Evaluation of Population Suppression by Irradiated Lepidoptera and Their Progeny

Bloem & Carpenter: Population suppression by irradiated Lepidoptera 165

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- in the ﬁeld. Nevertheless, two SIT programs are portant pests of major annual and perennial currently operating against pest Lepidoptera, crops, forests, and stored products throughout the namely the pink bollworm, Pectinophora gossyp- world. More than 25% of the species that appear iella (Saunders), program in the USA (Staten et on a list of the 300 most important exotic insects al. 1993), and the codling moth program in Can- that threaten the United States are in the order ada (Dyck et al. 1993; Bloem & Bloem 2000), and Lepidoptera (ESA 2001). In a supplement to that both of these programs have been very successful. list, where the 30 most serious threats to Agricul- One approach to reduce the negative effects of ture are named, 50% of the species are lepidopter- radio-resistance in Lepidoptera has been the use

ans (ESA 2001). Unfortunately, control of lepi- of inherited or F1 sterility. Proverbs & Newton

dopteran pests worldwide is achieved almost en- (1962) ﬁrst documented F1 sterility during the tirely through the use of synthetic insecticides. course of their studies on the codling moth. Sub-

This dependence on insecticides has contributed sequently, investigators have reported F1 sterility to the development of insecticide resistance in in many lepidopteran species of economic impor-

many of the most serious pests. Relevant exam- tance (LaChance 1985). Like SIT, F1 sterility in- ples include the codling moth, Cydia pomonella volves the mass rearing and release of genetically (Lepidoptera: Tortricidae) (Varela et al. 1993) and altered insects to insure that when matings occur the diamondback moth, Plutella xylostella (Lepi- in the field, a significant proportion of matings in- doptera: Plutellidae) (Shelton et al. 1993), where volve a treated, released insect. However, F1 ste- resistance has developed even against the micro- rility takes advantage of two unique genetic bial insecticide Bacillus thuringiensis (Tabash- phenomena in Lepidoptera. First, lepidopteran nick et al. 1990). Heavy reliance and frequent females generally are much more sensitive to ra- indiscriminant use of pesticides also has had a diation than are males of the same species. This significant negative impact on the environment. may allow the dose of radiation to be adjusted so Of particular importance to agriculture is the de- that treated females are completely sterile and struction of crop pollinators and natural enemies males are partially sterile. Second, when these that keep secondary pests in check (Edwards partially sterile males mate with fertile females 2000). Development of alternative tactics to the the radiation-induced deleterious effects are in- unilateral use of insecticides is a major emphasis herited by the F1 generation. As a result, egg of most local, national and international research hatch is reduced and the resulting (F1) offspring organizations concerned with pest control. are both highly sterile and predominately male.

The lower dose of radiation used in F1 sterility in- The Sterile Insect Technique and Lepidoptera creases the quality and competitiveness of the released insects (North 1975). In addition, because

Genetic pest suppression is unique among bio- F1 sterile progeny are produced in the field, the re- logical methods in that it involves the release of lease of partially sterile insects offers greater sup- genetically modified insects to control the same pressive potential than the release of fully sterile species (LaChance 1985). Sterile Insect Tech- insects (LaChance 1985) and is more compatible nique (SIT) programs have been quite successful with other pest control mechanisms or strategies against a number of pest Diptera (including the (Carpenter 1993). screwworm fly, Cochliomyia hominivorax, and the Knipling (1970) explored the theoretical appli-

Mediterranean fruit fly, Ceratitis capitata), and cation of F1 sterility for control of lepidopteran numerous mass rearing facilities have been con- pests. Using mathematical models, he suggested structed worldwide to support these programs. that when releasing partially sterile insects, the However, compared to dipterans, lepidopterans sterile-to-wild overflooding ratio could be as low generally are more expensive to rear and have a as ¼ of what is normally required for fully sterile propensity to fly greater distances. Additionally, insects. Population models developed by other re- lepidopterans are more resistant to the effects of searchers using data collected from several pest ionizing radiation than dipterans. As a conse- species (Carpenter 1993; Anisimov 1998) corrobo- quence, the greater amount of radiation required rate Knipling’s findings. to completely sterilize lepidopterans negatively Field releases of partially sterile insects have

impacts their competitiveness and performance demonstrated the potential of using F1 sterility to

166 Florida Entomologist 84(2) June 2001 control many lepidopterans, including the cab- ings (RCM) were held to allow participants to dis- bage looper, Trichoplusia ni (North & Holt 1969), cuss initial results and share ideas. The first RCM the corn earworm, Helicoverpa zea (Carpenter et was held in Jakarta, Indonesia (24-28 April, al. 1987; Carpenter & Gross 1993), the gypsy 1995), the second meeting was held in Vienna, moth, Lymantria dispar (Mastro 1993) and the Austria (2-6 September, 1996), and the final codling moth, Cydia pomonella (Proverbs et al. meeting was held in Penang, Malaysia (28 May-2 1978; Bloem et al. 1999b; Bloem et al. 2001). In June, 1998) in conjunction with the FAO/IAEA addition, many studies have shown that F1 steril- International Conference on “Area-Wide Control ity can be effectively combined with other biolog- of Insect Pests Integrating The Sterile Insect and ical controls such as pheromone disruption Related Nuclear and Other Techniques.” (Bloem et al. 2001), entomopathogens (Hamm & Twenty-five scientific teams from twenty-two Carpenter 1997), host plant resistance (Carpen- different countries (Bangladesh, China, Pakistan, ter & Wiseman 1992a, b) and natural enemies Myanmar, Syria, India, Java, Philippines, Mauri- (Carpenter et al. 1996; Greany & Carpenter 1999). tius, Vietnam, Tunisia, Bulgaria, Romania, Czech

As a result of these many studies, F1 sterility is Republic, Russia, Ukraine, Iran, Austria, United regarded as the most favorable genetic method States, Brazil, Cuba and Canada) participated in for most applications against Lepidoptera (for a this second CRP. Participants conducted research review, see Carpenter & Bartlett 1999). on important pests of annual and perennial crops and stored-product pests (see Tables 1 and 2). The The FAO/IAEA Sponsored research findings have been published in three Coordinated Research Programs (CRP’s) separate venues: as refereed publications in scientific journals, as part of a Technical Document or The Joint Division of Nuclear Techniques in meeting proceedings published by the Interna- Food and Agriculture of the Food and Agriculture tional Atomic Energy Agency, and as a block of four Organization (FAO) and the International Atomic manuscripts following this introductory article. Energy Agency (IAEA) promotes agricultural de- The manuscripts in this volume report important velopment through the peaceful use of atomic en- research findings from four different countries and ergy. This mission is accomplished through their on four different species. Ocampo (2001) describes Technical Cooperation Projects, Coordinated Re- the effects of a substerilizing dose of gamma radia- search Projects, publications, and meetings and tion (100 Gy) on the mating competitiveness and training courses. In response to the recommenda- mating propensity of the Old World cotton boll- tions of a group of consultants that met at the worm, Helicoverpa armigera, in the Philippines. IAEA in Vienna in 1984, the Insect and Pest Con- Seth & Sharma (2001b) report on the effects of dif- trol sub-program of the Joint FAO/IAEA Division ferent doses of radiation on the common cutworm, designed and initiated the first five-year (1987- Spodoptera litura reared on two different diets in 1991) Coordinated Research Program (CRP) on India. Koudelova & Cook (2001) examine the competitiveness of mutant and irradiated males of the “Radiation Induced F1 Sterility in Lepidoptera for Area-Wide Control.” Research by CRP participat- Mediterranean flour moth, Ephestia kuehniella, in ing scientists focused largely on modeling the ef- the laboratory by counting eupyrene (fertile) and fects of releasing partially sterile moths on the apyrene (non-fertile) sperm transferred to the fe- field dynamics of feral populations, conducting male during copulation. Finally, Nguyen Thi & laboratory studies to evaluate the relationship Nguyen Thanh (2001) report on the potential of between radiation dose and sterility and conduct- combining F1 sterility and the parasitoid Cotesia ing selected field-cage evaluations. Scientists plutellae in a system to manage the diamondback from ten countries participated in this CRP, and moth, Plutella xylostella, in Vietnam. the research results were published by the IAEA in 1993 (Anonymous 1993). As a result of the re- Major Findings and Impact of the Research search progress during the CRP, the participants Conducted During the F1 Sterility CRP recommended to the FAO/IAEA that a second Co- ordinated Research Program should be consid- The research conducted during this CRP re- ered which would emphasize field applications of vealed principles that were common to all species inherited or F1 sterility for lepidopteran pests. studied. These can be summarized into two major

A second CRP entitled “Evaluation of Popula- points: (1) F1 sterility is an effective and environ- tion Suppression by Irradiated Lepidoptera and mentally safe tactic for lepidopteran pest sup- Their Progeny” was therefore initiated in 1995 pression that is useful under a variety of with the objective of assessing the potential for environments and crop production strategies. (2)

controlling populations of pest Lepidoptera by re- F1 sterility is compatible with all pest control tac-

leasing irradiated moths and/or their progeny in tics. The combination of F1 sterility with phero- combination with other biological control meth- mones, natural enemies, host plant resistance, ods. This CRP concluded in 1998. During this entomopathogens and insecticides results in syn- time period, three Research Coordination Meet- ergistic pest population suppression.

Bloem & Carpenter: Population suppression by irradiated Lepidoptera 167

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 Common cutworm or Soybeans, sorghum, Seth et al. 1997 Tobacco caterpillar corn, vegetables Seth & Sharma 2001a, b Spodoptera exigua Beet armyworm Cotton, vegetables Carpenter et al. 1996 Spodoptera frugiperda Fall armyworm Forage grass, corn Carpenter et al. 1997 Proshold et al. 1998 Arthur et al. 2001 Helicoverpa armigera Corn earworm or Corn, cotton, tomato Pham Van et al. 1996 Cotton bollworm Ocampo et al. 2000 (Old World) Rimas et al. 2000 Ocampo 2001 Lu et al. 2001 Ocampo & deLeon 2001 Helicoverpa zea Corn earworm Corn, cotton, tomato Mannion et al. 1995 Hamm & Carpenter 1997 Proshold et al. 1998 Gelechiidae Pectinophora gossypiella Pink bollworm Cotton Ahmad et al. 2001a, b Diatraea saccharalis Sugarcane borer Sugarcane Garcia & Garcia 1996 Arthur et al. 2001 Tortricidae Cydia pomonella Codling moth Pome fruit, walnut Anisimov & Shvedov 1996 Chernyi et al. 1996 Bloem et al. 1999a, b, 2001 Mansour 2001 Cydia molesta Oriental fruit moth Stone & pome fruit Genchev 1996, 2001 Pyralidae Ectomyelois ceratoniae Carob or date moth Date, carob, pome- Dhouibi & Abderahmane granate 2001 Ephestia kuehniella Mediterranean ﬂour Stored grain Matolin & Marec 1998 moth Marec et al. 1999 Koudelova & Cook 2001 Crocidolomia binotalis Cabbage webworm Crucifer vegetables Sutrisno Apu 2001 Chilo suppressalis Asian rice stem borer Rice Esmaili et al. 1996 Crambidae Ostrinia furnacalis Asian corn borer Corn Wang et al. 2001 Ostrinia nubilalis European corn borer Corn Chernyi et al. 1996 Rosca & Barbulescu 1996 Plutellidae Plutella xylostella Diamondback moth Crucifer vegetables Dunhawoor 1996 Okine et al. 1998 Mitchell et al. 1999 Nguyen Thi & Nguyen Thanh 2001 Sutrisno Apu 2001 Yang et al. 2001 Maung 2001 Arctiidae Spilosoma obliqua Hairy jute caterpillar Jute Rahman et al. 2001

This CRP also highlighted several areas that those that would eliminate females at the egg or would benefit from further research and develop- early larval stage, would reduce rearing costs, ment to increase the economic viability of F1 ste- would increase the efficiency of rearing, irradia- rility programs. Development of diets using tion and release by 100% and would eliminate as- locally available ingredients would reduce rear- sortative mating of released moths in the field. ing costs, especially in locations with developing The FAO/IAEA sponsored CPR’s have had ma- economies. Improvements in mass rearing are jor impacts in the direction of future research for needed to take advantage of the economy of scale the control of lepidopteran pests. For example, ex- as evidenced in dipteran SIT programs. Develop- panded research and implementation programs ment of genetic sexing techniques, especially on F1 sterility in combination with natural ene-

168 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.

