partial sterility produces a more competitive


insect that will actively mate with wild females
and, as such, will effectively introduce heritable
sterility into the native population (Mastro &
Schwalbe 1988). Inherited sterility has been in-
vestigated in a number of lepidopteran pests such
as Helicoverpa zea (Carpenter & Gross 1993),
Spodoptera frugiperda (Carpenter et al. 1997),
Manduca sexta (Seth & Reynolds 1993) andEphe-
stia kuehniella (Marec et al. 1999).
Helicoverpa armigera, a closely related species
of H. zea (Laster & Hardee 1995), is a potential
candidate for population suppression by means of







Ocampo: Irradiation and mating in H. armigera


the F, sterility technique. Ocampo et al. (1996)
were able to induce partial sterility in this species
by irradiating pharate adult males with 100 and
150 Gy. When the treated males were mated to
untreated females, female fertility was greatly af-
fected. Percentage egg hatch in females mated to
100 Gy or 150 Gy treated males was only 40-60%
while that of females mated to untreated males
was 80-100% (V. Ocampo, unpublished data). In a
related study, Bella et al. (1997) reported that
when 100 Gy was used to treat H. armigera
males, the dose caused chromosomal transloca-
tions in 86% of the spermatocytes examined while
150 Gy caused chromosomal translocations in
98% of the spermatocytes in the testes of treated
males. Similarly, 100 Gy has been shown to be
most efficacious in the codling moth, Cydia
pomonella (Anisimov 1993) and the fall army-
worm, Spodoptera frugiperda (Carpenter et al.
1997). In these species, the dose induced a high
level of inherited sterility in the F, generation
without reducing competitiveness of parental
males treated.
Mating competitiveness is essential to the ef-
fectiveness of a sterile insect release program.
However, the refractory period of wild females
must not be adversely affected by mating with the
released males (Snow 1988). An irradiated male
must be able to transfer a full complement of
sperm and accessory gland fluid to the female,
and this complement must reach the spermath-
eca to elicit the female refractory period. Giebul-
towicz et al. (1990) showed that presence of sperm
in the spermatheca is necessary to induce the
switch from virgin to mated behavior in the gypsy
moth, Lymantria dispar. During the refractory
period, pheromone production by the female re-
mains low, oviposition behavior is triggered and
the female will refrain from remating. If sperm
and accessory gland fluid do not reach the sper-
matheca, pheromone production will increase and
the female will resume calling for mates.
In this article we report the results of experi-
ments conducted with H. armigera to determine
the effects of 100 Gy of gamma radiation on the
mating competitiveness of treated males and the
effect on the mating propensity of the females
with which they mate.

MATERIALS AND METHODS

Helicoverpa armigera used in these experi-
ments came from a laboratory colony maintained
at the Department of Entomology, University of
the Philippines Los Banos. Larvae were reared
under ambient laboratory conditions (27 + 2C) in
50 ml plastic cups and fed a soybean-corn based
diet as described in Ocampo et al. (2000). Pupae
were collected and sexed and stored at ambient
conditions until needed. Late pupae containing
pharate males were irradiated at a dose of 100 Gy


using a Cobalt60 Gammacell 220 irradiator deliver-
ing a dose of 4 Gy/min. Treated and untreated pu-
pae were held separately until adult emergence.
Newly emerged (<24 h-old) virgin male and fe-
male moths were used in all mating experiments.

Mating Competitiveness of Males

Male competitiveness was assessed in field-
cages (n = 4; 6m x 5m x 2.15m) placed over cab-
bage plants within 1-ha cabbage field. Three
types of males were used in the experiments.
Males treated with 100 Gy (TJ), as indicated
above; F, progeny males (TF, ), obtained from
crossing treated (100 Gy) P, males to untreated
virgin females, and untreated males (NJ). In or-
der to distinguish the male "types" the wing tips
of treated and F, males were differentially colored
with a felt-tip marker and untreated males were
left unmarked. Moths were allowed to acclimate
to field conditions for 3 hours (from 1700 hours
Philippine Standard Time (PST) until they were
released at 2000 hours PST). Males and females
were released at opposite ends of the cages. In
each cage mating pairs were collected once every
hour for five hours, from 2100 to 0200 hours PST,
coinciding with the peak of mating activity as re-
ported by Morallo-Rejesus and Alcala-Carilo
(1981). These workers also report that copulation
lasted an average of 30 minutes.
Mating competitiveness was measured at two
different release ratios. A 1:92 ratio, maintain-
ing equal numbers of male "types" while keeping
a density of 60 moths per cage. Field-cages in this
study received 30 NJ each, and the following
male treatments: 30 TJ; 30 NJ; 15 TJ + 15 NJ;
10 TJ + 10 TF,1 + 10 NJ. The next set of exper-
iments was conducted at a 4?:1 9 ratio, while
keeping a constant density of 50 moths per cage.
Field-cages in this study received 10 NY each,
and the following male treatments: 40 TJ; 40 N J;
30 TJ + 10 NJ. All combinations were replicated
once per night for four nights. Male types with the
highest number of recorded matings were consid-
ered the most competitive. Data were subjected to
analysis of variance and differences between
means were tested for significance using Waller-
Duncan's K-ratio t-test (Ott 1993).

Mating Propensity of Females

Mating propensity was assessed inside field-
cages (see above). Insects were released into sep-
arate cages in the following combinations: 100
untreated H. armigera females (NY) + 100
treated males (TJ?-100 Gy), and 100 NY + 100 un-
treated (NJ) males. Pairs in copula were collected
once per hour as described above. Females that
mated with treated (N2-T?) and untreated (NY
- NJ) males were differentially colored using a
felt-tip marker as above. On the following







Florida Entomologist 84(2)


evening, the mated females (from both groups)
were given the opportunity to remate with un-
treated males (Nd) by releasing them into field-
cages in the following combinations (at 19:16 ra-
tio): 30 NY-NJ + 30 NJ, 30 N$-T? + 30 NJ, 15
NY-NJ + 15 N$-T? + 30 NJ. Pairs in copula
were collected as above. All treatments were rep-
licated four times. Females were then dissected to
determine the number of spermatophores in the
bursa copulatrix. A twice-mated female should
have two spermatophores to confirm two success-
ful matings/copulations.

RESULTS
Mating Competitiveness of Males

The results of the mating competitiveness tests
are summarized in Table 1. At the 19:16 ratio,
the mean number of mating pairs collected in
cages containing treated (TS) and untreated
(NJ) males was not significantly different (F = 1.07,
d.f. = 6, a = 0.05). In addition, when both male
types were together in the same cage (30 N9:15
Td:15 NJ), both Td and NJ males appeared to
be equally competitive. The mean number of mat-
ings for NJ was 4.25 and this number was 3.76
for Td (F = 0.17, d.f. = 6, a = 0.05). Mating com-
petitiveness of F, male offspring (TF,1) was also
evaluated. Results show that the mean number of
matings for TF1S was 4.76, which did not differ
statistically from the number of matings with
other male types (F = 0.21, d.f. = 9, a = 0.05).
When experiments were conducted at a 46:19
ratio, there was no significant difference in the
mean number of mating pairs collected in cages
containing Td (7.00) or NJ (6.00) males (F = 0.46,
d.f. = 6, a = 0.05). When three times more treated
males than untreated males were released into
the field-cage (10 N9:10 Nd:30 T6) the number
of observed matings for the treated males signifi-
cantly exceeded the expected number (F = 216,
d.f. = 6, a = 0.05).


Mating Propensity of Females

The results of the mating propensity studies,
where virgin female H. armigera were first mated
to treated or untreated males and then re-ex-
posed to untreated males 24 hours later are
shown in Table 2. Thirty point eight percent of the
females first mated with treated males (N$-T?)
and 29.17% of the females first mated to un-
treated (NY-NS) males re-mated in this study.
The difference was not statistically significant (F
= 0.04, d.f. = 6, a = 0.05). When both types of fe-
males were re-exposed to untreated males in the
same field-cage (15 N$-T? + 15 NY-NJ + 30
NS), a higher percentage (38.3%) of females that
had initially mated with a treated male re-mated
than those initially mated with a untreated male
(31.7%), although the differences were not signif-
icant (F = 0.25, d.f. = 6, a = 0.05). Carpenter et al.
(1987) report similar trends for Helicoverpa zea, a
closely related species to H. armigera.

DISCUSSION

An important concern in sterile insect release
programs is that treated males destined for re-
lease retain the ability to perceive/orient to pher-
omone signals from females and, as such, are able
to compete with the wild males in locating and
mating with calling females in the field. Data pre-
sented herein suggest that partially sterilized
male H. armigera and their male progeny were as
competitive as untreated males in seeking and se-
curing mates in a field-cage situation. Males
treated with 100 Gy, F, males (from 100 Gy
treated fathers) and untreated males appeared to
be equally as competitive in mating with virgin
females when placed together in field-cages. It ap-
pears that a dose of 100 Gy of gamma radiation
does not cause sufficient physiological damage to
alter male mating behavior, but induces sufficient
genetic damage in the spermatocytes to reduce
sperm viability, as reported by Bella et al. (1997).


TABLE 1. MATING COMPETITIVENESS OF TREATED (100 GY-TC), UNTREATED (Nd) AND F, OFFSPRING (TF, ) OF HELI-
COVERPA ARMIGERA MALES FOR VIRGIN FEMALES INSIDE FIELD-CAGES (6M x 5M x 2.15M) IN A CABBAGE FIELD
AT UPLB, LAGUNA, PHILIPPINES, 1996.

Mean number of mating pairs (SD)'

Ratio Number and type of moths NxN NxT N x TF,

16:12 30 N2:30 N6 or 30 N2:30 T6 10.00 + 2.00a 12.75 + 4.92 a 2
30 NY:15 Td + 15 Nd 4.25 + 2.22a 3.76 + 0.96 a 2
30 N2:10 TJ + 10 TF16 + 10 Nd 6.25 + 4.50a 5.25 + 3.30 a 4.76 + 1.26 a
46:1Y 10 N2:40 Nd or 10 N:40 T6 6.00+ 2.31a 7.00 + 1.82 a 2
10 NN:30 T6 + 10 NJ 0.50+ 0.58a 6.50 + 0.58 b 2

Means + SD on the same row followed by the same letter are not significantly different (K ratio = 500).
Not applicable.


June 2001







Ocampo: Irradiation and mating in H. armigera


TABLE 2. MATING PROPENSITY OF ONCE-MATED FEMALE HELICOVERPA ARMIGERA MATED TO EITHER UNTREATED (NY
-Nd) OR TREATED (NY-T6) MALES AND EXPOSED TO UNTREATED MALES AFTER 24 HOURS INSIDE FIELD-
CAGES (6M x 5M x 2.15M) IN A CABBAGE FIELD AT UPLB, LAGUNA, PHILIPPINES, 1996.

Mean number of re-matings (SD) 24 h after the first mating (percent)'

Moths released into field-cages N -Nd x N N -Nd x N

30 N2-Nd:30 Nd or 30 N2-Td:30 Nd 8.75 3.86 (29.17) a 9.25 2.99 (30.83) a
15 N2-Td:15 NY-Nd:30 Nd 4.75 2.87 (31.67) a 5.75 2.75 (38.33) a

'Means + SD (percentage) on the same row followed by the same letter are not significantly different (K ratio = 500).


This dose also induced a high level of inherited
sterility in the F, male progeny of males treated
with 100 Gy without reducing their capacity to
seek and secure mates. The competitiveness of
other irradiated Lepidopterans has been reported
by Carpenter et al. (1989, 1997) and Anisimov
(1993). In these species, 100 Gy has also been
shown to be most efficacious. Carpenter et al.
(1989) further showed that the irradiated H. zea
males were competitive with normal laboratory
reared insects in their ability to survive under
field conditions.
Helicoverpa armigera has a tendency to mate
more than once. In our experiments, about 30% of
mated females re-mated regardless of the type of
male with which they had first mated (Table 2).
These findings agree with an earlier study
(Ocampo et al. 2000) that found 31-36% ofH. ar-
migera females mated twice. Our data also sug-
gests that females mated to treated (100 Gy)
males were no more attractive to untreated males
on the night following the first mating than were
females mated to untreated males. One explana-
tion for this finding might be that there is no dis-
cernible difference in the quality of the sperm
complement and accessory fluid transferred by
untreated and treated males during mating.
Thus, the type of male with which the female
mates first does not affect her mating propensity
and refractory period.


ACKNOWLEDGMENTS

This research was supported by Research Con-
tract No. 8156/RB under the FAO/IAEA Coordi-
nated Research Program entitled "Evaluation of
Population Suppression by Irradiated Lepidop-
tera and Their Progeny" of the International
Atomic Energy Agency, Vienna, Austria. The au-
thor is very grateful to the two anonymous re-
viewers for valuable suggestions on the
manuscript. The author would also like to thank
Ms. Josephine B. de Leon, Ms. Gaudencia M.
Anober and Ms. Rosita M. Crisologo for their
technical support during the conduct of field-cage
experiments.


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CARPENTER, J. E., A. N. SPARKS, S. D. PAIR, AND H. L.
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MASTRO, V. C., AND C. P. SCHWALBE. 1988. Status and
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June 2001







Nguyen Thi & Nguyen Thanh: F, sterility in DBM suppression


RADIATION INDUCED F, STERILITY IN PLUTELLA XYLOSTELLA
(LEPIDOPTERA: PLUTELLIDAE): POTENTIAL FOR POPULATION
SUPPRESSION IN THE FIELD

NGUYEN THI QUANG HOA AND NGUYEN THAN THUY TIEN
Department of Biology, Dalat University, 1 Phu Dong Thien Vuong St., Dalat, Viet Nam

ABSTRACT

The potential of using F1 sterility in a system to manage the diamondback moth (DBM), Plu-
tella xylostella (L.) (Lepidoptera: Plutellidae), was investigated in the laboratory and in
field-cages. When 6-day old male pupae were treated with 200 Gy of gamma radiation, 71.5%
developed into normal adults. However, radiation-induced reductions in fecundity and via-
bility were expressed during the P,, F, and F2 generations. Sterility exceeded 60% in the P,
and F2 generations and 90% in the F, generation. The sex ratio was skewed in favor of males
among F1 and F2 progeny. The percentages of metaphase spermatogonial cells with chromo-
somal aberrations were 86.9, 21.5 and 9.7 in the F,, F2, and F, respectively. No differences
were observed in the sperm transfer between irradiated and unirradiated males. When
treated males were released into field-cages at either a 5:1 or a 10:1 overflooding ratio with
unirradiated moths, there was a significant reduction in the number of F, and F2 adults
emerging in the field-cages as compared to the control. A 50-60% reduction in the F1 and 59-
68% in the F2 generation were observed. When irradiated females and males were released
at a 5:5:1:1 overflooding ratio with untreated DBM, the decrease in F, adult emergence was
not significantly different than for the control. However, adult emergence in the F2 genera-
tion was reduced by almost 90%. This degree of suppression was significantly greater than
that achieved in cages where only irradiated males had been released. The use ofF, sterility
in combination with releases of the parasitoid, Cotesia plutellae (Kurdjumov) (Hy-
menoptera: Braconidae), in field-cages resulted in a 40% decrease in the DBM population in
the F, and more than 90% in the F2 generation. Nevertheless, additional research is needed
to develop this system into an economically feasible strategy for managing early season pop-
ulations of DBM.

Key Words: diamondback moth, Cotesia plutellae, inherited sterility, black stripe pupal mutant

RESUME

El uso potential de la esterilidad F, como m6todo de control para Plutella xylostella (L.)
(DBM, "diamond back moth") se investigo en studios de laboratorio y de campo. Cuando pu-
pas macho de 6 dias de edad se irradiaron con 200 Gy de radiaci6n gamma, el 71.5% se de-
sarrollaron como palomillas aparentemente normales. Sin embargo, en las generaciones P,,
F, y F2 las reducciones en fecundidad y viabilidad fueron obvias. El nivel de esterilidad ex-
cedi6 el 60% en las generaciones P, y F2 y el 90% en la F,. En la progenie F, y F2, la tasa sexual
favoreci6 a los machos. El porcentaje de espermatogonias en metafase con aberraciones cro-
mosomicas fue de 86.9, 21.5 y 9.7 en las generaciones F,, F2, y F,, respectivamente. No se ob-
servaron diferencias entire machos irradiados y no irradiados en su habilidad en transferir
esperma. Cuando se liberaron machos irradiados en jaulas de campo junto con palomillas
fertiles en las tasas 5:1 o 10:1, el numero de adults en las generaciones F1 y F2 se redujo sig-
nificativamente. La reducci6n fue de 50-60% en la generaci6n F1 y 59-68% en la generaci6n
F2. Cuando se liberaron insects irradiados de ambos sexos en la tasa 5:5:1:1 con palomillas
fertiles, la reducci6n en el nivel poblacional de la generaci6n F, no fue diferente de lo obser-
vado en la jaula control. Sin embargo, se observe una reducci6n del 90% en la generaci6n F2.
El uso combinado de la esterilidad F, con liberaciones de Cotesia plutellae (Kurdjumov) (Hi-
men6ptera: Braconidae) en jaulas de campo causaron que la poblaci6n de DBM disminuyera
mas del 40% en la F, y mas de 90% en la generaci6n F2. Sin embargo, sugerimos que es ne-
cesario continuar las investigaciones para lograr que este sistema sea econ6micamente fac-
tible para el control de las infestaciones tempranas de DBM.


The use of inherited or F, sterility as a compo- (Lepidoptera: Plutellidae), is a serious pest of cru-
nent of population management programs for ciferous crops throughout the world. Costs for its
Lepidopterans has been studied by several re- control are estimated at US$1 billion annually
searchers (Knipling 1970; North & Holt 1971; (Talekar 1992). Omar & Mansor (1993) report that
LaChance 1985; Carpenter et al. 1987). The dia- radiation doses between 150 Gy and 200 Gy were
mondback moth (DBM), Plutella xylostella (L.) suitable for inducing inherited sterility in DBM







Florida Entomologist 84(2)


males. Sutrisno et al. (1993) and Sutrisno & Hoe-
daya (1993) suggested that doses of 175 Gy or 200
Gy could be considered in a DBM suppression pro-
gram. Preliminary results relating to the poten-
tial suppression of DBM populations in the field
by the F, sterility technique are reported herein.

MATERIALS AND METHODS

Laboratory Colonies

Diamondback moth. Field-collected DBM pu-
pae from cabbage were used to found the colony.
Emerging adults were paired in oviposition cages
and provided with a 10% sucrose solution.
Grooved aluminum foil egg sheets that had been
dipped in autoclaved cabbage juice were used as
oviposition substrates. Egg sheets were changed
daily. Neonates were reared on leaves of Tropae-
olum majus L. (Rhoedales: Tropaeolaceae), a wild
host of DBM. The colony was maintained at 20 +
2C, 70-80% RH and a 14L:10D photoperiod. The
black stripe pupal mutant (BSP) is a marker
present in our DBM laboratory colony (see below).
Parasitoids. A colony of Cotesia plutellae (Kur-
djumov) (Hymenoptera: Braconidae), reared on
DBM larvae is maintained at our laboratory in
Dalat. Laboratory conditions are the same as for
the DBM colony. The colony was founded with
field-collected material.

Effects of Gamma Radiation on DBM

Six day-old colony pupae were irradiated with
a dose of 200 Gy in a Co60 irradiator at the Dalat
Nuclear Research Institute (dose rate 43.2 Gy/
min). Insects were used in laboratory and field-
cage tests. Percent emergence, percentage of
moths without deformities and longevity of irra-
diated versus untreated DBM adults were re-
corded in the laboratory. An average of 200 pupae
were used in each of three replicates for the first
two parameters. A subset of 100 adults in each of
three replicates were used in the longevity study.
Treated DBM males and females (from the P1,
F, and F2 generations) were crossed with un-
treated counterparts and allowed to mate and lay
eggs. Fecundity, fertility (percent sterile eggs),
percent surviving to adulthood and sex ratio were
recorded for all crosses. For fecundity and fertility
data, an average of 20 moth pairs were used per
replicate and three replicates were completed.
For percent survival, an average of 100 larvae per
replicate were used.

Chromosomal Aberrations

Testes of fourth instar DBM larvae from the F1,
F2 and F3 generations were dissected in Belar's sa-
line solution and then squashed to release the
germ cells. The slides were stained with aceto-or-


cein, sealed with clear nail polish and stored in a
covered container in the refrigerator until exam-
ined for the presence of chromosomal aberrations.

Sperm Transfer

Irradiated and unirradiated DBM adult males
were allowed to mate with unirradiated virgin fe-
males inside laboratory cages. Mating pairs were
collected and mating was allowed to proceed until
the insects separated. Males were reintroduced
into the cages and exposed to virgin females after
24 h for 5 consecutive days. Females from each
mating pair were dissected after oviposition to de-
termine the presence of a spermatophore and to
assess the ratio of eupyrene to apyrene sperm
present in the spermatheca. The normal ratio of
eupyrene to apyrene is considered to be 2:1 for Tri-
choplusia ni according to North & Holt (1971). Any
ratio deviating from this is considered abnormal.

Effect of Mating Status and Type
and Ratio of Sperm on DBM Oviposition

Unirradiated males were allowed to mate with
unirradiated virgin females in laboratory cages.
Mating pairs were removed from the cages. After
separation, females were allowed to lay eggs in
the laboratory. When oviposition was completed
females were dissected to determine the presence
of sperm and the ratio of apyrene:eupyrene sperm
in the spermatheca. Virgin females were also held
for oviposition as controls.

BSP Mutant

Individuals in our DBM laboratory colony pos-
sess the black stripe pupa or BSP mutant. We
have isolated and maintained a vigorous and ho-
mozygous mutant culture using the method of
Bartlett & Raulston (1982). Mutant individuals
were inbred and a small colony was established.
We attempted to determine the mode of inherit-
ance of the BSP mutant using the method of
Walder (1988). Reciprocal crosses were made be-
tween mutant (BSP) females and males, and wild
type stock (BSP). Some F, adults from the initial
crossed were used in reciprocal back-crosses with
the mutant strain and the rest were inbred.

Evaluation of F, Sterility
for Suppression of DBM in Field-Cages

Cabbage was planted inside four field-cages
(2m x 2m x 4m); plants were irrigated regularly
and received no other treatments during the ex-
periments. Six-day old colony (BSP) pupae were
irradiated as indicated above and pupae were
sexed and allowed to emerge into separate con-
tainers. A wild strain (BSP+ = pupae with no black
stripe) was collected from the field as 4th instar


June 2001







Nguyen Thi & Nguyen Thanh: F, sterility in DBM suppression


larvae and allowed to pupate and emerge in the
laboratory. One-day old adults from both groups
were used in the experiment.
Field-cages received the following ratios of ir-
radiated BSP colony females (IF), irradiated BSP
colony males (IM), feral BSP females (UF) and
feral BSP' males (UM), respectively: Treatment A
= 0:0:1:1 (control), Treatment B = 0:5:1:1, Treat-
ment C = 0:10:1:1 and Treatment D = 5:5:1:1.
Cages were checked every two days and eggs de-
posited on the cabbage were tagged and counted.
The population increase in each field-cage was de-
termined by counting the number of eggs, pupae
and adults for two generations. Three replications
were completed.

Combined Releases of Irradiated DBM
and Cotesia plutellae in Field-Cages
Cotesia plutellae (CP) parasitoids from our col-
ony (see above), one day-old 200 Gy treated colony
(BSP) DBM adults (I) and unirradiated wild
(BSP') moths (U) were released into field-cages in
the following ratios: Treatment 1 = 0 CP: 0 I: 1 U
(control), Treatment 2 = 5 CP: 0 I: 1 U, Treatment
3 = 0 CP: 5 I: 1 U, and Treatment 4 = 2.5 CP: 2.5
I: 1 U. In Trt. 4, the release of one day-old irradi-
ated moths occurred at day one and was followed
ten days later with the release of one-day old par-
asitoid adults. Field-cages were checked every
two days and the cabbage leaves on which eggs
had been deposited were tagged and eggs were
counted. The size of the DBM population inside
the field-cages was determined by counting DBM
eggs, pupae and adults. The size of the parasitoid
population was determined by counting the num-
ber of parasitoid cocoons present in each cage.

Statistical Analysis
Data were subjected to either the pooled t-test
or to analysis of variance. Differences between


means were tested for significance using Tukey's
Honest Significant Difference (HSD) (Minitab 9.2;
Minitab Statistical Software). Deviations from ex-
pected ratios were tested by chi-square analysis.

RESULTS AND DISCUSSION

Effects of Gamma Radiation on DBM

The results of our study on the effect of 200 Gy
on percent emergence, percentage of adults with-
out deformities and longevity of DBM are pre-
sented in Table 1. In general, DBM females
appear to be more sensitive to gamma radiation
than males. When 6-day old pupae were treated
with 200 Gy, 71.5% of the male pupae developed
as normal adults and 65.3% of these survived to
the 6th day, while the corresponding percentages
for treated females were 70.6 and 46.5. Adult lon-
gevity of both males and females was reduced by
about one-third as compared to controls.
Table 2 shows the results of measuring fecun-
dity, sterility, percent survival and sex ratio of
DBM irradiated with 200 Gy and their F, and F2
progeny. Significant differences were found in all
measured parameters when compared to con-
trols. These significant effects persisted in the F,
and F2 progeny. In the P, generation, the mean
number of eggs laid by the controls was 154. This
number decreased to an average of 84 eggs when
the female was crossed to an irradiated male.
Further, the number of eggs produced by un-
treated females mated to F, males was reduced to
about one-fifth the number laid by the controls.
However, when F2 males were mated to untreated
females, the number of eggs produced per female
was similar to the number produced when irradi-
ated males were mated to normal females. The
sterility of treated males was 62.1%, while that of
F1 males was 94.8% and that of F, females was
91.1%. In the F2 generation sterility ranged from


TABLE 1. EMERGENCE, PERCENTAGE OF ADULTS WITHOUT DEFORMITIES AND LONGEVITY OF PLUTELLA XYLOSTELLA
ADULTS WHICH HAD BEEN IRRADIATED WITH 200 GY AS 6-DAY-OLD PUPAE.

Mean + SD

Percent adults Percent survival Longevity
Sex Percent emergence' without deformities' to day 62 (days)3

Control
Male 94.1 + 3.1' 89.3 + 2.7' 84.2 + 5.3' 12.3 + 2.4'
Female 95.2 + 1.8' 91.1 + 5.8' 85.1 + 4.6' 11.9 + 1.4'
200 Gy
Male 85.4 + 6.3' 71.5 + 7.2' 65.3 + 7.4b 8.9 + 0.8"
Female 83.2 + 4.1' 70.6 + 7.8' 46.5 + 8.5' 7.9 + 1.0'
Means followed by the same letter within a column for each treatment are not significantly different (P > 0.05; pooled t-test).
Average of 200 pupae, 3 replications.
Moths, which did not survive up to the sixth day usually, were not able to mate.
Average of 100 moths, 3 replications.







Florida Entomologist 84(2)


TABLE 2. FECUNDITY, STERILITY, PERCENT SURVIVAL AND SEX RATIO OF PLUTELLAXYLOSTELLA IRRADIATED WITH 200
GY AS 6-DAY-OLD PUPAE AND THEIR F, AND F2 PROGENY.

Mean + SD

Sterility
Fecundity (% of eggs that Survival Ratio of
Mating type (total # of eggs)' failed to hatch)' (%)2 males/females

P, generation
UM x UF 156 + 10' 12.7 + 7.8' 78.5 + 8.9b 1.01 + 0.01)
IM x UF 84 + 3b 62.1 + 6.8b 57.8 + 7.6' 1.98 + 0.07b
F1 generation
UM x UF 145 + 8b 10.3 + 0.6' 85.1 + 2.2' 0.97 + 0.05'
AF1M x UF 33 + 4' 94.8 1.3b 11.1 + 3.7' 1.78 0.20b
"F1F x UM 28 + 2' 91.1 + 2.0b 32.1+ 10.5b 1.63 + 0.17b
F2 generation
UM x UF 140 + 7b 11.4 + 3.4' 75.7 + 78' 1.03 + 0.14'
F2AM x UF 84 + 3 63.5+ 7.1b 64.7 + 9.7' 1.18 +0.11
F2AF x UM 80 + 7" 61.8 + 8.2b 65.3 + 13.3' 1.42 + 0.13b
F2BM x UF 87 + 6' 64.8 + 11.8b 62.9 + 12.3' 1.25 + 0.12ab
F2BF x UM 86 + 3 62.4 5.6b 70.1+ 10.6' 1.34+ 0.10b


I= irradiated, U= unirradiated.
Means followed by the same letter within a column for each treatment are not significantly different (P = 0.05


Average of 20 pairs of moths, 3 replications.
Percent survival of 100 neonate larvae to adult, 3 replications.
"Male and female progeny from each F, cross were followed separately.

61.8% to 64.8%. Survival to adulthood in progeny
of untreated moths varied between 75-85%, while
it was 57.8% in the F, generation, from 11.1% to
32.1% in the F2 and from 62.9% to 70.1% in the F,.
The sex ratio was biased in favor of males among
F1 and F2 progeny.
These results differ from those reported by Su-
trisno et al. (1993) and Sutrisno & Hoedaya
(1993). We report higher levels of sterility than
those reported by these authors. In addition, the
fecundity of untreated females mated to irradi-
ated, F, and F2 males was lower than for un-
treated controls in our experiments, while
Sutrisno et al. (1993) and Sutrisno & Hoedaya
(1993) report that fecundity in these crosses was
equal to that observed in the untreated controls.
The reasons for these differences remain unclear,


Tukey's Honest Significant Difference).


but might be explained by differences in DBM
strain, rearing methods and dose calibration.

Chromosomal Aberrations

Cytological examination of chromosomal aber-
rations in the primary spermatocytes in 4th-instar
larvae (Table 3) showed abnormal chromosomes
evident in 86.9%, 21.5% and 9.7% of larvae from
the F1, F2 and F3 generations respectively, while
none were evident in the untreated controls.
DBM have 31 pairs of chromosomes, individually
recognizable during metaphase-I. Reciprocal
translocations in the form of rings or chains, in-
volving four or six chromosomes were observed in
the F, generation. These particular aberrations
are the main cause of inherited sterility.


TABLE 3. PERCENTAGE OF METAPHASE NUCLEI WITH VISIBLE CHROMOSOMAL ABERRATIONS IN PROGENY OF PLUTELLA
XYLOSTELLA MALES WHICH HAD BEEN IRRADIATED WITH 200 GY AS 6-DAY-OLD PUPAE AND THEIR F1, F2 AND
F, MALE PROGENY.

No. with chromosomal Frequency of aberrations
Generation No. of nuclei examined' aberrations (%)

F, 92 80 86.9
F2 79 17 21.5
F, 72 7 9.7
Control 77 0 0.0

'Testes from mature larvae were dissected in Belar's solution and then squashed to release the germ cells. The slides were stained with aceto-orcein,
sealed and stored in the refrigerator until examined.


June 2001







Nguyen Thi & Nguyen Thanh: F, sterility in DBM suppression


Carpenter (1991) and Zhang et al. (1993) re-
port that incidence of visible chromosomal aber-
rations in F, and F2 larvae of Helicoverpa zea and
Ostrinia furnacalis is dose dependent, and that
aberrations occur most frequently in the F, and
progressively less frequently in subsequent gen-
erations. North (1975) reported that male prog-
eny of H. zea treated with 200 Gy showed at least
one translocation. Our observations on DBM
agree with results reported by these authors for
other Lepidoptera.

Sperm Transfer

Table 4 shows the percentage of matings on
each of five consecutive days by irradiated (200
Gy) and unirradiated DBM males. This percent-
age diminished for both groups with each succes-
sive mating. Lepidopteran males produce
apyrene and eupyrene sperm that is transferred
to the female during mating. Eupyrene sperm are
nucleate and therefore capable of fertilization,
while apyrene sperm are smaller and anucleate.
Both types are present in the spermatophore and
migrate to the spermathecae in the female after
copulation is complete (North & Holt 1971). The
percentage of treated and untreated DBM males
that transferred sperm in each of five consecutive
matings is given in Table 4. No reduction in
sperm transfer was evident among the first, sec-
ond and third matings for either male group. The
percentage of males that transferred a normal ra-
tio of eupyrene to apyrene sperm remained fairly
high during the first three consecutive matings
for both groups.