Type of Study Species and Reference

Diet Development and Insect Rearing 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. 2001b) Spilosoma obliqua (Rahman et al. 2001) Spodoptera litura (Seth & Sharma 2001a) Radiation Biology 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. 2001b) 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) Genetics and Genetic Sexing Ephestia kuehniella (Koudelova & Cook 2001; Marec 1998; Marec et al. 1999; Matolin & Marec 1998) Effects of Radiation on Sperm Development and Helicoverpa armigera (Rimas et al. 2000) Sperm Competitiveness Ostrinia furnacalis (Wang et al. 2001) Spodoptera frugiperda (Carpenter et al. 1997) Mating Competitiveness of Irradiated Insects Cydia pomonella (Bloem et al. 1999b, Mansour 2001) and Their Progeny 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)

Compatibility of F1 Sterility with Other Control Cydia pomonella (Bloem et al. 2001) Strategies 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)

Use of F1 Sterility to Enhance Parasitoids Helicoverpa zea (Proshold et al. 1998) Plutella xylostella (Mitchell et al. 1999) Spodoptera frugiperda (Proshold et al. 1998) Field Releases for Suppression 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) Population Models Cydia pomonella (Anisimov & Shvedov 1996; Anisimov 1998) Plutella xylostella (Carpenter 2000) mies are underway in Tunisia for suppression of grams for other lepidopteran pest species also are the carob moth, Ectomyelois ceratoniae, and on being considered. Furthermore, the research from the island of Mauritius for control of the diamond- the F1 sterility CRP’s contributed to the develop- back moth, Plutella xylostella. F1 sterility pro- ment of a new Coordinated Research Program on Bloem & Carpenter: Population suppression by irradiated Lepidoptera 169

“Evaluating the Use of Nuclear Techniques for (Lepidoptera: Tortricidae): Effect of substerilizing the Colonization and Production of Natural Ene- doses of radiation on field competitiveness. Environ. mies”. This new CRP was initiated in October Entomol. 28: 669-674. 1999, with scientiﬁc teams from fourteen coun- BLOEM, S., K. A. BLOEM, J. E. CARPENTER, AND C. O. tries. As one of the research objectives in this new CALKINS. 2001. Season-long releases of partially sterile males for control of codling moth, Cydia CRP, F1 sterility is being developed as a tactic to pomonella (Lepidoptera: Tortricidae), in Washing- study possible lepidopteran biocontrol agents for ton apples. Environ. Entomol. (In press). invasive noxious weeds (Carpenter et al. 2001). CARPENTER, J. E. 1993. Integration of inherited sterility and other pest management strategies for Helicov- REFERENCES CITED erpa zea, pp. 363-370. In Proc. Int. Symp. Manage- ment of insect pests: Nuclear and related molecular AHMAD, N., M. ASHRAF, T. HUSSAIN, AND B. FATIMA. and genetic techniques, IAEA/FAO, Vienna, Austria. 2001a. Integration of pheromones and biological con- CARPENTER, J. E. 2000. Area-wide integration of lepi-

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ity for control of corn earworm and fall armyworm diation-induced F1 sterility in Plutella xylostella (Lep- (Lepidoptera: Noctuidae). J. Entomol. Sci. 32: 48-153. idoptera: Plutellidae): Potential for population KNIPLING, E. F. 1970. Suppression of pest Lepidoptera suppression in the field. Florida Entomol. 84: 199-208. by releasing partially sterile males: a theoretical ap- NORTH, D. T. 1975. Inherited sterility in Lepidoptera. praisal. BioScience. 20: 456-470. Annu. Rev. Entomol. 20: 167-182. KOUDELOVA, J., AND P. A. COOK. 2001. Effect of gamma NORTH, D. T., AND G. G. HOLT. 1969. Population sup- radiation and sex-linked recessive lethal mutations pression by transmission of inherited sterility to on sperm transfer in Ephestia kuehniella (Lepi- progeny of irradiated cabbage loopers, Trichoplusia doptera: Pyralidae). Florida Entomol. 84: (In press). ni. Can. Entomol. 101: 513-520. Bloem & Carpenter: Population suppression by irradiated Lepidoptera 171

OCAMPO, V. R. 2001. Effect of a substerilizing dose of ra- SETH, R. K., AND V. P. SHARMA. 2001a. Growth, devel- diation on the mating competitiveness of Helicov- opment, reproductive competence and adult behav- erpa armigera (Lepidoptera: Noctuidae). Florida ior of Spodoptera litura (Lepidoptera: Noctuidae) Entomol. 84: In press. reared on different diets. In Proc. FAO/IAEA Final OCAMPO, V. R., AND J. B. DE LEON. 2001. The effects of Research Coordination Meeting, “Evaluation of Pop- radiation on the biology and reproduction of Helicov- ulation Suppression by Irradiated Lepidoptera and erpa armigera (Lepidoptera: Noctuidae). In Proc. Their Progeny”, 28-30 May 1998. Penang, Malaysia, FAO/IAEA Final Research Coordination Meeting, IAEA-D4-RC-561. “Evaluation of Population Suppression by Irradiated SETH, R. K., AND V. P. SHARMA. 2001b. Inherited steril- Lepidoptera and Their Progeny”, 28-30 May 1998. ity by substerilizing radiation in Spodoptera litura Penang, Malaysia, IAEA-D4-RC-561. (Lepidoptera: Noctuidae): Bio-efficacy and potential OCAMPO, V. R., M. T. TABIR, R. D. FULGENCIO, A. C. LIM, for pest suppression. Florida Entomol. 84: (In press). AND F. D. OROZCO. 2000. Diet development and SETH, R. K., V. P. SHARMA, D. K. RAO, AND J. J. KAUR. mass-rearing of the corn earworm Helicoverpa ar- 1997. Pupae as a physiological and morphogenetic

migera in the Philippines. The Philippine Agricul- indicator for assessing F1 sterility technique for tural Scientist. 83: 282-291. management of Spodoptera litura (Fabricius). J. Nu- OKINE, J. S., E. R. MITCHELL, J. E. CARPENTER, AND G. clear Agric. Biol. 26: 247-253. Y. HU. 1998. Oviposition response of Cotesia plutel- SHELTON, A. M., J. A. WYMAN, N. L. CUSHING, K. APFEL- lae (Hymenoptera: Braconidae) to sterile and normal BECK, T. J. DENNEHY, S. E. R. MAHR, AND S. D. diamondback (Lepidoptera: Noctuidae) larvae and EIGENBRODE. 1993. Insecticide resistance of dia- spatial dispersion of larvae on collard plants. Envi- mondback moth (Lepidoptera: Plutellidae) in North ron. Entomol. 27: 1520-1524. America. J. Econ. Entomol. 86: 11-19. PHAM VAN, L., Q. PHAM BIHN, AND S. NGUYEN VAN. STATEN, R. T., R. W. ROSANDER, AND D. F. KEAVENY. 1996. Life characteristics of Helicoverpa armigera on 1993. Genetic control of cotton insects: The pink boll- cotton, pp. 290-302. In Proc. FAO/IAEA First Re- worm as a working programme, pp. 269-284. In search Coordination Meeting, “Evaluation of Popu- Management of Insect Pests: Nuclear and Related lation Suppression by Irradiated Lepidoptera and Molecular and Genetic Techniques. Proc. FAO/IAEA Their Progeny”, 24-28 April 1995. Jakarta, Indone- Symp., Vienna, 1992, 669 pp.

sia, IAEA-D4-RC-561. SUTRISNO APU, S. 2001. The use of F1 sterility and par- PROSHOLD, F. I., H. R. GROSS, JR., AND J. E. CARPENTER. asitoids for suppression of lepidopteran pests of cru- 1998. Inundative release of Archytas marmoratus cifers in Indonesia. In Proc. FAO/IAEA Final (Diptera: Tachinidae) against the corn earworm and Research Coordination Meeting, “Evaluation of Pop- fall armyworm (Lepidoptera: Noctuidae) in whorl- ulation Suppression by Irradiated Lepidoptera and stage corn. J. Entomol. Sci. 33: 241-255. Their Progeny”, 28-30 May 1998. Penang, Malaysia, PROVERBS, M. D., AND J. R. NEWTON. 1962. Influence of IAEA-D4-RC-561. gamma radiation on the development and fertility of TABASHNICK, B. E., N. L. CUSHING, N. FINSON, AND M. the codling moth, Carpocapsa pomonella L. (Lepi- W. JOHNSON. 1990. Field development of resistance doptera: Olethreutidae). Can. J. Zool. 40: 401-420. to Bacillus thuringiensis in diamondback moth (Lep- PROVERBS, M. D., J. R. NEWTON, AND D. M. LOGAN. idoptera: Plutellidae). J. Econ. Entomol. 83: 1671-1676. 1978. Suppression of codling moth, Laspeyresia VARELA, L. G., S. C. WELTER, V. P. JONES, J. F. BRUN- pomonella (Lepidoptera: Olethreutidae), by release NER, AND H. RIEDL. 1993. Monitoring and character- of sterile and partially sterile moths. Can. Entomol. ization of insecticide resistance in codling moth 110: 1095-1102. (Lepidoptera: Tortricidae) in four western states. J. RAHMAN, R., M. M. RAHMAN, S. ISLAM, AND R. HUQUE. Econ. Entomol. 86: 1-10. 2001. Observations on the growth parameters of Spi- WANG, H. S., Q. R. LIU, D. G. LU, E. D. WANG, W. KANG, losoma obliqua (Lepidoptera: Arctiidae) reared on X. H. LIU, Y. J. LI, Q. L. HE, AND H. Q. ZHANG. 2001. artificial diets and reproductive competence of this Radiation-induced substerility of Ostrinia furnaca- irradiated pest and its progeny. In Proc. FAO/IAEA lis (Lepidoptera: Pyralidae) integrated with the re- Final Research Coordination Meeting, “Evaluation lease of Trichogramma ostriniae (Hymenoptera: of Population Suppression by Irradiated Lepi- Trichogrammatidae) for areawide control. In Proc. doptera and Their Progeny”, 28-30 May 1998. Pen- FAO/IAEA Final Research Coordination Meeting, ang, Malaysia, IAEA-D4-RC-561. “Evaluation of Population Suppression by Irradiated RIMAS, F. M., P. P. OCAMPO, AND V. R. OCAMPO. 2000. Lepidoptera and Their Progeny”, 28-30 May 1998. Effects of gamma radiation on the testicular cells of Penang, Malaysia, IAEA-D4-RC-561. corn earworm (Helicoverpa armigera). Philipp. Ent. YANG, R., D. XIA, W. GU, AND Y. ZHANG. 2001. Field tri- 14: 159-174. als in South China to control the diamondback moth, ROSCA, I., AND A. L. BARBULESCU. 1996. Results regard- Plutella xylostella (Lepidoptera: Plutellidae), using ing rearing of European corn borer (Ostrinia nubilalis) radiation-induced sterility. In Proc. FAO/IAEA Fi- and evaluations of the potential control by radiation nal Research Coordination Meeting, “Evaluation of induced F1 sterility in the field in Romania, pp. 246- Population Suppression by Irradiated Lepidoptera 272. In Proc. FAO/IAEA First Research Coordination and Their Progeny”, 28-30 May 1998. Penang, Ma- Meeting, “Evaluation of Population Suppression by laysia, IAEA-D4-RC-561. Irradiated Lepidoptera and Their Progeny”, 24-28 April 1995. Jakarta, Indonesia, IAEA-D4-RC-561.

172 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. KOUDELOVÁ1 AND P. A. COOK2 1Department of Genetics, Institute of Entomology, Branisovská˘ 31, CZ-370 05 Ceské 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 of lepidopteran populations. Herein we have examined competitiveness 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 mutations, 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 F1 descendants of the treated males, males were found to transfer either a relatively normal 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

RESUMEN La calidad y la competitividad del esperma es de suma importancia en la determinacion 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 estos 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), produjeron 50% menos cantidad de los dos tipos de esperma que los machos de tipo salvaje (WT- C). Sin embargo, la relacion entre la cantidad de esperma apyreno y eupyreno se mantuvo constante en ambos tipos de macho (9.5:1). La irradiacion de pupas maduras, heterocigotas para las mutaciones sl-2 o sl-15 con dosis entre 150 y 350 Gy mostro efectos dependientes de la dosis en cuanto a la cantidad de esperma transferido y el tiempo que los machos permanecen en copula. A medida que la dosis de irradiacion se incrementa, el volumen de esperma transferido decrece y el tiempo que los machos permanecen en copula aumenta. Asimismo,

se encontro que los descendientes de machos irradiados (F1) 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- important to quantify the competitiveness of the ited sterility in Lepidoptera (Anisimov et al. 1989; released males. Carpenter 1991), and the impact of gamma rays In Lepidoptera, the amount of sperm trans- at the chromosomal level is well known (Astaurov ferred during mating may affect reproductive suc- & Frolova 1935; LaChance 1967; Carpenter 1991). cess (Cook et al., in press), and as such has The effectiveness of radiation-induced inherited important consequences in the success of a sterile sterility in the Mediterranean flour moth (Ephes- male release program. Females that receive in- tia kuehniella), an extremely radio-resistant spe- sufficient amounts of sperm during mating may cies, is enhanced by the use of a genetic sexing remate in order to secure enough sperm to fertil- technique (Marec & Mirchi 1990; Marec et al. ize their complement of eggs. Even if females 1999). However, when evaluating the potential of have received enough sperm to fertilize the eggs, inherited sterility for population suppression it is they might be able to perceive that insufficient

Koudelová & Cook: Effect of radiation on sperm transfer in Ephestia kuehniella 173 sperm quantities were transferred during mating. In the Indian meal moth (Plodia interpunctella), 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 proportion of the females eggs. Similar results were reported 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 reported 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 compared to the first mating (Cook & Wedell 1996; Wedell & Cook 1999b). Gage & Cook (1994) examined the effect of high versus low protein larval diets on sperm number and size in P. interpunctella. Fig. 1. Eupyrene sperm bundle of Ephestia kueh- They found that the number but not the size of niella taken from the spermatophore and seen in sperm was reduced when male larvae suffered re- Nomarski interference contrast using Aristoplan microscope. Scale bar = 100 mm. source restrictions. However, in a study of sublethal viral larval infection in the same species, Sait et al. (1998) found that sub-lethally infected males, despite having lower fertility, did not have chemical degradation. It has also been suggested significantly fewer or smaller sperm. that apyrenes play a role in sperm competition Lepidopterans produce two types of sperm (Silberglied et al. 1984; Cook & Gage 1995). Also, cells, nucleated eupyrene (Figs. 1, 2) and smaller, there is good evidence that they protect a males anucleated apyrene sperm (Fig. 3). The two sperm reproductive investment by delaying female re- types result from two distinct modes of spermato- mating (Cook & Wedell 1999). Thus, apyrene genesis (Friedländer 1997), with apyrene out- sperm, as well as eupyrene sperm, must be con- numbering eupyrene sperm by about ten to one sidered when investigating male reproductive (Gage & Cook 1994; Cook & Wedell 1996, 1999; success. Marec et al. 1996). Both types are transferred to In this study we examined the effects of muta- the female during copulation via the spermato- tion and doses of gamma radiation on the number phore and both reach the site of sperm storage, of eupyrene and apyrene sperm transferred by the spermatheca. Apyrene sperm bundles dissoci- males of E. kuehniella in the P and F1 generations ate and become motile prior to male ejaculation in an effort to assess the sperm competitiveness of while eupyrene sperm remain in bundles (Fig. 1). the released males used in an F1 sterility manage- Until recently, the function of apyrene sperm was ment program for Ephestia kuehniella. unknown although several hypotheses have been postulated (reviewed by Silberglied et al. 1984). MATERIALS AND METHODS Osanai et al. (1987, 1989) suggested that apyrene sperm aid in the motility of eupyrene sperm bun- Insects dles inside the female. They characterized apyrene function as that of promoting dissociation of Insects were reared and experiments carried eupyrene bundles and separation of individual out in rooms held at constant temperature (25 ± eupyrene sperm both mechanically and by bio- 1°C), 12L:12D photoperiod and ambient relative

174 Florida Entomologist 84(2) June 2001

Fig. 2. Eupyrene sperm of Ephestia kuehniella after Fig. 3. Apyrene sperm of Ephestia kuehniella taken dissociation from the sperm bundle and seen in Nomar- from the spermatophore and seen in Nomarski interfer- ski interference contrast using Aristoplan microscope. ence contrast using Aristoplan microscope. Scale bar = Scale bar = 100 mm. 100 mm.