In Trichoplusia ni Holt & North (1970) report
that the ability of fully sterile males to transfer
sperm was considerably lower than in unirradi-
ated males. They found that when sperm were de-
posited into the bursa copulatrix, the eupyrene
sperm lacked motility, while the apyrene sperm
were able to migrate to the spermatheca. In con-
trast, treating DBM pupae with a substerilizing
dose of radiation (200 Gy) resulted in males able
to transfer normal ratios of eupyrene:apyrene
sperm. Our results for DBM are similar to those
reported by El-Naggar et al. (1984) onAgrotis yp-
silon, Carpenter et al. (1987) on Helicoverpa zea,
and Sallam & Ibrahim (1993) on Spodoptera lit-
toralis.

Effect of Mating Status and Type
and Ratio of Sperm on Oviposition

North & Holt (1971) reported that when T ni
females mated with an irradiated male they often
failed to deposit a normal number of eggs. They
suggested that this might be caused by inade-
quate sperm or accessory gland fluid transfer to
the female during mating. Table 5 shows the ef-
fect of mating status and type and ratio of sperm
on oviposition in DBM. Females that received a
normal sperm complement laid significantly
(twice as many) more eggs than those receiving
an abnormal sperm ratio. It appears that type
and quantity of sperm transferred significantly
influences fecundity in DBM females.
Holt & North (1970) reported that early in cop-
ulation the spermatophore in Trichoplusia ni
becomes filled with a clear fluid even in females


TABLE 4. ABILITY OF PLUTELLA XYLOSTELLA MALES IRRADIATED WITH 200 GY OF TO TRANSFER SPERM.

No. of Percent of males which
consecutive No. males % of mated males which transferred normal ratio
matings tested' Percent mated transferred sperm eupyrene:apyrene sperm2

Unirradiated male
1 92 91.3 92.8 89.7
2 84 75.0 92.2 83.3
3 63 69.8 90.9 80.0
4 44 34.1 53.3 32.5
5 15 33.3 0.0
Irradiated male
1 127 88.9 92.1 88.9
2 113 74.3 92.7 83.4
3 84 65.4 91.1 79.5
4 55 32.7 51.2 31.2
5 18 0.0

Irradiated and unirradiated males were allowed to mate with unirradiated virgin females on each of five consecutive days. Mating pairs were re-
moved from the cages. Mated females were dissected after oviposition to determine the presence of a spermatophore and the ratio of apyrene to eupyrene
sperm in the spermatheca.
The normal ratio of eupyrene to apyrene is considered to be 2 eupyrene:1 apyrene. Any ratio below 1:1 is considered abnormal (North & Holt 1971).







Florida Entomologist 84(2)


TABLE 5. EFFECT OF MATING STATUS AND TYPE AND RATIO OF SPERM ON PLUTELLA XYLOSTELLA OVIPOSITION IN THE
LABORATORY.

No. eggs/female
Sperm transferred No. females (Mean SD)

Normal eupyrene:apyrene' 77 234 + 26d
Abnormal eupyrene:apyrene1 55 112 + 16'
No sperm' 32 73 + 14b
None (virgin females) 42 47 + 11"

Means followed by a different letter within a column are significantly different (P = 0.05; Tukey's Honest Significant Difference).
Unirradiated males were allowed to mate with unirradiated virgin females in laboratory cages. The mating pairs were removed from the cages. When
the mated females had finished oviposition, they were dissected to determine the presence of sperm and the ratio of apyrene and eupyrene sperm in the
spermatheca. Virgin females were held for oviposition as controls.


that receive no sperm, and that this may elicit the
laying of a significant number of eggs. This obser-
vation appears to hold true for DBM. In our ex-
periments mated females that received no sperm
laid significantly more eggs than virgin females,
which suggests that fecundity in DBM is also
influenced by the secretions from the accessory
gland transferred during mating.

BSP Mutant

Reciprocal crosses made between BSP and/or
BSP' virgin females and males confirm that the
gene responsible for the mutation black stripe
pupa (BSP) is dominant and most likely located
on an autosome.

Evaluation of F, Sterility
for Suppression of DBM in Field-Cages

Evaluating the results of inherited sterility as
a method for population suppression is particu-
larly complex. Released partially sterile individu-
als mate with released untreated individuals to
produce partially sterile progeny. The progeny
continue to do the same until the experiment is
stopped and effects are ascertained. Several
marking techniques (such as radioisotopes, exter-
nally applied fluorescent powders and internal oil
soluble dyes) have been used to identify released
Lepidopteran insects. Unfortunately, these mark-
ers do not allow the descendants of a released
insect to be tracked through subsequent genera-
tions. Bartlett (1967) suggested that mutations
could be used as biological markers. Further-
more, he suggested that dominant and co-domi-
nant mutants are most useful, as they can
identify not only the released individuals but also
their progeny and descendants in subsequent
generations. The presence of the BSP mutant in
our DBM colony made it possible for us to exam-
ine the mating interactions of the irradiated DBM
through several generations.
The results of our field-cage experiments are
shown in Table 6. When treated males (Trts. B
and C) were released at either a 5:1 or a 10:1 over-


flooding ratio with unirradiated moths, there was
a significant reduction in the number of F, and F2
adults emerging in the field-cages as compared to
the control (Trt. A). A 50-60% reduction in the F1
and 59-68% in the F2 generation were observed
for field-cages receiving Trt. B and C, respectively.
When irradiated females and males were re-
leased at a 5:5:1:1 overflooding ratio (Trt. D), the
decrease in F, adult emergence was not signifi-
cantly different than for the control (<3%). How-
ever, adult emergence in the F2 generation was
reduced by almost 90%. This degree of suppres-
sion was significantly greater than that achieved
in cages where only irradiated males had been re-
leased. Nonetheless, 1.4 times more neonates
were produced by Treatment D in the F, genera-
tion as compared to the control, and these neo-
nates caused greater host plant damage.
Several investigators have formulated hypoth-
eses concerning the role that released irradiated
females might play in the suppression of popula-
tions subjected to the sterile insect technique
(Whitten & Taylor 1970, Allam & Galun 1976). If
partially sterile females are released they could
contribute a significant fraction of the progeny
produced in the target population. This increase
in the number of F, progeny may benefit a release
program when the released females carry lethal
chromosomal changes that will be transferred to
the target population. However, the major sup-
pression of the target population is deferred to
(but greatly enhanced in) the second generation.
North & Holt (1971) suggested that the maximum
economic efficiency in the use of inherited steril-
ity for the suppression of lepidopteran popula-
tions requires the release of both sexes of
partially sterile moths. Our results support the
above analyses and findings.
In our field-cage studies BSP individuals were
identified among the progeny in all cages except
the control. The ratio of BSP to BSP in the F, gen-
eration was 2.21, 3.41 and 8.94 in Treatments B,
C, and D, respectively. The ratio of BSP to BSP in
the F2 generation was 0.11, 0.09 and 0.07 in Trts.
B, C, and D, respectively (Table 6). All individuals
expressing the black stripe pupal mutant were


June 2001







Nguyen Thi & Nguyen Thanh: F, sterility in DBM suppression


TABLE 6. INFLUENCE OF RELEASING IRRADIATED (200 GY) AND UNIRRADIATED BSPPLUTELLAXYLOSTELLA ADULTS AT
DIFFERENT RATIOS ON THE DEMOGRAPHIC PARAMETERS OF THE F, AND F2 GENERATIONS IN FIELD-CAGES.

Mean + SD
Treatment
(No. of IF:IM:UF:UM per cage) No. eggs No. neonates No. pupae No. adults Ratio

F, generation
A-control 3243 + 767" 3037 + 64' 1015 + 167b 921 + 5b 0
(0:0:50:50)
B 2653+ 667" 2238 + 338b 542 + 124" 459 + 87" 2.21
(0:250:50:50) (50.16%)
C 2397+ 645" 1490 + 166" 415 + 90" 362 + 51" 3.41
(0:500:50:50) (60.69%)
D 8781+ 1138b 4325 + 206d 1120 + 194b 895 + 118b 8.94
(250:250:50:50) (2.82%)
F2 generation
A 8348 + 459b 7766 + 198' 2345 + 160' 2154 + 145' 0
B 4246 + 361" 3166 + 323' 923+ 74b 879+82b 0.11
(59.19%)
C 3619 + 212" 1523 + 105b 729 + 70"b 684 + 68b 0.09
(68.25%)
D 4370 + 466" 688 + 270" 648 + 47" 266 + 43" 0.07
(87.65%)

Means followed by the same letter within the same column are not significantly different (P = 0.05; Tukey's Honest Significant Difference).
'Values in parentheses indicate the percent decrease in the moth population as calculated by the formula: ((U-R)/ U) where U is the number of
emerged moths in the control population, and R is the number of moths which emerged in the treated population.
'BSP+ = wild moth and BSP = mutant moth.
Three replications were completed.


descendants of released DBM, and it was these
individuals that transmitted the sterility factors
into subsequent generations. In fact, the ratios of
BSP to BSP' in the various field-cages are the
ratios of substerile to fertile DBM in the F, and F2
generations. The presence of BSP-marked indi-
viduals is the most compelling evidence of the
mating interaction between released substerile
and wild moths. The usefulness of mutant mark-
ers as a tool to monitor the outcome of sexual
interactions between irradiated (BSP) and unir-
radiated (BSP) DBM has been demonstrated in
these experiments.
Even though our results would suggest that
male only releases should be considered for DBM,
no efficient technique is currently available to sep-
arate large numbers of male and female pupae. As
a consequence, simultaneous release of treated
males and females is unavoidable at this time. In
our experiments, population suppression in the F2
generation for bisexual releases was significantly
higher than that observed in the F2 for male only
releases, even at a ratio of 10:1. However a higher
amount of crop damage by F, larvae was observed
when both irradiated substerile male and female
DBM were released into the field-cages. One po-
tential solution to avoid excessive crop damage
would be to rear the DBM F, generation in the lab-
oratory and release F, adults instead of their irra-
diated parents. This would reduce crop damage


because of the lower fertility in the F, as compared
to the P, generation. However, the lower fertility
in the F, generation would call into question the
economic feasibility of this option.

Combined Releases of Irradiated DBM
and Cotesia plutellae in Field-Cages

Knipling (1979) suggested that combining par-
asitoid releases with sterile insect release might
yield both additive and synergistic effects. Al-
though the modes of action of both tactics are dif-
ferent, the effectiveness of the sterile insect
technique increases the ratio of adult parasitoids
to adult hosts, while the action of parasitoids in-
creases the ratio of sterile to fertile insects. Even
greater suppression could be expected if parasi-
toids are combined with the releases of partially
sterile insects. Carpenter (1993) demonstrated
that the economic benefits of combining inherited
sterility and parasitoids would be greatest when
the ratio of irradiated to unirradiated moths is
<10:1 and the ratio of parasitoid to hosts is <5:1.
Our objective was to investigate the potential
of using combined releases of partially sterile
DBM with releases of Cotesia plutellae, a specific
larval parasitoid, to suppress wild DBM popula-
tions. The results are summarized in Table 7. In
cages receiving partially sterile DBM (Trts. 3 and
4), the number of F, eggs was significantly (1.5







Florida Entomologist 84(2)


TABLE 7. INFLUENCE OF RELEASING COTESIA PLUTELLAE (CP), IRRADIATED (I) (200 GY) AND UNIRRADIATED (U) PLU-
TELLA XYLOSTELLA ADULTS AT DIFFERENT RATIOS ON THE DEMOGRAPHIC PARAMETERS OF THE F, AND F2
GENERATIONS IN FIELD-CAGES.

Mean + SD
Treatment'
CP:I: U No. eggs No. neonates No. pupae No. adults'

F, Generation
Treatment 1 298 + 67' 262 + 42' 127 + 33b 108 + 25'
(0:0:10)
Treatment 2 279 + 40' 252 + 34' 87 + 10ab 78 + 17'
(50:0:10) (27.78%)
Treatment 3 417 + 21b 255 + 41' 115 + 25ab 104 + 16'
(0:50:10) (0%)
Treatment 42 432 + 20b 262 + 41' 66 + 15' 63 + 15'
(25:25:10) (41.67%)
F2 Generation
Treatment 1 2796 + 415b 2534 + 204 1572 + 297d 1431 + 2214d
Treatment 2 2267 + 308b 2018 + 195b 1108 + 109 1002 + 86
(29.98%)
Treatment 3 1432 + 289' 288 + 56' 261 + 48b 257 + 51b
(82.04%)
Treatment 42 843 + 152' 195 + 8' 52 + 4' 40 + 5'
(96.58%)

Means followed by the same letter within a column are not significantly different (P = 0.05; Tukey's Honest Significant Difference).
CP = Cotesta plutellae; I = irradiated BSP moth; U = unirradiated BSP' moth.
Irradiated DBM were released on day 1 and were followed 10 days later with the release of Cotesta plutellae.
Values in parentheses indicate the percent decrease in the moth population as calculated by formula: (U-R)/U) where U is the number of moths which
emerged in the control population, and R is the number of moths which emerged in treated population.
Three replications were completed.


times) higher. However, because dominant lethal
mutations in the irradiated moths killed many
embryos during development, no significant dif-
ference was found in the number of neonates
among treatments. Thus, the level of host damage
caused by larvae in all four populations was al-
most identical. The number of adult moths was
lowest in Treatment 4, although differences
among treatments were not significant. However,
in the F, generation the number of eggs in Trts. 3
and 4 was significantly lower than the number of
eggs in Trts. 1 and 2. Treatment 4 had the lowest
number of eggs at 843. The number of neonates
for each of the treatments was significantly lower
than the number of neonates in the control. Fur-
thermore, the number of neonates in Trt. 3 (288)
and Trt. 4 (195) was significantly lower than the
number of neonates in Trt. 2 (2018). As a result,
the level of damage on host plants was the lowest
in cages treated with both irradiated males and
females and parasitoids.
Each of the treatments significantly sup-
pressed population growth as compared to the
control. However, population suppression was
significantly greater in cages receiving a combi-
nation of irradiated (200 Gy) DBM males and fe-
males followed by a single release of C. plutellae.
When only one tactic was used, population sup-
pression was significantly higher in cages receiv-


ing irradiated DBM than in those receiving
parasitoids.
The numbers of parasitoids, BSP, and BSP'
marked moths recovered from each of the treat-
ments are summarized in Table 8. In Treatment 2
(parasitoids only), the population of C. plutellae
decreased by about 20% in the F, generation and
increased by about 75% in the F2 generation.
However, in Trt. 4 (irradiated DBM + parasitoids)
the size of the parasitoid population increased by
about 50% in F, generation and declined by about
15% in the F2. These observations might suggest
that C. plutellae may have had a higher survival
rate on irradiated F, (BSP) larvae than on the
wild type (BSP) larvae. However the greater
number of C. plutellae in the F2 generation in Trt.
2 is largely due to lower number of DBM larvae in
Trt. 4 (195 neonates).
Our data suggest that the use of both tactics,
inherited sterility and releases of the parasitoid,
C. plutellae, may be feasible for managing early-
season populations of DBM. Partially sterile
DBM adults could be released to produce large
numbers of F, sterile larvae on early-season an-
nual hosts. These F, larvae could in turn serve as
hosts for C. plutellae and other parasitoids
present in the field. In this way the next genera-
tion of parasitoids would be increased, and any
surviving partially sterile larvae would become


June 2001







Nguyen Thi & Nguyen Thanh: F, sterility in DBM suppression


TABLE 8. NUMBER OF PARASITOIDS, BSP AND BSP* MARKED PLUTELLA XYLOSTELLA OBSERVED IN FOUR POPULATIONS
SUBJECTED TO ONE RELEASE OF IRRADIATED BSP MOTHS AND OF THE PARASITOID, COTESIA PLUTELLAE.

Mean + SD'

Population (CP:I:U)' Parasitoids BSP BSP'

F, generation
Treatment 1 (0:0:10) 108 + 25
Treatment 2 (50:0:10) 41 + 8 78 + 17
Treatment 3 (0:50:10) 93 + 12 11+ 5
Treatment 4 (25:25:10) 38 + 3 56 + 13 7 + 2
F2 generation
Treatment 1 1431 + 214
Treatment 2 70 + 6 1002 + 86
Treatment 3 11+ 5 246 + 45
Treatment 4 32 + 3 21 +4 28 + 7

'BSP+ = wild moth; BSP = mutant moth; I= irradiated BSP moth; U= unirradiated BSP+ moth; CP = Cotesta plutellae.
Three replications were conducted.


partially sterile adults (Carpenter et al. 1996).
Further studies are needed to measure the in-
field effects of these combined tactics on the early-
season population dynamics in DBM.

ACKNOWLEDGMENTS

We are grateful to Waldemar Klassen, Bill
Butt, Leo LaChance, James Carpenter and Jorge
Hendrichs for documents and recommendations.
This work was supported by the International
Atomic Energy Agency under Research Contract
No. 7174/RB and the Dalat Nuclear Research In-
stitute under Research Contract VH 10/92.

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BARTLETT, A. C. 1967. Genetic markers in the boll wee-
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BARTLETT, A. C., AND J. R. RAULSTON, JR. 1982. The
identification and use of genetic marker for popula-
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national Workshop. Taiwan, AVRDC. 603 pp.


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June 2001







Henne &Johnson: Parasitism of Scapteriscus in Louisiana


SEASONAL DISTRIBUTION AND PARASITISM OF SCAPTERISCUS SPP.
(ORTHOPTERA: GRYLLOTALPIDAE) IN SOUTHEASTERN LOUISIANA

D. C. HENNE AND S. J. JOHNSON
Department of Entomology, 402 Life Sciences Building
Louisiana State University Agricultural Center, Baton Rouge, LA 70803

ABSTRACT
Mole crickets of the genus Scapteriscus were accidentally introduced into the southern
United States almost a century ago and are considered to be economically important pests
in southern U.S. regions. Mole crickets were sampled using acoustic traps in Baton Rouge
and New Orleans, Louisiana in the fall of 1998, the spring and fall of 1999, and the spring
of 2000. In southeastern Louisiana Scapteriscus borellii has a seasonal flight period starting
in late February and continuing into June. A lesser flight period occurs in the fall, starting
in mid-September and continuing into November. S. vicinus was captured only from late
February to late April. We determined that S. borellii was being parasitized inside acoustic
traps by the tachinid fly, Ormia ochracea and that S. vicinus was parasitized by an anth-
omyiid fly, Acridomyia sp.

Key Words: acoustic attraction, mole crickets, Ormia, Tachinidae,Acridomryia

RESUME
Los grillos topo del genero Scapteriscus fueron introducidos accidentalmente en el sur de los
Estados Unidos hace un siglo y se consideran ser parasitos importantes econ6micamente en
regions surenas de EE.UU. Los grillos topo fueron muestreados usando trampas acusticas
en Baton Rouge y New Orleans, Louisiana en el otoho de 1998, la primavera y otoho de 1999,
y la primavera del 2000. Scapteriscus borelli tiene un period de vuelo estacional comen-
zando tarde en febrero y continuando hasta junio. Un period de vuelo mas corto ocurre en
el otono, comenzando a mediados de septiembre y continuando hasta noviembre. S. vicinus
fue capturado solo entire fines de febrero y fines de abril. Determinamos que S. borellii estaba
siendo parasitado dentro de las trampas acusticas por la mosca tachinida Ormia ochracea y
que S. vicinus fue parasitado por la mosca esp.Acridomyia.


Three species of mole crickets, Scapteriscus
vicinus Scudder, S. borellii Giglio-Tos and S. ab-
breviatus Scudder, were accidentally introduced
into the southern United States from South
America from around 1899 to 1926 (Walker &
Nickle 1981). Scapteriscus vicinus and S. borellii
have since spread throughout the coastal plain
from southeastern Texas to southeastern North
Carolina (Parkman et al. 1996), with isolated pop-
ulations of S. borellii reported from Arizona
(Nickle & Frank 1988) and California (Frank
1994). Scapteriscus spp. mole crickets, especially
S. vicinus, are considered to be serious pests of
turf and pasture grasses throughout the south-
eastern U.S. (Hudson et al. 1988).
Using traps with artificial calling songs of S.
borellii and S. vicinus, surveys of these two spe-
cies were conducted in Baton Rouge and New Or-
leans, Louisiana, in the fall of 1998, the spring
and fall of 1999, and the spring of 2000. This was
done in order to ascertain the population levels in
these areas and to determine the flight periods of
introduced mole crickets in southeastern Louisi-
ana prior to release of Ormia depleta (Wiede-
mann) (Diptera: Tachinidae) in these cities for
biological control. This fly is native to South


America, where it attacks mole crickets of the ge-
nus Scapteriscus (Fowler 1987). It has been re-
leased at many sites in Florida and is established
there (Frank et al. 1996).

MATERIALS AND METHODS

Acoustic traps that use artificially produced
calling songs of mole crickets capture large num-
bers of these insects, and they are used for popu-
lation surveys and behavioral studies (Walker
1988). Traps in this study were constructed ac-
cording to details given in Parkman and Frank
(1992) with some modifications. Our traps con-
sisted of 1 m diameter sheet metal funnels sus-
pended from metal yokes. The yokes were
supported by 10 cm x 10 cm x 100 cm wooden
posts buried into the ground to a depth of 30 cm.
A single 20 1 plastic bucket was attached to the
bottom of each funnel to hold captured insects.
Buckets were suspended above ground level to
minimize predation of captured mole crickets by
red imported fire ants (Solenopsis invicta Buren).
Holes were drilled through the bottom of the
buckets to allow rainwater to drain. Two sound
traps were deployed at Burden Research Station







Florida Entomologist 84(2)


in Baton Rouge (3124.46 N, 9106.74 W) during
fall 1998, the spring and fall of 1999 and spring
2000. A second set of traps was also deployed in
City Park, New Orleans (2959.70 N, 9005.33 W)
during fall 1999 and spring 2000. A different song
was played over each of the traps; one synthesized
the song of S. borellii males and the other the
song of S. vicinus males. The traps were sepa-
rated by at least 2 m as suggested by Walker
(1982). Artificial crickets were obtained from
Night Caller Artificial Crickets (Eco-Sim, Gaines-
ville, FL), and they were powered by 12V, 7.0
amp-hour, lead acid gel-cell rechargeable batter-
ies (Power Sonic PS-1270). Batteries were re-
charged once per week.
Traps were serviced at least every other day.
Captured adults of Scapteriscus spp. were placed
in zippered plastic bags which were placed in a
cooler for transport to the laboratory. Adults were
held individually in 15 dram snap-lid, plastic vi-
als (WWR Scientific, West Chester, PA). Each vial
contained at least 4 cm (~35 ml) of moist sand.
The mole crickets were checked each day for par-
asitism. Dead mole crickets were removed from
vials and the sand searched for fly puparia. Lar-
vae of 0. ochracea Bigot require approximately
one week to complete development in the labora-
tory at 23-25C (Wineriter & Walker 1990). After
three weeks, all puparia were removed from the
sand and placed inside 15 dram snap-lid vials
onto 3 cm x 3 cm moist paper towels until emer-
gence of adults. Adult flies were identified using
keys in Sabrosky (1953). Voucher specimens of O.
ochracea, G. rubens Scudder, S. borellii and S.
vicinus were deposited in the Louisiana State
University Arthropod Museum. Larvae of Acri-
domyia were identified using keys to immature
Diptera in Teskey et al. (1991).


RESULTS AND DISCUSSION

Seasonal Distribution

Numbers of S. borellii and S. vicinus captured
in fall 1998, spring and fall 1999, and spring 2000
are shown in Figures 1 and 2. The flight period for
S. borellii in southeastern Louisiana begins in
late February and lasts into June. A lesser flight
period occurs in the fall, starting in mid-Septem-
ber and lasting into November. For S. vicinus, the
flight period in southeastern Louisiana also begins
in late February and continues into late April. No
S. vicinus were captured at either of the two trap-
ping locations during fall of the years 1998 or
1999. Flight periods of S. borellii and S. vicinus in
southeastern Louisiana are similar to those re-
ported by Walker et al. (1983) for S. borellii and S.
vicinus at Gainesville, Florida (29040'N), except
that we never captured S. vicinus in our traps
during the fall months. In addition, numbers of


mole crickets captured in our sound traps were
much lower than those reported in Florida.
Trap abundance cycles of 7-12 days were ob-
served during the fall 1998 and 1999, and spring
1999 and 2000 trap data (Figs. 1 and 2). Ngo and
Beck (1982) also observed trap abundance cycles
lasting ca. 9 days for S. borellii in Florida and at-
tributed this phenomenon to egg laying cycles of
S. borellii females. Walker and Nation (1982) re-
ported that some S. borellii males will call during
the fall months and females can respond in signif-
icant numbers. They found that some S. borellii
females do mate during the fall, since 7 out of 25
females attracted to synthetic calling song in Oc-
tober to December had sperm in their spermathe-
cae. It is not generally believed, however, that egg
laying occurs during the fall.

Parasitism of S. borellii by 0. ochracea

Ormia ochracea adults were commonly col-
lected in the S. borellii traps but were never col-
lected from the S. vicinus traps during fall 1998
and 1999. 0. ochracea were often observed resting
inside trap buckets and caller housings. No 0.
ochracea were reared from ca. 200 Scapteriscus
spp. collected during each spring of 1999 and 2000.
This was expected because 0. ochracea adults
were never observed at our traps during spring
1999 and 2000. Of 88 S. borellii captured at New
Orleans and Baton Rouge between 7 October and
18 October 1999, 18 were parasitized by 0. ochra-
cea, yielding 34 puparia (range 1-4 puparia per
host), (Table 1). A total of 14 adults ofO. ochracea
closed from the puparia. Flies (n = ca. 5-10) were
regularly observed resting inside the caller hous-
ing during the day. When the S. borellii trap buck-
ets were opened to inspect the contents, several
dozen flies would escape. We were often able to col-
lect flies, usually 10-20, from inside the trap buck-
ets that were either dead or too weak to fly. Walker
(1993) found that calls of S. borellii and S. vicinus
attracted few and zero 0. ochracea, respectively.
Tachinid flies of the tribe Ormiini are orthop-
teran parasitoids specializing on crickets and katy-
dids (Walker 1986, 1989). They are known to occur
throughout the southeastern United States from
Florida to Texas (Walker 1989; Robert & Hoy
1994). Females of the phonotactic tachinid fly
0. ochracea are larviporous (Frank 1994), are
attracted to artificially produced songs of its host,
Gryllus integer and larviposit in the vicinity of
calling males (Cade 1975; Walker 1989). Walker
(1986, 1989) captured large numbers of 0O. ochra-
cea at traps using artificially produced Gryllus
rubens Scudder songs. Mangold (1978) found that
0. ochracea also was attracted to synthesized
songs of S. borellii males and was able to success-
fully rear 0. ochracea to adulthood on 1 of 5
S. borellii and on one G. rubens artificially in-
fested. Wineriter and Walker (1990) also reared


June 2001







Henne &Johnson: Parasitism of Scapteriscus in Louisiana


t


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p
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ft I*~
9~ rfl go go go go
00 in
- et N N N ri


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t 4
U


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80
70-
S60-
| 50-
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. 40 -
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S10
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N e C4 C 4 -. t


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Sampling date


Fig. 1. (A) Numbers of S. borellii captured in sound traps at Burden Research Station, Baton Rouge, LA. Traps
were operated from 16 September to 4 November, 1998, (B) Numbers of S. borellii and S. vicinus captured in sound
traps at Burden Research Station, Baton Rouge, LA. Traps were operated from 16 February to 2 June, 1999, (C)
Numbers of S. borellii captured in sound traps at Burden Research Station, Baton Rouge, LA. Traps were operated
from 14 September to 21 October, 1999.


S


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Florida Entomologist 84(2)


l1a


.

t
aa
a I
I I
i i
(


0. /. ';
O .A
s *~ a A.


- - S. vicinus

*- --S. borellii


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a t t -
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--- S. vicinus


-- - S. borellii


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.-". *
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Sampling date

Fig. 2. Numbers of Scapteriscus spp. captured in sound traps at (A) City Park, New Orleans, LA. Traps were op-
erated from 22 February to 1 May, 2000, (B) Burden Research Station, Baton Rouge, LA. Traps were operated from
16 February to 1 May, 2000.


0. ochracea from S. borellii. According to Hudson
et al. (1988), larvae of O. ochracea have been col-
lected in the field only from crickets of the genus
Gryllus. As Mangold (1978) points out, the rear-
ing of 0. ochracea on S. borellii does not indicate
that this is a natural host. Instances of parasit-
ism of S. borellii by 0. ochracea are probably an
artifact of the trapping technique (Walker & Win-
eriter 1991). Confinement of O. ochracea females


with S. borellii for several days in our traps in-
variably resulted in a portion of the mole crickets
being parasitized. Hundreds of first-instar larvae
of 0. ochracea also were collected from the trap
buckets, suggesting that larvipositing by 0.
ochracea females occurred inside the trap buck-
ets. Our results are in contrast to data reported in
Frank et al. (1996), where only one out of tens of
thousands of Scapteriscus mole crickets captured


30

20


15 +


i


'''-'~


June 2001


.i







Henne &Johnson: Parasitism of Scapteriscus in Louisiana


TABLE 1. NUMBERS OF SCAPTERISCUS BORELLII PARASITIZED BY ORMIA OCHRACEAE: BATON ROUGE (BR) AND NEW
ORLEANS (NO), LA. OCTOBER, 1999

No. Ormia ochraceae
Date Site No. S. borellii captured No. S. borellii parasitized adults obtained

7.x BR 8 3 (7)* 5
11.x BR 30 10(20) 6
14.x BR 27 2 (3) 2
18.x BR 13 1 (1) 1
12.x NO 10 2 (3) 0

Totals 88 18 (34) 14

= numbers of 0. ochracea puparia reared from S. borelllu in parentheses.


in a survey was parasitized, presumably by 0.
ochracea. The authors did not state what species
of Scapteriscus was parasitized.
Three female G. rubens were collected in the
S. borellii sound trap at Baton Rouge between 14-
18 October 1999. Mangold (1978) also reported
that several Gryllus spp. were attracted to artifi-
cial songs of S. borellii in Florida. Of particular in-
terest is the attraction of 0. ochracea and G.
rubens to artificial songs of S. borellii but not of S.
vicinus. The calling songs of S. borellii and G.
rubens are similar (Burk 1982). Males of
S. borellii broadcast songs at a carrier frequency
of 2.7 kHz, with a pulse rate of ~50 pulses s1
(Walker 1982). Males of G. rubens broadcast songs
at a carrier frequency of 4.8 kHz, also with ~50
pulses s-1. (Walker 1986). In addition, calling songs
of S. vicinus have a carrier frequency that falls in
between that of S. borellii and G. rubens (3.3 kHz),
but have a much faster pulse rate of ~130 s1
(Walker 1993). Robert et al. (1992) found that the
hearing organ of 0. ochracea females is most sen-
sitive to frequencies in the range of 4 to 6 kHz.
There is, however, no reason to suggest that 0.
ochracea females would not respond to carrier fre-
quencies outside these ranges. In fact, Walker
(1993) demonstrated that 0. ochracea females are
attracted to carrier frequencies between 2.4 and
6.8 kHz. According to Chapman (1982) there is no
evidence that frequency discrimination alone is
especially important to the insect. The pulse repe-
tition frequency of insect songs is probably a more
important factor in their recognition because im-
pulses occur in the auditory nerves in synchrony
with sound pulses. This may explain why 0.
ochracea and G. rubens were attracted to artificial
calling songs of S. borellii at our trapping stations
but not to those of S. vicinus. In Walker (1986), 0.
ochracea females were attracted to synthesized
calls of G. rubens but not G. firmus. The carrier
frequencies of these two species fall within the
most sensitive range of 0. ochracea females (4-6
kHz), but songs of G. firmus have a much slower
pulse rate of ~17 s-1 (Walker 1986). The pulse rate


of S. vicinus (~130 S-1) is much faster than both S.
borellii and G. rubens. In addition, the carrier fre-
quencies of S. borellii and S. vicinus are much
lower than G. rubens. As Walker (1993) points out,
our knowledge of cricket communication, and that
of their acoustically attracted parasitoids, is far
from complete and future studies should examine
other components of sound that may be important.

Parasitism of S. vicinus byAcridomyia sp.

In late March 2000, two S. vicinus that were
captured in an acoustic trap in New Orleans were
found to have been parasitized byAcridomyia sp.
(Diptera: Anthomyiidae). One of the parasitized
S. vicinus contained six maggots, the other con-
tained only one. Unfortunately, no adult flies were
obtained. Larvae of flies in this genus are para-
sitoids of grasshoppers (Acrididae) (Dahlem &
Thompson, 1991). Little else is known about mem-
bers of this genus. AreAcridomyia sp. attracted to
the calling songs of S. vicinus or were these two
mole crickets accidentally parasitized?

ACKNOWLEDGMENTS

The authors would like to thank Daniel J. Gill,
County Agent, Orleans Parish for his assistance
in arranging for the deployment of sound traps on
City Park property in New Orleans. Thanks are
also extended to Drs. Abner Hammond and Gregg
Henderson (Louisiana State University) for their
helpful comments and criticisms of an earlier
draft of this manuscript. Approved for publication
by the Director, Louisiana Agricultural Experi-
ment Station as Manuscript number 00-17-0632.

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phonotactic parasitoid and levels of parasitism
(Diptera: Tachinidae: Ormia ochracea). Florida En-
tomol. 74(4): 554-559.
WINERITER, S. A., AND T. J. WALKER. 1990. Rearing pho-
notactic parasitoid flies (Diptera: Tachinidae: Ormia
spp.). Entomophaga 35: 621-632.


June 2001







Ellis et al.: Evaluation of Serangium


EVALUATION OF SERANGIUM PARCESETOSUM (COLEOPTERA:
COCCINELLIDAE) FOR BIOLOGICAL CONTROL OF SILVERLEAF WHITEFLY,
BEMISIA ARGENTIFOLII (HOMOPTERA: ALEYRODIDAE), ON POINSETTIA

DONNA ELLIS', RICHARD McAvoY', LUMA ABU AYYASH', MELISA FLANAGAN', AND MATTHEW CIOMPERLIK2
1Department of Plant Science, University of Connecticut, Storrs, CT 06269-4163

2USDA-APHIS-PPQ Mission Plant Protection Center, Mission, TX 78572

ABSTRACT
Control of silverleaf whitefly (Bemisia argentifolii Bellows & Perring) on greenhouse poin-
settia with biological agents has been unreliable. Serangium parcesetosum Sicard, a coc-
cinellid predator, appears to have great potential for silverleaf whitefly control. In our study,
dynamic changes in B. argentifolii populations on caged poinsettia in response to S. parces-
etosum were monitored. Silverleaf whiteflies were introduced to caged poinsettias at 1 or 10
adults per plant and 6 weeks later S. parcesetosum were introduced at 0, 2 or 4 adults per
plant. Within 2 weeks of Serangium release whitefly mortality increased dramatically, and
for the ensuing 10 weeks whitefly levels remained at or near those observed at time of pred-
ator release. Beetle larvae were observed 2 to 10 weeks after Serangium release when prey
was initially high but not when prey was initially low. Thus, whitefly control was primarily
due to prolonged survival and continuous feeding of individual beetles. Our data suggest
that Serangium may work well in a multiple species biological control program for whiteflies
on poinsettia. However, further study is needed on multiple species interactions within the
host (pest/plant) species, and on release management strategies.

Key Words: Population dynamics, caged study, predator, prey

RESUME
El control de la mosca blanca (Bemisia argentifolii Bellows & Perring) en poinsetias de in-
vernadero con agents biol6gicos ha sito erratico. Serangium parcesetosum Sicard, un pre-
dador coccinelido, parece tener gran potential para el control de B. argentifolii. En nuestro
studio, cambios dinamicos en poblaciones de B. argentifolii en poinsetias enjauladas en res-
puesta a S. parcesetosum fueron observados. Las moscas blancas fueron liberadas en poin-
setias enjauladas de 1 a 10 adults por plant y 6 semanas despu6s S. parcesetosum fueron
liberados de 0,2, o 4 adults por plant. Dentro de 2 semanas desde la introducci6n de Se-
rangium la mortalidad de la mosca blanca incremento dramaticamente, y por las pr6ximas
10 semanas los niveles de moscas permanecieron en o cerca de aquellos observados al mo-
mento de introducci6n del predador. Larvas de escarabajos fueron observadas de 2 a 10 se-
manas despu6s de la liberaci6n de Serangium cuando el numero de presa estaba
inicialmente alto pero no cuando el numero de presa estaba inicialmente bajo. Por lo tanto,
el control de la mosca blanca fue debido principalmente a supervivencia prolongada y ali-
mentaci6n continue de escarabajos individuals. Nuestros datos sugieren que Serangium
pudiera servir bien en un program de control de species multiples de moscas blancas en
poinsettia. Sin embargo, mas investigaci6n es necesaria sobre las interacciones de species
multiples dentro la especie (plaga / planta, y en estrategias de control de liberaci6n.


Silverleaf whitefly, Bemisia argentifolii Bel-
lows & Perring (Homoptera: Aleyrodidae, also
known as the sweetpotato whitefly, B. tabaci
(Gennadius) Biotype B), is the most important ar-
thropod pest on greenhouse grown poinsettias
(Ecke et al. 1990). Poinsettia cuttings often arrive
infested with whitefly nymphs at levels well be-
low economic thresholds (Helgesen & Tauber 1977;
Hoddle et al. 1999) but whitefly populations rap-
idly increase to exceed economic thresholds in the
absence of effective controls.
Poinsettia is the single largest potted flower-
ing greenhouse crop grown in the U.S. in terms of
both number of pots produced (>59 million) and


annual wholesale value (>$220 million dollars)
(USDA 1997). It appears to be a good candidate-
crop for biological control because it is produced
as a monoculture and has few serious pest prob-
lems other than Bemisia argentifolii (Parrella et
al. 1991). In practice however, economic biological
control systems capable of suppressing B. argen-
tifolii to the low thresholds required for ornamen-
tal crops have been elusive (Parrella et al. 1991).
For example, Hoddle et al. (1997a) reported that
when Encarsia formosa Gahan (Hymenoptera:
Aphelinidae) was used as the control agent, 23 to
70% of poinsettia plants were infested with im-
mature whitefly at the end of the season. By com-







Florida Entomologist 84(2)


prison they observed 30% of poinsettias in
commercial retail outlets were infested with im-
mature whitefly. With the E. formosa Beltsville
strain, end-of-season whitefly infestation ranged
from 77 to 100% of plants under various release
rates compared to 28% of plants observed in com-
mercial retail outlets (Hoddle et al. 1997b). Heinz
& Parrella (1994) observed satisfactory whitefly
control with a combination of Encarsia luteola
Howard and Delphastus pusillus LeConte (Co-
leoptera: Coccinellidae) but at a cost 5-times
higher than insecticide-based control. With
weekly releases of Eretmocerus eremicus n. sp.
Rose & Zolnerowich (Hymenoptera: Aphelinidae),
Hoddle et al. (1998) reported that 73 and 83% of
plants were infested with immature whitefly at
the end of the season compared to only 28% of
plants in commercial retail outlets. These studies
underscore the need for continued evaluation of
promising new biological control agents.
Serangium parcesetosum Sicard is a coccinellid
predator that has demonstrated potential for the
biological control of silverleaf whitefly (Legaspi
et al. 1996). This species was originally collected
from India in 1929 for release as a biological con-
trol agent of citrus whitefly, Dialeurodes citri
Ashmead (Aleyrodidae), in the Union of Soviet
Socialist Republics (Kapur 1954; Timofeyeva &
Nhuan 1979). As a result of the success in that
biological control program, and because of its re-
discovery during foreign exploration in Podumbu,
India, S. parcesetosum is currently being re-
searched as a predator of silverleaf whitefly.
Serangium parcesetosum (herein referred to as
Serangium) has been known from the available
literature to feed mainly on citrus whitefly, al-
though in field trials in the U.S. it has also at-
tacked silverleaf whitefly (M. C., unpublished).
All of the known coccinellids belonging to the tribe
Serangiini are obligate predators of whiteflies, or
in a few cases, scale insects (Gordon 1985).
Laboratory studies to date show that both lar-
vae and adults of Serangium are voracious feed-
ers, capable of consuming large numbers of
immature silverleaf whiteflies in short periods of
time. Legaspi et al. (1996) showed adults con-
sumed approximately 400 whitefly nymphs in a
24h period. Serangium larvae consumed 25 to 50
whitefly eggs or nymphs in 24 h, depending on the
larval stage (M. C., unpublished). Furthermore,
Legaspi et al. (1996) determined the cumulative
lifetime predation rate to be approximately 5,000
whitefly nymphs per adult Serangium. These
data suggest that Serangium may have the poten-
tial to control silverleaf whitefly at moderate to
high levels. However, low whitefly infestation lev-
els may not be adequate to sustain Serangium re-
production or even adult survival.
In this study we investigate the effects of Se-
rangium release rates on the population dynam-
ics of B. argentifolii on caged poinsettia.


MATERIALS AND METHODS

Four rooted poinsettia (Euphorbia pulcher-
rima Willd. Ex. Klotzsch. cv. 'Freedom Red') cut-
tings were transplanted into individual 30 cm
pots on 16 July 1997 in the University of Connect-
icut research greenhouse range. Each exclusion
cage was constructed of a white organdy sleeve
supported by 75 cm bamboo plant stakes and
placed around each pot. Cages were sealed above
the four poinsettia plants and below the lip of
each pot. Velcro strips were used on two vertical
seams, one on each side of the cage, to facilitate
access to the plants.
After plants were established in the pots, sil-
verleaf whiteflies were introduced on the caged
poinsettia plants on 23 Aug. 1997 at a rate of ei-
ther 1 or 10 adults per plant (equivalent to 4 or 40
adult whiteflies per cage). On 3 Oct., Serangium
parcesetosum were introduced into cages at 0, 2
and 4 adults per plant (equivalent to 0, 8 and 16
Serangium per cage, respectively). The result was
a 2 x 3 factorial design with initial levels of either
1 or 10 whiteflies per plant and 0, 2, or 4 Seran-
gium per plant. Treatments were arranged in a
randomized complete block with five replications.
Poinsettias in separate cages, but without either
prey or Serangium, were used to evaluate the ef-
fects of whitefly on plant growth. Prey and Seran-
gium were shipped overnight from the USDA
APHIS Mission Plant Protection Center (Mission,
TX) in insulated containers with ice packs. A
small Hibiscus plant infested with whitefly pupae
was shipped just prior to emergence of the adults.
Whiteflies were held in a controlled temperature
chamber until adults emerged within 48 h. Newly
emerged whitefly adults were aspirated and then
transferred into the treatment cages. Serangium
were shipped as adults in paper cartons with or-
ganza lids for ventilation. Each container held 25
adults. A Hibiscus leaf with whiteflies was in-
cluded in each container for Serangium feeding in
transit. Immediately upon arrival, Serangium bee-
tles were introduced into treatment cages using a
fine camelhair paintbrush.

Monitoring Silverleaf Whitefly
and Serangium Populations

Two leaves per plant (8 leaves per cage) were
harvested weekly from 3 Oct. to 5 Dec. 1997.
Leaves were selected from the strata of the plant
canopy with the greatest number of late instar
whitefly nymphs; the sample strata was deter-
mined each week just prior to leaf harvest. A 25
cm2 section of each leaf was examined under a dis-
secting microscope and the number of whitefly
eggs and the number of live and dead nymphs and
pupae were recorded. Immature whitefly were
judged dead when they appeared discolored or
desiccated, or when the empty integument


June 2001







Ellis et al.: Evaluation of Serangium


showed evidence of Serangium feeding. As leaf
samples were harvested they were visually
checked for Serangium, and all larvae and adults
were returned to their respective cages.
At the conclusion of the study, 8 Dec. 1997, a
stratified leaf sample was collected from each
cage and whitefly population and percent mortal-
ity were determined. The stratified sample con-
sisted of two leaves per cage from each of four
locations on the plant: bracts, upper canopy, mid-
dle canopy and lower canopy. The two leaves per
stratum were collected randomly and examined
for whitefly life stages with a dissecting micro-
scope. The poinsettia plants were then destruc-
tively harvested and total laminar surface area
(leaves plus bracts) was determined for all plants
in each treatment using a LI-COR 1600 leaf area
meter (LI-COR, Inc. Lincoln, NE). Plants were
separated into bracts, leaves and stems and dried
in a forced-air oven at 70C for one week. Plant
dry mass was then recorded.

Data Analysis

The mean density of whitefly eggs, nymphs,
and pupae (per 25 cm2) was calculated for each
treatment. Percent whitefly mortality was calcu-
lated as follows [1]:
[(dead nymphs + dead pupae)/(live nymphs + live
pupae + dead nymphs + dead pupae)]*100
Equation [1]
Bartlett's test for homogeneity of variance was
conducted on all data (Bartlett, 1937). Non-nor-
mal data for whitefly and Serangium counts and
for plant measurements were transformed using
the formula: log10O (x + 1), with x representing de-
pendent variables. Non-normal data for whitefly
mortality were transformed using the formula:
Arcsine (x/100)%, with x representing percent
whitefly mortality. Analysis of variance was per-
formed using the Statistical Analysis System
(SAS Institute 1995). Insect population data were
analyzed as a factorial in a randomized complete
block design with five replicates. Plant data (lam-
inar surface area and shoot dry mass) were sub-
jected to a two-way analysis of variance with
seven treatments and five replicates. The treat-
ments included the six whitefly-Serangium com-
binations and a control with no predators or prey.

The Greenhouse Environment

Plants were grown under standard cultural
practices for poinsettias (Ecke et al. 1990). Envi-
ronmental conditions were recorded on a Camp-
bell Scientific 21x Datalogger (Campbell Scientific,
Logan, UT) at 1-minute intervals. LI-COR 190-
SA quantum sensors (LI-COR, Inc. Lincoln, NE)
were used to monitor photosynthetic photon flux
and copper-constantan thermocouples were used


to monitor temperature. Light intensity was mon-
itored in the greenhouse immediately above the
exclusion cages, and also above the plant canopy
within the cages. Air temperature was monitored
within the cages in the plant canopy and above
the canopy, and directly outside of the cages. For
the duration of the study, daily photosynthetic
photon flux (SE) averaged 9.1 0.5 mol.m-2.day-1
in the greenhouse above the cage and 8.0 + 0.5
mol.-m-day' above the plant canopy in the cage.
Air temperature in the cage and in the plant can-
opy (in the cage) averaged 21.3 0.1C and 21.4 +
0.2C (respectively) and the ambient air tempera-
ture in the greenhouse (outside the cage) aver-
aged 22.4 + 0.2C.

RESULTS

Initial silverleaf whitefly release rates greatly
affected final population densities of all whitefly
life stages (Figs. 1 & 2; df = 1,16; eggs F = 12.3; P
= 0.025, nymphs F = 32.8; P=0.005, pupae F = 10.7;
P = 0.031). This effect was most evident when
whitefly populations were left uncontrolled. For
example, the final density of nymphs was approx-
imately 10-times higher in cages with initial re-
lease rates of 10 whitefly adults per plant than in
cages with initial release rates of 1 whitefly adult
per plant (Fig. ib). A similar pattern was observed


180

1600
so
40
20


- ~


25

w~~~ ~ ~ ---------------- ----------------0-c4
Ia



DATE
1- Wmhtefly 0 Serengium -0--10 Whely 0 Serangium

Fig. la-c. Changes in silverleaf whitefly life stages on
poinsettia plants over time in the absence of a biological
control agent. Plants were initially inoculated with ei-
ther 1 or 10 whitefly adults per plant on 23 August.
Stages include; a) eggs, b) nymphs, and c) pupae. Verti-
cal bars denote standard error of the mean.


. ._ -_ .







Florida Entomologist 84(2)


25 a
-20
E 4

10


DATE
-I Whitelfy 2 Se .ngium .- Whiteffy 4 $er.tngm
-- Whi Miaffy 2 Seanghim --1 Whieflt 4 Serangium

Fig. 2a-c. Changes in silverleaf whitefly life stages on
poinsettia plants over time in response to different initial
Serangium parcesetosum release rates. Poinsettia plants
were initially inoculated with either 1 or 10 adult white-
flies per plant on 23 Aug., and 6 weeks later S. parce-
setosum was introduced at 2 or 4 adults per plant. White-
fly stages include; a) eggs, b) nymphs, and c) pupae.
Arrows denote Serangium introduction on 3 October. Ver-
tical bars denote standard error of the mean.


with respect to both whitefly eggs (Fig. la) and pu-
pae (Fig. ic) at the 1 and 10 whitefly release rates.
A single release of adult Serangium beetles
was extremely effective at stopping the growth of
whitefly populations on poinsettias (Fig. 2). Six
weeks after Serangium were introduced (13 Nov.),
whitefly population densities were dramatically
higher in cages without Serangium (Fig. 1) than
in cages with Serangium (Fig. 2) [df = 2,16; eggs
F = 13.9; P = 0.0003, nymphs F = 19.3; P = 0.0001,
pupae F = 9.4; P = 0.002]. Serangium effectively
maintained immature whitefly densities at or
near those observed at the time of predator intro-
duction (Fig. 2). For example, nymphal prey den-
sities were 0.7 and 8.0 per 25 cm2 of leaf surface in
the 1 and 10 whitefly cages (respectively) when
Serangium were introduced (Fig. 2b). These pop-
ulations remained nearly constant when exposed
to either the 2 or 4 beetles per plant release rates
(Fig. 2b), while nymphal densities increased up to
70 fold in the ensuing 10 week period without Se-
rangium (Fig. ib). Within both the 1 and 10
whitefly treatments, similar final prey densities
were observed for the high and low Serangium
treatments (Fig. 2).
A dramatic increase in prey mortality was ob-
served within 14 days (16 Oct.) of Serangium re-


lease (Fig. 3; df = 2,16; F = 49.5; P = 0.0001). In
cages with an initial release rate of 10 whitefly
adults per plant, mortality reached 57 and 69%
for the 2 and 4 Serangium treatments (respec-
tively) on 16 Oct. (Fig. 3a). Whitefly mortality in
these treatments peaked at about 85% during the
23 Oct. to 6 Nov. time period and was approxi-
mately 55% at final harvest. Over the 10 week ex-
perimental period, prey mortality averaged 60%
(+4.4% SE) with Serangium present in cages with
the 10 whitefly treatments.
In cages with an initial whitefly release rate of
1 adult per plant, the increase in prey mortality
was less dramatic than in the 10 whitefly treat-
ments (Fig. 3a). For example, mortality was only
10 and 38% for the 2 and 4 Serangium treatments
(respectively) on 16 Oct. but reached approxi-
mately 50 and 80% on 30 Oct. At final harvest in
the 1 whitefly treatment, prey mortality rates
were about 20 and 60% with 2 and 4 Serangium
(respectively). Over the 10 week experimental pe-
riod, whitefly mortality averaged 24% (3.9% SE)
with an inoculation of 2 Serangium per plant and
52% (3.9% SE) with 4 Serangium per plant. In
contrast, mortality in cages without Serangium


*- I WNIteffy 2 Serungum t*- I Wntetly4 Seranqginm
-4- 10 Whtffy 2 Sfraag~iu -t-10 Whiiefty4 S-gn-m1
4as -____ _i-- -- --- *- - -- -. ------- ._ -- --
b

S36

2o.
-20
0 i




DATE
U- t1 WlitMyO Soangium -6-O1WhietflyOSoas.gium

Fig. 3a-b. Changes in silverleaf whitefly mortality (%)
on poinsettia plants over time in response to Serangium
parcesetosum release rates of 2 or 4 adults per plant (a),
or in the absence of biological control agents (b). Poinset-
tia plants were initially inoculated with either 1 or 10
adult whiteflies per plant on 23 Aug., and 6 weeks later
S. parcesetosum was introduced at 0, 2, or 4 adults per
plant. Percent whitefly mortality was calculated using
the equation: [(dead nymphs + dead pupae)/(live nymphs
+ live pupae + dead nymphs + dead pupae)l*100. Arrows
denote Serangium introduction on 3 October. Vertical
bars denote standard error of the mean.


June 2001







Ellis et al.: Evaluation of Serangium


averaged 4.2% (0.6% SE) in the 10 whitefly
cages and 6.8% (2.3% SE) in the 1 whitefly cages
during the final 10 weeks of the study (Fig. 3b).
At final harvest, live immature whiteflies were
observed throughout the plant canopy (Table 1).
In cages without Serangium, approximately 70%
of whiteflies were located in the upper leaf strata
and on the red-colored poinsettia bracts. With
Serangium present prey were more uniformly
distributed in the leaf/bract strata in the 1 white-
fly cages than in the 10 whitefly cages. This may
indicate that Serangium needed to move more
quickly to the upper canopy to find prey when
prey populations were low but not when popula-
tions were high. In the 1 whitefly treatment cages
with Serangium, the average live immature
whitefly counts observed throughout the canopy
were about 16% of those without predators (Table
1). In the 10 whitefly treatment cages with Seran-
gium, the average live immature prey counts
were about 10% of those without predators. Aver-
age live immature prey counts were similar for
both the 2 and 4 Serangium treatments in all four
strata of the plant canopy with an initial prey
release of 1 per plant (Table 1). Only small differ-
ences in the number of live immature whiteflies
in the middle and lower leaf strata were observed
between the 2 and 4 Serangium treatments with
an initial whitefly release of 10 per plant.
At final harvest, the highest prey mortality
was observed in the lower half of the plant canopy
(middle and lower leaf strata v. upper leaf and
bract strata) (Table 1). Throughout the plant can-
opy, the highest mortality consistently occurred
in cages with Serangium and in cages with high
initial whitefly release rates (Table 1).
Very few Serangium were recovered from
cages at time of final harvest and the total num-
ber (larvae + adults) of Serangium recovered did
not vary with treatment (P<0.05). In cages with
an initial inoculation rate of 1 prey, only adult Se-
rangium were recovered and at an average den-
sity of 0.2 and 1.2 per cage for the 2 and 4
Serangium release rate treatments, respectively.
In cages with an initial inoculation rate of 10
prey, 2 and 2.4 Serangium per cage were recov-
ered from the 2 and 4 Serangium release rate
treatments (respectively) and Serangium larva
(1.2 per cage) were only observed in the 2 Se-
rangium release rate treatment. During the final
weeks of this study, whitefly mortality rates de-
clined in all Serangium treatments (Fig. 3a) and
simultaneously whitefly populations increased
(Fig. 2). These data suggest that low Serangium
counts at final harvest were not indicative of the
predator levels that prevailed during the first
eight weeks following Serangium release when
maximum prey control was observed (Fig. 2).
Poinsettia growth was unaffected by whitefly
populations in this study. Total laminar surface
area (1.6 0.03 m2/cage, df = 6, 23; F = 1.99; P =


0.11) and shoot dry mass (84.7 + 1.9 g/cage, df= 6,
23; F = 0.86; P = 0.54) were similar for plants in
all treatments.

DISCUSSION

Consumers have a low tolerance for insect
pests on greenhouse ornamentals like poinsettia.
Consequently, high standards must be set when
evaluating the effectiveness of natural enemies
and their management. Inundative releases of
Encarsia formosa can produce satisfactory re-
sults if introduced in sufficient numbers before
whitefly populations begin to build (Hoddle et al.
1997a,b). However, in instances where Encarsia
fail to control whiteflies, alternative measures are
required in order to maintain a salable plant
(Parrella et al. 1991; Heinz & Parrella 1994).
In our study, silverleaf whitefly reached dam-
aging populations 6-8 weeks after introduction
when left uncontrolled even when initial popula-
tions were low (1 adult per plant). Heinz & Par-
rella (1994) observed a dramatic increase in
whitefly populations on greenhouse grown plants
(both inside and outside of exclusion cages) after
nine weeks exposure to whiteflies even in the
presence of weekly releases ofE. luteola. However,
a series of three weekly releases of the predatory
beetle Delphastus pusillus (1 beetle per plant per
week) effectively checked whitefly population
growth until the study was ended 3 weeks after
the final release. In our study, a single release of 2
Serangium per plant effectively checked further
increases in prey population for up to 10 weeks.
Heinz & Parrella (1994) recovered several
adult D. pusillus 3 weeks after the last release,
but no evidence of successful predator reproduc-
tion was reported. Hoelmer et.al. (1993) reported
that D. pusillus required 100-150 whitefly eggs
per day to initiate and sustain oviposition. In our
study, Serangium larvae were first observed 2
weeks after adults were released in cages with
high initial whitefly levels (data not shown) but
not in cages with low initial whitefly levels. In
cages with high initial prey levels, Serangium lar-
vae were recovered as late as 10 weeks after
adults were introduced. Cohen & Brummett's
(1997) data suggest that Serangium could con-
sume a sufficient number of whitefly immatures
during a daily 10 hour feeding to meet the mini-
mum methionine requirement for normal growth
and development. This calculation assumes an
average handling time of 1 minute per prey item,
and as with D. pusillus (Hoelmer et al. 1993), it
appears from our study that Serangium can only
reproduce (without nutritional augmentation)
under high prey populations.
Legaspi et al. (1996) reported that the average
life-span of Serangium ranged from 75 days at
20C to 25 days at 300C. In our study, typical com-
mercial cropping practices were used, poinsettia
















TABLE 1. DENSITY OF LIVE IMMATURE SILVERLEAF WHITEFLY AND INCIDENCE OF WHITEFLY MORTALITY AT FOUR LEVELS IN THE POINSETTIA CANOPY AT FINAL HARVEST.


Initial release rate treatments


Whitefly


Serangium


Sample strata in poinsettia canopy


Bracts


Upper leaves


Middle leaves


Whitefly' Mortality Whitefly Mortality Whitefly Mortality Whitefly Mortality
(No. per plant) (No. SE) (% + SE) (No. SE) (% + SE) (No. + SE) (% + SE) (No. SE) (% + SE)

1 0 8.2 +4.0 0 49.3+ 18.5 6.7 +3.3 13.5 + 5.2 8.3 + 3.3 7.4 + 4.8 0
1 2 1.5 + 1.0 5.8 + 4.9 3.5 + 1.6 5.7 + 3.5 2.9 + 1.7 24.2 + 10.9 1.9 + 1.8 10.0 + 10
1 4 1.8 + 1.1 5.0+ 5.0 2.8 +2.0 37.1+ 11.4 3.5 + 3.0 49.5 + 17.9 1.3 + 0.4 31.4 +9.8
10 0 104+ 48 0 477+ 100 14 + 4.5 123 + 30.6 17.1+ 4.1 168 + 24.7 4.0 + 1.6
10 2 18.8 + 10.2 16.2 + 9.9 37.2 + 25.5 52.8 + 8.9 6.6 + 0.9 79 + 2.0 4.7 + 1.5 69.7 + 9.6
10 4 16.7 + 4.5 6.4 + 4.0 33.7 + 18.5 38.2 + 12.7 16.8 + 6.3 66 + 11.4 8.4 + 1.0 64.8 + 11.5

Statistical effects

F-value F-value F-value F-value F-value F-value F-value F-value
Source of variation df (P-value) (P-value) (P-value) (P-value) (P-value) (P-value) (P-value) (P-value)

Whitefly 1 17.2 1.2 32.4 7.1 29.4 11.3 47.9 34.7
(0.014) (0.334) (0.005) (0.056) (0.006) (0.028) (0.002) (0.004)
Serangium 2 6.5 1.3 22.42 4.7 7.0 7.1 16.0 17.0
(0.009) (0.308) (<0.001) (0.025) (0.007) (0.007) (<0.001) (<0.001)
Whitefly x Serangium 2 0.9 0.4 14.6 5.2 0.8 2.2 5.7 5.8
interaction (0.426) (0.704) (<0.001) (0.02) (0.456) (0.145) (0.014) (0.014)

"Silverleaf whitefly counts are expressed per 25cm' of leaf surface.


Lower leaves







Ellis et al.: Evaluation of Serangium


canopy temperature averaged 21.6C and the
crop matured in a normal time period. Legaspi
et.al. (1996) reported a mean life-time cumulative
predation of 4909 whitefly (eggs and immature
stages) for Serangium at a mean temperature of
20C. Even without reproductive success, the sin-
gle Serangium release in our study effectively
prevented prey populations from increasing over
a 10-week period (Fig. 1). It appears that this suc-
cess was largely due to the prolonged survival and
continuous feeding of individual adult beetles.
Due to the relatively high number of whiteflies
needed to sustain Serangium reproduction and
the extremely low pest levels tolerated on orna-
mental crops, it is unlikely that Serangium could
function effectively as the sole biological control
agent on a crop like poinsettia. However, Seran-
gium would be especially useful for suppressing
localized pest population increases or 'hot spots'
in the greenhouse, or as the primary biological
control agent on crops such as greenhouse tomato
where pest population tolerance levels are higher
than for ornamental crops. Based on our data it
appears that Serangium might be best suited for
inclusion in a multiple species biological control
approach to silverleaf whitefly management on
ornamental crops. As an obligate whitefly preda-
tor with a voracious feeding potential, Serangium
is capable of checking rapid increases in whitefly
populations (based on the caged studies herein),
thus potentially enabling whitefly parasitoid spe-
cies such as Eretmocerus or Encarsia to suppress
whiteflies to acceptable thresholds. Heinz & Nel-
son (1996) found that D. pusillus provided the
greatest suppression of silverleaf whitefly when
used in conjunction with one or more species of
Encarsia. In order to determine if Serangium
would be effective in such a role, interspecific in-
teractions between predator and parasite within
the host species (pest/plant), as well as release
management strategies, must be investigated.

ACKNOWLEDGMENTS

Storrs Agricultural Experiment Station scien-
tific contribution #1906. This research was
funded, in part, by the University of Connecticut
Storrs Agricultural Experiment Station and
USDA APHIS.

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COHEN, A. C. AND D. L. BRUMMETT. 1997. The non-
abundant nutrient (NAN) concept as a determinant
of predator-prey fitness. Entomophaga 42: 85-91.
ECKE, P. J., 0. A. MATKIN, AND D. E. HARTLEY. 1990. In-
sects and other pests, pp. 139-166. In Ecke, Matkin,
and Hartley (eds.). The Poinsettia Manual. Published
by Paul Ecke. Poinsettias, Encinitas, CA. 267 pp.


GORDON, R. D. 1985. The Coccinellidae (Coleoptera) of
America North of Mexico. J. New York Entomol. Soc.
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HEINZ, K. M. AND M. P. PARRELLA. 1994. Biological con-
trol of Bemisia argentifolii (Homoptera: Aleyrodi-
dae) infesting Euphorbia pulcherrima: Evaluations
of releases of Encarsia luteola (Hymenoptera: Aphe-
linidae) and Delphastus pusillus (Coleoptera: Coc-
cinellidae). Environ. Entomol. 23: 1346-1353.
HEINZ, K. M. AND J. M. NELSON. 1996. Interspecific in-
teractions among natural enemies of Bemisia in an
inundative biological control program. Biol. Control
6: 384-393.
HELGESEN, R. G. AND M. J. TAUBER. 1977. The whitefly-
Encarsia system: A model for biological control on
short-term greenhouse crops. U.S. Agricultural Re-
search Service Northeast Region. 85: 71-73.
HODDLE, M., R. VAN DRIESCHE, AND J. SANDERSON.
1997a. Biological control of Bemisia argentifolii (Ho-
moptera: Aleyrodidae) on poinsettia with inundative
releases of Encarsia formosa (Hymenoptera: Aphe-
linidae): Are higher release rates necessarily better?
Biol. Control 10: 166-179.
HODDLE, M. S., R. G. VAN DRIESCHE, AND J.P. SANDER-
SON. 1997b. Biological control of Bemisia argentifolii
(Homoptera: Aleyrodidae) on poinsettia with inun-
dative releases of Encarsia formosa Beltsville strain
(Hymenoptera: Aphelinidae): Can parasitoid repro-
duction augment inundative releases? J. Econ. Ento-
mol. 90: 910-924.
HODDLE, M. S., R. G. VAN DRIESCHE, J. P. SANDERSON,
AND 0. P. J. M. MINKENBERG. 1998. Biological con-
trol of Bemisia argentifolii (Hemiptera: Aleyrodidae)
on poinsettia with inundative releases of Eret-
mocerus eremicus Hymenoptera: Aphelinidae): do re-
lease rates affect parasitism? Bull. Entomol. Res. 88:
47-58.
HODDLE, M. S., R. G. VAN DRIESCHE, AND J. P. SANDER-
SON. 1999. Biology and use of the whitefly parasitoid
Encarsia formosa. Annu. Rev. Entomol. 43: 645-669.
HOELMER, K. A., L. S. OSBORNE AND R. K. YOKOMI.
1993. Reproduction and feeding behavior of Delphas-
tus pusillus (Coleoptera: Coccinellidae), a predator
of Bemisia tabaci (Homoptera: Aleyrodidae). J. Econ.
Entomol. 86: 322-329.
KAPUR, A. P. 1954. A new species of Coccinellidae (Co-
leoptera) predacious on the citrus white-fly in India.
Rec. of the Ind. Mus. 52: 189-193.
LEGASPI, J. C., B. C. LEGASPI, R. L. MEAGHER, JR. AND
M. A. CIOMPERLIK. 1996. Evaluation of Serangium
parcesetosum (Coleoptera: Coccinellidae) as a biolog-
ical control agent of the silverleaf whitefly (Homop-
tera: Aleyrodidae). Environ. Entomol. 25: 1421-1427.
PARRELLA, M. P., T. D. PAINE, J. A. BETHKE, K. L. ROBB
AND J. HALL. 1991. Evaluation of Encarsia formosa
(Hymenoptera: Aphelinidae) for biological control of
sweetpotato whitefly (Homoptera: Aleyrodidae) on
poinsettia. Environ. Entomol. 20: 713-719.
SAS INSTITUTE. 1995. SAS/STAT version 6. SAS Insti-
tute, Cary, NC.
TIMOFEYEVA, T. V. AND H. D. NHUAN. 1979. Morphology
and biology of the Indian ladybird Serangium
parcesetosum Sicard (Coleoptera: Coccinellidae) pre-
dacious on the citrus whitefly in Adzharia. Entomol.
Rev. 57: 210-214.
USDA 1997. Floriculture Crops. 1997 Summary USDA,
National Agricultural Statistics Service, Washing-
ton, D.C.