humidity. The Mediterranean ﬂour moth, Ephes- Irradiation tia kuehniella Zeller (Lepidoptera: Pyralidae) has been in laboratory culture since 1984 and the Irradiation was performed at the Entomology strain is considered to be wild-type (WT-C) (for Unit of the FAO/IAEA Laboratories in Seibers- details see Marec 1990). A balanced lethal strain, dorf, Austria, using a Co60 Gammacell 220 (AE of BL-2, was constructed in 1990 (Marec 1991) and Canada Ltd.) with dose rates ranging between 50- has been reared since 1990 in single pair cultures. 60 Gy/min. The sl/+ male pupae were placed in BL-2 males are trans-heterozygous for two non- plastic petri-dishes (5 cm diam.) In the ﬁrst ex- allelic sex-linked recessive lethal mutations, sl-2 periment we examined the effect of dose on the P and sl-15. The genetic structure of the BL-2 strain generation. Pupae were treated with 150, 175, is described in Marec et al. (1999). The number 200, 250 or 350 Gy of gamma radiation. In the and ratio of eupyrene and apyrene sperm (see be- second experiment we examined the effect on the low) and the total time in copula for BL-2 and WT- F1 generation. A sixth dose was added in this ex- C males mated to WT-C females was recorded in periment (300 Gy). As a control for the irradia- the laboratory. tion procedure, untreated sl/+ and WT-C males BL-2 males were individually mated to virgin were subjected to the same handling as irradiated WT-C females one generation before irradiation. males. Pupae were placed in containers at the Heterozygous males of two genotypes (sl-2 or sl- conditions mentioned above and adults were col- 15) were produced (both male types are desig- lected as they emerged. nated here as sl/+), whereas female zygotes died during embryogenesis (since they were hemizy- Sperm Counts gous for the lethal sl-2 or sl-15 mutations). Six to eight day-old pupae were removed from their co- Males were allowed to mate with WT-C virgin coons and irradiated. females. For each mating, the total time in copula

Koudelová & Cook: Effect of radiation on sperm transfer in Ephestia kuehniella 175 and the number of eupyrene and apyrene sperm for BL-2 and WT-C males is presented in Table 1. transferred was recorded. Sperm were counted us- The number of both eupyrene and apyrene sperm ing the methods outlined in Gage & Cook (1994) were significantly lower in males from the BL-2 and Cook & Wedell (1996). Immediately after mat- strain. However, the ratio of apyrene to eupyrene ing, the female was dissected and the spermato- sperm was the same in both male groups (9.5:1). phore removed from the bursa copulatrix and The total time in copula was slightly (although placed in a drop of modified Barth saline solution not significantly; P = 0.0774) longer for the mu- on a glass slide (Gurdon 1991). Using a dissecting tant male strain (BL-2) than for the wild type microscope (40× magnification), the spermato- (WT-C) males. phore was ruptured to release the sperm. The eu- Radiation dose significantly affected both eupyrene sperm bundles were counted and the pyrene (Fig. 4-P < 0.05, F = 3.844) and apyrene number of eupyrene sperm was obtained by mul- (Fig. 5-P < 0.05, F = 2.953) sperm numbers in the P tiplying the number of bundles by 256 (which is generation as males were treated with increasing the number of sperm per bundle present after doses of gamma radiation. When males were both mitotic and meiotic divisions are completed). treated with 175 Gy the number of eupyrene (Fig. To count the apyrene sperm, the sample was 4) and apyrene (Fig. 5) sperm was higher than for washed off from the glass slide with more Barth both controls (sl/+ and WT-C). At doses above 175 saline and diluted with distilled water. Six 10 µl Gy the amount of sperm decreased significantly sub samples were placed on slides and allowed to to reach a 25% reduction at 350 Gy for both sperm dry. Each was examined using dark-field phase types (F = 3.85, P = 0.013 and F = 2.95, P = 0.009 contrast microscopy (100× magnification) and the for eupyrene and apyrene sperm, respectively). number of apyrene sperm in each dried sample There were no significant differences in the was counted. The sperm ratio was expressed as a apyrene to eupyrene sperm ratios among treat- quotient of apyrene to eupyrene sperm. ments (Fig. 6) (F = 1.25, P = 0.28). The total time in copula for control and treated P generation Statistical Analysis males is shown in Figure 7. There was a significant treatment effect, with males treated at 350 Data was analyzed using Instat and GraphPad Gy remaining in copula for the longest period of Prism software. The number and ratio of eu- time (F = 5.55, P = 0.0001). pyrene and apyrene sperm and the total time in No adult progeny developed from crosses in- copula for the BL-2 and WT-C males was analyzed volving P males treated with 300 and 350 Gy. As

with one way analysis of variance (ANOVA). Data such, no data is included for the F1 generation at

collected for the F1 generation appeared to deviate these doses. In the remaining F1 male groups from normality. As such, Kruskal-Wallis one-way (150-250 Gy), no dose-response was evident when analysis of variance by ranks was used to decide if the mean numbers of sperm in the spermatophore samples came from populations with the same were compared. However the numbers of eu- median (Siegel & Castellan 1988). This non-para- pyrene to apyrene sperm suggested a bimodal dis- metric method has the advantage of not requiring tribution but could not be tested for normality the assumption of normally distributed error. To- because of insufﬁcient sample size. Therefore, a

tal time in copula for the F1 generation was com- Kruskall-Wallis test was used to compare medi- pared with one-way ANOVA. ans (Table 2). The number of sperm transferred

by F1 generation males was highly variable. Fig- RESULTS ures 8 and 9 illustrate the distribution of numbers of both sperm types at various doses. These Data on the number of eupyrene and apyrene distributions clearly show the difference between sperm, sperm ratio and the total time in copula 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 ratio1 (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

1The difference between strains was not significant at P = 0.05 (compared using ANOVA). *Marginally signiﬁcant at P = 0.0774. ** P < 0.001.

176 Florida Entomologist 84(2) June 2001

Fig. 4. Eupyrene sperm numbers transferred in P generation of irradiated sl/+ males of Ephestia kuehniella. Untreated controls: sl/+, WT-C. Bars indicate Fig. 6. Apyrene to eupyrene sperm ratio in the P gen- standard deviation. eration for irradiated sl/+ males of Ephestia kuehniella. Untreated controls: sl/+, WT-C. Bars indicate standard deviation. numbers of both sperm types transferred. Apy-

rene to eupyrene sperm ratios in the F1 genera- sory gland secretion present, only apyrene sperm tion were lower in the controls (sl/+ and WT-C) present in bursa copulatrix, and (3) no spermato- than in the treatments (Table 2). Some of the phore or sperm present. These defects were ob- males from each treated group exhibited very served in 7.3% of the total (110 males) studied high apyrene to eupyrene ratios as shown in Fig. and occurred only in treated males.

10. The total time in copula in F1 males did not follow a dose-dependent pattern (Fig. 11-F = 4.605, DISCUSSION P = 0.0009). When compared with one-way ANOVA, signiﬁcant differences were found In order to predict the amount of sperm trans- among controls and treated groups. Males treated ferred by Ephestia kuehniella males, we analyzed with 175 Gy remained in copula for the longest sperm numbers in parents of irradiated sl/+ time (the mean was 160 minutes). males, heterozygous for either the sl-2 or sl-15

In addition, some F1 males exhibited various mutations, and in BL-2 males which carried both abnormalities in spermatophore production, in- mutations. We discovered that mutations had an cluding spermatophore bulb formation, sperm effect on the amount of sperm in the spermato- transfer to the spermatophore and accessory phore but not on the apyrene to eupyrene sperm gland ﬂuid transfer to the bursa copulatrix. We ratio (Table 1). A 9.5:1 apyrene to eupyrene ratio

recognized three types of copulatory defects in F1 was found in the control (WT-C) males and in the males: (1) no accessory gland ﬂuid present in the BL-2 mutant males, and this ratio is similar to bursa copulatrix; (2) no spermatophore or acces- that reported for other Lepidoptera. For example,

Fig. 5. Apyrene sperm numbers transferred in P generation of irradiated sl/+ males of Ephestia kuehniella. Fig. 7. Total time in copula in the P generation for ir- Untreated controls: sl/+, WT-C. Bars indicate standard radiated sl/+ males of Ephestia kuehniella. Untreated deviation. controls: sl/+, WT-C; bars indicate standard deviation. Koudelová & Cook: Effect of radiation on sperm transfer in Ephestia kuehniella 177

TABLE 2. EUPYRENE AND APYRENE SPERM NUMBERS AND APYRENE: EUPYRENE SPERM RATIO IN F1 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 statistics1 23.43** 12.58* 27.16***

1Medians vary significantly at: *, P < 0.05; **, P < 0.001; ***, P < 0.0001. a 9:1 ratio was reported for Plodia interpunctella late treated males of Bombyx mori had problems

by Gage & Cook (1994); a 5:1 ratio for Spodoptera in the production and transfer of sperm. F1 males litura by Etman & Hooper (1979) and a 9.6:1 ratio of B. mori produced normal numbers of apyrene for Manduca sexta by J. G. Shepherd, unpub- but fewer numbers of eupyrene sperm. Basically,

lished data in Silberglied et al. (1984). inherited (or F1) sterility is caused by radiation- Several authors have suggested that anucle- induced chromosomal aberrations that affect the ated apyrene sperm play a role in sperm competi- regular segregation of chromosomes, thus gener- tion (Silberglied et al. 1984; Osanai et al. 1987; ating unbalanced gametes (Anisimov et al. 1989) Cook & Gage 1995). Cook & Weddel (1999) found which can lead to changes during copulation. In

that female Pieris napi that had more apyrene our experiments, F1 males showed great variabil- sperm in their storage organ were more likely to ity in the numbers of sperm transferred to fe- delay remating than females with less apyrene males (Table 2). In the range of doses tested (150- sperm. Marec et al. (1999) examined the effect of 250 Gy) some males showed sperm numbers that irradiation on mating success and fecundity of sl/ were similar to those found in the untreated con- + E. kuehniella and found that radiation dose did trols. However, at the same doses, some males not produce any major physiological changes that transferred reduced eupyrene and increased apy- would decrease reproductive capacity in treated rene sperm when compared to controls. These re- males. Holt & North (1970) studied the effect of sults agree with those of a cytogenetic study gamma radiation on sperm distribution and cop- (Tothová & Marec, subm.) which showed variabil- ulation in Trichoplusia ni. They reported that ity in the number and type of chromosome aberra-

there was no difference in sperm volume ejacu- tions inherited from irradiated male parents in F1 lated but found differences in the number of males of E. kuehniella. This variability was mag- sperm present in the spermatophore and con- nified by abnormal pairing of chromosomes dur- cluded that irradiation affects the complex pro- ing meiosis. This abnormal pairing most probably cess of copulation. In the present study, gamma influenced chromosome segregation in meiosis I, radiation reduced the number of sperm trans- finally resulting in either genetically unbalanced ferred in treated E. kuehniella males of the P gen- or balanced gametes. However, the level of steril- eration at all doses except for 175 Gy. At higher ity predicted according to the observed frequency doses, the number of eupyrene and apyrene of chromosome aberrations was much higher than sperm were reduced and the total time in copula the level of inherited sterility found by Marec et for these males was increased. We suggest that to- al. (1999). Thus, it has been suggested that there tal time in copula was dose and sperm volume-de- is a regulation mechanism which enables the pendent (i.e., the higher the treatment dose and moths to correct the predicted unbalanced state the lower the sperm number, the longer the total towards balanced segregation of chromosomes time in copula). Holt & North (1970) also found during meiosis I (Tothová & Marec, subm.).

that irradiated males copulated for longer times In the F1 males we observed no clear dose- than did untreated controls. dependence between sperm transfer and the dose We suggested that the impact of irradiation of gamma radiation. In general, the mean num-

would be greater in the progeny (F1) of treated sl/ ber of eupyrene sperm transferred decreased, + Ephestia kuehniella males than in the parental whereas the mean number of apyrene sperm

(P) generation. North (1975) suggested that F1 transferred increased. This resulted in a high ra- males originating from a partially sterile male tio of apyrene to eupyrene sperm, which ﬂuctu- parent in Pectinophora gossypiellla or from apho- ated between values similar to those of controls 178 Florida Entomologist 84(2) June 2001

Fig. 8. Frequency distribution of eupyrene sperm numbers transferred by control (sl/+, WT-C) and treated males of F1 generation Ephestia kuehniella. Koudelová & Cook: Effect of radiation on sperm transfer in Ephestia kuehniella 179

Fig. 9. Frequency distribution of apyrene sperm numbers transferred by control (sl/+, WT-C) and treated males of F1 generation Ephestia kuehniella. 180 Florida Entomologist 84(2) June 2001

Fig. 11. Total time in copula in the F1 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 F1 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 F1 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 apyrene sperm is stored in females that do not remate. As such, males that are able to transfer more apyrene sperm might reduce the possibility of sperm competition. We observed that some

treated F1 males were able to transfer large

amounts of apyrene sperm. Matings with these F1 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 F1 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 F1 Fig. 10. Frequency distribution of apyrene to eu- males, the number of apyrene sperm was higher pyrene sperm ratios in control (sl/+, WT-C) and treated than in both controls and this number had a males of F1 generation Ephestia kuehniella. broader frequency distribution. Therefore we suggest that 175 Gy may be the best dose to treat

Ephestia kuehniella for F1 sterility induction. We conclude that males inheriting many chro- and those which could be clustered into four mosomal aberrations may change either the groups: (1) higher than 10:1, (2) higher than 20:1, mechanisms regulating spermatogenesis or those (3) higher than 30:1 and (4) the ratios that ex- underlying copulation and sperm transfer to the ceeded the value 100:1. At 200 and 250 Gy the spermatophore. In this study we found that apy- most frequent ratios ﬂuctuated around the value rene sperm are transferred in higher numbers in 30:1. Several authors have reported reduced eu- irradiated males and in very high numbers in pyrene to apyrene ratios in the F1 progeny of lep- their progeny. Given that apyrene sperm num- idopteran species. Proshold et al. (1993) reported bers are related to female remating in untreated Koudelová & Cook: Effect of radiation on sperm transfer in Ephestia kuehniella 181