Florida Entomologist 84(2)


June 2001


SURVEY OF TERMITES IN THE DELTA EXPERIMENTAL FOREST
OF MISSISSIPPI

CHANGLU WANG AND JANINE POWELL
Stoneville Research Quarantine Facility, USDA ARS, Stoneville, MS 38776

ABSTRACT

Termites were surveyed in the Delta Experimental Forest in west central Mississippi in
1998. Logs, branches, and stumps along three 200-m long, 6-m wide transects were investi-
gated at each of the three study plots. Two subterranean termite species in the family Rhi-
notermitidae, viz., Reticulitermes flavipes (Kollar) and Reticulitermes virginicus (Banks),
were recorded. Reticulitermes flavipes was the common species and constituted 81.3% of the
termite occurrences. Of the 685 pieces of wood surveyed, 16.5% had termites. The percentage
of the two termite species varied among plots. The percentage of wood materials with signs
of termite activity or foraging termites present was positively correlated with the diameter
of the wood materials (R = 0.85). The chances of a log, branch, or stump being attacked by
these termites increases by 1.3% as the diameter of the wood material increases 1 cm. The
percentage of dead wood with sign of termite activity ranged from 11.6% to 67.2% among the
sampled plots. Termites were significantly less abundant at Plot 3, which might correspond
to a lower elevation and a higher soil moisture.

Key Words: Reticulitermes flavipes, Reticulitermes virginicus, forest, relative abundance

RESUME

Una inspection de termitas se llevo a cabo en el bosque del Delta Experimental Forest al cen-
tro-oeste de Mississippi en 1998. Se seleccionaron 3 lotes de 200 m de largo por 6 m de ancho
y en cada uno de ellos se colectaron troncos, ramas y tocones. Se anotaron dos species de ter-
mitas subterraneas en la familiar Rhinotermitidae: Reticulitermes flavipes (Kollar) y Reticu-
litermes virginicus (Banks). R. flavipes fue la especie mas comun y constituyo el 81.3% de las
ocurrencias de termitas. De los 685 pedazos de madera inspeccionados, 16.5% tenian termi-
tas. El porcentaje de las dos species de termitas vari6 entire lotes. El porcentaje de materia-
les de madera con senas de actividad de termitas o la presencia de termitas forrajeras fue
correlacionada positivamente con el diametro de los materials de madera (R = 0.85). La pro-
babilidad de un tronco, rama o toc6n ser atacado por estas termitas incremento un 1.3% al
incrementar el diametro de estos materials por 1 cm. El porcentaje de madera muerta con
senas de actividad de termitas vario entire 11.6 a 67.2% entire los lotes muestreados. Las ter-
mitas fueron significativamente menos abundante en el Lote 3, lo cual puede corresponder
a una elevaci6n mas baja y una humedad de suelo mas alta.


Termites play an important role in forest eco-
systems, especially in tropical rain forests, where
they occur in extremely dense populations (Har-
ris 1966; Gray 1972; Matsumoto 1976). Termites
promote organic matter decomposition, alter soil
properties, and provide food to other animals
(Wood & Sands 1978; La Fage & Nutting 1978).
Although their ecological importance is well
known, termites have rarely been quantitatively
studied in forests of the United States (Gentry &
Whitford 1982; Howard et al. 1982).
Like any other insects, the abundance of ter-
mites is influenced by the distribution and quality
of their food materials. However, there is very little
information regarding the relationship between
termites and size or distribution of wood materials
(Gentry & Whitford 1982; Jones et al. 1995).
In Mississippi, Reticulitermes flavipes (Kollar)
and Reticulitermes virginicus (Banks) appear to
be the most common termite species. In 1998, we


conducted a survey of termites in an experimental
forest. In this study, we tried to determine the spe-
cies composition and their foraging activities as-
sociated with dead wood in a forest environment.
The objectives of this study were to quantitatively
describe termite distribution patterns and feeding
activities in different forest ecosystems.

MATERIALS AND METHODS

Study Sites

This study was carried out at the Delta Exper-
imental Forest near Stoneville, Mississippi. The
study site is mostly level with gentle slopes (<2)
at some sections. The soil type is fine-textured
Sharkey clay soil with clay particle content varies
from 60 to 85% (Krinard & Johnson 1985). It ex-
pands considerably when wet in the winter and







Wang & Powell: Survey of termites


cracks profusely when dry in the summer. The
study sites were sometimes under water in the
wet winter months (December-February). Aver-
age air temperature in January and July from
30-year data is 5.1C and 27.6C, respectively
(Boykin 1995). The average annual precipitation
is 1326 mm.
Three 200 ha experimental plots were delin-
eated for the survey based on differences in tree
stand.
Plot 1 is a sweetgum (Liquidambar styraciflua
L.) plantation established in 1964-65 at 3 x 3-m
spacing. The diameter at breast height is 23.4 +
1.2 (mean + standard error) cm. Some sugarber-
ries (Celtis laevigata Willdenow) have grown into
small to large trees. Common shrubs were: sugar-
berry, hawthorn (Crataegus spp.), willow oak
(Quercus phellos L.), Chinese privet (Ligustrum
sinense Loureiro), and blackberry (Rhubus sp.).
Plot 2 is a naturally regenerated tree stand.
Major trees species were: red oaks (Quercus spp.),
sweetgum, sugarberry, overcup oak (Quercus
lyrata Walter), green ash (Fraxius pennsylvanica
Marshall), and elms (Ulmus spp.). Common un-
derstory shrubs were: Chinse privet, swamp dog-
wood (Cornus stricta Lamarck), Eastern
swampprivet (Forestiera acuminata (Michaux)
Poiret), American snowball .Sr' .. americana
Lamarck), green ash, elms, deciduous holly (Ilex
decidua Walter), and blackberry.
Plot 3 is an even-aged stand of natural regen-
eration resulting from a clearcut in 1937. Major
tree species were: green ash, cottonwood (Populus
deltoides Marshall), sugarberry, sweetgum, red
oaks, black willow, elms, and white oaks. Under-
story shrubs were similar to Plot 2 but at a much
lower density. This plot is at a slightly lower ele-
vation than plots 1 and 2 and is more likely to be
flooded in wet winter months.
Soil water content of the plots was measured
on September 15, 1999. The precipitation during
one month prior to the sampling date was 35.3
mm. The precipitation during the same period of
1998 was 68.1 mm. Nine soil samples were taken
from the three transects of each plot. The soil was
taken from 0-9 cm of the "A" layer. Then they
were dried in an oven at 105C for 48 hrs. Soil
moisture content was determined by the differ-
ence between fresh soil weight and dry soil weight
over fresh soil weight.

Survey

A survey of termites was conducted from Octo-
ber 20 through November 25, 1998 in plots 1, 2,
and 3, in that order. Weather data in the three
plots during the survey were: average maximum
air temperature 22.9, 29.5, and 17.6C, respec-
tively; average minimum air temperature 8.6,
13.6, and 8.1C, respectively; average daily rainfall
0.3, 1.5, 6.4 mm, respectively (Stoneville weather


station, http://www.deltaweather.msstate.edu/). At
each plot, three 200-m long, 6-m wide transects in
north-south direction were delineated. Diameter
at the large end of all logs, branches, and stumps
>3 cm in diameter and partially or entirely within
each transect was measured. Each was examined
for the presence of termites or signs of termite ac-
tivity. Termite galleries, fecal debris, and/or body
parts were noted as signs of termite activity. De-
termining whether termite galleries exist in wood
was usually not difficult. Galleries made by other
arthropods had a more regular shape and not con-
nected to each other into great lengths or width.
Soil particles often exist in termite galleries or on
surface of the wood. There were occasions when
the wood materials were too decayed to determine
whether or not termites had existed before. These
wood materials were not included in the analysis.
Results were recorded as three categories: I) with
live termites, II) with sign of termite activity, or
III) without termites and no sign of termite activ-
ity. When live termites were found, soldiers were
collected for species determination. Host trees
were not identified. Termite soldiers were exam-
ined under an Olympus SZX12 dissecting scope.
Typically, two or three termite soldiers which are
representative of the soldiers in size and mor-
phology were examined. More soldiers were ex-
amined if necessary to determine the species.
Termites were identified to species using the key
provided by Scheffrahn & Su (1994), and identifi-
cations of representative specimens were verified
by Rudolf Scheffrahn. Voucher specimens have
been deposited in collections of Stoneville Re-
search Quarantine Facility, USDA Agricultural
Research Service, Stoneville, MS.

Statistical Analysis

The presence or absence of live termites or
signs of termite activity in dead wood materials
were recorded as 1 or 0, respectively. The wood
materials were grouped into 6 categories accord-
ing to diameter. The actual diameter of the 6 cat-
egories ranged from 3-5.9, 6-8.9, 9-11.9, 12-14.9,
15-17.9, and >18 cm, respectively. Analysis of
Variance (ANOVA) was performed to test for dif-
ferences between diameter groups and plots us-
ing PROC MIXED of the SAS software (SAS
Institute 1999). Then the data were further ana-
lyzed for the trend between diameter of the wood
materials and presence of termites or signs of ter-
mite activity. R2 of the regression equation was
determined by the ratio of the sum of squares for
diameter groups as a trend with 1 degree of free-
dom divided by the sum of squares for diameter
groups with 5 degrees of freedom. Soil moisture
content data of the plots were compared by
Tukey's Studentized Range Test after ANOVA.
All analyses were performed by SAS software
(SAS Institute 1999).







Florida Entomologist 84(2)


RESULTS AND DISCUSSION

Species Composition and Distribution of Termites

Among the 685 branches, logs, and stumps ex-
amined, 114 (16.5%) had live termites. Two ter-
mite species were found, Reticulitermes flavipes
(Kollar) and R. virginicus (Banks). Species were
not identified in 7 samples because no soldiers
were collected. Among the 107 identified termite
samples, R. flavipes and R. virginicus were found
in 12.7% and 2.9% of the samples, respectively
(Table 1). Reticulitermes flavipes was most fre-
quently encountered and occurred in 81.3% of the
termite samples. This is similar to observations
by Howard et al. (1982) in southern Mississippi.
The relative abundance ofR. virginicus in plots 1-
3 was 7.7%, 21.4%, and 53.8%, respectively The
high percentage in Plot 3 was not precise because
termites in 7 of the 20 samples were not identi-
fied. However, the percentage of R. virginicus in
Plot 3 was at least 35.0%, which was still higher
than the other plots. This higher percentage
might relate with higher soil moisture. Soil mois-
ture in Plot 3 (21.4 0.8%) was significantly
higher than that in Plot 1 (19.8 0.8%) measured
on September 15, 1999 (F = 12.90; df = 2, 24; P =
0.0002). As reported by Howard et al. (1982), R.
flavipes tends to occupy higher, more arid places
in southern Mississippi.
The percentage of samples with signs of ter-
mite activity in plots 1-3 was 60.0%, 67.2%, and
11.6%, respectively. The percentage in plots 1 and
2 was significantly higher than Plot 3 (F = 132.76;
df = 2, 508; P < 0.0001) (Fig. 1). The much lower
termite activity that observed in Plot 3 might in-
dicate unsuitable conditions for termites, espe-
cially forR. flavipes. Plot 3 tended to be flooded for
a longer period of time and more frequently than
plots 1 and 2 during the winter months. This
might have created an unsuitable condition for
termites to survive in the winter.
Live termites were found in 24.8%, 22.2%, and
7.0% of the logs, branches, and stumps in plots 1-
3, respectively. The percentage of wood materials


with termites in Plot 3 was significantly lower
than that in plots 1 and 2 (Fig. 1) (F = 23.04; df =
2, 674; P < 0.0001). This pattern is same as the re-
sult on the percentage of wood with signs of ter-
mite activity. The lower temperatures during the
survey might have an impact on the low number
of termite occurrences in Plot 3. However, the dif-
ference between percentage of wood with termites
and that with signs of termite damage in Plot 3 is
proportionally smaller than those in plots 1 and 2
(Fig. 1). This suggests that the influence of tem-
perature on termite occurrences in Plot 3 was not
significantly higher than that in plots 1 and 2.

Relationship between Termite Activity
and Size of Wood Materials

Most of the wood samples in the three plots
were <12 cm in diameter. The percentage of wood
samples with diameter >12 cm in the plots 1-3
was 1.8%, 5.1%, and 19.7%, respectively. A cause-
effect relationship existed between percentage of
samples with signs of termite activity and diame-
ter of the wood (F = 20.34; df = 1, 508; P < 0.0001;
R2 = 0.73) (Fig. 2). The chance of a sample being
infested by termites increased 1.3 + 0.3% as the
diameter of the wood increased 1 cm. Perhaps this
is because larger wood materials tend to maintain
moisture, which is important for termite survival
and foraging. According to the regression equa-
tion, Percentage = 0.327 + 0.013 diameter, even a
very small dead branch such as 1 cm in diameter
will have a 34% chance of having been attacked
by termites.
The percentage of samples with termites
present was also positively correlated with diame-
ter of the wood material (F = 30.82; df = 1, 674; P
< 0.0001;R2 = 0.72) (Fig. 3). The chance of a sample
with termites present increased 1.3 + 0.2% as the
diameter of the wood materials increased 1 cm.
In conclusion, we found two species of termites
in the Delta Experimental Forest near Stoneville,
Mississippi. Reticulitermes flavipes was the most
common termite species. The occurrence of ter-
mites varied with site conditions. Selection of


TABLE 1. RELATIVE ABUNDANCE OF 2 TERMITE SPECIES IN 3 PLOTS OF THE DELTA EXPERIMENTAL FOREST, MISSISSIPPI.

Wood materials with termites

R. flavipes R. virginicus Reticulitermes spp.
Number of
wood materials Percentage Percentage Percentage
Plot examined Number (%) Number (%) Number (%)

1 210 48 22.9 4 1.9 0 -
2 189 33 17.5 9 4.8 0 -
3 286 6 2.1 7 2.4 7 2.4

Total 685 87 12.7 20 2.9 7 1.0


June 2001







Wang & Powell: Survey of termites


90 -
80 - - - - - - - - - - -





'wit 70 - - - - A v ..0 - - - -

Fig. 1. Percentage of dead wood samples with te. .- - -
I 2 J
Plot
_.. with Sign ofTennite Activity .. with Live Termites

Fig. 1. Percentage of dead wood samples with ter-
mites or with signs of termite activity in Delta Experi-
mental Forest, Mississippi.


wood materials by the two termite species was
positively correlated with the diameter of the
wood materials. This has practical significance
for termite control and baiting studies. It provides
an estimate of the probability of termite attack on
wood materials.
Gentry & Whitford (1982) sampled lowland
hardwood forests and pine plantations in South
Carolina (similar latitude as in our study location),
and found that the occurrences of R. flavipes and
R. virginicus varied among different habitats. Reti-
culitermes flavipes represented 26.6-60% of the ter-
mites, which was lower than what we observed on
the 3 sites on the Delta Experimental Forest. In
their study, nearly all the wood greater than 2 cm
diameter had signs of termite usage. The termite
population or activity in their study location was
much higher than in our study location.
We measured the length of every piece of wood
in this survey. The wood materials were grouped
into 4 categories according to their length. The


70
< 60
50
o 40
30



o
10




Fi
ples a
(Perce
512).


50 -------------- ----- - ------ ----- ..-.- .
5 ....- -... ---------------......- ------.....



0 - - - . . . . .


4.5 7.5 10.5 13.5
Diameter of Wood (cm)


16.5 19.5


Fig. 3. Relationship between diameter of wood sam-
ples and the percentage with termites present (Percent-
age = 0.044 + 0.013 x diameter, R2 = 0.72, n = 678).



actual length of the 4 categories ranges from <3, 3-
5.9, 6-8.9, and >9 m, respectively. Although the
length of wood had significant effect on the per-
centage of wood with live termites (F = 11.23; df =
3, 666; P < 0.0001), it did not have significant effect
on the percentage of wood with signs of termite ac-
tivity (F = 0.67; df = 3, 502; P = 0.57). Along
branch with small diameter may be less likely to
be attacked by termites than a short branch with
large diameter. Therefore, length alone is not a
good predictor for estimating the probability of ter-
mite attack on the wood materials.
Jones et al. (1995) found a positive relation-
ship (R2 = 0.50) between volume of wood and dry
wood termite colony size on Mona Island, Puerto
Rico. In a laboratory experiment, Hedlund and
Henderson (1999) found termite consumption
rate increased as the wood volume increased. Ter-
mites foraged less actively when larger wood ex-
isted. These findings support our conclusion that
larger wood tends to be more attractive for ter-
mites to feed on. So, size of the wood materials
measured by diameter or volume is important for
termite foraging activities.


ACKNOWLEDGMENTS

---------------..-------...-----------------......... We thank Fannie Williams from our research
unit for her assistance in field work. We are grate-
-.-------.-...--...--.-..-.-...........- ful to Deborah Boykin (USDA Agricultural Re-
......................................... search Service) for help in the statistical analysis,
and Rudolf Scheffrahn, University of Florida for
...' 7. .. verifying identifications. Steve Meadows from
4.5 7.5 10.5 13.5 16.5 19.5 USDA Forest Service provided the vegetation in-
Diameter of Wood (cm) formation of the study plots. The manuscript was
g. 2. Relationship between diameter of wood sam- improved by E. W. Riddick, J. A. Morales-Ramos,
nd the percentage with signs of termite activity W. D. Woodson, T. E. Nebeker, and three anony-
ntage = 0.327 + 0.013 x diameter, R' = 0.73, n = mous reviewers. This research was funded
through USDA Agricultural Research Service.







Florida Entomologist 84(2)


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June 2001







McFarland & Hoy: Survival ofD. citri and parasitoids


SURVIVAL OF DIAPHORINA CITRI (HOMOPTERA: PSYLLIDAE),
AND ITS TWO PARASITOIDS, TAMARIXIA RADIATA
(HYMENOPTERA: EULOPHIDAE) AND DIAPHORENCYRTUS
ALIGARHENSIS (HYMENOPTERA: ENCYRTIDAE), UNDER DIFFERENT
RELATIVE HUMIDITIES AND TEMPERATURE REGIMES

CLINT D. MCFARLAND AND MARJORIE A. HOY
Department of Entomology and Nematology, University of Florida
P.O. Box 110620, Gainesville, FL 32611-0620

ABSTRACT

The ability of an exotic citrus pest, Diaphorina citri Kuwayama, and its two parasitoids,
Tamarixia radiata (Waterston) and Diaphorencyrtus aligarhensis (Shafee, Alam & Agar-
wal), to survive under different relative humidities (7%, 33%, 53%, 75% and 97%) at 25 and
30 C was compared. The data obtained may help to predict potential climatic limitations to
their establishment in Florida and indicate whether the pest has different climatic toler-
ances compared to these parasitoid populations. Adult survival was evaluated under rela-
tive humidities maintained by saturated salt solutions. D. citri survived longer than the
parasitoid populations at all experimental conditions, suggesting it has a lower net water
loss rate. The T radiata (Taiwan) population showed the greatest moisture requirement at
all temperatures and relative humidities tested. The T radiata (Vietnam) population sur-
vived longer than the Taiwan, suggesting that the two populations may perform differently
in different geographical regions. D. aligarhensis and T radiata (Vietnam) survived similar
lengths of time, except at the higher relative humidities, so the moisture requirements for
these two populations are comparable.

Key Words: Asian citrus psylla, relative humidity tolerances, saturated salt solutions, bio-
logical control

RESUME

La habilidad de la plaga ex6tica del citrico, Diaphorina citri Kuwayama, y sus dos parasitoi-
des, Tamarixia radiata (Waterson) y Diaphorencyrtus aligarhensis (Shafee, Alam & Aga-
rwal) de sobrevivir bajo diferentes humedades relatives (7%, 33%, 53%, 75% y 97%) a 25 y
30 C fueron comparadas. Los datos obtenidos pueden ayudar a predecir las limitaciones po-
tenciales climaticas a su establecimiento en la Florida e indican si la plaga tiene diferentes
tolerancias climaticas en comparaci6n a estas poblaciones parasitoides. La supervivencia de
adults fue evaluada bajo humedades relatives mantenidas por soluciones de sal saturada.
D. citri sobrevivi6 mas tiempo que las poblaciones parasitoides bajo todas las condiciones ex-
perimentales, sugiriendo que tiene una velocidad mas baja de perdida de agua neta. La po-
blaci6n T radiata (Taiwan) demostr6 el mayor requerimiento de humedad en todas las
temperatures y humedades relatives probadas. La poblaci6n T radiata (Vietnam) sobrevivi6
mas que la de Taiwan, sugiriendo que las dos poblaciones pueden ejecutar de manera dis-
tinta en diferentes regions geograficas. D. aligarhensis y T radiata (Vietnam) sobrevivieron
duraciones de tiempo similares, except bajo humedades relatives altas, indicando que los
requerimientos de humedad para estas dos poblaciones son comparable.


The Asian citrus psylla, Diaphorina citri Ku-
wayama, was found for the first time in June 1998
by personnel in the Division of Plant Industry,
Florida Department of Agriculture and Consumer
Services (Hoy & Nguyen 1998; Halbert 1998;
Mead 1977). It has successfully established in
Florida and it will probably colonize all citrus-
growing areas in Florida and may spread to Lou-
isiana, Texas, Arizona and California citrus.
D. citri damages citrus by depleting sap from
the plant and, because its saliva is toxic, growing
shoots are distorted and may die. It also causes
damage by excreting honeydew, which allows the
growth of sooty mold (Chien & Chu 1996). How-


ever, the worst threat is thatD. citri is an efficient
vector of the bacterium, Liberobacter asiaticum,
which causes greening disease. This is the most
serious disease of citrus in the world, causing re-
duced production and eventual death of the trees
(McClean & Schwarz 1970).
No effective eradication efforts are known for
D. citri, but classical biological control of the psyl-
lid vector should contribute to suppression of
their populations. Two parasitoids have been im-
ported (Hoy et al. 1999; Hoy & Nguyen 2000).
Tamarixia radiata (Waterston) is an ectoparasi-
toid imported from Taiwan and Vietnam, while
Diaphorencyrtus aligarhensis (Shafee, Alam &







Florida Entomologist 84(2)


Agarwal) is an endoparasitoid of D. citri obtained
from Taiwan. Approximately 12,000 T radiata
were released into Florida between 15 July and 1
December 1999. D. aligarhensis was approved for
release on 15 March 2000 and 5000 were released
during the 2000 growing season (Hoy, unpublished).
The success of classical biological control pro-
grams may be determined by the use of the appro-
priate natural enemy ecotypes. Moisture require-
ments may be especially important in determining
suitability of an imported biological control agent
for release and establishment in a new geographic
area. Only a few experiments have been conducted
to compare the water balance or the temperature-
relative humidity relations in a pest and its para-
sitoids. Yoder & Hoy (1998) found the citrus leaf-
miner and two populations of its endoparasitoid
Ageniaspis citricola Logvinovskaya both require a
moisture-rich environment although the citrus
leafminer is less dependent on high relative hu-
midities than its parasitoid,A. citricola.
The objective of this study is to compare, under
laboratory conditions, the effect of temperature
and relative humidity on the survival of an exotic
citrus pest, D. citri and its two parasitoids T radi-
ata and D. aligarhensis. This information will
help in mass rearing these parasitoids and may
help to predict potential climatic limitations to
their establishment in Florida.

MATERIALS AND METHODS
Rearing
D. citri was reared on Murraya paniculata
(Chien et al. 1989), ornamental orange jasmine,
in the quarantine facility at the University of
Florida, Gainesville (Skelley & Hoy, unpublished).
The plants were pruned, fertilized and held under
a photoperiod of 16:8 (L:D) at 21 to 24C to stim-
ulate the growth of new flushes because adult
psyllids lay eggs only on newly sprouted leaf
buds. Trees that had aphid or scale infestations
were sprayed with an application of oil or pyre-
thrum (Prentiss, Inc., Sandersville, GA).
D. citri was reared in quarantine under a pho-
toperiod of 18:6 (L:D) at 26C and 60% relative
humidity (RH). T radiata colonies from Taiwan
and Vietnam and D. aligarhensis from Taiwan
were reared in separate cages and rooms in quar-
antine to reduce the risk of contamination. Both
parasitoid species were reared at 27C and 60%
RH under a photoperiod of 18:6 (L:D) in cages (76
cm x 46 cm x 66 cm) constructed with 1.27 cm
diameter polyvinyl chloride pipe and covered with
a fine nylon mesh.

Specimen Collection
Adults used for experiments were collected at
5 PM on the day of their emergence. To ensure
that all four populations emerged on the same


day, the psyllids, T. radiata and D. aligarhensis
were set up 15 days, 12 days and 18 days, respec-
tively, before the start of the experiments. The
day before starting an experiment, all old psyllids
and parasitoids were collected to ensure that only
new individuals would be present in the cages.
D. citri is collected by aspirating adults off the
plant, while T. radiata can be collected off the top
of the mesh cage. D. aligarhensis is aspirated
most often from the bottom of the cage. Individual
adults were placed in groups of five into 28.35 g
condiment cups (Plastics, Inc. St. Paul, MN). The
lid of the cup was covered with mesh glued to it
with clear nail polish. All containers were cleaned
with 70% EtOH before being reused.

Pretreatment Conditions
After collection, all individuals were pre-
treated for 21 hours by placing them into bell jars
placed in an incubator (Percival I-30B) pro-
grammed with a photoperiod of 18:6 (L:D) at
17C. RH within the bell jars was maintained by
a saturated salt solution, Mg(NO,)2 6H20, which
yields a 56% RH (Winston & Bates 1960; O'Brien
1948; Carr & Harris 1949). Specimens were given
no water or honey. Pretreatment was expected to
remove adsorbed water from the cuticular surface
and to ensure a uniform physiological state. Pre-
treatment minimizes the effects of ingestion, re-
production, excretion and defecation (Arlian &
Ekstrand 1975). The survival rate should reflect
the relative changes in water balance of the in-
sects before desiccation (Wharton 1985).

Experimental Conditions
At 2 PM specimens were transferred into
stainless steel chambers (30.5 cmx 30.5 cm x 30.5
cm) (Fisher Scientific, Pittsburgh, PA) held
within a single incubator (Percival 1-35 series) at
either 25 or 30C with a photoperiod of 18:6 (L:D).
Plastic cups containing five adults were placed
into a chamber with either 7, 33, 53, 75 or 97%
RH maintained by saturated salt solutions. Re-
agent grade salts (Sigma, St. Louis, MO) dis-
solved in distilled water were used: 200 ml of
saturated salt solution was placed into chambers
with the three higher RH, and 100 ml of salt solu-
tion was used in the two lower RH because they
are hygroscopic substances and should be formu-
lated as a paste to maintain the desired RH.
These salts were chosen because they maintain
the same RH at 25 and 30C (Winston & Bates
1960). The chemicals used were sodium hydroxide
(7% RH), magnesium chloride hexahydrate (33%
RH), magnesium nitrate hexahydrate (53% RH),
sodium chloride (75% RH) and potassium sulfate
(97% RH). RH in each chamber was confirmed by
a hygrometer (Thomas Scientific, Swedesboro,
NJ), as well as the salt crystal method outlined by
Winston & Bates (1960), which utilizes the fact


June 2001







McFarland & Hoy: Survival ofD. citri and parasitoids


that a salt crystal will pick up moisture and deli-
quesce in relative humidities which are at or
above that maintained by its saturated solution.
A very small crystal is introduced into the closed
chamber and observed under magnification; if the
RH is below that for the salt, there will be no
change. If the RH is very close, the deliquescence
will be only partial; if just above there will be a
narrow rim of liquid around the mass. Experi-
mental units were placed randomly into each RH
chamber.

Survivability Tests

Survival of individuals was recorded every
three hours. RH chambers were sampled sepa-
rately and specimens were out of the chamber for
a maximum often minutes. Insects found on their
dorsal surface that failed to respond to probing
were considered dead. All dead specimens were
sexed.

Statistics

Four replicates were tested at 25C under each
RH on separate dates, while three replicates were
run at 30C and each RH condition. At 25C, a to-
tal of 400 individuals (100 per experiment) were
tested for D. citri and the two T radiata popula-
tions. Only 300 individuals (75 per experiment) of
the D. aligarhensis population were tested at
25C. At 30C, a total of 300 individuals (100 per
experiment) were tested for all four populations.
Mean percentage survival (SEM) was calculated
for all four populations at each temperature-RH
combination. LTso values were calculated by add-
ing the time it took to get 50% mortality under
each condition on each date. A grand mean and
SEM was determined for each population at each
temperature-RH combination. LTso values were
compared statistically by z test statistics.

RESULTS AND DISCUSSION

The survival of D. citri, D. aligarhensis and
T radiata populations increased with increasing
RH (Figs. 1, 2). When the temperature was in-
creased from 25 to 30C, adult survival of all pop-
ulations decreased, as expected (Figs. 1, 2). At 75
and 97% RH at both temperatures, D. citri sur-
vived longer than the parasitoid populations, sug-
gesting it has a lower net water loss rate (Figs.
1D, 1E, 2D, 2E).
The T radiata (Taiwan) population showed the
greatest moisture requirement at all tempera-
tures and RH tested. The T radiata (Vietnam)
population survived longer than the Taiwan pop-
ulation at 25C and 53, 75 and 97% RH as well as
all RH tested at 30C (Figs. 1C, 1D, 1E, 2). This
suggests that the two populations may perform
differently in different geographical regions.


D. aligarhensis and T radiata (Vietnam) sur-
vived similar lengths of time under all RH at 25
and 30C, so the moisture requirements for these
two populations appear comparable (Figs. 1, 2).
LT1o and LT90o values can be found easily from
the figures, but the differences are small and not
many inferences about the populations can be made
at the ends of the curves. The LT59 values of the
pest D. citri were always higher than the LT5o val-
ues of its parasitoid populations at 25C (Fig. 1).
LT o values at 25C for D. aligarhensis are dif-
ferent from LT5o values for the T radiata (Taiwan)
population (P < 0.05) (Table 1). D. aligarhensis
and T radiata (Vietnam) had different LTso val-
ues, except at 53 and 97% RH (z = 0.10, P > 0.05;
z = 0.75, P > 0.05) (Table 1). The LT5o values of the
two T radiata populations are not different at 7
and 33% RH (z = 1.55, P > 0.05); z = 0.10, P > 0.05)
(Table 1).
All parasitoids held at 25C were dead by 40.5
hours, whereas a few psyllids survived up to 94.5
hours (Fig. 1). There were no differences between
survival of males and females of the D. citri and
T radiata populations, perhaps because there is no
apparent difference in size. The D. aligarhensis
population is made up of females only.
The LT5 values of the pest were equal to or
greater than the LT5o values of its parasitoids at
30C (Table 1). As the RH increased to 97%, the
LT o of the D. citri population was different from
that of D. aligarhensis and the T. radiata (Viet-
nam) population (P < 0.05) (Table 1). The LT5o val-
ues of the T. radiata (Taiwan) population were
different (P < 0.05) from theD. citri, D. aligarhen-
sis and T. radiata (Vietnam) populations at all
RH tested (Table 1). Again, there were no differ-
ences in survival rates of males and females. All
parasitoids held at 30C were dead by approxi-
mately 40.5 hours, but a few psyllids managed to
survive up to 52.5 hours (Fig. 2).
The data suggest that, especially at 75 and
97% RH at 25C and 97% RH at 30C, some
D. citri will have up to two times longer to find
food and water in the field compared to its parasi-
toids (Figs. 1D, 1E, 2E).D. citri also survived well
at the lower RH of 7 and 33%, suggesting that it
could disperse and survive in the more arid condi-
tions found in western citrus-growing regions in
the United States (Figs. 1A, 1B, 2A, 2B).
The RH responses of the Vietnam population
of T radiata were different from those of the Tai-
wan population, especially at 30C, suggesting
that the two populations of T radiata may be
ecotypes (populations varying in their ability to
survive in different climates).
This information is helpful in rearing these
parasitoids for release in biological control pro-
grams because it indicates there are no large dif-
ferences in pest and natural enemy responses to
the RH and temperatures likely to be encoun-
tered in the rearing facility. The ability ofD. citri








Florida Entomologist 84(2)


1.00

0.80

0.60

0.40

0.20

0.00

1 00

0.80

0.60

0.40

0.20

0.00

1.00

0.80

7).60

I ).40

S).20

+ 1.00
(0)
+1 1.00

380


0.00

1.00

0.80

0.60

0.40

0.20

0.00


A
7% RH


B
33% RH


June 2001


- D. citri
--D. aligarhensis
-.- T radiate (Taiwan)
-- -- T radiata (Vietnam)


* - D. citri
- D. aligarhensis
---- T. radiata (Taiwan)
-- T- radiata (Vietnam)


C D- citri 080
53% RHD aligarhensis
--53% RH T. radiata (Taiwan) 060
0- T radiata (Vietnam) 0.40

* 0.20

K~i Q- 0* 0-a- GO-ao-O 0O00

1.00

D --a - D. e, 0.80

75% RH -A-D aligarhensis
--- T. radiata (Taiwan) 0 60
* 4- T. radiate (Vietnam)


E - D. ciri
97% RH .--0 aligarhensis
-4-- T. radiata (Taiwan)
* -- -e- radiata (Vietnam)

KI .
U, i


Ole I. 0 00


No. hours post treatment at 25C


Fig. 1. Survival curves of the Asian citrus psylla, Diaphorina citri, and its two parasitoids, Diaphorencyrtus ali-
garhensis imported from Taiwan and Tamarixia radiata imported from Taiwan and Vietnam, at 25'C and 7% RH
(A), 33% RH (B), 53% RH (C), 75% RH (D), and 97% RH (E).








McFarland & Hoy: Survival ofD. citri and parasitoids


A

7% RH


,- I D. citri
---D. aligarhensis
-#- T radiata (Taiwan)
- -e- T. radiata (Vietnam)


B -*.--D. citri

33% RH -o. aligarhe
T. radiata (
- T- T radiata (






C - D. citri

53% RH -*-D aligarhen
T, radiata (T,
-- T, radiata (V



o0.E-0e0-0*0


M ., 0.00

1.00

0.80
nsis
0.60
Taiwan)
Vietnam) 0.40

0.20

a-qao OMO0

1.00

0.80
sis
aiwan) 060
ietnam) 0.40

0.20

3B .o-0 0.00

1.0C


0.00

1,00

0.80

0.60

0.40

.0.20

.0.00

1.00

1.80

).60

).40

).20

S).00

1.00 o


0.40

0.20

0.00


E

97% RH


- D. citn
-A-D. aligarhensis
- T radiata (Taiwan)
- - T radiata (Vietnam)


- D. cifri
-A--D aligarhensis
-4-- T. radiata (Taiwan)


0.40 a 0.40

0.20 0.20

0.00 0.00
0o In C LC 4) 4) ' a t B tn q? 0 iaLA U C LlA u? u
N -o toQ^- a o cA d 't ac wN g w
C4N CA) t 'q If In CD N. N Wo 305

No. hours post treatment at 30C

Fig. 2. Survival curves of the Asian citrus psylla, Diaphorina citri, and its two parasitoids, Diaphorencyrtus ali-
garhensis imported from Taiwan and Tamarixia radiata imported from Taiwan and Vietnam, at 30'C and 7% RH
(A), 33% RH (B), 53% RH (C), 75% RH (D), and 97% RH (E).


D

75% RH


O.BC


0.6(

0.4C

0.2(

a 0.0(







Florida Entomologist 84(2)


TABLE 1. SURVIVAL OF D. CITRI AND ITS PARASITOIDS T. RADIATA AND D. ALIGARHENSIS AT 25 AND 30'C AND FIVE REL-
ATIVE HUMIDITIES.

LT,, values (SEM) in hours at relative humidity (%) of

Species 7 33 53 75 97

---------- ------------------------------ at 25C -----------------------------
D. citri 24.9 28.3 28.5 37.0 43.2
(2.4) (2.0) (2.4) (2.8) (3.2)
D. aligarhensis 21.3 21.8 24.2 26.1 25.5
(1.8) (1.6) (1.5) (1.8) (2.1)
T radiata Taiwan 15.3 18.1 16.5 18.4 22.5
(1.0) (1.3) (1.0) (2.5) (1.4)
T radiata Vietnam 16.2 18.1 25.6 22.4 26.1
(1.2) (1.0) (1.9) (1.8) (1.5)

--------- --------------------------- at 30C -----------------------------
D. citri 12.1 18.0 20.4 23.2 32.2
(1.1) (1.4) (1.8) (1.5) (2.5)
D. aligarhensis 12.1 14.3 18.0 18.6 22.3
(0.8) (1.1) (1.2) (1.2) (1.8)
T radiata Taiwan 6.2 9.2 11.7 14.0 18.2
(0.4) (0.8) (0.9) (0.9) (1.1)
T radiata Vietnam 12.3 17.0 23.3 21.8 25.2
(0.8) (1.5) (2.1) (1.6) (1.7)


to survive longer at 97% RH than its parasitoids
is interesting and difficult to interpret (Figs. 1E,
2E). It could mean that D. citri is better adapted
to very high RH than its natural enemies. The
data obtained compare the intrinsic water loss
rates of these four populations under extreme
conditions of no food or water, and the ability of
D. citri, D. aligarhensis and T radiata popula-
tions to disperse to more optimal sites was pre-
cluded. In citrus groves D. citri can feed on host
plants to obtain water and food. Likewise, the
parasitoids can host feed for moisture and nutri-
ents, feed on honeydew, drink droplets of water
and disperse to find microhabitats more suitable
for survival.
Yoder & Hoy (1998) found two populations of
A. citricola and their host, the citrus leafminer,
both require a moisture-rich environment al-
though the citrus leafminer is less dependent on
high RH than its parasitoids. The results of this
study were similar, with D. citri and its parasi-
toids requiring a moisture-rich environment, al-
though D. citri does not depend on high RH as
much as its parasitoids, especially the T radiata
(Taiwan) population. D. citri survived longer at
higher temperatures and lower RH than the T ra-
diata (Taiwan) and the D. aligarhensis popula-
tions (Figs. 2A, 2B).
Hoy et al. (2000) found slight physiological dif-
ferences in the Taiwan and Australian ecotypes of
A. citricola, which indicated genetic differences
exist. Subsequent analysis of Random-Amplified-
Polymorphic-DNA Polymerase-Chain-Reaction


(RAPD-PCR) and DNA sequence analyses of two
Actin genes led to the conclusion that the two pop-
ulations actually are cryptic species, even though
these two populations of A. citricola can not be
separated on the basis of morphological charac-
teristics (Hoy et al. 2000). Additional analysis of
the two populations of T radiata could establish
whether they are cryptic species.

ACKNOWLEDGMENTS

We thank John Capinera and James Nation for
providing advice on the manuscript, Lucy Skelley
for her assistance in rearing Murraya paniculata,
psyllids and parasitoids, and Ramon Littell for
his help with the statistical analyses. This work
was funded in part by the Davies, Fischer and
Eckes Endowment. This is University of Florida
Agricultural Experiment Station Journal Series
No. R-07835.

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CHIEN, C. C., S. C. CHIU, AND S. C. KU. 1989. Biological
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McFarland & Hoy: Survival ofD. citri and parasitoids


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Florida Entomologist 84(2)


FALL DRAGONFLY (ODONATA) AND BUTTERFLY (LEPIDOPTERA)
MIGRATION AT ST. JOSEPH PENINSULA, GULF COUNTY, FLORIDA

GARY L. SPRANDEL
Florida Fish and Wildlife Conservation Commission, 620 Meridian St., Tallahassee, FL 32399-1600 USA

ABSTRACT
I describe the fall 1999 migration of 5 Lepidoptera and 4 Odonata species north along St. Jo-
seph Peninsula, Gulf County, in the Florida Panhandle. Highest counts were for the Gulf
fritillary butterfly (Agraulis vanilla (L.), Lepidoptera: Nymphalidae) which accounted for
58% of the insects counted; the highest rate was 3,162/h, with an estimate of total season mi-
gration of over 250,000 individuals. The common green darner dragonfly (Anax junius
(Drury 1773) Odonata: Aeshnidae) was the next most common with a maximum rate of
3,297/h. The median and peak period for these two species was the first week in October. The
observed flight pattern may demonstrate a reluctance to cross open water.

Key Words: Odonata, dragonfly, Anax junius, common green darner, butterfly, Lepidoptera,
Gulf fritillary, Agraulis vanilla, migration, dispersal

RESUME
Se describe la migraci6n de 5 species Lepid6ptera y 4 species Odonata en el otono de 1999
hacia el norte a lo largo de la peninsula de St. Joseph, condado del Golfo, en la region noroeste
de la Florida. Las cuentas mas altas fueron de la mariposaAgraulis vanilla (L.) (Lepid6p-
tera: Nymphalidae), la cual constituyo el 58% de los insects contados; la cuenta mas alta fue
de 3,162/h, con un estimado de migraci6n estacional total de mas de 250,000 individuos. La
lib6lulaAnaxjunis (Drury 1773) (Odonata: aeshnidae) le sigue en numero con una cuenta to-
tal de 3,297/h. El medio y period cumbre para estas dos species fue la primera semana de
Octubre. El patron de vuelo observado puede demostrar una aversion a cruzar el agua.


Due to its southern location, Florida may be
either a migration pathway, stopover, or desti-
nation for migrant insects. Migrating butterflies
usually fly close enough to the ground and often
in sufficient numbers to attract public attention.
Large scale fall movements of the monarch but-
terfly (Danaus plexippus (L.), Lepidoptera:
Nymphalidae) have been documented westward
along the Gulf Coast (Urquhart & Urquhart 1978,
Van Hook & Hermann 1999). Additionally, 5 spe-
cies of butterflies have been reported migrating in
a generally southward direction in peninsular
Florida (Walker 1979; Walker 1991). Localized
fall movements of massive numbers of the com-
mon green darner dragonfly (Anaxjunius (Drury
1773) Odonata: Aeshnidae) have been docu-
mented on the Atlantic coast of Florida (Russell et
al. 1998). This paper presents fall migration
observations for both Odonata and Lepidoptera at
a location along the Gulf Coast in the Florida
Panhandle.

MATERIALS AND METHODS

Both butterflies (Lepidoptera) and dragonflies
(Odonata) were counted on St. Joseph Peninsula,
Gulf County, Florida from 14 Aug through 7 Dec,
1999. The peninsula is 24 km long, oriented
north-south, roughly parallel to the mainland


(Fig. 1). The count location was on an elevated, 50
m long boardwalk bisecting the peninsula, at Ea-
gle Harbor (29045.98'N, 8524.29'W) in St. Joseph
Peninsula State Park. At the boardwalk, the pen-
insula has a width of 200 m and is 11 km from the
north tip (Fig. 1).
There are only a few small freshwater wet-
lands on the peninsula that support small breed-
ing populations (<50 pair) of Odonata including
A. junius, black saddlebags (Tramea lacerata
Hagen 1861, Odonata: Libellulidae) and Carolina
saddlebags (Tramea carolina (L.), Odonata: Libel-
lulidae). There were low numbers of breeding but-
terflies including D. plexippus and Gulf fritillary
butterfly (Agraulis vanilla (L.), Lepidoptera:
Nymphalidae) on the peninsula.
Counts were made in 2, 5-minute periods per
hour, 1-2 days per week, from 3 to 11 hours each
day. All species with >3 cm wing span were counted.
Flight direction, estimated height, predation,
perching, tandem flights, and copulation, were re-
corded. Weather conditions, particularly frontal
boundary, and wind direction were recorded.
Hourly flight rates (in any flight direction) were
calculated from the average of the 2, 5-minute
counts in an hour. Highest observed hourly migra-
tion rates for the fall for each species were re-
ported. Linear interpolation between the observed
hourly flight rates, first between hours in a day,


June 2001






Sprandel: Migration at St. Joseph Peninsula


St. Joe Beach


St. Joseph
Peninsula


flight
direction


Gulf of
Mexico





N





5 km


GULF
COUNTY


prevailing winds


Eagle


Bias


Fig. 1. Location of the migration count site (*) for Odonata and Lepidoptera on the St. Joseph Peninsula, Gulf
County, Florida 14 Aug-7 Dec 1999.


and then between days, gave an hourly rate for magnitude. The date ranges when the middle 50%
each unobserved daylight hour from 14 Aug to 7 of the individuals of a species migrate were calcu-
Dec. All observed and interpolated hourly rates lated (that is, the period between the dates on
were summed to get an index of total fall flight which migration was 25 and 75% complete).


.... ..,- ".. ... "







Florida Entomologist 84(2)


RESULTS AND DISCUSSION

A total of 12,616 insects were counted in 222,
5-minute count intervals on 19 days from 14 Aug
to 7 Dec. Most (98.7%) were heading north along
the peninsula (Fig. 1), with the remainder either
patrolling (Odonata) or heading south (both Odo-
nata and Lepidoptera). The most common mi-
grant was the Gulf fritillary butterfly,A. vanilla,
followed by the common green darner dragonfly,
A. junius (Table 1). All insects appeared to use
powered flight rather than soaring. Although
there were peak flights during the first week of
October (Fig. 2), there was a steady and persis-
tent flight on each clear day.
One possible flight pattern is that the insects
head south down the continent, fly west when
reaching the Gulf, and are then funneled north
via the peninsula. The northeast prevailing wind
direction in the fall (Winsberg 1990) would pro-
vide a further push from the mainland onto the
peninsula (Fig. 1). Local sea breezes, would com-
plicate this general pattern. The insects may ex-
hibit some risk avoidance by not crossing the open
Gulf. This migration route has been documented
for 3-5,000 hawks annually, with the heaviest
flights 3-4 days after the passage of northern cold
fronts when the wind is from the northeast (Sted-
man 1984). After reaching the north tip of the
peninsula, it is unknown whether the insects fly
the 2-km distance to the mainland or head south-
west over the Gulf (to uncertain landfall). The low
number of south-flying insects counted suggests
that they are not back-tracking down the penin-
sula. Observations from the peninsula may not


necessarily reflect the direction of a potentially
larger migration on the mainland.
There have been observations from boats (e.g.,
Lowery 1946) and oil platforms (Baust et al. 1981,
Russell 1999) of some dragonflies and butterflies
flying over the Gulf. My observations of monarch
migration match previously recorded western
movement of eastern populations (Urquhart &
Urquhart 1978; Van Hook & Hermann 1999); and
destinations may be either the Gulf Coast states,
or Mexico. These data do not suggest a peninsular
Florida destination for all Gulf fritillary (A. vanil-
lae) and cloudless sulphur butterflies (Phoebis
sennae (L) Lepidoptera: Pieridae), as suggested
by other studies (Walker 1979; Walker 1991). Pre-
vious reports of the long-tailed skipper (Urbanus
proteus L., Lepidoptera: Hesperiidae) at Carra-
belle Beach (80 km east), indicated flight direc-
tions in both coastal directions (roughly east and
west, Walker and Littell 1994). Destination and
migration patterns of Odonates are generally un-
known (May 1992) though northward movement
of A. junius in summer and scarcity of adult
A. junius and T lacerata in winter in Florida have
been suggested (Paulson 1966; Dunkle 1989).
The peak day on 2 Oct (Figs. 2, 3) was 3 days
after the passage of a cold front. Some studies
have reported the passage of cold fronts as an im-
petus for migration (Russell et al. 1998), but the
following weekend also had high numbers, de-
spite no frontal activity. During drizzle the flight
dropped off to zero, but on the 2 overcast days (18
Sep, 27 Sep) the high A. vanilla rates were still
48 and 2,472/h; while for A. junius the high rates
were 336 and 252/h respectively.


TABLE 1. MIGRATION LEVELS DURING FALL 1999 OF DRAGONFLIES (COMMON GREEN DARNER, BLACK SADDLEBAGS,
WANDERING GLIDER, CAROLINA SADDLEBAGS) AND BUTTERFLIES (GULF FRITILLARY, LONG-TAILED SKIPPER,
CLOUDLESS SULFUR, MONARCH, BUCKEYE) AT ST. JOSEPH PENINSULA, FLORIDA.

Highest hourly rate' 50% period3
Species (date and hour) % total2 (median) Est. total4

Odonata
Anaxjunius 3,297 (2 Oct, 7 pm) 23.2% 27 Sep-6 Oct (1 Oct) 77,970
Tramea lacerata 228 (2 Oct, 11 am) 3.3% 13 Sep-16 Oct (Sep 30) 20,938
Pantala flavescens 138 (16 Oct, 4 pm) 1.8% 17 Sep-8 Nov (2 Nov) 9,052
Tramea carolina 108 (11 Sep, 1 pm) 1.0% 12 Sep-2 Nov (2 Oct) 4,762

Lepidoptera
Agraulis vanillae 3,162 (3 Oct, 10 am) 57.8% 28 Sep-17 Oct (6 Oct) 273,201
Urbanus proteus 522 (21 Oct, 2 pm) 7.4% 2 Oct-23 Oct (14 Oct) 43,908
Phoebis sennae 114 (3 Oct, 10 am) 2.2% 11 Sep-11 Oct (1 Oct) 11,447
Danaus plexippus 138 (6 Nov, 12 pm) 2.1% 2 Oct-8 Nov (22 Oct) 12,244
Junonia coenia 42 (2 Oct, 2 pm) 1.0% 8 Oct-9 Nov (23 Oct) 6,088

Average rate from 2 5-minute counts/h, and date and hour of the count in parenthesis.
Percentage of total count of all insects for this species.
'The 50% period is the period between 25% and 75% completion of seasonal migration.
'Estimated total is based on linear interpolation between observed hourly rates both between hours and days.


June 2001







Sprandel: Migration at St. Joseph Peninsula


LF


Survey Date
Fig. 2. Estimated daily survey totals for Gulf fritil-
lary butterfly (Agraulis vanillae) and common green
darner (Anaxjunius) from 14 Aug-7 Dec 1999 on the St.
Joseph Peninsula, Gulf County, Florida.

For both the Lepidoptera and Odonata the pe-
riod when the middle 50% of the seasonal flight
occurred varied by species (Table 1) with the ear-
liest being P. sennae and the latest being the
buckeye, Junonia coenia (L., Lepidoptera:
Nymphalidae). The whole extent of the flight pe-
riod was much larger. For example, the flights
ranged from 14 Aug to 28 Nov for the dragonflies
A.junius and the wandering glider Pantala flave-
scens (F., Odonata: Libellulidae). Flights began on
5 Sep for all butterflies and extended to 28 Nov for
D. plexippus and J coenia. By 7 Dec, A. vanillae
was the only species observed.


3500


3000

2500

2000


7 8 9 10 11 12 13 14 15 16 17 18 19
Survey Hour
Fig. 3. Hourly flight rates by hour of the day (07:00-
19:00) on 2 Oct 1999 for migrating Gulf fritillary butterfly
(Agraulis vanilla) and common green darner (Anax
junius) on the St. Joseph Peninsula, Gulf County, Florida.


Flight height of the Odonata was about 2-m
over the boardwalk, for a total of 9-m over the
base of the fore dune. The Lepidoptera were
within 2-3 m of the ground, but on encountering
the boardwalk most flew over it. The flights were
normally on the leeward side of the fore dune.
Though there wasn't a consistent daily pat-
tern, flights usually began increasing by 9 am
(e.g., Fig. 3) The occasional peak hourly rates of
A. junius in the evening (e.g., Fig. 3) may repre-
sent an evening descent from above the height of
direct observation (Corbet 1999).
Behaviors other than flight included 51 in-
stances of predation by A. junius on A. vanillae,
stable fly Sr. .. calcitrans (L), Diptera: Mus-
cidae, see Wright 1945; Fye et al. 1980), mosqui-
toes (Diptera: Cilicidae), long-tailed skipper
(U proteus), buckeye butterfly J coenia, and
black saddlebags dragonfly T lacerata. The only
other instances of predation were twice by T lac-
erata on stable flies, and once by a wandering
glider dragonfly (P. flavescens) on the buckeye
butterfly (J coenia). No predation on dragonflies
was observed despite abundance of potentially
predatory birds including merlin (Falco colum-
barius), American kestrel (Falco sparverius) (e.g.,
Walter 1996) barn swallow (Hirundo rustica),
tree swallow (Tachycineta bicolor), and logger-
head shrike (Lanius ludovicianus). Only dragon-
flies were observed preying on Lepidoptera, as
described above. There was some mortality from
vehicles on the peninsula road of both butterflies
and dragonflies. Some butterflies paused within
100 m north of the boardwalk on flowering plants.
There were 28 pairs ofA. junius flying in tan-
dem (latest date 3 Oct). There were 9 pairs of
T lacerata flying in tandem (as late at 20 Nov)
and one instance of flying in the wheel position in-
dicating copulation (20 Nov). There was one in-
stance of a paired P. flavescens in the wheel
position on 6 Nov. This indicates that at least
some Odonates were reproductively mature. No
mating among the butterflies was observed.
The estimated seasonal totals ranged from
4,700 for T carolina, to 273,000 for A. vanillae
(Table 1). There was an average 40% variance of
the two counts in a given hour. The temporal vari-
ability within the day (e.g., Fig. 3) and sharp
peaks between days (Fig. 2), indicate that the lin-
ear interpolation method should not be viewed as
an absolute number, but rather as an index of the
relative abundance of the migrants. These migra-
tion levels are less than the millions in massive
migratory swarms (May 1995; Russell et al. 1998)
of Odonates, and less than the estimated 30 mil-
lion seasonally migrating Lepidoptera in penin-
sular Florida (Walker 1991). This study does
indicate a slow and steady migration for both
dragonflies and butterflies at this location. Al-
though none of these species are considered
threatened (Logan 1997) or rare (Deyrup & Franz


. Common green darner
g Gulffrittilary




*







Florida Entomologist 84(2)


1994), their migrations may be endangered
(Brower & Malcom 1991). Further studies along
obvious migration corridors, and observations
from the tip of St. Joseph Peninsula, would help
piece together flight patterns. More intensive sur-
veys over multiple years would help determine if
there were synoptic or local weather correlates to
migration. Protection along migration paths
might include avoiding use of insecticides, and
not mowing nectar plants. The impact of coastal
development on these routes is unknown.

ACKNOWLEDGMENTS

Michael May alerted me to the general interest
in these phenomena. Wendy Brill, Jerrell J. Daigle,
Michael May, Tom Walker, and George Wallace
reviewed earlier drafts of this manuscript.

REFERENCES CITED

BAUST, J. G., A. H. BENTON, AND G. D. AUMANN. 1981.
The influence of off-shore platforms on insect disperals
and migration. Bull. Entomol. Soc. America 27:23-25.
BROWER, L. P., AND S. B. MALCOM. 1991. Animal migra-
tions: endangered phenomena. American Zool.
31:265-276.
CORBET, P. S. 1999. Dragonflies: behavior and ecology of
Odonata, Cornell University Press, Ithaca, NY.
DEYRUP, M., AND R. FRANZ (eds.). 1994. Rare and en-
dangered biota of Florida. Vol. IV-invertebrates.
University Press of Florida, Gainesville.
DUNKLE, S.W. 1989. Dragonflies of the Florida penin-
sula, Bermuda, and the Bahamas, Sci. Pub. Nature
Guide No. 1, Gainesville, FL.
FYE, R. L., J. BROWN, J. RUFF, AND L. BUSCHMAN. 1980.
A survey of northwest Florida for potential Stable fly
breeding. Florida Entomol. 63(2): 246-251.
LOGAN, T. H. 1997. Florida's endangered species, threat-
ened species, and species of special concern: official
lists. Florida Game and Fresh Water Fish Commis-
sion, Tallahassee.


LOWERY, G. H. JR. 1946. Evidence of trans-Gulf migra-
tion. Auk 63: 175-211.
MAY, M. L. 1992. Migrating dragonflies in North Amer-
ica. Argia 4(2): 4-8
MAY, M. L. 1995. Dependence of flight behavior and heat
production on air temperature in the green darner
dragonflyAnaxjunius (Odonata: Aeshnidae). J. Exp.
Bio. 198: 2385-2392.
PAULSON, D. R. 1966. Dragonflies (Odonata: Anisop-
tera) of South Florida. Ph.D. Dissertation, Univ. of
Miami, 603 pp.
RUSSELL. R. W., M. L. MAY, K. L. SOLTESZ, AND J. W.
FITZPATRICK. 1998. Massive swarm migrations of
dragonflies (Odonata) in Eastern North America.
American Midland Nat. 140: 325-342.
RUSSELL. R. W. 1999. Migration Over the Gulf. Louisiana
Museum of Natural Science. http://transgulf. org/.
STEDMAN, S. J. 1984. St. Joseph Peninsula hawk migra-
tion. Dep. Nat. Res., Tallahassee, FL.
URQUHART, F. A., AND N. R. URQUHART. 1978. Autum-
nal migration routes of the eastern population of the
monarch butterfly (Danaus p. plexippus L.; Dan-
aidae; Lepidoptera) in North America to the over-
wintering site in the Neovolcanic Plateau of Mexico.
Canadian J. Zool. 56: 1759-1764.
VAN HOOK, T. V., AND S. HERMANN. 1999. Monarch fall
migratory population monitoring at St. Marks Na-
tional Wildlife Refuge: 1985-86, 1988-89, 1991-92,
1997-98. Prelim. rept. to Florida Fish and Wild.
Cons. Comm., Tallahassee.
WALKER, T. J. 1979. Migrating lepidoptera: are butter-
flies better than moths? Florida Entomol. 63: 79-98.
WALKER, T. J. 1991. Butterfly migration from and to
peninsular Florida. Ecol. Entomol. 16:241-252.
WALKER, T. J. AND R. C. LITTELL. 1994. Orientation of
fall-migrating butterflies in North Peninsular Flor-
ida and source areas. Ethology 98: 60-84.
WALTER, S. 1996. Fort Tilden dragonfly migration
watch. Argia 8(1): 8-13.
WINSBERG, M. D. 1990. Florida weather. University of
Central Florida Press, Orlando.
WRIGHT, M., 1945, Dragonflies predaceous on the
Stablefly, Stomoxys calcitrans (L.), Florida Entomol.
28: 11-13.


June 2001







Liu & Yue: Effects of Temperature on Lipaphis erysimi


COMPARISON OF SOME LIFE HISTORY PARAMETERS BETWEEN ALATE
AND APTEROUS FORMS OF TURNIP APHID (HOMOPTERA: APHIDIDAE)
ON CABBAGE UNDER CONSTANT TEMPERATURES

TONG-XIAN LIU AND BISONG YUE'
Vegetable IPM Laboratory, Texas Agricultural Experiment Station, Texas A&M University
2415 East Highway 83, Weslaco, TX 78596-8399

'Permanent address: Department of Biology, Sichuan University, Chengdu, Sichuan, China 610064

ABSTRACT
Development, longevity, survivorship and fecundity of the alate and apterous forms of the
turnip aphid, Lipaphis erysimi (Kaltenbach), were studied on cabbage under constant tem-
peratures in the laboratory. The developmental durations for alate nymphs were 15.8, 9.5,
8.0 and 5.4 d at 15, 20, 25 and 30 C, respectively, and those for apterous nymphs were 13.9,
6.8, 6.1 and 5.0 d. Alate nymphs developed 1.9-3.0 d longer at 15, 20 and 25 C than the apter-
ous nymphs, but the developmental durations between the alate and apterous forms were
not significantly different at 30 C. The longevities of alate adults were 12.6, 17.7, 17.6, and
17.2 d at 15, 20, 25, and 30 C, respectively, compared with 25.3, 21.3, 17.5 and 11.7 d, re-
spectively, for apterous aphids under the corresponding temperature regimes. Fecundity
was also significantly less for alate adults than for apterous adults. Alate adults produced an
average of 7.9, 37.9, 39.0, and 11.9 nymphs in their lifespan at 15, 20, 25 and 30 C, respec-
tively, compared with 52.5, 90.8, 83, and 29.7 nymphs per apterous adult at the same tem-
perature regimes.

Key Words: Lipaphis erysimi, turnip aphid, alate aphid, apterous aphid, development, re-
production, cabbage, vegetable

RESiMEN

Bajo temperatures constantes en lanratorio, se estudi6 el desarrollo, la longevidad, la super-
vivencia y la fecundidad en col de las formas alada y aptera del afido del nabo Lipaphis ery-
simi (Kaltenbach). El tiempo de desarrollo en ninfas aladas fue de 15.8, 9.5, 8.0 y 5.4 dias a
15, 20, 25 y 30 C respectivamente, mientras que en ninfas apteras fue de 13.9, 6.8, 6.1 y 5.0
dias. Las ninfas aladas se desarrolaron de 1.9-3.0 dias mas rdpido a 15, 20 y 25 C que las
ninfas apteras, pero el tiempo de desarrollo entire las formas alada y aptera no fue significa-
tivamente diferente a 30 C. La longevidad de los adults alados fue de 12.6, 17.7, 17.6 y 17.2
dias a 15, 20, 25 y 30 C respectivamente, comparada con 25.3, 21.3, 17.5 y 11.7 dias para los
afidos apteros bajo los r6gimenes correspondientes de temperature. La fecundidad tambien
fue perceptiblemente menor para los adults alados que para los adults aptreos. Los adul-
tos alados produce un promedio de 7.9, 37.9, 39.0 y 11.9 ninfas en su lapso de vida a 15, 20,
25 y 30 C respectivamente, comaparado con 52.5, 90.8, 83.0 y 29.7 ninfas por adulto aptero
bajo el mismo regimen de temperatures.


The turnip aphid, Lipaphis erysimi (Kalten-
bach), is a worldwide pest on Brassica crops
(Begum 1995; Liu et al. 1997; Prasad 1988; Yue &
Liu 2000). The biology of apterous L. erysimi on
several Brassica vegetables were well documented
(Ahlawat & Chenulu 1982, Amjad and Peters
1992; Chander & Phadke 1994; Castle et al. 1992;
Prasad & Phadke 1984; Edelson et al. 1993; Set-
okuchi & Muma 1993; Singh et al. 1983; Singh &
Sachan 1995). The nymphs and adults suck the
sap from leaves, young shoots, inflorescence and
young pods, resulting in chlorophyll reduction or
even plant death. Additionally, alate L. erysimi
can transmit some important plant virus diseases,
such as sugar cane mosaic virus, cucumber mosaic
virus and bean yellow mosaic potyvirus (Ahlawat
& Chenulu 1982; Castle et al. 1992).


The life history parameters, such as develop-
ment, longevity, survivorship and fecundity of the
alate form ofL. erysimi were not well documented,
and these biological characteristics and parame-
ters are essential for effective aphid management
(Halbert et al. 1981). In this paper, we report the
effects of four different constant temperature re-
gimes on development, survivorship, longevity
and fecundity of both the alate and apterous forms
ofL. erysimi on cabbage in the laboratory.