Lepidoptera, the effect of receiving such high apy- FRIEDLÄNDER, M. 1997. Control of the eupyrene-apyrene sperm numbers on female behavior merits rene sperm dimorphism in Lepidoptera. J. Insect further study. Physiol. 43: 1085-1092. GAGE, M. J. G. 1991. Risk of sperm competition directly affects ejaculate size in the Mediterranean fruit fly. ACKNOWLEDGMENTS Anim. Behav. 42: 1036-1037. GAGE, M. J. G., AND R. R. BAKER. 1991. Ejaculate size We thank Ms. Ivana Kollárová for technical varies with sociosexual situation in an insect. Ecol. support and Dr. Rostislav Zemek for help in the Entomol. 16: 331-337. statistical analysis. We are grateful to Dr. Fran- GAGE, M. J. G., AND P. A. COOK. 1994. Sperm size and tisek Marec for discussion of genetics questions. numbers? Effects of nutritional stress upon eu- The research was supported by the research con- pyrene and apyrene sperm production strategies in tract no. 7161/RB of the International Atomic En- the moth Plodia interpunctella (Lepidoptera: Pyral- ergy Agency (Vienna) and by grant no. A6007609 idae). Functional Ecol. 8: 594-599. of the Czech Academy of Sciences (Prague). GURDON, G. B. 1991. Nuclear transplantation in Xeno- pus, pp. 299-306. In B. K. Kay and H. B. Peng (eds.). Methods in Cell Biology 36: Xenopus laevis: Practical REFERENCES CITED Uses in Cell and Molecular Biology, Chapter 16. Ac- ademic Press, London. 718 pp. ANISIMOV, A. I., N. V. LAZURKINA, AND A. N. SHVEDOV. HOLT, G. G., AND D. T. NORTH. 1970. Effects of gamma 1989. Influence of radiation-induced genetic damage irradiation on the mechanisms of sperm transfer in on the suppressive effect of inherited sterility in the Trichoplusia ni. J. Insect Physiol. 16: 2211-2222. codling moth (Lepidoptera: Tortricidae). Ann. Ento- LACHANCE, L. E. 1967. The induction of dominant lethal mol. Soc. Amer. 82: 769-777. mutations in insect by ionizing radiation and chem- ASTAUROV, B. I., AND S. L. FROLOVA. 1935. Artificial mu- icals—as related to the sterile-male technique of in- tations in the silkworm (Bombyx mori L.). V. Steril- sect control, pp. 617-650. In J. W. Wright and R. Pal ity and spermatogenic anomalies in the progeny of (eds.). Genetics of Insect Vectors of Disease. Elsevier, irradiated moths concerning some questions of gen- Amsterdam. 794 pp. eral biological and mutagenic action of X-rays. Biol. LACHANCE, L. E., R. D. RICHARD, AND R. L. RUUD. 1977. J. 4: 861-894 (in Russian). Movement of eupyrene sperm bundles from the tes- BAKER, R. R., AND M. A. BELLIS. 1989. Number of sperm tis and storage in the ductus ejaculatoris duplex of in human ejaculates varies in accordance with sperm the male pink bollworm: Effects of age, strain irradi- competition theory. Animal Behav. 37: 867-869. ation and light. Ann. Entomol. Soc. Amer. 70: 647- BELLIS, M. A., R. R. BAKER, AND M. J. G. GAGE. 1990. 651. Variation in rat ejaculates consistent with the kami- MAREC, F. 1990. Genetic control of pest Lepidoptera: In- kaze sperm hypothesis. J. Mammol. 71: 479-480. duction of sex-linked recessive lethal mutations in CARPENTER, J. E. 1991. Effect of radiation dose on the Ephestia kuehniella (Pyralidae). Acta Entomol. Bo- incidence of visible chromosomal aberrations in hemoslov. 87: 445-458. Helicoverpa zea (Lepidoptera: Noctuidae). Environ. MAREC, F. 1991. Genetic control of pest Lepidoptera: Entomol. 20: 1457-1459. Construction of a balanced lethal strain in Ephestia CHENG, W. Y., AND D. T. NORTH. 1972. Inherited steril- kuehniella. Entomol. Exp. Appl. 61: 271-283.

ity in the F1 progeny of irradiated male pink boll- MAREC, F., I. KOLLÁROVÁ, AND J. PAVELKA. 1999. Radi- worms. J. Econ. Entomol. 65: 1271-1275. ation induced inherited sterility combined with a ge- COOK, P. A. 1999. Sperm numbers and female fertility in netic sexing system in Ephestia kuehniella. Ann. the moth Plodia interpunctella (Hubner) (Lepi- Entomol. Soc. Amer. 92: 250-259. doptera; Pyralidae). J. Insect Behav. 12: 767-779. MAREC, F., AND R. MIRCHI. 1990. Genetic control of the COOK, P. A., AND M. J. G. GAGE. 1995. Effects of risks of pest Lepidoptera: Gamma-ray induction of translo- sperm competition on the numbers of eupyrene and cations between sex chromosomes of Ephestia kueh- apyrene sperm ejaculated by the moth Plodia inter- niella Zeller (Lepidoptera: Pyralidae). J. Stored punctella (Lepidoptera: Pyralidae). Behav. Ecol. So- Prod. Res. 26: 109-116. ciobiol. 36: 261-268. MAREC, F., J. PAVELKA, J. KOUDELOVÁ, V. KÌIVAN, AND COOK, P. A., AND N. WEDELL. 1996. Ejaculate dynamics P. A. COOK. 1996. Suppression of model populations in butterﬂies: a strategy for maximizing fertilization of Ephestia kuehniella by releasing males balanced success? Proc. R. Soc. London B. 263: 1047-1051. for two sex-linked recessive lethal mutations, pp. 39- COOK, P. A., AND N. WEDELL. 1999. Non-fertile sperm 51. In Working Material, First FAO/IAEA Research delay female remating. Nature. 397: 486. Coordination Meeting, Evaluation of Population COOK, P. A., I. F. HARVEY, AND G. A. PARKER. 1997. Pre- Suppression by Irradiated Lepidoptera and Their dicting variation in sperm precedence. Philos. Trans. Progeny, Jakarta, Indonesia, 24-28 April 1995. Inter- R. Soc. London. B 352: 771-780. national Atomic Energy Agency, Vienna. 321 pp. COOK, P. A., N. WEDELL, AND M. J. G. GAGE. 2000. Re- NORTH, D. T. 1975. Inherited sterility in Lepidoptera. productive strategies in Lepidoptera. In K. W. Wolf Annu. Rev. Entomol. 20: 167-182. (ed.). Double Spermatogenesis: Theory and Experi- OSANAI, M., H. KASUGA, AND T. AIGAKI. 1987. Physio- ments. Harwood Academic Publishers, Reading. In logical role of apyrene spermatozoa of Bombyx mori. press. Experientia. 43: 593-596. ETMAN, A. A. M., AND G. H. S. HOOPER. 1979. Develop- OSANAI, M., H. KASUGA, AND T. AIGAKI. 1989. Isolations mental and reproductive biology of Spodoptera lit- of eupyrene sperm bundles and apyrene spermato- ura (Lepidoptera: Noctuidae). J. Aust. Entomol. Soc. zoa from seminal ﬂuid of the silkmoth, Bombyx mori. 18: 363-372. J. Insect Physiol. 35: 401-408. 182 Florida Entomologist 84(2) June 2001

PARKER, G. A. 1970. Sperm competition and its evolu- SIMMONS, L. W., M. CRAIG, T. LLORENS, M. SCHINZIG, tionary consequences in the insects. Biol.Rev. 45: AND D. HOSKEN. 1993. Bushcricket spermatophores 525-567. vary in accord with sperm competition and parental PROSHOLD, F. I., V. C. MASTRO, AND G. L. BERNON. investment theory. Proc. R. Soc. London B. 251: 183- 1993. Sperm transfer by gypsy moths (Lepidoptera: 186. Lymantriidae) from irradiated males: implication TOTHOVÁ, A., AND F. MAREC. 2000. Chromosomal prin-

for control by inherited sterility. J. Econ. Entomol. ciple of radiation-induced F1 sterility in Ephestia 86: 1104-1108. kuehniella (Lepidoptera: Pyralidae). Genome (sub- SAIT, S. M., M. J. G. GAGE, AND P. A. COOK. 1998. Ef- mitted). fects of a fertility-reducing baculovirus on sperm WEDELL, N., AND P. A. COOK. 1998. Determinants of pa- numbers and sizes in the Indian meal moth, Plodia ternity in a butterfly. Proc. R. Soc. London. B 265: interpunctella. Functional Ecol. 12: 56-62. 625-630. SIEGEL, S., AND N. J. CASTELLAN, JR. 1988. Nonpara- WEDELL, N., AND P. A. COOK. 1999a. Butterflies tailor metric Statistics for the Behavioral Sciences, 2nd their ejaculate in response to sperm competition risk Edition. Mc Graw-Hill Book Company, New York. and intensity. Proc. R. Soc. London B 266: 1033- 399 pp. 1039. SILBERGLIED, R. E., J. G. SHEPHERD, AND J. L. DICKIN- WEDELL, N., AND P. A. COOK. 1999b. Strategic sperm al- SON. 1984. Eunuchs: The role of apyrene sperm in location in the small white butterfly Pieris rapae Lepidoptera? Amer. Naturalist. 123: 255-265. (Lepidoptera: Pieridae). Functional Ecol. 13: 85-93.

Seth & Sharma: Inherited sterility in Spodoptera litura 183

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 substerilizing doses of gamma radiation, 100 Gy and 130 Gy, and examined for inherited steril-

ity. Irradiation affected mating success in the parental (P) and F1 generations. F1 sterility was

higher than P sterility, and F1 males inherited more sterility than did F1 females. F1 progeny

developed at a slower rate compared with controls. F1 survival to adulthood decreased with

increasing dose of radiation. Sex ratio in F1 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 F1 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 F1 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 of F1 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, F1 sterility, synthetic diet, embryonic lethality, population dynamics

RESUMEN Insectos de la especie Spodoptera litura provenientes de colonias mantenidas en plantas 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 F1. Se encontro que la es-

terilidad en la generacion F1 es mas alta que en la generacion P y los machos F1 heredaron mas esterilidad que las hembras de la misma generacion. Asimismo, se encontro que la ve-

locidad de desarrollo de la generacion F1 es mas lenta que en el grupo control (no irradiado), y la supervivencia al estado adulto se redujo a medida que los insectos fueron expuestos a dosis de radiacion mas altas. Finalmente, la tasa sexual se vio favoracida hacia el sexo macho

en la generacion F1. Se construyeron tablas de vida para ambas colonias y se investigo el

efecto de la radiacion sobre varios parametros de estas poblaciones. La tasa reproductiva (R0) se redujo signiﬁcativamente y el efecto fue mas severo en parejas de insectos de la genera-

cion F1. Asimismo, se encontro una correlacion negativa entre la dosis de radiacion y la for- macion de hevecillos embrionados en la generacion P. Esta corralacion fue positiva en la

generacion F1 cuando la radiacion se aplico solamente a los machos. Se detecto mortalidad temprana dentro de los huevecillos no eclosionados provenientes de cruces en la generacion

P y se detecto muerte tardia de los huevecillos embrionados en cruces en la generacion F1.

En este articulo discutimos estos efectos con enfasis a la aplicacion de la esterilidad F1 como metodo de control para esta especie.

Spodoptera litura (Fabricius) (Lepidoptera: insects in this order are radio-resistant, presum- Noctuidae), the common cutworm, is an economi- ably due to their holokinetic chromosomal conﬁg- cally serious and polyphagous pest in India. This uration (Bauer 1967). Therefore, lepidopterans pest is reported to attack a wide range of food require large doses of radiation for sterilization, plants (112 cultivated plants belonging to 44 fam- leading to somatic damage and reduced competi- ilies worldwide and 60 plants in India) (Lefroy tiveness in the irradiated insect. 1908; Moussa et al. 1960; Thobbi 1961; Chari & A favored alternative to using fully sterile

Patel 1983). A multifaceted approach is required moths in SIT is the use of F1 sterility. F1 survivor for the control of this pest because it has devel- progeny of sub-sterile parental (P) male moths oped resistance against a range of insecticides result when sub-sterilizing doses of radiation are and because other control measures are inade- applied to the P males. The resulting F1 progeny quate when applied alone (Ramakrishnan et al. are more sterile than the irradiated parent, and 1984; Armes et al. 1997). The sterile insect tech- the irradiated moths are more competitive as a re- nique (SIT) has been used for Lepidoptera but sult of receiving a lower dose of radiation. Inherited