MATERIAL AND METHODS


Host Plants


Cabbage, Brassica oleracea var. capitata L.
(Grand Slam Hybrid), was seeded in styrofoam







Florida Entomologist 84(2)


germination trays with 5 seeds per cell (2.5 by 2.5
by 7.5 cm) in a greenhouse. Seedlings were
thinned when the plants were 2.5-cm high leav-
ing one healthy plant per cell. These seedlings
were transplanted individually to plastic pots (15
cm in diam.) when they were =8-cm high with 5-6
leaves. Some of these seedlings were maintained
in the greenhouse, and others were maintained in
an insectary for feeding the aphid colony.

Development, Survivorship, Longevity and Fecundity

The aphid colony has been maintained on cab-
bage in a greenhouse for >1 year. Both alate and
apterous adult aphids were collected from the
greenhouse colony, and were transferred onto pot-
ted cabbage plants in an air-controlled insectary
at 25 + 2C and 55-60% relative humidity (RH)
under a photoperiod of 12:12 (L:D) h. Detached
cabbage leaves were used to rear L. erysimi in all
experiments. Clear plastic petri dishes (12.5 cm
by 1.2 cm) were used as aphid rearing arenas.
Eight layers of paper tissues were put on the bot-
tom of the petri dishes, and the paper tissues
were saturated with water for sufficient moisture.
A cabbage leaf disk ( 8.0 cm in diam) with the
adaxial surface facing up was placed on the wa-
ter-saturated paper tissue in each petri dish. At
the time of the experiment, neonate nymphs (<24
h old 1st instars) were collected from the labora-
tory colony using a small camel hair brush (#000)
and placed into each rearing arena. Thirty to 40
aphids were used for each treatment. Four con-
stant temperatures, 15, 20, 25, and 30C, were
maintained in growth chambers (Percival, Boone,
IA) with a photoperiod of 14:10 (L:D) h and 50-
75% RH. Development, molting, survival and
number of newborn nymphs were recorded daily
until the females died. Newborn nymphs were re-
moved after the daily recording until the death of
the adult. Leaf disks were replaced at the first
sign of deterioration, normally at 4-5 d intervals.

Data Analysis

Aphids were excluded from the data set if they
died within 24 h or they were never observed
feeding. Developmental duration, longevity and
reproduction of both alate and apterous L. ery-
simi under the four constant temperatures were
analyzed using a two-way analysis of variance
(ANOVA) (temperatures x aphid forms), and the
means were separated using the least significant
difference (LSD) test at P = 0.05 upon a signifi-
cant F-test (SAS Institute 1996).

RESULTS AND DISCUSSION

Nymph Development

Temperature significantly affected the develop-
ment for both alate and apterous nymphs (Fig. 1).


16 -
, i
+1 14
12






0
sD .
25 -



o l
410


100
UJ
+1
60

a 40


0-


Nymph Development I
Alateaphids
7/7 Apterous aphids






Adult Longevity









Fecundity


-
15


20 25
Temperature (C)


30


Fig. 1. Development, longevity and fecundity of both
alate and apterous alate Lipaphis erysimi on cabbage
under four constant temperatures in the laboratory.


At the four temperatures, alate nymphs devel-
oped significantly faster at higher temperatures
than at lower temperatures (F = 18.6; df = 3, 102;
P = 0.0001). The developmental duration was
shortest at 30C (5.4 d), followed by at 25C (8.0
d), at 20C (9.5 d), and the longest at 15C (15.8
d). Similarly, apterous nymphs also developed
faster at higher temperatures than at lower tem-
peratures (F = 9.3; df= 3, 101;P = 0.0002). The de-
velopmental duration was shortest at 30C (5.0
d), followed by at 25C (6.1 d), at 20C (6.8 d), and
the longest at 15C (13.9 d). Alate nymphs devel-
oped significantly longer than apterous nymphs
at 15, 20 and 25C (F = 11.2- 23.14; df= 1, 102;P
= 0.0025-0.0001). Those under 30C did not show
significant differences in developmental dura-
tions (F = 1.13; df= 1, 102;P = 0.5741).

Survivorship and Longevity

All nymphs in both alate and apterous forms
survived to adulthood (Figs. 1 and 2). The longev-
ities of both alate and apterous adults were signif-
icantly affected by temperature. The longevity of
alate adults was significantly shorter at 15C
than those at the other three temperatures (F =
7.13; df= 3, 104; P = 0.0471). In contrast, the lon-


June 2001







Liu & Yue: Effects of Temperature on Lipaphis erysimi


100 ------ -------- .
I -.- Alate-survival 6
-60 -- Apterous-survival
S ---- Alate-fecundity 5
-D-- Apterous-fecundity
20 -1
i .___ __ ...-\
100 xxc a?0 c

60 > ~
40




C 2 S i B
0 4



20 --2-




20- \



80 7

0 5 10 15 20 25 30 35 40 45 50
Days since birth
Fig. 2. Survival rates and natality of both alate and
apterous Lipaphis erysimi on cabbage at different tem-
peratures in the laboratory.


gevities of apterous adults declined as the tem-
peratures increased from 150C to 300C. The
longest longevity (25.3 d) was obtained at 150C,
followed by 21.3 d at 200C, 17.5 d at 250C, and the
shortest, 11.7 d at 300C. Between the two forms,
alate adults lived a significantly shorter period
than apterous adults (F = 9.87-12.23; df = 1, 102;
P = 0.0012-0.0001) at 15, 20 and 300C except for
those at 250C (F = 0.97; df = 1, 102; P = 0.8754).
The largest difference in longevity between the
two forms was found at 150C at which the apter-
ous adults lived r2-fold longer than that of alate
adults (25.3 d vs. 12.6 d respectively). At 200C, the
apterous adults lived 3.6 d longer than the alate
adults. The longevities of both forms were almost
the same at 250C. In contrast, the apterous adults
lived 5.5 d shorter than the alate adults (17.2 dvs.
11.7 d) at 300C.
Similar effects were also reported by DeLoach
(1974) and Liu (1991). Aphid development and re-
production typically increase from zero at a low
temperature threshold, reach a maximum at the
most favorable temperature, then decrease rap-
idly to zero at a lethal threshold. Like other
aphids in general, L. erysimi reared at tempera-
tures above the upper or below the lower thresh-
olds develop more slowly than those under the


most favorable conditions. The nymphs developed
fastest at 30C, and slightly slower at 25 and
20C. DeLoach (1974) found that L. erysimi on
turnip could continue to reproduce at 30C, but
failed to do so at 35C. Similarly, Liu (1991) found
the most favorable temperature forL. erysimi was
26C. At 8.3 and 35C, few nymphs developed to
adults, but none produced any offspring. At 11.3
and 32.8C, few adults successfully reproduced.
Therefore, the lower and upper threshold temper-
ature for L. erysimi should be higher than 8C
and lower than 33C, with the most favorable
temperature between 25 and 27C. DeLoach
(1974) also reported great mortality for some
young L. erysimi nymphs because these young
nymphs failed to become well-established on the
new host plants after having been transferred
from the original host plants.

Fecundity

Temperature played a significant role for aphid
reproduction (Figs. 1 and 2). Alate adults produced
the most nymphs at 25C (39.0 nymphs/adult)
and 20C (37.9 nymphs/adult), followed by these
(11.9 nymphs/adult) at 30C, and the fewest at
15C (7.9 nymphs/adult). Apterous adults pro-
duced most nymphs at 20C (90.8 nymphs/adult),
followed by 83.0 nymphs/adult at 25C, 52.5
nymphs/adult at 15C, and the fewest, 29.7
nymphs/adult at 30C. Between the two forms,
apterous adults produced 6.6-, 2.4-, 2.1- and 2.5-
fold more nymphs per adult than alate adults of
the corresponding temperature.
Both alate and apterous adults started to pro-
duce nymphs 1 or 2 d after the last molting, so the
adults from last molting to the first reproduction
were only 1-2 d old (Fig. 2). Both fecundity and re-
productive period varied at the four temperature
regimes. Generally, the alate aphids had lower
daily reproductive rates and shorter period of re-
production than the apterous forms at the same
temperature. At 15C, reproductive periods lasted
12 d at 15C, compared with 24 d at 20 and 25C,
respectively, and only 11 d at 30C. Daily fecundity
at the peak reproduction period was 3-4 nymphs
at 20 and 25C, <2 nymphs at 30C and 15C.
The reproductive periods of apterous lasted 34
d at 15C, compared with 24 d at 20C, 25 d at
25C, and only 11 d at 30C. An aphid could pro-
duce 8-9 nymphs per d at the peak reproduction
period at 20 and 25C, 6-7 nymphs at 30C, and 2-
3 nymphs at 15C.
Our results clearly indicate that many biologi-
cal parameters, including nymph development,
adult longevity and fecundity differed between
alate and apterous forms. Similar results were re-
ported by Takaoka (1973) who reviewed that
longer nymph period, longer adult longevity and
lower fecundity of alate virginoparae were found
in many species of aphids compared to those of







Florida Entomologist 84(2)


their apterous form. Information from this study
will aid in predicting the population dynamics of
L. erysimi on cole crops in south Texas. The sus-
ceptibility to low (<10C) and high temperature
(>35C) may present a partial explanation for the
low field populations ofL. erysimi in the summer
and in the winter months when temperatures are
often >30 and <10C, respectively. Meanwhile,
the information onL. erysimi obtained under con-
stant temperatures may not be directly applicable
for field populations that may be affected by fluc-
tuating temperatures (Liu & Meng, 1990) and
other biotic and abiotic factors.

ACKNOWLEDGMENTS

We thank B. Legaspi (USDA-ARS, Weslaco,
TX) for reviewing the early version of this manu-
script, and M. Morales, J. Martinez, M. De Leon
and J. Martinez, Jr. for their technical assistance.
Publication of this paper was approved by the
Center Director of Texas Agricultural Research
Center at Weslaco, and the Head of the Depart-
ment of Entomology, Texas A&M University at
College Station.

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the transmission of radish mosaic virus by the aphid,
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LIU, S. S., X. G. WANG, X. J. WU, Z. H. SHI, Q. H. CHEN,
AND H. X. HU. 1997. Population fluctuation of aphids
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PRASAD, S. K., AND K. G. PHADKE. 1984. Yield loss in
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aphid, Lipaphis erysimi Kalt. Indian J. Entomol. 46:
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June 2001







Landolt: Cabbage looper orientation to potato plants


MOTH EXPERIENCE AND NOT PLANT INJURY AFFECTED FEMALE
CABBAGE LOOPER MOTH (LEPIDOPTERA: NOCTUIDAE)
ORIENTATION TO POTATO PLANTS

PETER J. LANDOLT
U. S. Department of Agriculture, Agricultural Research Service, 5230 Konnowac Pass Rd., Wapato, WA 98951

ABSTRACT

Naive mated female cabbage looper moths, Trichoplusia ni Hubner, responded in a flight
tunnel to potted potato plants (Solanum tuberosum). Percentages of moths attracted to un-
injured potato plants, mechanically-damaged potato plants, and potato plants treated with
regurgitant from larvae of the Colorado potato beetle, Leptinotarsa decemlineata Say, were
similar, indicating no effect of plant treatment. Attraction of female cabbage looper moths to
potato plants was increased following prior contact (experience) by the moth with a potato
plant. This increase in responsiveness to potato plants with experienced moths occurred
whether the plants were uninjured, mechanically damaged, or treated with Colorado potato
beetle larval regurgitant. Moths preconditioned on potato plants treated with regurgitant
exhibited similar rates of attraction to mechanically-damaged plants and to regurgitant-
treated plants. However, moths preconditioned on mechanically-damaged plants were more
responsive to mechanically damaged plants compared to regurgitant-treated plants.

Key Words: Trichoplusia, attraction, host-finding, learning, kairomone

RESUME

Polillas de repollo Trichoplusia ni Htibner, sin experiencia previa, respondieron en un tunel
de vuela a plants de papa en tiestos (Solanum tuberosum). Los porcentajes de polillas atraf-
das a plants de papa sin daho, plants de papa con daho mecanico, y plants de papa tra-
tadas con regurgitante de larvas del escarabajo de papa de Colorado, Leptinotarsa
decemlineata Say, fueron similares, sindicando la falta de efecto al tratamiento de la plant.
La atracci6n de hembras de polilla a plants de papa fue incrementada siguiendo contact
previo (experiencia) de la polilla con la plant de papa. Este incremento en respuesta a plan-
tas de papa con polillas con experiencia ocurri6 aun si las plants no tenfan daho, tenfan
daho mecanico, o tratadas con regurgitante larval del escarabajo de papa de Colorado. Poli-
llas preacondicionadas a plants tratadas con regurgitante exhibieron tiempos similares de
atracci6n a plants mecanicamente dahadas y a plants tratadas con regurgitante. Sin em-
bargo, polillas preacondicionadas a plants mecanicamente dahadas respondieron mas a
plants mecanicamente dahadas en comparaci6n con plants tratadas con regurgitante.


The cabbage looper, Trichoplusia ni (Hiubner)
is a polyphagous herbivore that can feed on a di-
versity of plant taxa (Sutherland & Greene 1984).
The cabbage looper moth exhibits weak attraction
responses to a wide range of host plants, a behav-
ior that may bring it to a host habitat (Landolt
1989). This response is enhanced by conspecific
larval feeding or mechanical damage to the plant
(Landolt 1993). Attraction of cabbage looper
moths to plants is also enhanced following contact
with a host plant (experience), indicating learn-
ing of host plant odors (Landolt & Molina 1996).
Cabbage looper moths previously exposed to cel-
ery (Apium graveolens) or cotton (Gossypium hir-
sutum) were more likely to orient to that same
species of plant rather than to the other species of
host plant (Landolt and Molina 1996). These pre-
vious studies did not consider possible differen-
tial roles of constitutive (stored and released) and
induced (produced de novo) odor chemistry pro-
duced by plants in response to injury. That is, we


do not know if cabbage looper moths are more
strongly attracted to injured plants because of
constitutive or induced plant odors, or how cab-
bage looper moth learning of host plant odor
(Landolt & Molina 1996) might be affected by
these changes in host plant chemistry.
The cabbage looper larvae are found on potato
plants and complete development on potato foliage
(Sutherland & Greene 1984; Landolt, unpublished
data), qualifying potato as a host plant, although it
is not considered to be a common pest of potato. Po-
tato plants respond to mechanical injury, feeding
by Colorado potato beetle, Leptinotarsa decemlin-
eata (Say), and applications of Colorado potato
beetle regurgitant, in part by increasing the re-
lease of volatile chemicals (Bolter et al. 1997).
Treatment of potato foliage with regurgitant from
larvae of the Colorado potato beetle or from cab-
bage looper larvae makes potato plants more at-
tractive to Colorado potato beetle females (Landolt
et al. 1999). The Colorado potato beetle is a spe-







Florida Entomologist 84(2)


cialist on species of Solanum and might be ex-
pected to be adapted to the defensive chemicals,
including volatiles, produced by induced potato
plants. Karban and Baldwin (1997) suggest that
specialist herbivores might respond positively and
generalist herbivores might respond negatively to
induced host plant kairomones. It would be of in-
terest then to determine how a polyphagous herbi-
vore such as the cabbage looper responds to plants
following such treatments.
The objectives of these studies were to deter-
mine if cabbage looper moths are attracted to po-
tato plants, if they are attracted more strongly to
potato plants that are mechanically injured (to re-
lease constitutive odorants), and if they are more
weakly attracted to potato plants that are treated
to produce and release induced odorants. Also, ef-
fects of previous exposure or experience with
plants on cabbage looper moth attraction to unin-
jured and injured potato plants was examined,
with the expectation that moth attraction re-
sponses might be enhanced following experience
with undamaged and mechanically-damaged
plants, but might not be enhanced following expe-
rience with induced plants.

MATERIALS AND METHODS

General Methods

Russet-Burbank potato plants were used in all
experiments. Plants were grown in a glass green-
house from September through April in Yakima,
Washington, but with supplemental lighting from
sodium lamps and 400 watt metal halide lamps
providing a 14:10 L:D photoperiod. Plants were
started with potato eyes in soil in 15 cm diam pots.
Soil was a mix of sand, peat moss and fertilizer.
Plants were used in experiments when 4 to 6 weeks
old (30-35 cm in height) and before blooming.
Three to five days before experiments, plants
were placed in a controlled environment room at
25C and 50% RH. Lighting was supplied with
overhead 400 watt metal halide lamps on a light
cycle coinciding with the light cycle for the cab-
bage looper moths.
Cabbage looper moths were obtained from a
colony established in 1997 at the Yakima Agricul-
tural Research Laboratory, with stock that origi-
nated from northern Florida in 1987. Pupae were
sorted by sex and placed in cages with dishes of
sugar water on cotton balls and with water dis-
pensers on the cage tops. Pupae were moved daily
to new cages to provide cohorts of moths of dis-
crete ages. All pupae and adult moths (including
mating cages, see below) were kept in an environ-
mentally controlled room with reversed light cy-
cle (lights off at 0900 h and lights on at 1700 h),
24C, and 60% RH.
To obtain mated females, groups of 30 males
(3-6 d old) and 25 females (2-3 d old) were placed


together in screened cages (45 x 45 x 45 cm) at the
beginning of the scotophase. Moths were provided
sugar water and water as in the emergence cages.
Females were removed from mating cages near
the end of the subsequent photophase and were
placed in another cage. Assays were conducted
during the first 3 h of the following scotophase.
For each set of assays, a subset of female moths
were recaptured and were dissected to verify that
they mated, evidenced by the presence of a sper-
matophore in the bursa copulatrix.
In all experiments, mated female moth re-
sponses to potted plants were evaluated in a flight
tunnel that was similar to that described by
Landolt and Molina (1996). Charcoal-impreg-
nated fiberglass filters were used at both ends of
the tunnel to minimize contamination of the tun-
nel and experimental room with plant and other
odors. The experimental room was equipped with
overhead red incandescent lamps to facilitate ob-
servation of moths. Flight tunnel room conditions
were 22-24C and 50-70% RH. Plants tested for
attractiveness to moths were placed at the center
of the upwind end of the flight tunnel. Moths were
released from open 30 ml polystyrene vials near
the center of the downwind end of the flight tun-
nel and were observed for two minutes. Released
moths were scored for upwind oriented flight (zig-
zagging upwind flights within the probable
boundaries of the odor plume) and for contact
with the plant. A replicate consisted of a series of
5-10 moths tested per plant. No plant was used
more than once either in a conditioning treatment
or in an assay.
Four experiments were conducted to evaluate
moth attraction to potato plants.

Naive Moths to Uninjured Potato Plants

The first experiment tested the hypothesis
that naive moths are attracted to uninjured po-
tato plants. Naive mated female moths were as-
sayed for attraction to an uninjured potato plant
or to a pot of soil (as an experimental control). Ten
replicates were conducted, with 80 moths tested
to plants and 80 moths tested to pots of soil.

Naive Moths to Injured Potato Plants

The second experiment evaluated the effects of
plant injury on attraction of naive moths to potato
plants. Two non-competitive tests were conducted
using naive female moths. These two tests were
1) a comparison of moth responses to uninjured po-
tato plants and to potato plants that were damaged
mechanically and 2) a comparison of moth re-
sponses to uninjured potato plants and potato
plants treated with regurgitant from larvae of the
Colorado potato beetle. To incur mechanical dam-
age to the potato plants, three leaves on a potato
plant were cut with scissors, lengthwise about 3 cm


June 2001







Landolt: Cabbage looper orientation to potato plants


along the main leaf axis. This was done one hour
before assays were started. Treatment of plants
with beetle regurgitant were made to three leaves
of each plant. Leaves were first scraped with a ra-
zor blade (about one cm2). A fourth or fifth instar
beetle larva was gently squeezed until it produced
a droplet of regurgitant at the mouthparts which
was then applied to the scraped area of leaf. This
was done 24 h before assays were started.
Nine bioassay replicates were conducted, with
90 moths tested per treatment and 90 per control.
In both studies, the sequence of plants tested
(treatment and control) was reversed daily for the
9 days.

Experienced Moths to Uninjured
and Injured Potato Plants

The third experiment evaluated the effects of
prior contact (experience) with potato plants on
moth attraction to potato plants. Three tests were
conducted to compare the responses of naive ver-
sus experienced mated female moths to potato
plants that were either uninjured, were mechani-
cally-damaged, or were treated with Colorado po-
tato beetle regurgitant. For each assay, mated
females were divided into 2 equal groups when
separated from males in the mating cages. One
group was placed in a larger screened cage (45 x
45 x 45 cm) with a potted potato plant, at the on-
set of the scotophase (experienced moth group).
The other group was placed in a similar cage with
no plant (naive moth group). Both cages were
kept in the controlled environment room used to
hold plants for bioassays, on a 14:10 L:D light cy-
cle, 50% RH and 25C for 24 h. Female moths
were then removed from these cages and held in
30 x 30 x 30 cm screened cages in the controlled
environment room holding moths and no plants,
with water and sugar water, until used in bio-
assays in the following scotophase. A comparison
was first made of naive versus experienced moths
to an undamaged plant. Naive moths were tested
for attraction to and contact with an uninjured
potato plant, followed by the testing of experi-
enced moths tested to the same plant. The follow-
ing day, using a new plant, a set of experienced
moths were tested for responses to an uninjured
plant, followed by the testing of naive moths. This
was continued for 12 assay sets, with 80 naive
and 80 experienced moths tested to uninjured
potato plants. This protocol was then followed to
compare naive versus experienced moth re-
sponses to a mechanically damaged plant, with a
total of 80 naive and 80 experienced moths tested
to mechanically damaged plants in 16 bioassay
sets. A comparison was then made of naive versus
experienced moth responses to regurgitant treated
potato plants, with 60 naive and 60 experienced
moths tested to regurgitant treated potato plants
in 10 bioassay sets.


Discrimination of Uninjured vs Injured Potato
by Experienced Moths

The fourth experiment evaluated the ability of
experienced moths to discriminate between me-
chanically-damaged plants and plants that had
been treated with Colorado potato beetle regur-
gitant. The objective of this experiment was to
determine if moths preconditioned on one type of
injured potato plant (mechanically-damaged or
regurgitant-treated) would respond better to the
type of plant they were preconditioned with, com-
pared to the other type of plant. There were 4
treatment regimes: 1) moths that were placed
with mechanically-damaged plants in one scoto-
phase and were then tested for responses to
mechanically-damaged plants in the following
scotophase, 2) moths that were placed with
mechanically-damaged plants in one scotophase
and were then tested for responses to regur-
gitant-treated plants in the following scotophase,
3) moths that were placed with regurgitant-
treated plants in one scotophase and were then
tested for responses to mechanically-damaged
plants in the following scotophase, and 4) moths
that were placed with regurgitant-treated plants
in one scotophase and were then tested for re-
sponses to regurgitant-treated plants in the fol-
lowing scotophase.
Mated females were subdivided into 2 equal
groups when initially separated from males in the
mating cages, near the end of a photophase. These
female moths were placed either in a cage with a
mechanically-damaged plant (cut with scissors as
before) or in a cage with a regurgitant-treated
plant (Colorado potato beetle larval regurgitant
applied to scrapes on 3 leaves 24 h before). After
24 h, during the last 2 h of the photophase, these
moths were then removed from the cages contain-
ing plants and were placed in 2 clean cages and
held another 2-4 h until they were used in bio-
assays. For the bioassay, a mechanically-damaged
plant was placed in the flight tunnel and 5 moths
that had been preconditioned with a mechani-
cally-damaged plant and 5 moths that had been
preconditioned with a regurgitant-treated plant
were then tested alternately for responses to the
same plant. A regurgitant-treated plant was then
placed in the flight tunnel and 5 moths that had
been preconditioned with a mechanically-dam-
aged plant and 5 moths that had been precondi-
tioned with a regurgitant-treated plant were
tested alternately for responses to this plant. This
protocol was followed on 10 different days, provid-
ing a total of 50 moths tested for each treatment
category.
For experiments 1, 2 and 3, percentage re-
sponse data for treatment pairs were analyzed by
a paired t test to determine if responses differed
between treatments or between treatment and
control, with a significance level of P < 0.05. Per-







Florida Entomologist 84(2)


centage response data for the treatment regimes
of experiment 4 were compared using Tukey's
Test following an ANOVA.

RESULTS

Naive Moths to Uninjured Potato Plants

The percentages of naive moths that flew up-
wind towards uninjured Russet Burbank potato
plants were significantly greater than the per-
centages of moths responding to a pot of soil (Ta-
ble 1). A significant percentage of moths tested
also landed on uninjured potato plants and none
landed on pots of soil (Table 1).

Moths to Injured Potato Plants

Percentages of naive moths attracted to me-
chanically-damaged and uninjured plants were
not significantly different (Table 1). Percentages
of naive moths attracted to uninjured plants were
not significantly different from those attracted to
regurgitant-treated plants. There were also no
significant differences between percentages of
moths landing on plants, for either of the treat-
ment comparisons (Table 1).

Experienced Moths to Uninjured
and Injured Potato Plants

Moths preconditioned (experienced) on potato
plants were attracted significantly more often to
potato plants, than were naive moths (Table 2).
Increased attraction of preconditioned moths to
potato plants was observed when those plants
were uninjured, mechanically-damaged, or
treated with Colorado potato beetle regurgitant
(Table 2). Additionally, increased landing was ob-
served in moths that were preconditioned either
on uninjured plants or on plants with mechanical
damage (Table 2). Percentages of preconditioned
moths landing on regurgitant-treated plants
were numerically but not statistically greater


than percentages of naive moths landing on those
same plants.

Discrimination of Uninjured vs Injured Potato
by Experienced Moths

Greatest attraction response rates were seen
for moths preconditioned on mechanically-dam-
aged plants and were assayed to other mechani-
cally-damaged plants (Fig. 1). The attraction of
moths that were preconditioned on regurgitant-
treated plants to either mechanically-damaged or
regurgitant-treated plants was similar. These re-
sponses, both attraction and landing on plants,
were also similar to those of moths precondi-
tioned on mechanically-damaged plants and as-
sayed to regurgitant-treated plants (Fig. 1).

DISCUSSION

Results indicate significant but weak attrac-
tion of naive cabbage looper moths to Russet Bur-
bank potato plants. This finding is similar to the
weak attraction responses of naive cabbage
looper moths to cabbage (Brassica oleracea), cel-
ery, tomato (Lycopersicon esculentum), and soy-
bean (Glycine max) plants (Landolt 1989). This
low level attraction to a wide variety of plants
(Landolt 1989) may be a host-habitat finding
strategy rather than a host-finding behavior, as
was suggested previously (Landolt 1993).
There was no significant enhancement of at-
traction of cabbage looper moths to potato plants
cut with scissors to produce mechanical damage.
Because mechanical damage causes short term
increased emission of volatile chemicals from po-
tato (Bolter et al. 1997), a heightened response by
cabbage looper moths was expected. Similar me-
chanical damage to foliage of cotton plants (Gos-
sypium hirsutum) increased their attractiveness
to cabbage looper moths (Landolt 1993) and simi-
lar damage to cabbage plants increased their at-
tractiveness to Mamestra brassicae (L.) moths
(Rojas 1999). However, this effect was not observed


TABLE 1. MEAN (SE) PERCENTAGES OF CABBAGE LOOPER FEMALES THAT WERE ATTRACTED TO AND CONTACTED PO-
TATO PLANTS IN A NO-CHOICE TEST CONDUCTED IN A FLIGHT TUNNEL.

% Attracted % Contact

Treatment comparisons n* x + SE t p x + SE t p

Pot of Soil 10 0.0+ 0.0 2.58 0.01 0.0+ 0.0 2.14 0.03
Untreated plant 10 20.0 + 7.7 14.0 + 6.5
Untreated plant 9 25.7 + 3.7 1.25 0.24 5.8 + 2.7 0.90 0.39
Mechanically-damaged plant 9 32.1 +5.9 8.1 +2.7
Untreated plant 9 20.0 + 6.7 0.96 0.37 6.7 + 1.7 1.04 0.33
Regurgitant-treated plant 9 14.4 + 3.8 10.2 + 4.1

*n = number of replicates with 5 moths tested per replicate.


June 2001







Landolt: Cabbage looper orientation to potato plants


TABLE 2. MEAN (SE) PERCENTAGES OF NAIVE AND EXPERIENCED CABBAGE LOOPER FEMALES ATTRACTED TO AND
CONTACTING POTATO PLANTS IN A NO-CHOICE FLIGHT TUNNEL ASSAY.

% Attracted % Contact

Moth treatment n* x + SE t p x + SE t p

Undamaged plants
Naive 12 25.9 + 6.6 3.12 <0.01 9.2 + 5.4 2.30 0.02
Experienced 12 44.3 + 5.7 21.9 + 6.6
Mechanically-damaged plants
Naive 16 27.5 + 4.9 7.25 <0.01 8.8 + 4.1 4.48 <0.01
Experienced 16 55.4 + 4.2 24.8 + 4.2
Regurgitant-treated plants
Naive 15 42.5 + 6.0 2.19 0.03 25.1 + 5.1 1.58 0.07
Experienced 15 62.1 +4.1 40.0 + 7.4

*n = number of assay replicates with 5 moths tested per assay replicate.


with cabbage looper attraction to mechanically-
damaged cabbage plants (Landolt 1993). It is not
yet evident why orientation to mechanically-dam-
aged plants may be enhanced in some cases and
not in others.
There was also no enhancement of moth at-
traction to potato plants treated with regurgitant
of larvae of the Colorado potato beetle. This treat-
ment is known to stimulate the production and
prolonged release of odorants from potato (Bolter
et al. 1997) and makes potato plants more attrac-


C:C C:R R:C R:R
PRECONDITIONED:TESTED PLANTS
Fig. 1. Mean (SE) percentages of mated female cab-
bage loopers attracted to (solid bars) and contacting
(open bars) Russet Burbank potato plants in a flight
tunnel, after preconditioning contact with other potato
plants during the previous scotophase. The first treat-
ment letter indicates the type of plant the moths were
preconditioned with and the second letter indicates the
type of plant the moths were presented with in the flight
tunnel assays. Plants for preconditioning and for flight
tunnel assays were either mechanically-damaged (C) or
were regurgitant-treated (R). Bars with the same letter
are not significantly different by Tukey's Test (P < 0.05).


tive to female Colorado potato beetle (Landolt et
al. 1999). In previous studies, conspecific larval
damage made cotton plants more attractive to
cabbage looper moths (Landolt 1993) and made
cabbage plants more attractive to M. brassicae
moths (Rojas 1999). However, such damage to
cabbage plants made them less attractive to cab-
bage looper moths (Landolt 1993) and such dam-
age to chrysanthemum plants made them less
attractive to M. brassicae moths (Rojas 1999). As
with the varying responses of moths to mechani-
cally-damaged plants, the varying responses of
moths to insect damaged and regurgitant-treated
plants calls for an explanation.
Results of experiments 3 and 4 indicate a pos-
itive effect of cabbage looper moth experience
with potato plants prior to testing moths in flight
tunnel assays. This enhancement of attraction re-
sponses occurred regardless of whether the plant
was uninjured, mechanically-damaged or regur-
gitant-treated. Similarly, greater numbers of
moths contacted the plants if they had prior expe-
rience with the same type of plants, compared to
moths with no such experience, with the excep-
tion of regurgitant treated plants. Such an effect
of preconditioning is considered a form of learn-
ing, as described by Papaj and Prokopy (1989).
Landolt and Molina (1996) reported strong en-
hancement of host attraction responses by mated
female cabbage looper moths following prior expe-
rience with host plants, using cotton, celery, and
soybean plants. In those studies it was shown
that brief contact with the plant was all that was
required to strongly increase the subsequent re-
sponse of the moth to that plant species. Evidence
of learning of host plant odor chemistry also ex-
ists for the moths Helicoverpa armigera (Hiibner)
(Cunningham et al. 1998), Heliothis virescens
(Hartlieb 1996) and Spodoptera littoralis (Boisdu-







Florida Entomologist 84(2)


val) (Fan et al. 1997) and for the phytophagous
beetles L. decemlineata (Say) (Visser & Thiery
1986) and Diaprepes abbreviatus (L.) (Harari &
Landolt 1999). In contrast, Rojas and Wyatt
(1999) noted decreased responsiveness to host
plants by Mamestra brassicae L. following earlier
contact with host plants.
In previous studies (Landolt and Molina 1996),
it was shown that cabbage looper moth learning
of host plant odor was specific to the plant species
used in preconditioning. When moths had prior
contact with cotton plants, they were more
strongly attracted to other cotton plants and not
to celery plants. Similarly, when moths had prior
contact with celery plants, they were more
strongly attracted to other celery plants and not
to cotton plants. In the studies described herein,
this discrimination between plants did not occur
when moths had prior contact with mechanically-
damaged potato plants or regurgitant-treated
potato plants and were tested for attraction to
both types of potato plants. There was some en-
hancement of response to those plants following
moth preconditioning on mechanically-damaged
plants, but moths responded similarly to the two
types of injury. Apparently, cotton and celery
plants have strongly divergent odor chemistries
with little, if any, qualitative overlap, making it
more likely that an insect could learn and dis-
criminate separate odors. However, the odor
chemistries of mechanically-damaged (to release
constitutive odors) and regurgitant-treated (to re-
lease induced odors) potato plants overlap exten-
sively (Bolter et al. 1997) and moths that are
preconditioned on potato plants with one type of
injury may be unable to discriminate between
plants with either type of injury.
It was hypothesized that the cabbage looper
moth, as a generalist herbivore, might show en-
hanced attraction to mechanically-damaged
plants because of increased emission of volatiles
and might show decreased attraction to Colorado
potato beetle regurgitant-treated plants because
of the induction of defensive chemistry associated
with that treatment to potato (Bolter et al 1997)
and similar treatments with lepidopterous larvae
to other plants (Tumlinson et al. 1992; Turlings et
al. 1995). In the case of cabbage looper orientation
to potato odor, those expectations appear incor-
rect because observed response rates were similar
to uninjured and injured potato, both by naive
and by experienced moths.
Host-finding by the cabbage looper moth ap-
pears to involve several behavioral strategies,
host-habitat location, responses to some plant in-
jury volatiles, and learning of host plant odor. Ev-
idence indicates a weak response to a variety of
plant species that may bring them into the vicin-
ity of possible hosts (Landolt 1989). Injury to
plants may (cotton) or may not (cabbage, potato)
increase a plant's attractiveness to cabbage


looper (Landolt 1993). Additional work is needed
to determine the chemical bases for the response
differences observed among plants. Prior contact
with a plant increases the subsequent responsive-
ness of the moth to the same type of plant, indi-
cating associative learning of host plant odor
when contact is made with plant foliage. The in-
ability of the moth to selectively respond to potato
plants with different types of injury still leaves
open the question of the relative significance of
constitutive versus induced odorants to cabbage
looper host finding with other species of plants.