184 Florida Entomologist 84(2) June 2001

sterility in the progeny of treated males has been tural Research Institute, New Delhi, using a Co60 shown to have potential in suppressing popula- source at the dose rate of about 13.5 Gy/min. On tions of lepidopteran pests (North & Holt 1969; the basis of our initial studies (Seth & Sehgal, Knipling 1970; North 1975; LaChance 1985). 1993), two gamma-radiation doses, 100 Gy and Previous studies of substerilizing gamma-radia- 130 Gy, were selected for this study. tion doses on the growth, bioenergetics and repro-

ductive behavior of S. litura in the F1 progeny of treated moths indicated the potential of managing Reproductive Performance and Viability this pest by using inherited sterility (Seth & Sehgal of Irradiated Moths and Their Progeny 1993). In this study we evaluated the reproductive performance and mating behavior of S. litura in re- Various reproductive parameters were as- sponse to two substerilizing doses (100 Gy and 130 sessed by pairing treated insects (irradiated P males and F moths derived from the treated P Gy) when reared on two different diets. 1 males crossed with normal females) with their normal (N) counterparts from the stock culture. MATERIALS AND METHODS Eggs from single-pair matings were counted daily and the number hatching was monitored in 10 Insect Rearing replicated samples of 80-100 eggs each (up to first 3 days of egg laying). Corrected sterility and con- Spodoptera litura was reared on two diets, the trol of reproduction of insect population due to ir- natural food, castor leaves (Ricinus communis) radiation were determined according to the and a meridic diet containing chickpea seeds and methods described by Abbott (1925) and Seth & sinigrin as a phagostimulant (Table 1). Insects Reynolds (1993). were held at ambient environmental conditions, Experiments on mating success were con- 26.8 ± 1°C temperature, 75 ± 5% R.H. and a pho- ducted in laboratory cages (each cage having 10- toperiod of 12L:12D in the insectary. Larvae de- 15 pairs, comprising one replicate). The mating veloping on the castor leaves were allowed to success of moths was assessed by dissection of fe- pupate in moist, loose soil. Larvae developing on males immediately after death. The presence of a the chickpea diet pupated in the diet container. spermatophore in the bursa copulatrix indicated that the female had mated; the number of sper- Irradiation of Insects matophores indicated the mating frequency. The viability of treated moths, and the sur- Irradiation of 0-24-h old adult males was con- vival and developmental pattern of F1 progeny ducted in the Genetics Division, Indian Agricul- were determined. Diurnal observations on the insect behavior and the growth index for each treatment was calculated. TABLE 1. CONSTITUENTS OF THE SEMI-SYNTHETIC DIET PRO- POSED FOR REARING OF SPODOPTERA LITURA. Population Characteristics Ingredients Amount Various features of population dynamics were Agar 25 g studied by constructing life tables for S. litura on Deionized water 750 ml castor leaves and chickpea diet. The female life ta- Casein 44 g bles of P and F1 generation were constructed as de- Ground chickpea seeds 93.50 g scribed by Birch (1948), and elaborated by Howe Wessons salts 12.50 g (1953), Morris & Miller (1954), and Atwal & Bains Cholesterol 1.25 g (1989). For ascertaining the life table characteris- Yeast (dried, brewer’s) 19 g tics, a defined size of population was established Methyl-p-hydroxybenzoate 1.25 g in field-cages for a particular cross. Then seven Sugar 39 g batches of 200-250 eggs collected from each cross Sorbic acid 2 g were reared to determine age specific mortality in Deionized water 400 ml different life stages and successful adult emer- 4 M KOH 6.25 ml gence. The life tables gave the probability at birth Corn oil 2.50 ml of a female being alive at age x, designated as lx (l0 Linseed oil 2.50 ml = 1). The age schedule for female births denoted Formaldehyde (10%) 5.50 ml the mean number of female offspring produced per Sinigrin (1%) 3.53 ml unit time by a female of age x, designated as mx. Antibiotic and vitamin mixture1 7.50 g The net reproductive rate (R0) was calculated from Choline chloride 1.25 g lx and mx. Mean length of generation time (Tc), innate capacity for increase in number (rm) and fi- 1Composition: chloramphenicol (2 g), streptomycin (4 g), tetracycline λ (36 g), ascorbic acid (80 g), Evion (vitamin E; 0.2 g; Merck Co.), vitamin nite rate of increase ( ) were calculated from the mixture (2 g; Roche Co.). data generated in the life tables.

Seth & Sharma: Inherited sterility in Spodoptera litura 185

Embryonic Development arcsine √x before ANOVA. Means were separated at the 5% significance level by least significant The embryonic development in F1 and F2 eggs difference (LSD) test (Snedecor & Cochran 1989). was studied to understand the stage at which irradiation induced lethality was manifested. The developmental state of embryos was assessed in RESULTS dechorionated eggs. The eggs were treated with 3- 5% NaClO for 30-40 min, fixed with 10% formalin Reproductive Performance and Viability for 8-12 h, and stained with lactoacetic-orcein or of Irradiated Moths and Their Progeny Feulgen stain. The stained embryos were examined under the microscope. The embryonic devel- For the untreated controls, the pre-oviposition opment was classified as stage I (cleavage/early period ranged from 1.65 to 1.72 d on the castor development), stage II (germ band stage), and leaf diet and 1.69 to 1.70 d on the chickpea. The stage III (embryo stage). Some embryos were re- dose of radiation did not significantly affect pre- corded as abnormal. oviposition period for the P and F1 crosses in either diet. The oviposition period was not affected Statistical Analysis in P crosses, but was significantly reduced in most

F1 crosses (Table 2). Also, the radiation treat- The effect of radiation on various parameters ments caused a signiﬁcant reduction in fecundity in the two diets was subjected to analysis of vari- of the mated female during P crosses and F1 ance (ANOVA). Data were usually obtained in crosses, with the greatest reduction in F1 crosses. replicates of ten, unless otherwise speciﬁed in the Even normal females mated with treated males text. Percentage data were transformed using (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 F1 PROGENY.

Preoviposition Oviposition Eggs per Life span (days) Dose Cross period period female Diet (Gy) type1 (days) (days) (no.) Male Female

P crosses Castor leaf 0 Nm × 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 × 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 × Nf 1.89 ± 0.13 a 7.21 ± 0.25 a 1795 ± 93 b 9.4 ± 0.4 a 9.1 ± 0.5 a

F1 crosses 0 Nm × Nf 1.72 ± 0.08 a 7.62 ± 0.31 a 1990 ± 49 a 10.3 ± 0.4 a 9.3 ± 0.3 a × ± ± ± ± ± 100 F1m 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 F1f 1.72 0.19 a 7.01 0.09 a 1693 80 b 9.8 0.2 a 9.3 0.3 a × ± ± ± ± ± F1m F1f 1.60 0.24 a 6.22 0.49 b 1520 56 b 9.6 0.4 a 8.3 0.4 b × ± ± ± ± ± 130 F1m 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 F1f 1.73 0.11 a 6.29 0.42 b 1499 69 b 8.8 0.3 b 8.7 0.9 ab × ± ± ± ± ± F1m F1f 1.85 0.24 a 6.22 0.59 b 1219 30 c 8.8 0.3 b 8.4 0.1 b P crosses Chickpea diet 0 Nm × 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 Pm × Nf 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 × Nf 1.88 ± 0.11 a 6.66 ± 0.42 a 1727 ± 69 b 8.9 ± 0.3 a 8.6 ± 0.2 a

F1 crosses 0 Nm × 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 F1m Nf 1.63 0.17 a 7.09 0.08 a 1649 70 b 8.8 0.4 a 8.5 0.4 b × ± ± ± ± ± Nm F1f 1.62 0.16 a 6.90 0.37 ab 1592 84 b 8.6 0.4 a 8.5 0.4 b × ± ± ± ± ± F1m F1f 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 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 F1f 1.75 0.17 a 5.95 0.26 bc 1363 75 cd 8.5 0.4 b 7.9 0.2 bc × ± ± ± ± ± F1m F1f 1.84 0.20 a 5.83 0.22 c 1099 53 e 7.0 0.3 c 7.1 0.3 c

1 ± N, normal; P, treated parent; F1, 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. 186 Florida Entomologist 84(2) June 2001 ample, the reduction in oviposition in P crosses, moths from either diet. Radiation-induced sup- with respect to untreated crosses, was 6.2% at pression effects were examined in terms of cor- 100 Gy and 14.1% at 130 Gy on the chickpea diet. rected sterility and control of reproduction (Table × This reduction in oviposition was further in- 3). At 100 Gy on chickpea diet, the F1 male N fe- creased in the F1 crosses (23-30% at 100 Gy, and male cross gave 76.5% of the total eggs laid by 39-49% at 130 Gy on chickpea diet). Both doses controls (see Table 2) and resulted in 28.3% egg

significantly reduced F1 male and female longev- hatch as compared with 78.6% hatch in controls. ity when the chickpea diet was used. Although Therefore, this cross exhibited 63.9% sterility, the same trend was evident when moths were and 72.3% control of reproduction. For both the reared on castor leaves, the reduced longevity for diets tested, the control of reproduction in F1 males at the 100 Gy dose was not significant. males (mated with normal females) was more A significant, dose-dependent effect was ob- than 71% at 100 Gy and about 83-87% at 130 Gy. served on the mating success of P and F1 moths reared on both diets. The treated males were less Growth and Survival of F1 Progeny successful at mating than untreated males (Table

3). For example, at 130 Gy on chickpea diet, the The developmental time of F1 eggs, larvae, pu-

mating success of P males and F1 males was pae and adults reared on the two diets were signif- 76.4% and 72.7%, respectively, as compared with icantly affected by the radiation treatment given 89.6% in the control. The mating percentage was to the male parent (crossed with a normal female).

more adversely affected in F1 female crosses and Their developmental rate was slower than that of

F1 self crosses. However, the radiation did not sig- the control progeny (Table 4); the total develop- niﬁcantly affect the mating frequency of the 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) type1 (no. ± SE) (%) (%) (%) (%)

P crosses Castor leaf 0 Nm × Nf 1.7 ± 0.1 a 92.8 ± 2.2 a 89.7 ± 2.1 a 0 0 100 Pm × Nf 1.9 ± 0.3 a 84.0 ± 3.1 b 52.3 ± 2.0 b 41.1 ± 1.6 48.0 ± 1.9 130 Pm × Nf 1.9 ± 0.3 a 72.6 ± 3.8 c 44.8 ± 2.5 c 49.5 ± 2.8 56.6 ± 3.2

F1 crosses 0 Nm × Nf 1.7 ± 0.1 a 94.1 ± 1.9 a 88.9 ± 1.4 a 0 0 × ± ± ± ± ± 100 F1m 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 F1f 1.8 0.2 a 73.2 2.2 cd 44.5 2.9 b 49.9 3.3 59.4 3.9 × ± ± ± ± ± F1m F1f 1.7 0.3 a 71.4 2.9 de 28.3 3.1 d 68.2 7.4 76.8 8.4 × ± ± ± ± ± 130 F1m 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 F1f 1.7 0.3 a 66.0 2.4 ef 35.9 2.2 c 59.6 3.7 71.0 4.4 × ± ± ± ± ± F1m F1f 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 × Nf 1.7 ± 0.1 a 89.2 ± 2.2 a 84.4 ± 2.9 a 0 0 100 Pm × Nf 1.8 ± 0.3 a 81.5 ± 2.2 b 46.2 ± 4.1 b 41.2 ± 3.7 48.6 ± 4.3 130 Pm × Nf 1.8 ± 0.2 a 76.4 ± 3.2 b 41.6 ± 2.1 b 47.1 ± 2.4 57.6 ± 2.9

F1 crosses 0 Nm × Nf 1.7 ± 0.2 a 89.6 ± 2.4 a 78.6 ± 3.0 a 0 0 × ± ± ± ± ± 100 F1m 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 F1f 1.6 0.1 a 75.4 1.5 bc 43.2 2.9 b 45.0 3.0 59.4 4.0 × ± ± ± ± ± F1m F1f 1.5 0.1 a 65.9 2.8 de 22.3 2.0 cd 71.6 6.4 80.4 7.1 × ± ± ± ± ± 130 F1m 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 F1f 2.4 0.1 a 60.9 1.3 ef 26.7 2.2 c 66.1 5.4 78.5 6.5 × ± ± ± ± ± F1m F1f 2.0 0.2 a 56.1 3.1 f 14.6 2.1 e 81.4 8.7 90.6 8.1

1 N, normal; P, treated parent; F1, progeny of treated males; m, male, f, female. 2For 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. Seth & Sharma: Inherited sterility in Spodoptera litura 187 (M:F) Sex ratio 2 0.1 c 1:0.64 b 0.1 c 1:0.65 c 0.1 a 1:1.03 a 0.1 b 0.1 a 1:0.69 ab 1:0.99 a 0.1 b 1:0.71 b ± ± ± ± ± ± 1 2.2 c 1.6 2.5 a 3.0 2.8 b 3.4 b 2.2 1.8 2.8 a 2.8 3.1 b 2.0 ± % Growth index ± ± ± ± ± . x. √ DIETS Adult eclosion

1 TWO

2.9 c 56.3 4.6 a 82.3 2.7 b 64.0 3.3 a 79.2 4.5 b 60.9 2.1 b 51.7 ± ± ± ± ± ± posttest). % pupation REARED

re transformed using arcsine LITURA

0.4 a 89.3 0.6 ab 71.2 0.7 a 90.2 0.8 b 62.8 1.0 ab 68.4 0.3 b 58.8 ± ± ± ± ± ± SPODOPTERA

0.2 a 27.2 0.2 ab 29.2 0.2 a 28.3 0.2 b 30.1 0.3 ab 30.7 0.3 b 31.5 ± ± ± ± ± ± IRRADIATED

0.3 a 7.9 0.5 ab 8.5 0.4 a 8.4 0.4 b 8.6 0.4 b 8.5 0.3 b 9.1 ± ± ± ± ± ± PROGENY

1 F

Developmental period (days) OF

0.1 a 15.8 0.1 b 16.7 0.1 a 16.2 0.1 b 17.3 0.1 b 17.9 0.1 b 18.7 SURVIVAL ± ± ± ± ± ±

Egg Larva Pupa Total AND

PROFILE

100 3.9 130100 4.2 3.9 130 4.0 (Gy) Dose EVELOPMENTAL SE followed by the same letter in a column within each followed by LSD diet are not signiﬁcantly different at P < 0.05 (ANOVA ± 4. D Observed in group of 25 individuals comprising each replicate, n = 7. For statistical analysis by ANOVA, the percentage data we Growth index = % adult eclosion/total developmental period. 1 2 Means ABLE Castor leaf 0 3.6 Diet Chickpea dietChickpea 0 3.6 T 188 Florida Entomologist 84(2) June 2001 creased by about 7-9% at 100 Gy and 10-13% at or embryonic death. Certain fertilized eggs exhib-

130 Gy. The F1 insects experienced signiﬁcantly ited embryonic development but were incapable of more larval and pupal mortality than the controls, hatching. The development of such zygotes was ar- and also exhibited a higher rate of pupal and adult rested at different levels of embryogenesis showing malformation than the controls. The proportion of partial to complete embryonation. Some of these

F1 insects surviving to adults decreased with the eggs could reach the “black head” stage wherein, increasing dose on both diets tested. About 61- blackish brown colored head cuticle of the pharate

64% of the F1 progeny of 100 Gy treated male par- 1st instar larva was visible through translucent ents emerged as adults, whereas 79-82% adults chorion of the egg. This type of egg was categorized emerged in the controls. The F1 growth index was as an unhatched embryonated egg. At 130 Gy on

signiﬁcantly decreased according to the dose of ra- the chickpea diet, early mortality (EM) of F1 eggs diation administered to the male parent. The sex (derived from P crosses) was 36.9% with no detect-

ratio of the F1 generation was signiﬁcantly skewed able embryonic development. Late embryonic le- towards males as a result of the radiation treat- thality (LEL) was 22.4%, mainly due to embryonic

ments. For example, F1 females constituted about stages blocked at cleavage or early development

41% of the population at 100 Gy and about 50% of stage. In F2 eggs (from F1 crosses) at the same dose, the population in the control. EM constituted 16.8% and LEL constituted 65.8%. Notably, more than 75% of LEL was restricted to Population Characteristics germ band and embryo stages (Table 6, Fig. 1). The formation of embryos was signiﬁcantly reduced in