ACKNOWLEDGMENTS
Technical assistance was provided by L. Bid-
dick, P. Chapman, R. S. Hofstetter, and D. Larson.
L. Finch, J. Jewett, and C. Albano provided cab-
bage looper moths for use in these experiments.
This work was supported in part by funding from
the Washington State Potato Commission. Cab-
bage looper eggs were kindly provided by Ken
Haynes, Dept. Entomology, Lexington, KY, for es-
tablishment of the colony at the Yakima Agricul-
tural Research Laboratory. Helpful comments to
improve the manuscript were made by C. 0.
Calkins, N. Epsky, H. C. Reed, D. C. Robacker, and
M. Zlotina.

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BOLTER, C. J., M. DICKE, J. J. A. VAN LOOP, J. H. VIS-
SER, AND M. A. POSTHUMUS. 1997. Attraction of Col-
orado potato beetle to herbivore-damaged plants
during herbivory and after its termination. J. Chem.
Ecol. 23: 1003-1023.
CUNNINGHAM, J. P. M. F. A. MALLOW, D. J. WRIGHT,
AND M. P. ZALUCKI. 1998. Learning in host selection
in Helicoverpa armigera (Hubner) (Lepidoptera:
Noctuidae). Animal Behav. 55: 227-234.
FAN, R., P. ANDERSON, AND B. S. HANSSON. 1997. Be-
havioral analysis of olfactory conditioning in the
moth Spodoptera littoralis (Boisd.) (Lepidoptera:
Noctuidae). J. Exp. Biol. 200: 2969-2976.
HARARI, A. R., AND P. J. LANDOLT. 1999. Feeding expe-
rience enhances attraction of female Diaprepes ab-
breviatus (L.) (Coleoptera: Curculionidae) to food
plant odors. J. Insect Behav. 12: 415-422.
HARTLIEB, E. 1996. Olfactory conditioning in the moth
Heliothis virescens. Naturwissenshaften 83: 87-88.
KARBAN, R., AND I. T. BALDWIN. 1997. Induced re-
sponses to herbivory. University of Chicago Press,
Chicago. 319 pp.
LANDOLT, P. J. 1989. Attraction of the cabbage looper to
host plants and host plant odor in the laboratory. En-
tomol. Exp. Appl. 53: 117-124.
LANDOLT, P. J. 1993. Effects of host plant leaf damage on
cabbage looper moth attraction and oviposition. En-
tomol. Exp. Appl. 67:79-85.
LANDOLT, P. J., AND 0. MOLINA. 1996. Host-finding by
cabbage looper moths (Lepidoptera: Noctuidae):
Learning of host odor upon contact with host foliage.
J. Insect Behav. 9: 899-908.
LANDOLT, P. J., D. LARSON, J. H. TUMLINSON, AND H.
ALBORN. 1999. Attraction of Colorado potato beetle


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(Coleoptera: Chrysomelidae) to damaged and chemi-
cally induced potato plants. Environ. Entomol. 28:
973-978.
PAPAJ, D. R., AND R. J. PROKOPY. 1989. Ecological and
evolutionary aspects of learning in polyphagous in-
sects. Annual Review of Entomology. 34: 1281-1288.
ROJAS, J. C. 1999. Influence of host plant damage on the
host-finding behavior of Mamestra brassicae (Lepi-
doptera: Noctuidae). Environ. Entomol. 28: 588-593.
ROJAS, J. C., AND T. D. WYATT. 1999. The role of pre-
and post-imaginal experience in the host-finding and
oviposition behavior of the cabbage moth. Physiol.
Entomol. 24.
SUTHERLAND, D. W. S., AND G. L. GREENE. 1984. Culti-
vated and wild host plants, pp. 1-13. In P. D. Lingren
and G. L. Greene (eds.). Suppression and Manage-


ment of Cabbage Looper Populations. USDA Tech.
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TUMLINSON, J. H., T. C. J. TURLINGS, AND W. J. LEWIS.
1992. The semiochemical complexes that mediate
insect parasitoid foraging. Agric. Zool. Reviews. 5:
221-252.
TURLINGS, T. C. J., J. H. LOUGHRIN, P. J. MCCALL, U. S.
R. ROSE, W. J. LEWIS, AND J. H. TUMLINSON. 1995.
How caterpillar-damaged plants protect themselves
by attracting parasitic wasps. Proc. National Acad.
Sciences of the USA. 92: 4169-4172.
VISSER, J. H., AND D. THIERY. 1986. Effects of feeding
experience on the odour-conditioned anemotaxes
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198-200.







Florida Entomologist 84(2)


June 2001


EFFECTS OF FIRE ANTS (HYMENOPTERA: FORMICIDAE)
ON HATCHING TURTLES AND PREVALENCE OF FIRE ANTS
ON SEA TURTLE NESTING BEACHES IN FLORIDA

CRAIG R. ALLEN', ELIZABETH A. FORYS2, KENNETH G. RICE3, AND DANIEL P. WOJCIK4
1U.S. Geological Survey, Biological Resources Division, South Carolina Cooperative Fish and Wildlife
Research Unit, Clemson University, Clemson, SC 29634

2Department of Environmental Science, Eckerd College, St. Petersburg, FL 33711

3U.S. Geological Survey, Biological Resources Division, Florida Caribbean Science Center
Restoration Ecology Branch, Everglades National Park Field Station, Homestead, FL 33034

4U.S. Department of Agriculture, Center for Medical, Agricultural and Veterinary Entomology
Gainesville, FL 32608

ABSTRACT

Red imported fire ants (Solenopsis invicta Buren) have increasingly been observed in logger-
head (Caretta caretta L.) and green (Chelonia mydas L.) sea turtle nests in Florida, and in
the nests of freshwater turtles. They may be attracted to the disturbance, mucous and mois-
ture associated with turtle nesting and establish foraging tunnels into turtle nests shortly
after egg-laying, thus increasing the vulnerability of hatchlings to fire ant predation. We
conducted experiments on a freshwater turtle (Pseudemys nelsoni Carr) to determine the po-
tential impacts of S. invicta on turtle hatchlings. Over 70% of hatchlings were killed by S. in-
victa during pipping or shortly after hatching. To determine the extent of S. invicta
infestation of sea turtle nesting beaches, we sampled known nesting beaches throughout the
state of Florida. Beach surveys indicated that S. invicta are present and often abundant on
most beaches and dunes along the Florida coast.

Key Words: Caretta caretta, endangered species, fire ant, invasive species, Pseudemys nel-
soni, Solenopsis invicta, turtles

RESUME

Se han observado hormigas bravas (Solenopsis invicta Buren) cada vez mas en nidos de tor-
tugas marinas (Caretta caretta L.) y (Chelonia mydas L.) en la Florida, y en los nidos de tor-
tugas de agua dulce. Estas hormigas pueden ser atraidas al disturbio, mucosidad y humedad
asociada con anidaje de tortugas y establecen tuneles de forraje hacia nidos de tortuga poco
despues de la puesta de huevos, asi incrementando la vulnerabilidad de los reci6n nacidos a
predaci6n por la hormiga brava. Llevamos a cabo experiments con una tortuga de agua dulce
(Pseudemys nelsoni Carr) para determinar el impact potential de S. invicta en tortugas re-
ci6n nacidas. Mas del 70% de los reci6n nacidos fueron muertos por S. invicta durante el pro-
ceso de salir del cascaron o poco despues de salir. Para determinar la extension de infestaci6n
de S. invicta en playas donde anidan tortugas marina, muestreamos playas conocidas por te-
ner nidos por todo el estado de la Florida. Examenes de playas indicaron que S. invicta esta
present y much la mayoria de las playas y dunas a lo largo de la costa Floridana.


There has been considerable concern and de-
bate over the potential impact of fire ants on nest-
ing sea turtles, but little quantitative evidence
exits. Information on the impact of Solenopsis in-
victa (Buren) on hatchling turtles has been largely
incidental and anecdotal. Fire ants may impact
turtle populations directly by preying on hatch-
lings and/or indirectly by stinging hatchlings,
resulting in reduced weight gain and survival.
Wilmers et al. (1996) documented an increase
in the presence of fire ants in green (Chelonia my-
das L.) and loggerhead (Caretta caretta L.) turtle
nests on undeveloped island beaches off Florida.


Red imported fire ants were observed feeding on
pipped eggs, and stinging, killing and subse-
quently feeding on turtle hatchlings (Wilmers et
al. 1996). Moulis (1997) documented a 15% de-
crease in hatchling release rate for loggerhead sea
turtles emerging from nests infested with fire ants
as compared to uninfested nests. The ultimate ef-
fect of this predation on sea turtle populations and
the magnitude of the problem else-where is un-
known. The nesting period (May-August) for log-
gerheads in the eastern United States (Johnson et
al. 1996) corresponds with a concentrated period
of brood production in S. invicta. During that







Allen et al.: Fire ant impacts on turtles


time, protein needs for fire ants are maximal (So-
rensen et al. 1983). For secure turtle populations,
high juvenile mortality may not affect population
size (Congdon & Gibbons 1990), but for small pop-
ulations, decreases in annual cohort size may af-
fect population viability (Heppell et al. 1996).
Our objectives were to assess the potential im-
pacts of the invasive non-indigenous ant, S. in-
victa, on hatching turtles by using the eggs of a
freshwater species (Florida red-bellied turtle,
Pseudemys nelsoni Carr) in a controlled experi-
mental setting. Pseudomys nelsoni often lays its
eggs in alligator nests, approximately 20% of which
are infested with fire ants in central Florida (Allen
et al. 1997). In addition, we determined the geo-
graphic extent ofS. invicta occurrence on sea turtle
nesting beaches throughout the state of Florida by
sampling beaches with baits attractive to ants.

MATERIALS AND METHODS

To assess the potential for S. invicta impact on
the eggs and hatchlings of turtles we conducted a
laboratory experiment using P. nelsoni eggs. The
eggs of this freshwater turtle species are elliptical
and approximately 2.5 cm long, occur in clutches
of about 15, and are found regularly in American
alligator nests in Florida. Although the shape of
eggs in this species is different from the generally
round shape of sea turtle eggs, and egg and clutch
size vary among turtle species, we have observed
no differences in attractiveness among eggs of
several different species regardless of size or
shape. Pseudemys nelsoni and many other turtle
species, including sea turtles, share the trait of
emerging from the nest only after most or all of
the clutch has hatched.
Ten clutches of P. nelsoni eggs were collected
(1996) from Lake Apopka in central Florida and
transferred to ten 61 x 36 x 13 cm enclosures at
the U.S.D.A.-A.R.S. Imported Fire Ant Labora-
tory, in Gainesville, Florida. Five clutches served
as controls. For both control and treated clutches,
eggs were placed in sphagnum nesting material
adjacent to a shallow pan of water, which allowed
individual hatchlings immediate access to water
upon emergence. Treated clutches were main-
tained identically to control clutches, but were ex-
posed to a field-collected mound of S. invicta at
the opposing end of each enclosure. This con-
trolled situation simulated the natural conditions
for the many P. nelsoni clutches which share alli-
gator nests with fire ant mounds (Allen et al.
1997). The enclosures allowed fire ants to forage
among the clutch as may occur within natural
turtle nests (Wilmers et al. 1996; Allen et al.
1997). Fire ants were provided with honey as a
food source. Eggs were observed twice daily as
they approached hatching and constantly as pip-
ping commenced. Surviving turtles were trans-
ferred to the Florida Game and Fresh Water Fish


Commission incubation facilities (Gainesville,
FL) with food supplied ad libitum, and measured
weekly for 6 weeks to determine if differences in
weight gain between treated and untreated
groups existed (Allen et al. 1997).
To determine the presence of S. invicta on sea-
turtle nesting beaches, we sampled for fire ants at
18 known sea turtle nesting beaches throughout
Florida. Collection localities are given in Table 1.
Transects consisting of approximately 20 samples
at 10-m intervals were established along dune
lines. Multiple transects (2 to 7) were sampled at
each locality. Baits on all transects consisted of
ground beef, except for those in Duval, St. Johns
and Volusia counties, which consisted of a sugar-
based bait attractive to a variety of ants, newly
formulated by the U.S.D.A.-A.R.S. Baits were left
in the field for approximately 1 h before being col-
lected and transported to U.S.D.A.-A.R.S. facili-
ties in Gainesville, Florida, for sorting and
identification of ant species.

RESULTS

Pseudemys nelsoni clutch size varied from 6-16
eggs, but only 2-11 of the eggs in a given clutch
ultimately hatched. In control groups, 59% of the
eggs did not hatch, and in those exposed to fire
ants 37% did not hatch. The cause or causes of
inviability were not determined but may be at-
tributed to flooding or crushing by the attendant
female alligator prior to collection. During and
after hatching, 100% of hatching turtles in con-
trol groups (17) survived, and were eventually
released. The proportion successfully hatching in
clutches exposed to fire ants (10 of 35) was signif-
icantly less (median = 33%, range 0-55%; Mann-
Whitney Rank Sum Test t = 40.0, df = 8, P =
0.008). Approximately half of the mortalities oc-
curred while the hatchlings were still in the egg,
while most others died less than 1 h after emer-
gence. Fire ants did not breach turtle eggs, but
entered the eggs as soon as a pipped hole was
present. Too few individuals exposed to fire ants
survived to assess differences in weight gain
between the two groups.
We collected 734 ant bait samples and a total
of 31,392 ants from Florida sea turtle nesting
beaches. About 40% of the collected ants (12,658)
were S. invicta. Fire ants were detected foraging
along dune lines on sea turtle nesting beaches in
all regions of the state, and were detected on 13 of
18 specific sites (Table 1). Within those 18 sites,
fire ant occurrence on baits varied from 0 to 63%,
and represented from 0 to 97% of the individuals
collected. Fire ant occurrence followed no obvious
geographic pattern. Ants were abundant at some
very remote locations (e.g., Boca Grande Key near
Key West) but were uncommon on beaches at some
locations that have undergone extensive human
disturbance (e.g., northeastern Florida beaches).







Florida Entomologist 84(2)


TABLE 1. RESULTS OF FIRE ANT SAMPLING IN SEA TURTLE NESTING HABITAT IN FLORIDA.


% baits
No. with
baits S. invicta


East Coast North


Panhandle
Southwest







Westcentral

Southeast


Keys


Crescent Beach
Daytona Beach
Appollo State Park
Cape San Blas
Clam Pass Park South
Vanderbilt Beach
Park Shore Beach
Collier Co., Gullivan Key
Collier Co., Turtle Key
Collier Co., "B" Key
West Panther Key
Pinellas Co., Sand Key
Pinellas Co., Passe Grille
Indian River Co., Sebastian Inlet
Palm Beach Co.,
J. D. MacArthur State Park
Boca Grande
Marquesas
Matecumbe


30 0 0 612 0
30 7 52 90 37
30 0 0 658 0
40 18 867 1,732 33


869
691
142
3
0
4,480
0


98
1,103
30
1675
1163
127
987


60 35 3,570 1,143 76
60 7 188 2,546 7
60 5 153 798 16


60 0


1,613
29
1


0 1,174 0


2,392
582
1,914


Totals 734 15 12,658 18,824 29


DISCUSSION

Our surveys found S. invicta present on most
beaches, at both the wrack and dune lines. Our
and other observations indicate that fire ants often
are present in sea turtle nest cavities (e.g., Wilm-
ers et al. 1996; Parris et al. 2001). The egg-laying
process may initially attract fire ants because it
represents a local disturbance and food source.
Mucous associated with the egg laying process is
an attractive food for fire ants and sea turtle nest
cavities provide a desirable micro-climate for fire
ants. It appears that fire ants cannot breach intact
sea or freshwater turtle eggs (Wojcik & Allen, un-
published data). However, once fire ants build sub-
terranean foraging trails to a site that has
provided food, such as turtle nest cavities, they
maintain those foraging tunnels. Additionally,
post-laying disturbances caused by predators such
as raccoons (Procyon lotor L.) or ghost crabs (Ocy-
pode sp.) that fracture some eggs may attract fire
ants to nest cavities. Thus, fire ants may maintain
a presence in the nest cavity until hatching.
Our experiments with P. nelsoni eggs indicate
that turtle hatchlings are both highly attractive
and vulnerable to fire ants. Presumably, endan-
gered species such as sea turtles or gopher tor-
toises attempting to hatch from nests with
established fire ant foraging tunnels are equally
as vulnerable. Fire ants often use the burrow


aprons of gopher tortoises as colony sites (personal
observation). Nearly half of the P. nelsoni killed by
S. invicta successfully exited from their eggs and
reached the water before succumbing to the effects
of envenomization. In a natural setting, these in-
dividuals would have been considered to have
hatched successfully, thus under-estimating fire
ant-induced mortality by about 50%. Our labora-
tory research only documented direct mortality of
hatchlings. However, other research has shown se-
rious indirect effects on individual animals (Amer-
ican alligators, Alligator mississippiensis Daudin,
Allen et al. 1997; northern bobwhite, Colinus vir-
ginianus L., Giuliano et al. 1996) stung non-le-
thally by S. invicta. These effects included the loss
of digits and appendages, and reduced weight
gain, both likely to affect survival in the wild.
This work indicates that hatching turtles are
very vulnerable to predation by S. invicta and
that S. invicta is now a common component of the
ant community of sea turtle nesting beaches.
However, population-level impacts are unknown.
Moulis (1997) documented a 15% decrease in
hatchling release rate for sea turtles (C. caretta)
emerging from nests infested with fire ants as
compared to uninfested nests, but predation by
vertebrate predators (e.g., raccoons) can vary be-
tween 5 to 90% (Ratnaswamy et al. 1997).
Fire ant populations are increasing in terms of
both the spatial extent of infestation (Cokendol-


Region


No.
S. invicta


No.
other
ants


%
S. invicta


June 2001







Allen et al.: Fire ant impacts on turtles


pher & Phillips 1989; Callcott & Collins 1996)
and population densities of sites already infested
(Wojcik 1994). Due to the sensitivity of using sea
turtle hatchlings as experimental units, this re-
search did not document the extent of S. invicta
utilization of sea turtle nesting cavities or inter-
actions between sea turtles and fire ants in natu-
ral settings. However, our experiment with P.
nelsoni was conservative because hatchlings had
immediate access to water upon emergence,
whereas in the wild, hatchlings of many species
may spend several hours in the nest cavity before
emergence. We conclude that there is a very real
potential for negative impacts by S. invicta on
hatchling turtles.
Additional investigation of the effect of S. in-
victa on hatching sea turtles is vital because fire
ants occur throughout sea turtle nesting habitat
in the United States, they are increasing in abun-
dance, and they clearly have the potential to neg-
atively impact hatchlings of sea turtles, other
turtle species, and many other species in terres-
trial ecosystems. In the case of sea turtles, further
research is also needed to determine why certain
beaches possessed higher densities of S. invicta,
and what beach management activities (e.g.,
beach renourishment, degree of disturbance, hu-
man activity levels) may influence S. invicta in-
festation and spread.

ACKNOWLEDGMENTS
We wish to extend thanks to J. P. Hosford, M.
Lamont, and A. Quistorff for aiding in the collec-
tion of beach samples. We thank J. Sullenger and
B. Mayfield for collecting, processing and identify-
ing the samples from beaches. We thank T. Wilm-
ers for providing access to the Marquesas, and to
T. Hopkins for providing access to beaches and
housing during sampling in southwest Florida.
We thank A. Garmestani for providing access to
beaches in the Ten Thousand Islands. We wish to
thank R. Carthy, D. Epperson, and R. Moulis for
reviewing an earlier draft of this manuscript. Our
laboratory work with turtles was approved by the
University of Florida Animal Care and Use Com-
mittee. The South Carolina Cooperative Fish and
Wildlife Research Unit is jointly supported by a
cooperative agreement among the USGS/BRD,
the South Carolina Department of Natural Re-
sources, Clemson University, and the Wildlife
Management Institute.


REFERENCES CITED

ALLEN, C. R., K. G. RICE, D. P. WOJCIK, AND H. F. PER-
CIVAL. 1997. Effect of red imported fire ant envenom-
ization on neonatal American alligators. J. Herpetol.
31: 318-321.
CALLCOTT, A. A., AND H. L. COLLINS. 1996. Invasion and
range expansion of the imported fire ants (Hy-
menoptera: Formicidae) in North America from
1918-1995. Florida Entomol. 79: 240-251.
COKENDOLPHER, J. C., AND S. A. PHILLIPS, JR. 1989.
Rate of spread of the red imported fire ant, Solenop-
sis invicta (Hymenoptera: Formicidae) in Texas.
Southwest. Nat. 34: 443-449.
CONGDON, J. D., AND J. W. GIBBONS. 1990. The evolu-
tion of turtle life histories, pp. 45-54. In J. W. Gibbons
(ed.). Life History and Ecology of the Slider Turtle.
Smithsonian Institution Press, Washington, D.C.
GIULIANO, W. M., C. R. ALLEN, R. S. LUTZ, AND S. DEMA-
RAIS. 1996. Effects of imported fire ants on northern
bobwhite chick survival and body mass. J. Wildl.
Manage. 60: 309-313.
HEPPELL, S. S., L. B. CROWDER, AND D. T. CROUSE.
1996. Models to evaluate headstarting as a manage-
ment tool for long-lived turtles. Ecol. Appl. 6: 556-565.
JOHNSON, K. A. BJORNDAL, AND A. B. BOLTON. 1996. Ef-
fects of organized turtle watches on loggerhead
(Caretta caretta) nesting behavior and hatchling pro-
duction in Florida. Cons. Biol. 10: 570-577.
MOULIS, R. A. 1997. Predation by the imported fire ant
(Solenopsis invicta) on loggerhead sea turtle
(Caretta caretta) nests on Wassaw National Wildlife
Refuge, Georgia. Chelonian Cons. Biol. 2: 433-436.
PARRIS, L. N., M. M. LAMONT, AND R. R. CARTHY. 2001.
Observations of predation by red imported fire ants
(Solenopsis invicta) on hatching loggerhead sea tur-
tles (Caretta caretta). Herp. Rev. (in press).
RATNASWAMY, M. J., R. J. WARREN, M. T. KRAMER, AND
M. D. ADAM. 1997. Comparisons of lethal and nonle-
thal techniques to reduce raccoon depredation of sea
turtle nests. J. Wildl. Manage. 61: 368-376.
SORENSEN, A. A., T. M. BUSCH, AND S. B. VINSON. 1983.
Behavior of worker subcastes in the fire ant, Sole-
nopsis invicta, in response to proteinaceous food.
Physiol. Entomol. 8: 83-92.
WILMERS, T. J., E. S. WILMERS, M. MILLER, AND P.
WELLS. 1996. Imported fire ants (Solenopsis invicta):
a growing menace to sea turtle nests in Key West
National Wildlife Refuge, pp. 341-343. In J. A. Kein-
ath, D. E. Barnard, J. A. Musick, and B. A. Bell (eds.).
Proc. Fifteenth Annual Workshop on Sea Turtle Biol-
ogy and Conservation.
WOJCIK, D. P. 1994. Impact of the red imported fire ant
on native ant species in Florida, pp. 269-281. In D. F.
Williams (ed.). Exotic Ants: Biology, Impact, and
Control of Introduced Species. Westview Press, Boul-
der, CO.







Florida Entomologist 84(2)


June 2001


POTENTIAL FIRE ANT (HYMENOPTERA: FORMICIDAE) IMPACT ON THE
ENDANGERED SCHAUS SWALLOWTAIL (LEPIDOPTERA: PAPILIONIDAE)

ELIZABETH A. FORYS1', ANNA QUISTORFF2, AND CRAIG R. ALLEN3.
1Natural Sciences Collegium, Eckerd College, St. Petersburg, FL 33711

2South Carolina Cooperative Fish and Wildlife Research Unit, Clemson, SC 29634

3U.S. Geological Survey, South Carolina Cooperative Fish and Wildlife Research Unit
Clemson University, Clemson, SC 29634

ABSTRACT

The Schaus swallowtail, Papilio aristodemus ponceanus, historically occurred in tropical
hardwood hammocks from South Miami to the upper Florida Keys and is currently listed as
federally endangered. Much of the remaining hardwood hammock habitat is fragmented by
roads and human development that may alter the microhabitat within the hammocks and
increase the probability of invasion by non-native predators and competitors. One non-indig-
enous species that has recently invaded the Florida Keys, and that may impact the Schaus
swallowtail is the red imported fire ant (Solenopsis invicta Buren). We estimated abundance
of red imported fire ants in Schaus swallowtail habitat on Key Largo, and the decrease in red
imported fire ants resulting from the application of chemical ant baits. In addition, we con-
ducted laboratory experiments to determine how vulnerable swallowtail life stages are to
red imported fire ant predation. We found red imported fire ants at 50% of transects in the
hardwood hammock, up to 40 m from hammock edge. Chemical treatments were only par-
tially effective in decreasing red imported fire ant abundance, and the effect was short-lived.
All immature swallowtail life stages were vulnerable to predation by red imported fire ants.
Habitat restoration that decreases red imported fire ant abundance may be the most cost-
effective and long-term method of decreasing impacts from red imported fire ants.

Key Words: Florida, habitat loss, invasive species, non-indigenous species, Papilio aristode-
mus, Solenopsis invicta

RESUME

El Papilio aristodemus ponceanus ocurria historicamente en bosques tropicales de madera
dura ("tropical hardwood hammocks") desde el sur de Miami hasta en norte de los Cayos de
la Florida, y esta actualmente listado como bajo peligro por el gobierno federal. La mayoria
de habitat de este bosque tropical esta fragmentado por calls y desarrollo human que
puede alterar el microhabitat dentro de los bosques e incrementar la probabilidad de inva-
si6n por predadores y competidores ex6ticos. Una especie no indigena que recientemente ha
invadido los Cayos Floridanos, y que puede impactar a P. aristodemus es la hormiga brava
roja (Solenopsis invicta Buren). Estimamos la abundancia de S. invicta en habitats de P
aristodemus en Cayo Largo, y la reducci6n en S. invicta como resultado de la aplicaci6n de
trampas quimicas de hormigas. En adici6n, llevamos a cabo experiments de laboratorio
para determinar que tan vulnerable son las etapas de vida de P. aristodemus a predaci6n por
S. invicta. Encontramos S. invicta en un 50% de lotes en los bosques, hasta 40 m del borde
del bosque. Tratamientos quimicos fueron solo parcialmente efectivos para disminuir la
abundancia de S. invicta, y el efecto fue de corta duraci6n. Todas las etapas de vida de P. aris-
todemus fueron vulnerable a predaci6n por S. invicta. La restauraci6n del habitat que re-
ducen la abundancia de S. invicta puede ser el m6todo mas efectivo en costo y a largo plazo
para reducir los impacts de S. invicta.


The Schaus Swallowtail, Papilio (Heraclides) within the hardwood hammock, but will fly in
aristodemus ponceanus, is a large dark brown and clearings and along roads (Rutkowski 1971,
yellow butterfly that historically occurred in Brown 1976). Nectaring activity occurs on >30
hardwood hammocks from South Miami to the species of wild plants along the margins of the
upper keys of Florida (Emmel 1995). Hardwood hammock but rarely occurs in areas open to direct
hammocks are closed-canopy broad-leaved forests sunlight (Rutkowski 1971). Adult females lay
with a high diversity (>150 species) of both ever- eggs on a small number of host tree species that
green and semi-evergreen tropical tree species. occur primarily on the edges and in tree gaps of
Adult swallowtails spend most of their time hardwood hammocks in portions of Monroe and







Forys et al.: Fire ant impacts on swallowtails


Dade Counties, Florida, including torchwood,
Amyris elemifera (L.), and wild lime, Zanthoxy-
lum fagara (L.) Sarg., (Emmel 1986, 1995). Both
of these trees are relatively small and tend to pro-
duce suckers around the base. It appears that
leaves on these suckers may be the preferred ovi-
position locations for Schaus (Bagget 1982; Em-
mel 1995). Young torchwood and wild lime leaves
are the primary food of most Schaus caterpillars.
Like other butterflies, the Schaus is an important
pollinator of native plants, serves as a food source
for insectivorous species, and contributes to the
biological diversity of the Florida Keys.
The Schaus swallowtail was listed as Federally
endangered in 1984 because of population de-
clines caused by the destruction of its tropical
hardwood hammock habitat, mosquito control
practices, and over-harvesting by collectors. Rein-
troductions occurred between 1995 and 1997 (U.S.
Fish and Wildlife Service 1999) and in 1998 the
Schaus swallowtail was documented on 13 areas
on the mainland and the Upper and Middle Keys.
Currently, efforts are underway to protect the
remaining hardwood hammocks in south Florida
from commercial and residential development as
well as from pesticide spraying (U.S. Fish and
Wildlife Service 1999). Even if these measures
are successful, much of the existing habitat is al-
ready fragmented by roads and human develop-
ment that may alter the microhabitat within the
hammocks (Saunders et al. 1991) and increase
the probability of invasion by non-native preda-
tors and competitors (Usher 1988).
An invasive non-native species that may have
a negative impact on the Schaus swallowtail is
the red imported fire ant (Solenopsis invicta Bu-
ren). Red imported fire ants were first recorded in
the upper Florida Keys in 1976 (Callcott & Col-
lins 1996), but were considered to be restricted to
disturbed areas (Deyrup et al. 1988; Porter 1992).
During a Keys-wide ant survey in 1996, S. invicta
was identified on 10 of the 14 major keys (Forys et
al. 1999). Solenopsis invicta was found in every
major habitat type including hardwood ham-
mocks, pinelands, salt and freshwater marshes,
and disturbed areas (e.g., roadsides, parking lots)
(Forys et al. 1999). While little research has been
conducted on red imported fire ant predation on
Lepidoptera, the presence of S. invicta in the trop-
ical hardwood hammocks is of particular concern
for the Schaus swallowtail because red imported
fire ants are known to prey on a wide range of
other invertebrates (Porter & Savignano 1990).
Schaus swallowtail eggs, larvae, and pupae may
be vulnerable because they occur on tree species
(e.g., torchwood, wild lime) that generally occupy
habitat edge where red imported fire ant infesta-
tions tend to be the highest.
The objective of this study was to 1) measure
the abundance of red imported fire ants in Schaus
swallowtail habitat, 2) determine if swallowtail


eggs, larvae and pupae are readily discovered and
consumed by red imported fire ants, and 3) ex-
plore the effectiveness of fire ant control in areas
important to Schaus swallowtail reproduction.