Increase in the total developmental period of F1 eggs (100 Gy and 130 Gy), but was not statistically different from the controls in F eggs at both F1 progeny delayed the commencement of oviposi- 2 tion with further debilitating effects on adult sur- doses. However, the percentage zygotic viability in

vival, life expectancy, oviposition rate and female P and F1 crosses was signiﬁcantly reduced by the births with respect to the pivotal age group. The dose of radiation (Table 6). EM appeared predomi- nantly in F eggs, whereas F eggs showed sterility reproductive rate (R0) was significantly decreased 1 2 as a consequence of radiation treatment, with the mainly due to LEL. effect being more pronounced in F1 crosses than in P crosses (Table 5). For instance, R0 for untreated DISCUSSION insects was 785.2 on castor and 740.2 on chickpea diet. On chickpea diet, R was decreased by 16.4% 0 The phenomenon of F1 sterility in S. litura was at 100 Gy and 36.6% at 130 Gy in P generation. R0 examined over a range of substerilizing doses was further reduced by about 70% at 100 Gy and (Seth & Sehgal 1993). As a result of the reproduc- about 80% at 130 Gy in F1 crosses, with respect to tive performance and somatic damage caused by the control. the radiation, we selected two doses (100 Gy and

The mean generation time (Tc) was 31.3 days 130 Gy) for a more in-depth study. The reduced re- on castor and 32.3 days on chickpea diet for un- productive performance of the treated moths re-

treated insects. In F1 crosses, Tc was significantly sulted from the combined effects of reduced higher, indicating a protraction of 2-4 days as longevity, fecundity, mating success, and fertility. compared with the control. The Tc delay showed a The mating percentage was more adversely af- positive correlation with doses of radiation (Table fected in F1 females crossed with normal males 5). The intrinsic rate of increase (rm) in untreated and in F1 self crosses as compared with the F1 insects was 0.21 on castor and 0.20 on chickpea males paired with normal females. A greater re- diet. The effect of irradiation on the intrinsic rate duction in reproductive performance was ob- of increase was not apparent in P crosses, but was served at the 130 Gy dose than the 100 Gy dose. significant in F1 crosses, where there was a reduc- The effects of radiation dose on reproductive per- tion in the rm value (23-25% at 100 Gy and 31- formance were not significantly influenced by 36% at 130 Gy). Irradiation also affected the finite diet. Similar debilitating effects of irradiation on rate of increase of this insect population. For ex- the reproduction of moths have been reported by ample, on chickpea diet at 130 Gy, it was reduced several workers (North & Holt 1968; Proshold & to 1.15 in F1 male crosses from 1.23 in the control. Bartell 1970; Cheng & North 1972; North 1975; A similar pattern was observed on castor leaves. LaChance et al. 1976; Carpenter et al. 1983; LaChance 1985; Sallam & Ibrahim 1993; Omar & Mansor 1993; Ismail 1994; Carpenter et al. 1987; Embryonic Development of F1 and F2 Zygotes Makee & Saour 1997). Poor reproductive perfor-

In F1 and F2 generations, the embryonic devel- mance and low fertility in treated S. litura could opment was disrupted at different stages that re- be due to one or more of the following reasons: (i) ﬂected a speciﬁc pattern of radiation-mediated poor ability to mate (El-Sayed & Graves 1969), (ii)

lethality in P and F1 moths (Table 6). The reduced failure to produce and transfer as many sper-

egg hatch from P and F1 males crossed with normal matophores as normal males (Rule et al. 1965; females may have been caused by infertility of eggs Flint & Kressin 1969), (iii) transfer spermato- Seth & Sharma: Inherited sterility in Spodoptera litura 189 rm e of increase Finite rate Finite ) = antilog λ ( c /T 0 R e for increase rm = log Innate capacity . DIETS

0 /R x TWO

m x xl ON

∑ posttest); n = 7. = c length (days) T Mean generation REARED

PROGENY

1 x 835.0 b 30.90 a 0.2065 a 1.229 a 774.2 b 30.94 a 0.2068 a 693.4 b 32.19 a 1.229 a 0.1996 ab 1.220 a 328.7 c 31.85 a 0.1931 ab 1.213 a 683.3 a 31.30 a 0.2129 a 738.8 a 32.30 a 1.237 a 0.2045 a 1.226 a m F 355.1 c 153.1 d 34.12 c 34.76 c 131.7 de 0.1571 bc 34.76 bc 101.9 f 0.1463 cd 1.170 bc 35.97 c 0.1552 b 1.157 cd 0.1384 c 1.167 bc 1.148 c 180.5 c 110.7 e 33.76 bc 34.36 c 0.1578 bc 76.4 g 0.1371 d 1.170 bc 35.34 c 1.146 d 0.1308 c 1.139 d 161.0 e 35.01 c 0.1526 b 1.164 bc 292.0 c 150.2 d 32.92 b 34.62 c 220.7 d 0.1648 bc 34.82 bc 120.1 f 0.1479 cd 1.179 bc 35.92 c 0.1568 b 1.159 cd 0.1399 c 1.169 bc 1.150 c x ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± xl ∑ THEIR

AND

2 LITURA

x m x l 27.0 b 18279.8 25.4 b 18643.5 28.5 b 19898.6 36.7 c 14941.7 10.3 c 7262.3 7.3 d 5626.6 19.3 d 7669.1 11.1 e 5226.3 27.4 a 24583.0 6.1 c 6975.2 12.1 e 38.7 a 3819.7 23913.1 8.9 f 3602.7 10.2 c 7489.2 7.6 d 5805.4 21.4 d 8207.3 13.1 e 5461.3 13.3 d 7321.7 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ∑ = 0 R PODOPTERA S

Net reproductive rate 1 f 206.5 f 111.1 f 209.1+ f 101.9 f 212.8 f 161.8 f 220.5 f 145.0 1 1 1 1 1 1 1 1 IRRADIATED F F F F Nf Nf 227.4 167.7 Nf Nf 235.6 152.0

Nf 785.2 Nf 740.2 F F F F Nf 591.4 Nf 602.4 Nf 618.1 Nf 469.0 × × × × × × × × × × × × × × × × × × OF

m m m m m m m m l 1 l 1 l 1 l 1 F F F F F F F F Nm Nm Nm Nm , progeny of treated males; m, male, f, female. 1 130 Pm 100 Pm 100 Pm 130 Pm (Gy) Cross type Dose CHARACTERISTICS

, female offsprings produced per unit time; x, pivotal age. x OPULATION SE followed by the same letter in a column within each followed by LSD diet are not signiﬁcantly different at P < 0.05 (ANOVA ± 5. P , female survival; m x N, normal; P, treated parent; F l 1 2 Means ABLE Castor leaf 0 dietChickpea Nm 0 Nm Diet T 190 Florida Entomologist 84(2) June 2001 4 3.1 c 4.6 c 3.1 c 1.9 b 2.2 b 1.9 b 1.4 b 0.7 a 4.4 d 0.2 a ± ± ± ± ± ± ± ± ± ± (%) eggs Unhatched Unhatched 64.3 74.6 65.3 32.0 35.8 34.5 28.8 10.8 78.8 embryonated 4 4.4 3.2 c 3.7 bc 2.4 c 4.7 bc 4.2 a 4.1 ab 4.5 a 4.9 ab 3.0 a 4.7 ± ± ± ± ± ± ± ± ± ± (%) Embryo formation 0.1 66.4 0.2 71.5 0.2 63.6 0.1 73.5 0.3 88.6 0.3 86.1 0.2 88.5 0.4 83.5 0.03 88.2a ± ± ± ± ± ± ± ± ± . LITURA

2.1 4.0 2.2 7.1 1.8 3.8 2.3 6.8 0.2 2.3 0.1 4.2 0.1 3.1 0.1 0.8 0.1 3.3 0.0 0 93.3 ± ± ± ± ± ± ± ± ± ± PODOPTERA S (%)

3 OF

0.6 39.4 0.4 3.2 0.2 2.8 0.1 1.5 0.1 2.2 0.1 2.0 0.4 37.0 0.4 36.5 0.2 47.0 0.0 0.7 ± ± ± ± ± ± ± ± ± ± the stage out of total unhatched eggs; Stage I, cleavage or early developmental phase; SE followed by the same letter in a column case of each diet are not significantly different at P MOTHS ±

1 F Eggs showing Embryonic stage of unhatched eggs Embryonic stage of unhatched ; means . AND 0.7 4.2 0.8 3.1 0.5 2.5 0.2 6.4 0.2 1.6 0.3 7.6 0.2 10.3 0.4 9.6 0.3 6.5 0.1 0.9 ± ± ± ± ± ± ± ± ± ± P BY

Stage I Stage II Stage III Abnormal 1.6 9.4 1.5 14.1 1.4 14.3 0.7 5.0 1.7 13.4 0.6 8.4 0.7 7.5 0.6 7.7 1.0 5.5 0.2 2.8 OVIPOSITED ± ± ± ± ± ± ± ± ± ±

No EGGS

development IN

2 (no.) female (44.8%) (60.8)(46.2%) (17.0) (52.9) (11.5)(41.6%) (26.2) (5.7) (62.3) (7.8) (4.1) (24.4) (5.2) (5.3) (7.8) (2.5) (5.3) (52.3%) (55.5) (28.0) (5.2) (4.1) (6.9) (31.6%) (16.6)(21.8%)(78.6%) (12.2) (17.9) (11.1) (9.6)(30.7%) (54.0) (16.5) (13.2) (5.8) (11.1) (50.2) (13.8) (9.1) (52.6) (5.4) (17.5%) (20.1) (6.7) (7.9) (57.0) (8.3) (88.9%) VIABILITY

Eggs per ZYGOTE x before ANOVA, but data shown are back transformations

1 √ Nf 1755 Nf 11.5 1464 13.2 Nf 1750 Nf 11.6 1394 16.8 Nf 2155 11.2 Nf 2088 6.5 Nf 1795 33.1 Nf 1886 Nf 28.9 1727 36.9 Nf 1847 26.3 × × × × × × × × × × AND

type m m m m Cross 1 1 1 1 F F F F , progeny of treated males; m, male; f, female. 1 130 Pm 100 Pm 130 Pm 100 Pm (Gy) Dose DEVELOPMENT

MBRYONIC 6. E N, normal moth; P, treated parent; F Ovipositional data computed out of mated females; in parentheses represent % egg hatch. Data represent % inviable eggs in the particular stage out of total laid; data parentheses Percentage data were transformed using arcsine 1 2 3 4 ABLE Chickpea dietChickpea 0 Nm < 0.05 (ANOVA followed by LSD post-test); n = 7; each replicate comprising of 80-100 eggs. Castor leaf 0 Nm II, germ band phase; Stage III, embryo phase. Diet T Seth & Sharma: Inherited sterility in Spodoptera litura 191

growth index showed a decrease as a consequence

of irradiation of male parents. The sex ratio in F1 generation was skewed towards male as compared with a 1:1 ratio in the control group of insects reared on castor leaves as well as chickpea diet. Sex distortion appears to be general phenomenon 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 chromosome in females but not in ZZ males (Marec 1990). The effect of irradiation on population characteristics was not observed in the P generation, especially 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 F1 and F2 generations, particularly the production of females. The production of more males than females, and the reduction in female longevity due to sublethal radiation ultimately reduced the net reproductive rate and the potential rate of increase. Our data suggest that both inability of irradiated males to fertilize eggs and dominant lethal mutations (DLM) induced in sperm were responsible 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 unfertilized, indicating physiological damage in irradi- Fig. 1. Effect of substerilizing gamma-radiation ated males that reduced the ability to transfer doses on the development of eggs derived from the sperm. Alternatively, these eggs could have

crosses of treated Spodoptera litura males and their F1 ceased their development at an early stage due to progeny, reared on castor leaves and semi-synthetic induced DLM (see discussion in Marec et al. diet. Bars show mean ± SE; EM, early mortality in eggs; 1999). Whereas in F1 crosses, sterility was largely LEL, late embryonic lethality. standard error of the a result of induced chromosomal aberrations. mean (SE). N, normal moth; P, treated parent; F , prog- 1 Therefore, eggs from these crosses showed embry- eny of treated males; m, male; f, female. ns, non-significant at P < 0.05; *, significant at P < 0.05; **, significant onic development but were unable to hatch, indi- at P < 0.01 (t-test conducted between and LEL in each cating late egg lethality (LEL) as the main cause cross). of egg inviability. Similar findings were made by Proshold & Bartell (1970) in the tobacco budworm, Heliothis virescens, and by Bughio (1988) phores that contain little or no sperm; (iv) abnor- in Chilo partellus. LEL was clearly expressed by mal sperm structure, which fails to fertilize the F1 males, whereas EM was expressed less, unlike eggs (Ashrafi & Roppel 1973), or (v) inheritance of in P males of the codling moths (Anisimov et al. special chromosome rearrangements (LaChance 1989). Similarly, Seth & Reynolds (1993) reported

1985; Anisimov et al. 1989). that the main cause of sterility in F1 generation of

The developmental rate of F1 larvae originat- Manduca sexta was the induction by radiation of ing from the crosses between treated males and lethal mutations that arrested the development normal females of S. litura was slower than that in late embryonic life. Also, Marec et al. (1999) of controls, and this delay in development was observed sterile eggs (eggs with early embryonic greater when males were treated with 130 Gy. Be- lethality as well as unfertilized eggs) and inviable cause insect development and differentiation are eggs (in which embryos died during different controlled by hormones (Gilbert 1964), the pro- stages of embryogenesis) while studying the tracted development of F1 larvae might be due to gamma radiation induced sterility combined with alteration in hormonal or enzymatic production genetic sexing in Ephestia kuehniella. caused by chromosomal rearrangements, as indi- In view of the overall reproductive perfor- cated by Proshold & Bartell (1970, 1972). The F1 mance of P and F1 moths, and developmental be- 192 Florida Entomologist 84(2) June 2001

havior of F1 insects, our ﬁndings suggest the use son of inherited deleterious effects in progeny from of 100 Gy as an effective dose for the suppression irradiated males and females. J. Econ. Entomol. 79: of S. litura populations by the release of partially 46-49. CARPENTER, J. E., J. R. YOUNG, A. N. SPARKS, H. L. sterile males. This dose gives better viability of F1 insects among normal population and thus, a CROMROY, AND M. A. CHOWDHURY. 1987. Corn earworm (Lepidoptera: Noctuidae): effects of substeril- higher inherited sterility effect. Although the izing doses of radiation and inherited sterility on higher dose (130 Gy) could be more effective in P reproduction. J. Econ. Entomol. 80: 483-489. crosses, it would less effective in the F1 crosses. It CHARI, M. S., AND N. G. PATEL. 1983. Cotton leafworm is worth noting that degree of irradiation impact Spodoptera litura (Fabr.): its biology and integrated on the net reproductive rate of P and F1 crosses control measures. Cotton Development 13: 7-8. observed in their life tables was almost similar to CHENG, W., AND D. T. NORTH. 1972. Inherited sterility in the F progeny of irradiated male pink boll worms. the control of reproductive potential calculated. 1 Proper sperm competition is required for the J. Econ. Entomol. 65: 1272-1275. treated insects to induce reproductive suppres- EL-SAYED, E. I., AND J. B. GRAVES. 1969. Effects of gamma radiation on the tobacco budworm. II. Irradi- sion of the feral pest population by using F1 steril- ation of moths. J. Econ. Entomol. 62: 289- 293. ity. Studies on sperm formation, transfer and FLINT, H. M., AND E. L. KRESSIN. 1969. Transfer of their competitiveness are in progress (RKS). sperm by irradiated Heliothis virescens (Lepi- doptera: Noctuidae) and relationship to fecundity. ACKNOWLEDGMENTS Can. Entomol. 101: 500-507. GILBERT, L. I. 1964. Physiology of growth and develop- The author (RKS) is grateful to the Interna- ment: Endocrine aspects, pp. 150-225. In M. Rock- tional Atomic Energy Agency for supporting this stein (ed.). The Physiology of Insects, Vol. I. research work under Research Contract No. Academic Press, New York. 640 pp. IAEA/IND-7162/RB in a coordinated research HOWE, R. W. 1953. The rapid determination of the intrinsic rate of increase of an insect population. Ann. programme. Thanks are due to technical assis- Appl. Biol. 40: 134-151 tance offered by D. K. Rao and J. J. Kaur. ISMAIL, B. B. 1994. Radiation-induced changes in reproductive ability of diamond back moth (Lepidoptera, REFERENCES CITED Plutellidae). J. Econ. Entomol. 87: 1190-1197. KNIPLING, E. F. 1970. Suppression of pest Lepidoptera ABBOTT, W. S. 1925. A method for computing the effective- by releasing partially sterile males: A theoretical ap- ness of an insecticide. J. Econ. Entomol. 18: 265-267. praisal. BioScience 20: 465-470. ANISIMOV, A. I., N. V. LAZURKINA, AND A. N. SHVEDOV. LACHANCE, L. E. 1985. Genetic methods for the control 1989. Inﬂuence of radiation induced genetic damage of lepidopteran species: status and potential. ARS- on the suppressive effect of inherited sterility in the 28, USDA, Washington, DC. 40 pp. codling moth (Lepidoptera: Tortricidae). Ann. Ento- LACHANCE, L. E., R. D. RICHARD, AND C. BELICH. 1976.

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194 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 Baños, College, Laguna 4031, Philippines

ABSTRACT In this article we report the results of experiments conducted with Helicoverpa armigera (Hübner) 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 1(:1& and a 4(:1& ratio were used while keeping a constant density of moths per cage. The mean number 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 untreated males and then re-exposed to untreated males 24 hours later. No statistical differences 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-exposed 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 untreated male (31.7%), although the differences were not significant.

Key Words: F1 sterility, partial sterility, remating, corn earworm

RESUMEN En este articulo se reportan los resultados de experimentos con la especie Helicoverpa armigera. El objetivo de estos experimentos 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 liberacion dentro de jaulas de campo colocadas en una plantacion de repollo. Las tasas de liberacion utilizadas fueron 1 macho:1 hembra y 4 machos:1 hembra mientras se mantuvo el numero de insectos constante dentro de las jaulas. El numero promedio de copulas que se recobraron en cada jaula no fue signiﬁcativamente distinto, lo que sugiere que los machos irradiados pueden competir efectivamente con los machos fertiles. En estudios sobre propensidad de re-copula, hembras virgenes fueron apareadas con machos fertiles o irradiados y luego fueron re-expuestas a machos fertiles despues de 24 horas. No se encontro una diferencia estadistica en el porcentaje de re-copulas entre los grupos de hembras. Treinta punto ocho porciento de las hembras que primero se aparearon con machos irradiados re-copularon 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 (inicialmente apareadas con distintos tipos de machos) simultaneamente a machos fertiles despues 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 insect that will actively mate with wild females (Hubner), is one of the most destructive pests of and, as such, will effectively introduce heritable agricultural crops in the Philippines. It is a highly sterility into the native population (Mastro & polyphagous insect, feeding on 84 different plant Schwalbe 1988). Inherited sterility has been in- species (Gabriel 1997). One environmentally ac- vestigated in a number of lepidopteran pests such ceptable control strategy currently being explored as Helicoverpa zea (Carpenter & Gross 1993), for the control of this insect is the release of irra- Spodoptera frugiperda (Carpenter et al. 1997), diated partially sterile males capable of trans- Manduca sexta (Seth & Reynolds 1993) and Ephe- ferring sterility to the next generation. The stia kuehniella (Marec et al. 1999). advantages of using partial, inherited or F1 steril- Helicoverpa armigera, a closely related species ity over classical sterile insect release methods is of H. zea (Laster & Hardee 1995), is a potential that partial sterility produces a more competitive candidate for population suppression by means of

Ocampo: Irradiation and mating in H. armigera 195

60 the F1 sterility technique. Ocampo et al. (1996) using a Cobalt Gammacell 220 irradiator deliver- were able to induce partial sterility in this species ing a dose of 4 Gy/min. Treated and untreated pu- by irradiating pharate adult males with 100 and pae were held separately until adult emergence. 150 Gy. When the treated males were mated to Newly emerged (<24 h-old) virgin male and fe- untreated females, female fertility was greatly af- male moths were used in all mating experiments. fected. Percentage egg hatch in females mated to 100 Gy or 150 Gy treated males was only 40-60% Mating Competitiveness of Males while that of females mated to untreated males was 80-100% (V. Ocampo, unpublished data). In a Male competitiveness was assessed in field- related study, Bella et al. (1997) reported that cages (n = 4; 6m × 5m × 2.15m) placed over cab- when 100 Gy was used to treat H. armigera bage plants within 1-ha cabbage field. Three males, the dose caused chromosomal transloca- types of males were used in the experiments. tions in 86% of the spermatocytes examined while Males treated with 100 Gy (T(), as indicated 150 Gy caused chromosomal translocations in above; F progeny males (TF (), obtained from 98% of the spermatocytes in the testes of treated 1 1 crossing treated (100 Gy) P1 males to untreated males. Similarly, 100 Gy has been shown to be virgin females, and untreated males (N(). In or- most efficacious in the codling moth, Cydia der to distinguish the male “types” the wing tips pomonella (Anisimov 1993) and the fall army- of treated and F1 males were differentially colored worm, Spodoptera frugiperda (Carpenter et al. with a felt-tip marker and untreated males were 1997). In these species, the dose induced a high left unmarked. Moths were allowed to acclimate level of inherited sterility in the F1 generation to field conditions for 3 hours (from 1700 hours without reducing competitiveness of parental Philippine Standard Time (PST) until they were males treated. released at 2000 hours PST). Males and females Mating competitiveness is essential to the ef- were released at opposite ends of the cages. In fectiveness of a sterile insect release program. each cage mating pairs were collected once every However, the refractory period of wild females hour for five hours, from 2100 to 0200 hours PST, must not be adversely affected by mating with the coinciding with the peak of mating activity as re- released males (Snow 1988). An irradiated male ported by Morallo-Rejesus and Alcala-Carilo must be able to transfer a full complement of (1981). These workers also report that copulation sperm and accessory gland fluid to the female, lasted an average of 30 minutes. and this complement must reach the spermath- Mating competitiveness was measured at two eca to elicit the female refractory period. Giebul- different release ratios. A 1(:& ratio, maintain- towicz et al. (1990) showed that presence of sperm ing equal numbers of male “types” while keeping in the spermatheca is necessary to induce the a density of 60 moths per cage. Field-cages in this switch from virgin to mated behavior in the gypsy study received 30 N( each, and the following moth, Lymantria dispar. During the refractory male treatments: 30 T(; 30 N(; 15 T( + 15 N(; period, pheromone production by the female re- 10 T( + 10 TF1( + 10 N(. The next set of exper- mains low, oviposition behavior is triggered and iments was conducted at a 4(:1& ratio, while the female will refrain from remating. If sperm keeping a constant density of 50 moths per cage. and accessory gland fluid do not reach the sper- Field-cages in this study received 10 N& each, matheca, pheromone production will increase and and the following male treatments: 40 T(; 40 N(; the female will resume calling for mates. 30 T( + 10 N(. All combinations were replicated In this article we report the results of experi- once per night for four nights. Male types with the ments conducted with H. armigera to determine highest number of recorded matings were consid- the effects of 100 Gy of gamma radiation on the ered the most competitive. Data were subjected to mating competitiveness of treated males and the analysis of variance and differences between effect on the mating propensity of the females means were tested for significance using Waller- with which they mate. Duncan’s K-ratio t-test (Ott 1993).

MATERIALS AND METHODS Mating Propensity of Females

Helicoverpa armigera used in these experi- Mating propensity was assessed inside ﬁeld- ments came from a laboratory colony maintained cages (see above). Insects were released into sep- at the Department of Entomology, University of arate cages in the following combinations: 100 the Philippines Los Baños. Larvae were reared untreated H. armigera females (N&) + 100 under ambient laboratory conditions (27 ± 2°C) in treated males (T(-100 Gy), and 100 N& + 100 un- 50 ml plastic cups and fed a soybean-corn based treated (N() males. Pairs in copula were collected diet as described in Ocampo et al. (2000). Pupae once per hour as described above. Females that were collected and sexed and stored at ambient mated with treated (N&-T() and untreated (N& conditions until needed. Late pupae containing - N() males were differentially colored using a pharate males were irradiated at a dose of 100 Gy felt-tip marker as above. On the following

196 Florida Entomologist 84(2) June 2001 evening, the mated females (from both groups) Mating Propensity of Females were given the opportunity to remate with untreated males (N() by releasing them into field- The results of the mating propensity studies, cages in the following combinations (at 1&:1( ra- where virgin female H. armigera were first mated tio): 30 N&-N( + 30 N(, 30 N&-T( + 30 N(, 15 to treated or untreated males and then re-ex- N&-N( + 15 N&-T( + 30 N(. Pairs in copula posed to untreated males 24 hours later are were collected as above. All treatments were rep- shown in Table 2. Thirty point eight percent of the licated four times. Females were then dissected to females first mated with treated males (N&-T() determine the number of spermatophores in the and 29.17% of the females first mated to un- bursa copulatrix. A twice-mated female should treated (N&-N() males re-mated in this study. have two spermatophores to confirm two success- The difference was not statistically significant (F ful matings/copulations. = 0.04, d.f. = 6, α = 0.05). When both types of females were re-exposed to untreated males in the same field-cage (15 N&-T( + 15 N&-N( + 30 RESULTS N(), a higher percentage (38.3%) of females that had initially mated with a treated male re-mated Mating Competitiveness of Males than those initially mated with a untreated male (31.7%), although the differences were not signif- The results of the mating competitiveness tests α are summarized in Table 1. At the 1&:1( ratio, icant (F = 0.25, d.f. = 6, = 0.05). Carpenter et al. the mean number of mating pairs collected in (1987) report similar trends for Helicoverpa zea, a cages containing treated (T() and untreated closely related species to H. armigera. (N() males was not significantly different (F = 1.07, α d.f. = 6, = 0.05). In addition, when both male DISCUSSION types were together in the same cage (30 N&:15 T(:15 N(), both T( and N( males appeared to An important concern in sterile insect release be equally competitive. The mean number of mat- programs is that treated males destined for re- ings for N( was 4.25 and this number was 3.76 lease retain the ability to perceive/orient to pher- for T( (F = 0.17, d.f. = 6, α = 0.05). Mating com- omone signals from females and, as such, are able

petitiveness of F1 male offspring (TF1() was also to compete with the wild males in locating and evaluated. Results show that the mean number of mating with calling females in the field. Data pre- matings for TF1( was 4.76, which did not differ sented herein suggest that partially sterilized statistically from the number of matings with male H. armigera and their male progeny were as other male types (F = 0.21, d.f. = 9, α = 0.05). competitive as untreated males in seeking and se- When experiments were conducted at a 4(:1& curing mates in a field-cage situation. Males ratio, there was no significant difference in the treated with 100 Gy, F1 males (from 100 Gy mean number of mating pairs collected in cages treated fathers) and untreated males appeared to containing T( (7.00) or N( (6.00) males (F = 0.46, be equally as competitive in mating with virgin d.f. = 6, α = 0.05). When three times more treated females when placed together in field-cages. It ap- males than untreated males were released into pears that a dose of 100 Gy of gamma radiation the field-cage (10 N&:10 N(:30 T() the number does not cause sufficient physiological damage to of observed matings for the treated males signifi- alter male mating behavior, but induces sufficient cantly exceeded the expected number (F = 216, genetic damage in the spermatocytes to reduce d.f. = 6, α = 0.05). sperm viability, as reported by Bella et al. (1997).

TABLE 1. MATING COMPETITIVENESS OF TREATED (100 GY-T(), UNTREATED (N() AND F1 OFFSPRING (TF1() OF HELI- COVERPA ARMIGERA MALES FOR VIRGIN FEMALES INSIDE FIELD-CAGES (6M × 5M × 2.15M) IN A CABBAGE FIELD AT UPLB, LAGUNA, PHILIPPINES, 1996.