MATERIALS AND METHODS

Red Imported Fire Ant Foraging

To determine fire ant abundance and ability to
forage into closed canopy tropical hardwood ham-
mock, we established bait transects on North Key
Largo, Florida. North Key Largo is a long and
narrow (<1 km) island at the northernmost por-
tion of the Florida Keys. There are several resi-
dential developments, but most of North Key
Largo is federally and state owned. In the center
of the key, a well-traveled highway (SR 905) bi-
sects the hardwood hammock. Recently, Schaus
swallowtails have been successfully reintroduced
in this area (U.S. Fish and Wildlife Service 1999).
Twenty transects were placed perpendicular to
SR 905 into the Key Largo hammock. Ten of the
transects were on the federally-owned north side
of SR 905 and the other 10 were directly across
the road on the state-owned south side of SR 905.
Habitat on both sides of SR 905 was similar.
Transects were separated by 100 m and consisted
of 10 sampling stations spaced 5 m apart begin-
ning at known fire ant infested areas (the road-
side) and continuing perpendicular into the intact
hardwood hammock.
At each of the 10 sampling stations along all 20
transects, we placed two terrestrial and two arbo-
real baits, with members of each pair separated
by >1 m. The paired terrestrial baits (one honey,
one hamburger meat) were placed directly on the
ground on pieces of aluminum foil. The paired
arboreal baits were placed in plastic condiment
cups with 5-10, 3-6 mm holes punched throughout
the cup. Both arboreal cups were placed 1-1.5 m
from the ground in a tree as close to the terres-
trial bait as was possible.

Predation on Swallowtails

To determine the attractiveness and vulnera-
bility of Schaus swallowtails to red imported fire
ants we conducted an experiment using eggs, lar-
vae, and pupae of the giant swallowtail (P. cre-
sphontes) Cramer as a surrogate species. The
giant swallowtail is a common Key's resident that
is similar to the Schaus swallowtail in its distri-
bution and natural history. Both species of swal-
lowtails occur in the Florida Keys, utilize species
of Rutaceae as host plants, and lay eggs singly on
leaves. The larvae of both eat new leaves, do not
use nests, pupae of both hibernate and are similar
in structure and appearance (Scott 1986). The gi-
ant swallowtail reproduces more frequently (has
multiple broods in one year) and adults can be







Florida Entomologist 84(2)


found throughout the year, while the Schaus
swallowtail has only one set of brood a year and
adults are found mid March through mid Septem-
ber (Emmel 1995).
Ten eggs, larvae (third and fourth instar), and
pupae were purchased from a commercial butter-
fly farm (Robert Brown, Butterfly Paradise, 19940
Adams Rd., Ft. Myers, FL). Each egg, larva, and
pupa was individually attached 1 m off the ground
on a wild lime tree placed in enclosures with ac-
tive red imported fire ant colony. For comparison
we also placed 10 balls of hamburger meat of
equal size to the larvae and pupae one meter off
the ground on a wild lime tree in an additional 10
enclosures with an active red imported fire ant
colonies. The eggs had been laid on wild lime
leaves and we attached these leaves to the wild
lime tree using gardeners wire. The pupae were
placed in small porous cups and were attached to
the wild lime tree. The larvae were directly placed
on leaves. The enclosures were monitored for
three hours and both the length of time for the fire
ants to discover (make first contact) and entirely
consume the hamburger balls and swallowtail
eggs, larvae, and pupae were recorded. A t-test
was used to compare the time to discovery for each
swallowtail life stage and the hamburger meat.
No statistical comparisons were made between
time to consumption for the life stages and ham-
burger meat because the life stage differed in
shape and consistency from the meat.

Effectiveness of Fire Ant Control

Five, 50-m sections of the shoulder of the main
road that bisects the Key Largo hammock
(SR905) were treated in July, 1997, using a fire
ant bait, Amdro, broadcast from the back of a
four-wheeler and leaving 5, 50-m untreated areas
as controls. Areas >100 m were left between treat-
ment and control areas. The active ingredient in
Amdro is hydramethylnon, a metabolic inhibi-
tor that usually kills queens, workers and the col-
ony within 2-4 weeks (Williams 1994).
To measure the effectiveness of this treatment,
red imported fire ant abundance was monitored


by placing hamburger and honey baits every 5 m
along 50-m transects extending into the ham-
mock at the midpoint of each treatment and con-
trol areas before and after treatment. These
transects were surveyed twice before treatment
in March and July 1997, and three times after
treatment in October and December 1997 and
March 1998. We compared the number of red im-
ported fire ants collected at each hammock
transect in the treated and untreated areas for
each survey using a Mann-Whitney U Test be-
cause the data was not normally distributed and/
or variances were not equal.

RESULTS

Red Imported Fire Ant Foraging

In the North Key Largo hammock, red im-
ported fire ants were identified on 8 of the 10
transects from the south side of SR905 and 2 of
the 10 transects from the north side of SR905.
Most of the baits with red imported fire ants were
near the road. The maximum foraging distance
into the hammock was 40 m. Red imported fire
ants were detected arboreally at 3 transects on
the south side of the road, up to a distance of 25 m
into the hammock.

Predation on Swallowtails

Red imported fire ants predated all of the im-
mature swallowtail life stages. Fire ants discov-
ered the butterfly stages faster than the
hamburger meat although this difference was sig-
nificant only for the larval life stage (t = 4.66, d.f.
= 18, P < 0.0001) (Table 1). The larval stage was
the first to be discovered by fire ants and was con-
sumed the fastest (Table 1). However, three larvae
escaped predation during the three h experiment
by moving to higher branches of the wild lime tree
after being initially detected by a fire ant. All of
the pupae and eggs were discovered and con-
sumed by fire ants. It took ants the longest to con-
sume the pupal life stage because the fire ants
had to first breach the hard exterior of the pupa.


TABLE 1. AVERAGE LENGTH OF TIME IN MINUTES, WITH STANDARD DEVIATIONS IN PARENTHESES, FOR RED IMPORTED
FIRE ANTS TO DISCOVER AND COMPLETELY CONSUME DIFFERENT LIFE STAGES OF THE GIANT SWALLOWTAIL
COMPARED TO RAW MEAT.

Percent
Life stage (or meat) Time to discovery Time to full consumption escaping discovery

Eggs 30.8 (37.2) 18.2 (4.8) 0
Larvae 6.7* (5.4) 10.3 (1.9) 30
Pupae 46.2 (22.5) 93.8 (28.6) 0
hamburger meat 48.3 (27.7) 32.2 (5.6) 10

*P < 0.05.


June 2001







Forys et al.: Fire ant impacts on swallowtails


Effectiveness of Fire Ant Control

Before treating with Amdro, the average num-
ber of red imported fire ants in the proposed treat-
ment areas and untreated areas did not differ
significantly (March 1997: T = 92.5, P = 0.36, n1 =
10, n2 = 10; July 1997: T = 101.0, P = 0.78, nI = 10,
n2 = 10). In October, 1997, three months after the
July treatment, there were no red imported fire
ants at any of the bait transects in the treated area
and this was significantly fewer than in the un-
treated areas (T = 75.0, P = 0.03, nI = 10, n2 = 10).
However, five and eight months after the treat-
ment, the number of red imported fire ants was not
significantly lower in the treated areas (December
1997: T = 92.0, P = 0.34, n, = 10, n2 = 10; March
1998: T = 109.5, P = 0.76, n1 = 10, n2 = 10). Overall,
the number of red imported fire ants declined in
both treated and untreated areas after the July
1997 treatment, probably due to the unusually dry
conditions during that time period (Fig. 1).

DISCUSSION

The lab experiment we conducted provides ev-
idence of the potential for red imported fire ant
predation on the Giant swallowtail, and suggests
that other species of butterflies that occur in the
southeastern United States also may be vulnera-
ble, including Schaus swallowtail. Fire ants pre-
dated on all terrestrial life history stages of the
swallowtail, although the mobility of larvae af-


80


3 60
o 40

3i 40


f Treated
Untreated


20-


0
397


7-
-7- -- 0 -
7/97 10/97 12/97


3/98


Survey
Fig. 1. The average number of red imported fire ants
(Solenopsis invicta) on baits at the 10 transects treated
with Amdro to kill fire ants and the 10 left untreated.
The July 1997 sampling date occurred immediately
before the Amdro treatment. An "*" indicates that the
average number of ants significantly differed (p < 0.05)
between the treated and untreated transects.


forded some protection. The high rate of preda-
tion was surprising due to the suite of anti-
predator behaviors that swallowtails exhibit such
as laying one egg per leaf, secretive behavior of
larvae, and the production of foul-smelling scents
from the osmeteria when larvae are disturbed
(Rutkowski 1971). The ability of the red imported
fire ant to penetrate the pupae was particularly
disturbing. While these results were found in a
laboratory experiment on a related species, it is
probable that red imported fire ants impact
Schaus swallowtails in nature. A manipulative
field experiment with red imported fire ant popu-
lation reductions and careful monitoring of
Schaus swallowtail life stages and populations
would provide definitive evidence.
Recently, Schaus swallowtail reintroductions
occurred in state and federally owned hammocks
on northern Key Largo (Emmel 1995; U.S. Fish
and Wildlife Service 1999). We found that red im-
ported fire ants were abundant on both the edges
of the north Key Largo hammock and up to 40m
into the interior. Fire ants were more abundant
terrestrially than arboreally, but fire ants did for-
age arboreally. Because of the abundance of red
imported fire ants and the results of the predation
experiment, it is possible that fire ants are a
threat to the long-term success of Schaus swal-
lowtail reintroductions.
To further increase the chance of survival of
the reintroduced Schaus swallowtail populations,
red imported fire ant populations should be re-
duced either through use of fire ant baits or
through habitat restoration. However, treatment
of the road shoulders with Amdro in North Key
Largo was not as successful as similar treatments
elsewhere (Allen et al. 1995). Red imported fire
ants were significantly reduced for only three
months in the treated area (Fig. 1). While most of
the red imported fire ant mounds were on the
road shoulder, there may have been mounds
within the hardwood hammock that were not af-
fected by treatments which helped to recolonize
the treated areas. Some of these colonies may
have occurred on old paved roads that remain in
the Key Largo hammock. These roads are aban-
doned and mostly overgrown with vegetation, but
they may still serve as favorable fire ant habitat.
Even mowed paths <2 m in width inside the
hammock may increase red imported fire ant den-
sities. The south side of the Key Largo hammock
consistently had more red imported fire ants than
the north. A utility path that runs between power
poles on the south side of SR905 may account for
the differences seen in the abundance of red im-
ported fire ants on the south and north sides of
the road.
Previous studies in this area have indicated
that roads bisecting the hammock are especially
attractive colonization sites for fire ants (Forys et
al. 1999). The removal and restoration of aban-


7
T .. . . .


!







Florida Entomologist 84(2)


doned roads and access paths, and limiting dis-
turbance of road shoulders, will probably lower
fire ant populations in the area. Reducing the
abundance of red imported fire ants in the Key
Largo hammock would be beneficial to the
Schaus, as well as a suite of other rare inverte-
brate and vertebrate species that may be suscep-
tible to predation (e.g., Key Largo cotton mouse,
Peromyscus gossypinus; Key Largo woodrat,
Neotoma floridana small; Florida tree snails,
Liguus fasciatus).

ACKNOWLEDGMENTS

This project was funded by the Florida Game
and Fresh Water Fish Commission's Nongame
Wildlife Program (NG95-018). B. Stieglitz and R.
Skinner helped us by providing access and timely
permits to refuge and state owned land. P. Frank
and S. Klett provided input on study design. J.
Sullenger and B. Mayfield sorted and identified
all of the ants. D. P. Wojcik provided ultimate ant
identification and helped in the field. H. Collins
and associates helped design the bait treatment.
J. Hosford and M. Pratt assisted with data collec-
tion and entry. An earlier version of this paper
was improved by incorporating comments from D.
Cook, J. Isely, and J. Zettler. The South Carolina
Cooperative Fish and Wildlife Research Unit is
jointly supported by a cooperative agreement
among the USGS/BRD, the South Carolina De-
partment of Natural Resources, Clemson Univer-
sity, and the Wildlife Management Institute.

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June 2001







Mao et al: Sweeptotato weevil host plant resistance


EFFECT OF SWEETPOTATO GENOTYPE, STORAGE TIME
AND PRODUCTION SITE ON FEEDING AND OVIPOSITION BEHAVIOR
OF THE SWEETPOTATO WEEVIL, CYLAS FORMICARIUS
(COLEOPTERA: APOINIDAE)

LIXIN MAO, RICHARD N. STORY, ABNER M. HAMMOND AND DON R. LABONTE
Dept. of Entomology and Dept. of Horticulture, Louisiana State University, Agricultural Center
Baton Rouge, LA 70803

1Current address: 1100 Robert E. Lee Blvd. New Orleans, LA 70124

ABSTRACT
The effect of sweetpotato genotype, storage time and production site on Cylas formicarius
(Fab.) feeding and oviposition was investigated. Sweetpotato genotype had a significant ef-
fect on feeding and oviposition rates in both no-choice and choice arenas. Beauregard and
Centennial were uniformly susceptible across all age groups. W-250 had the least number of
feeding punctures and eggs at 7 and 25 days after harvest. At 85 days after harvest, W-244
had the least number of feeding punctures and eggs, while W-250 was not significantly dif-
ferent from Beauregard and Centennial. Roots of the same genotype grown in different lo-
cations differed in the number of feeding punctures and eggs. These results suggest that
antixenosis is responsible for at least part of the sweetpotato weevil resistance. Storage time
and production sites appeared to affect the expression of the resistance, but the outcomes de-
pended on the genotypes.

Key Words: host plant resistance, antixenosis, storage time, production site

RESUME

Fue investigado el efecto de genotipo del camote, la duraci6n de almacenamiento y el lugar
de producci6n sobre la alimentaci6n y oviposici6n de Cylas formicarius (Fab.). El genotipo del
camote tuvo un efecto significativo sobre la alimentaci6n y velocidad de oviposici6n dadas las
opciones de raiz y no raiz. Beauregard y Centennial fueron igualmente susceptibles entire los
genotipos evaluados. W-250 tuvo un menor numero de agujeros y huevos a 7 y 25 dias des-
pu6s del cosechado. A 85 dias despu6s de cosechado, W-244 tuvo menos agujeros y huevos,
mientras que W-250 no tuvo diferencias significativas con Beauregard y Centennial. Races
del mismo genotipo sembradas en diferentes localidades tuvieron diferente numero de agu-
jeros y huevos. Los resultados sugieren que antixenosis es responsible al menos en parte por
la resistencia del picudo del camote. La resistencia al picudo del camote parece ser afectada
por el tiempo de almacenaje y las localidades de producci6n, pero los resultados finales de-
penden mas en los genotipos.


Sweetpotato weevil (SPW), Cylas formicarius
(Fab.), is a major constraint to sweetpotato [Ipo-
moea batatas (L.) Lam.] production worldwide
(Chalfant et al. 1990; Jansson & Raman 1991). It
attacks sweetpotato both in the field and during
storage. Adults make feeding and oviposition
punctures on the root surface that can reduce root
quality and market value. Larval tunneling in
roots induces terpenoid production that renders
even slightly damaged roots unfit for human and
animal consumption (Cockerham et al. 1954; Uri-
tani et al. 1975). Due to the concealed nature of
the feeding habit, control of SPW is difficult. The
use of resistant sweetpotato cultivars is a poten-
tially viable option that could be an economical
component in the integrated management of SPW
(Martin & Jones 1986; Collins et al. 1991).
Many studies have been conducted on SPW re-
sistance in sweetpotato indicating variable resis-


tance in the field (Rolston et al. 1979; Mullen et
al. 1980b, 1981, 1982, 1985; Taleker 1987b) and
laboratory (Mullen et al. 1980a; Barlow & Rolston
1981; Nottingham et al. 1987, 1989; Ratnayake
1995; Story et al. 1996, 1999a, b, c). However, lit-
tle success has been realized in the development
of resistant cultivars, partly because of inconsis-
tencies in the performance of selected breeding
lines (Talekar 1987a, b) and a lack of understand-
ing of the resistance mechanisms.
The expression of insect resistance can be in-
fluenced by many environmental factors (Smith
1989). Identification of these factors would help to
explain the inconsistent performance of resistant
genotypes. Such information would also be useful
in facilitating the development of resistant culti-
vars and understanding the underlying mecha-
nisms of resistance. In temperate growing areas
like the United States, storage roots are cured by







Florida Entomologist 84(2)


keeping them in a specially designed facility
maintained at about 30C and 85% to 90% RH for
4 to 7 days. Cured roots are often evaluated over a
period of several months in sweetpotato breeding
programs. Thus curing and storage time may af-
fect the outcome of SPW resistance evaluations
because physical and chemical changes occur in
the roots (Bouwkamp 1985). In addition, sweetpo-
tato is grown throughout a wide geographic
range. Wide variations in SPW resistance be-
tween production sites have been observed in field
plots (Talekar 1987b). However, until this study,
no comparative study has been done under con-
trolled laboratory conditions to determine the ef-
fect of storage and production site on SPW feeding
and oviposition. We evaluated 4 sweetpotato gen-
otypes ("Beauregard", "Centennial", "W-244", and
"W-250") to determine the effects of storage and
production site on SPW feeding and oviposition.

MATERIALS AND METHODS

Insect rearing

A SPW colony was established from a field col-
lected population (about 500 insects) and main-
tained in the laboratory on storage roots of
Beauregard in plastic containers (5.6 L) with
screen covers at 28 + 2C and 85 10% RH. In
preparing experimental insects, 5 fresh storage
roots (US #1) were exposed to about 1000 adults
(male and female) for 5 days, then were removed
and kept under the conditions described above.
Emerging adults (male and female) were col-
lected weekly and held with fresh storage roots.
Female adults 3-4 weeks old were used in the bio-
assays to ensure adequate egg-laying capability
(Wilson et al. 1988).

Bioassay

The assay technique was an adaptation of one
previously described by Mullen et al. (1980a) and
has been used in several SPW feeding and oviposi-
tion studies (Nottingham et al. 1987; Wilson et al.
1988). It consisted of a 24-well tissue culture plate
(12.5 x 8.5 x 2.0 cm; Falcon@) placed in a rectangu-
lar clear plastic container (17 x 12 x 6 cm). Cores
were cut from selected roots with a cork borer (1.6
cm diameter) and inserted into the wells so that
only the surface of the root periderm was exposed.
The cores had the same diameter as the wells, pro-
viding a close fit. Female adults were kept without
food for 3 hours before being introduced into the
arena at the rate of 2 weevils per root core. A moist
cotton ball was placed in the container to maintain
90-100% RH and prevent desiccation of the root
cores. After 24 hours the number of feeding punc-
tures on each core was recorded, and after 48
hours the number of eggs was counted. All tests
were conducted at 28 5C, 85 10% RH under


total darkness to eliminate light as a variable.
Cores from only one genotype were presented to
the weevils in no-choice tests. In choice tests, one
core from each genotype was randomly arranged
on the plate and presented to the insects.
Four sweetpotato genotypes were chosen ac-
cording to their performance in no-choice whole-
root laboratory evaluations (Story et al. 1996).
W-244 and W-250 were breeding lines shown to be
resistant to SPW. Beauregard and Centennial
were two susceptible cultivars. To determine the
effect of curing and storage time, bioassays were
conducted with roots of the following groups: non-
cured 7 days after harvest (DAH), cured 25 DAH,
and cured 85 DAH. Storage roots were produced
using standard practices at Burden Research
Plantation, Baton Rouge, Louisiana. Slips were
planted on July 5, 1996 with 0.3 m spacing in 20-
plant plots with rows separated by 1.2 m. Storage
roots were harvested on November 1, 1996, cured
(30C, 90% RH for 7 days), and stored at 15 + 2C.
At each test date, both no-choice and choice tests
were conducted with complete randomized exper-
imental designs and 8 replications (8 US#1 roots
for each genotype).
Three sweetpotato growing regions were cho-
sen to evaluate the effect of production site on the
expression of SPW resistance. They were Baton
Rouge, Louisiana (LA), Edisto, South Carolina
(SC), and Pontotoc, Mississippi (MS). Storage
roots were produced at each site using similar
production practices. Non-cured 7 DAH roots of
all 4 genotypes from LA and MS and cured 25
DAH roots of 3 genotypes (Beauregard, Centen-
nial and W-250) from LA and SC were used. No-
choice tests were conducted with 8 replications.

Data Analysis

All data (average number of feeding punctures
or number of eggs per root core) were analyzed
with the PC SAS General Linear Model (GLM) pro-
cedure (SAS Version 6.12 1990), followed by Tukey
multiple range tests for mean separations. The ef-
fect of storage time was tested as a fixed block ef-
fect by pooling data from all 3 age groups. Curing
effect was tested using a contrast statement. Pro-
duction site effect was analyzed as a fixed block ef-
fect in a randomized complete block design. In all
tests the significance level was a = 0.05.

RESULTS

Genotype, Curing and Storage Time Effects

Significant differences in both choice and no-
choice tests in feeding and oviposition were found
among the 4 genotypes. In both no-choice and
choice tests, W-250 had the lowest number of feed-
ing punctures and eggs at 7 and 25 DAH (Table 1).
At 85 DAH, W-244 had the least numbers of feed-


June 2001







Mao et al: Sweeptotato weevil host plant resistance


TABLE 1. EFFECT OF GENOTYPE AND STORAGE TIME (DAH = DAYS AFTER HARVEST) ON THE NUMBER OF FEEDING PUNC-
TURES AND THE NUMBER OF EGGS OF SWEETPOTATO WEEVIL UNDER NO-CHOICE AND CHOICE TEST CONDITIONS.

No-choice test Choice test

Genotype Feeding puncture' Eggs' Feeding puncture' Eggs'

Non-cured 7 DAH roots
Beauregard 17.8 b 9.8 b 15.8 b 7.6 a
Centennial 23.1 a 12.4 a 21.8 a 8.9 a
W-250 9.0 c 5.7 c 3.4 d 2.0 c
W-244 20.9 ab 9.0 b 9.7 c 3.9 b
Cured 25 DAH roots
Beauregard 19.2 a 7.7 a 14.5 a 5.5 a
Centennial 20.4 a 7.6 a 18.3 a 6.6 a
W-250 11.9 b 4.0 c 4.7 b 1.8 b
W-244 17.4 a 6.2 b 13.5 a 6.3 a
Cured 85 DAH roots
Beauregard 23.3 a 10.5 a 20.1 a 7.0 a
Centennial 24.5 a 12.8 a 18.9 a 7.1 a
W-250 19.6 a 11.1 a 13.6 a 5.6 a
W-244 10.0 b 5.6 b 4.5 b 2.7 b

'Means followed by the same letter within a column of each storage time category are not significantly different (p > 0.05, Tukey).


ing punctures and eggs while W-250 was not sig-
nificantly different from Beauregard and
Centennial (Table 1). The curing process did not
have a significant effect on feeding and oviposi-
tion in both choice and no-choice tests. Storage
time had a significant effect on the number of
eggs deposited in no-choice tests (F = 61.52, df =
2,84, P = .0001), but not in choice tests. Storage
time did not have an effect on feeding punctures.
Cultivar and storage time interaction effect was
significant in all cases. W-250 had some resis-
tance relative to the susceptible cultivars when
the roots were non-cured 7 DAH and cured 25
DAH, but the resistance factors were diminished
in cured 85 DAH roots (Table 1). The opposite
trend was found with W-244, in which significant
differences in the number of punctures and eggs
were detected only with cured 85 DAH roots when
they were compared with the susceptible culti-


vars. Beauregard and Centennial were uniformly
susceptible across the three root age groups.

Production Site Effects

Non-Cured Roots. A significant production site
effect was found for the number of feeding punc-
tures (F = 5.72, df = 1,56, P = 0.0202) on the non-
cured roots from Louisiana and Mississippi where
Mississippi roots received higher number of feed-
ing punctures than Louisiana roots (Table 2).
However, the number of eggs deposited was not
significantly different (F = 0.05, df = 1,56, P =
0.8915). The interaction effect of genotype and
production site was highly significant for both
feeding and oviposition (F = 4.63, df = 3,56, P =
0.0058, F = 6.33, df = 3,56, P = 0.0009, respec-
tively), indicating that the feeding and oviposi-
tion rates among the 4 genotypes were different


TABLE 2. EFFECT OF GENOTYPE AND PRODUCTION SITE (LOUISIANA, MISSISSIPPI) ON THE NUMBER OF FEEDING PUNC-
TURES AND EGGS OF SWEETPOTATO WEEVIL ON NON-CURED ROOTS UNDER NO-CHOICE TEST CONDITIONS.1

Feeding puncture2 Eggs2

Genotype Louisiana Mississippi Louisiana Mississippi

Beauregard 15.8 b 24.3 a 7.6 a 8.5 a
Centennial 21.8 a 16.9 b 8.9 a 6.0 b
W-250 3.4 d 9.5 c 2.0 c 4.4 b
W-244 9.7 c 11.2 c 3.9 b 3.9 b

The tests were conducted using non-cured roots 7 days after harvest.
Means followed by the same letter within production site are not significantly different (p > 0.05, Tukey).







Florida Entomologist 84(2)


between these two sites (Table 2). For Louisiana
grown roots, all 4 genotypes were significantly
different from each other in number of feeding
punctures, while no significant difference was
found between Beauregard and Centennial in the
number of eggs laid. Centennial had the most
feeding punctures and eggs. For Mississippi
grown roots, significant differences were found
between the two susceptible cultivars in both
number of feeding punctures and eggs. Signifi-
cant differences were not found between W-244
and W-250 but they were different from suscepti-
ble cultivars. Beauregard was the preferred geno-
type for both feeding and oviposition.
Cured Roots. A significant production site ef-
fect was found between the number of eggs depos-
ited (F = 4.38, df = 1,42, P = 0.0424) in Louisiana
and South Carolina cured roots where South
Carolina roots had higher number of eggs (Table
3). The number of feeding punctures was not sig-
nificantly affected by production site (F = 1.90, df
= 1,42, P = 0.1723). Although no statistically sig-
nificant production site and genotype interaction
effects were found, there was a trend toward dif-
ferent performance of W-250 from these two loca-
tions. Roots grown in Louisiana had significantly
fewer punctures and eggs for W-250 when com-
pared to Beauregard and Centennial. However,
no differences were detected among the 3 geno-
types grown in South Carolina.

DISCUSSION

Plant resistance to insects may be due to anti-
biosis, antixenosis (nonpreference), tolerance, or
escape. All these types have been reported in
sweetpotato resistance to SPW (Waddill &
Conover 1978; Barlow & Rolston 1981; Mullen et
al. 1981; Talekar 1987b; Ratnayake 1995). This
study evaluated antixenosis effects (plants lack
the characteristics that attract insects and are
avoided by insects) on feeding and oviposition by
female adult of SPW. We found that Beauregard
and Centennial were preferred by SPW with re-
spect to both feeding and oviposition. These re-
sults are consistent with previous reports that
have shown the susceptibility of Beauregard (Rat-


nayake 1995; Story et al. 1996) and Centennial
(Mullen et al. 1980b; Nottingham et al. 1989;
Rolston et al. 1979) in both field and laboratory
tests. W-244 and W-250 are two breeding lines
with resistance to SPW (Ratnayake 1995; Story et
al. 1999a). The lower numbers of feeding punc-
tures and eggs on the roots of these two lines sug-
gests that antixenosis was responsible for at least
part of SPW resistance and that the resistant fac-
tor(s) may have a broad spectrum. Talekar (1987a)
argued against the feasibility of nonpreference in
sweetpotato. He pointed out that it had little value
because weevils lack choices among sweetpotato
genotypes in commercial plantings. We found that
SPW exhibited feeding and oviposition differences
among sweetpotato genotypes under no-choice
conditions, suggesting the possibility of utilizing
antixenosis in SPW management.
No-choice and choice are the two experimental
settings for evaluating plant resistance to insects.
Sometimes, results from these two kinds of tests
appear to be contradictory. Resistant genotypes
identified under choice conditions can receive
more feeding damage than susceptible genotypes
when insects are forced to feed on only one geno-
type (Tingey 1986). Usually, under choice condi-
tions, susceptible plants receive higher levels of
damage than resistant plants when compared to
the results of no-choice conditions. This results in
a larger variance among genotypes under the
choice conditions. We found no significant differ-
ences in number of feeding punctures (F,= 1.2513,
df, = df, = 95, P = 0.13818) and eggs (F = 1.2781,
df, = df, = 95, P = 0.11679) between choice and no-
choice tests when testing for equal variance as de-
scribed by Sokal and Rohlf (1981).
Curing and storage are common postharvest
procedures for sweetpotato in temperate growing
areas. During these processes many physical and
chemical changes may occur in the roots. For ex-
ample, curing promotes wound periderm forma-
tion on injured surfaces, thus reducing decay and
water loss (Bouwkamp 1985). Storage has been
reported to induce changes in carbohydrate com-
position, enzyme activities and cell wall compo-
nents (Takahata et al. 1995; Walter & Palma
1996). Our study shows that curing had no effect


TABLE 3. EFFECT OF GENOTYPE AND PRODUCTION SITE (LOUISIANA, SOUTH CAROLINA) ON THE NUMBER OF FEEDING
PUNCTURES AND EGGS OF SWEETPOTATO WEEVIL ON CURED ROOTS UNDER NO-CHOICE TEST CONDITIONS.1

Feeding puncture2 Eggs2

Genotype Louisiana South Carolina Louisiana South Carolina

Beauregard 14.5 a 15.5 a 5.5 a 6.1 a
Centennial 16.3 a 14.2 a 6.6 a 6.8 a
W-250 4.7 c 13.1 a 1.8 b 5.7 a

The tests were conducted using cured roots 25 days after harvest.
Means followed by the same letter within production site are not significantly different (p > 0.05, Tukey).


June 2001







Mao et al: Sweeptotato weevil host plant resistance


on SPW feeding and oviposition behaviors. How-
ever, as storage time lengthened, SPW feeding
and oviposition rates changed among genotypes.
This suggests that storage time may influence the
expression of SPW resistance, but the effect dif-
fers with each genotype.
The importance of environmental factors in
the expression of SPW resistance has been noted
by Talekar (1987 b). Our study also had some sig-
nificant production site effects and the interac-
tions of genotype and production site on SPW
feeding and oviposition. Previous studies have re-
lated SPW resistance to the presence and concen-
tration of a pentacyclic triterpene, boehmeryl
acetate, in the periderm tissues of sweetpotato
roots. This chemical has been identified as a SPW
oviposition stimulant (Son 1989; Wilson et al.
1989). Our study suggests the possibility of the
presence of deterrent(s) or repellent(s) in the re-
sistant genotypes or a reduction of boehmeryl ac-
etate. Environmental factors very likely influence
such phytochemicals and hence alter the level of
resistance.
In conclusion, SPW exhibited different feeding
and oviposition preferences among sweetpotato
genotypes. Curing, storage time, and production
site influenced SPW feeding and oviposition be-
havior. When screening for SPW resistance, all
conditions associated with testing materials
(storage roots) and environmental conditions
should be kept as consistent as possible. Poten-
tially resistant lines should be evaluated under
multiple sets of environmental conditions over a
period of several years.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Paul Thompson
of the Pontotoc Research and Extension Center,
Mississippi State University, and Dr. Janice Bo-
hac of the U.S. Vegetable Laboratory, Charleston,
South Carolina for providing storage roots for
testing. Jeff Murray is thanked for his assistance
in conducting these studies. Approved for publica-
tion by the Director of the Louisiana Agricultural
Experiment Station as manuscript number 00-
17-0223.

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