Mean number of mating pairs (±SD)1

× × × Ratio Number and type of moths N NN TN TF1

1(:1& 30 N&:30 N( or 30 N&:30 T( 10.00 ± 2.00a 12.75 ± 4.92 a –2 30 N&:15 T( + 15 N( 4.25 ± 2.22a 3.76 ± 0.96 a –2 ± ± ± 30 N&:10 T( + 10 TF1( + 10 N( 6.25 4.50a 5.25 3.30 a 4.76 1.26 a 4(:1& 10 N&:40 N( or 10 N&:40 T( 6.00 ± 2.31a 7.00 ± 1.82 a –2 10 N&:30 T( + 10 N( 0.50 ± 0.58a 6.50 ± 0.58 b –2

1Means ± SD on the same row followed by the same letter are not significantly different (K ratio = 500). 2Not applicable. Ocampo: Irradiation and mating in H. armigera 197

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

Mean number of re-matings (±SD) 24 h after the ﬁrst mating (percent)1

Moths released into ﬁeld-cages N&-N( × N( N&-N( × N(

30 N&-N(:30 N( or 30 N&-T(:30 N( 8.75 ± 3.86 (29.17) a 9.25 ± 2.99 (30.83) a 15 N&-T(:15 N&-N(:30 N( 4.75 ± 2.87 (31.67) a 5.75 ± 2.75 (38.33) a

1Means ± 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 REFERENCES CITED sterility in the F1 male progeny of males treated with 100 Gy without reducing their capacity to ANISIMOV, A. I. 1993. Study of mechanisms and possibil- seek and secure mates. The competitiveness of ities of using F1 sterility for genetic control of codling other irradiated Lepidopterans has been reported moth, pp. 135-155. In Proc. Final Research Coordi- nation Meeting: Radiation Induced F Sterility in by Carpenter et al. (1989, 1997) and Anisimov 1 Lepidoptera for Area-Wide Control, Phoenix, Ari- (1993). In these species, 100 Gy has also been zona, 1991. International Atomic Energy Agency, shown to be most efficacious. Carpenter et al. Vienna, STI/PUB/929. (1989) further showed that the irradiated H. zea BELLA, A. P., G. L. JERUSALEM, A. A. BARRION, V. R. males were competitive with normal laboratory OCAMPO, AND J. B. DE LEON. 1997. Gamma-ray reared insects in their ability to survive under induction of translocations in the corn earworm, field conditions. Helicoverpa armigera (Hübner) (Lepidoptera: Noc- Helicoverpa armigera has a tendency to mate tuidae). Philippine Ent. 11: 65-69. more than once. In our experiments, about 30% of CARPENTER, J. E., A. N. SPARKS, AND H. L. CROMROY. mated females re-mated regardless of the type of 1987. Corn earworm (Lepidoptera: Noctuidae): In- fluence of irradiation and mating history on the mat- male with which they had first mated (Table 2). ing propensity of females. J. Econ. Entomol. 80: These findings agree with an earlier study 1233-1237. (Ocampo et al. 2000) that found 31-36% of H. ar- CARPENTER, J. E., A. N. SPARKS, S. D. PAIR, AND H. L. migera females mated twice. Our data also sug- CROMROY. 1989. Heliothis zea (Lepidoptera: Noctu- gests that females mated to treated (100 Gy) idae): Effects of radiation and inherited sterility on males were no more attractive to untreated males mating competitiveness. J. Econ. Entomol. 82: 109-113. on the night following the first mating than were CARPENTER, J. E., AND H. R. GROSS. 1993. Suppression females mated to untreated males. One explana- of feral Helicoverpa zea (Lepidoptera: Noctuidae) tion for this finding might be that there is no dis- populations following the infusion of inherited sterility from release substerile males. Environ. Entomol. cernible difference in the quality of the sperm 22: 1084-1091. complement and accessory fluid transferred by CARPENTER, J. E., HIDRAYANI, AND B. MULLINIX. 1997. untreated and treated males during mating. Effect of substerilizing doses of radiation on sperm Thus, the type of male with which the female precedence in fall armyworm (Lepidoptera: Noctu- mates first does not affect her mating propensity idae). J. Econ. Entomol. 90: 444-448. and refractory period. GABRIEL, B. P. 1997. Insects and Mites Injurious to Phil- ippine Crop Plants. National Crop Protection Cen- ter. UPLB, College, Laguna. 171 pp. GIEBULTOWICZ, J. M., A. K. RAINA, AND E. C. UEBEL. ACKNOWLEDGMENTS 1990. Mated-like behavior in senescent virgin females of gypsy moth, Lymantria dispar. J. Insect This research was supported by Research Con- Physiol. 36: 495-498. tract No. 8156/RB under the FAO/IAEA Coordi- LASTER, M. L., AND D. D. HARDEE. 1995. Inter-mating nated Research Program entitled “Evaluation of compatibility between North American Helicoverpa Population Suppression by Irradiated Lepidop- zea and Helicoverpa armigera (Lepidoptera: Noctu- tera and Their Progeny” of the International idae) from Russia. J. Econ. Entomol. 88: 77-80. Atomic Energy Agency, Vienna, Austria. The au- MAREC, F., I. KOLLAROVA, AND J. PAVELKA. 1999. Radi- thor is very grateful to the two anonymous re- ation-induced inherited sterility combined with a ge- viewers for valuable suggestions on the netic sexing system in Ephestia kuehniella (Lepidoptera: Pyralidae). Ann. Entomol. Soc. Amer- manuscript. The author would also like to thank ica 92: 250-259. Ms. Josephine B. de Leon, Ms. Gaudencia M. MASTRO, V. C., AND C. P. SCHWALBE. 1988. Status and Añober and Ms. Rosita M. Crisologo for their potential of F1 sterility for control of noxious Lepi- technical support during the conduct of field-cage doptera, pp. 15-40. In Proc. Symp.: Modern Insect experiments. Control: Nuclear Techniques and Biotechnology. 198 Florida Entomologist 84(2) June 2001

IAEA/FAO, Vienna, Austria, 16-20 November 1987. armigera (Hübner) (Lepidoptera: Noctuidae) in the International Atomic Energy Agency, Vienna. Philippines. Philippine Agric. Sci. 83: 282-291. MORALLO-REJESUS, B., AND E. ALCALA-CARILO. 1981. OTT, R. L. 1993. Introduction to the analysis of variance The attacus juvenile hormone studies. II. Steriliza- and multiple comparisons, pp. 769-841. In An Intro- tion of Helicoverpa armigera armigera (Hübner) duction to Statistical Methods and Data Analysis with attacus juvenile hormone and dimilin. Philip- 4th edition. Duxbury Press, Belmont, CA. 1051 pp. pine Entomol. 4: 389-403. SETH, R. K., AND S. E. REYNOLDS. 1993. Induction of in- OCAMPO, V. R., J. B. DE LEON, AND M. T. TABUR. 1996. herited sterility in the tobacco hornworm Manduca Mass-rearing and effects of substerilizing doses of ra- sexta (Lepidoptera: Sphingidae) by substerilizing diation on the corn earworm, Helicoverpa armigera doses of ionizing radiation. Bull. Entomol. Res. 83: (Hübner) and its progeny, p. 51. In Working Material, 227-235. Second FAO/IAEA Research Coordination Meeting: SNOW, J. W. 1988. Radiation, insects and eradication in Evaluation of Population Suppression by Irradiated North America. An overview from screwworm to Lepidoptera and Their Progeny, Vienna, Austria, 2-6 bollworm, pp. 8-10. In Proc. Symp.: Modern Insect September 1996. IAEA-D4-RC-561. 57 pp. Control: Nuclear Techniques and Biotechnology, OCAMPO, V. R., M. T. TABUR, R. D. FULGENCIO, A. C. Vienna, Austria, 16-20 November 1987. Interna- LIM, AND F. D. OROZCO. 2000. Diet development and tional Atomic Energy Agency, Vienna. mass rearing of the corn earworm, Helicoverpa

Nguyen Thi & Nguyen Thanh: F1 sterility in DBM suppression 199

RADIATION INDUCED F1 STERILITY IN PLUTELLA XYLOSTELLA (LEPIDOPTERA: PLUTELLIDAE): POTENTIAL FOR POPULATION SUPPRESSION IN THE FIELD

NGUYEN THI QUANG HOA AND NGUYEN THANH 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 P1, F1, and F2 generations. Sterility exceeded 60% in the P1

and F2 generations and 90% in the F1 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 F1, F2, and F3, 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 F1 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 Fl adult emergence was

not signiﬁcantly different than for the control. However, adult emergence in the F2 generation 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 of F1 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 F1 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 populations of DBM.

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

RESUMEN

El uso potencial de la esterilidad F1 como método de control para Plutella xylostella (L.) (DBM, “diamond back moth”) se investigo en estudios de laboratorio y de campo. Cuando pupas macho de 6 días de edad se irradiaron con 200 Gy de radiación gamma, el 71.5% se de-

sarrollaron como palomillas aparentemente normales. Sin embargo, en las generaciones P1,

F1 y F2 las reducciones en fecundidad y viabilidad fueron obvias. El nivel de esterilidad ex-

cedió el 60% en las generaciones P1 y F2 y el 90% en la F1. En la progenie F1 y F2, la tasa sexual favoreció 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 F1, F2, y F3, respectivamente. No se ob- servaron diferencias entre machos irradiados y no irradiados en su habilidad en transferir esperma. Cuando se liberaron machos irradiados en jaulas de campo junto con palomillas

fértiles en las tasas 5:1 o 10:1, el numero de adultos en las generaciones F1 y F2 se redujo sig-

niﬁcativamente. La reducción fue de 50-60% en la generación F1 y 59-68% en la generación

F2. Cuando se liberaron insectos irradiados de ambos sexos en la tasa 5:5:1:1 con palomillas

fértiles, la reducción en el nivel poblacional de la generación F1 no fue diferente de lo obser-

vado en la jaula control. Sin embargo, se observo una reducción del 90% en la generación F2.

El uso combinado de la esterilidad F1 con liberaciones de Cotesia plutellae (Kurdjumov) (Hi- menóptera: Braconidae) en jaulas de campo causaron que la población de DBM disminuyera

mas del 40% en la F1 y mas de 90% en la generación F2. Sin embargo, sugerimos que es ne- cesario continuar las investigaciones para lograr que este sistema sea económicamente fac- tible para el control de las infestaciones tempranas de DBM.

The use of inherited or F1 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

200 Florida Entomologist 84(2) June 2001

males. Sutrisno et al. (1993) and Sutrisno & Hoe- cein, sealed with clear nail polish and stored in a daya (1993) suggested that doses of 175 Gy or 200 covered container in the refrigerator until exam- Gy could be considered in a DBM suppression pro- ined for the presence of chromosomal aberrations. gram. Preliminary results relating to the potential suppression of DBM populations in the ﬁeld Sperm Transfer

by the F1 sterility technique are reported herein. Irradiated and unirradiated DBM adult males MATERIALS AND METHODS were allowed to mate with unirradiated virgin females inside laboratory cages. Mating pairs were Laboratory Colonies collected and mating was allowed to proceed until the insects separated. Males were reintroduced Diamondback moth. Field-collected DBM pu- into the cages and exposed to virgin females after pae from cabbage were used to found the colony. 24 h for 5 consecutive days. Females from each Emerging adults were paired in oviposition cages mating pair were dissected after oviposition to de- and provided with a 10% sucrose solution. termine the presence of a spermatophore and to Grooved aluminum foil egg sheets that had been assess the ratio of eupyrene to apyrene sperm dipped in autoclaved cabbage juice were used as present in the spermatheca. The normal ratio of oviposition substrates. Egg sheets were changed eupyrene to apyrene is considered to be 2:1 for Tri- daily. Neonates were reared on leaves of Tropae- choplusia ni according to North & Holt (1971). Any olum majus L. (Rhoedales: Tropaeolaceae), a wild ratio deviating from this is considered abnormal. host of DBM. The colony was maintained at 20 ± 2°C, 70-80% RH and a 14L:10D photoperiod. The Effect of Mating Status and Type black stripe pupal mutant (BSP) is a marker and Ratio of Sperm on DBM Oviposition present in our DBM laboratory colony (see below). Parasitoids. A colony of Cotesia plutellae (Kur- Unirradiated males were allowed to mate with djumov) (Hymenoptera: Braconidae), reared on unirradiated virgin females in laboratory cages. DBM larvae is maintained at our laboratory in Mating pairs were removed from the cages. After Dalat. Laboratory conditions are the same as for separation, females were allowed to lay eggs in the DBM colony. The colony was founded with the laboratory. When oviposition was completed field-collected material. females were dissected to determine the presence of sperm and the ratio of apyrene:eupyrene sperm Effects of Gamma Radiation on DBM in the spermatheca. Virgin females were also held for oviposition as controls. Six day-old colony pupae were irradiated with a dose of 200 Gy in a Co60 irradiator at the Dalat BSP Mutant Nuclear Research Institute (dose rate 43.2 Gy/ min). Insects were used in laboratory and field- Individuals in our DBM laboratory colony pos- cage tests. Percent emergence, percentage of sess the black stripe pupa or BSP mutant. We moths without deformities and longevity of irra- have isolated and maintained a vigorous and ho- diated versus untreated DBM adults were re- mozygous mutant culture using the method of corded in the laboratory. An average of 200 pupae Bartlett & Raulston (1982). Mutant individuals were used in each of three replicates for the first were inbred and a small colony was established. two parameters. A subset of 100 adults in each of We attempted to determine the mode of inherit- three replicates were used in the longevity study. ance of the BSP mutant using the method of Walder (1988). Reciprocal crosses were made be- Treated DBM males and females (from the P1, F and F generations) were crossed with un- tween mutant (BSP) females and males, and wild 1 2 + treated counterparts and allowed to mate and lay type stock (BSP ). Some F1 adults from the initial eggs. Fecundity, fertility (percent sterile eggs), crossed were used in reciprocal back-crosses with percent surviving to adulthood and sex ratio were the mutant strain and the rest were inbred. recorded for all crosses. For fecundity and fertility data, an average of 20 moth pairs were used per Evaluation of F1 Sterility replicate and three replicates were completed. for Suppression of DBM in Field-Cages For percent survival, an average of 100 larvae per replicate were used. Cabbage was planted inside four field-cages (2m × 2m × 4m); plants were irrigated regularly Chromosomal Aberrations and received no other treatments during the experiments. Six-day old colony (BSP) pupae were

Testes of fourth instar DBM larvae from the F1, irradiated as indicated above and pupae were

F2 and F3 generations were dissected in Belar’s sa- sexed and allowed to emerge into separate con- line solution and then squashed to release the tainers. A wild strain (BSP+ = pupae with no black germ cells. The slides were stained with aceto-or- stripe) was collected from the ﬁeld as 4th instar

Nguyen Thi & Nguyen Thanh: F1 sterility in DBM suppression 201 larvae and allowed to pupate and emerge in the means were tested for significance using Tukey’s laboratory. One-day old adults from both groups Honest Significant Difference (HSD) (Minitab 9.2; were used in the experiment. Minitab Statistical Software). Deviations from ex- Field-cages received the following ratios of ir- pected ratios were tested by chi-square analysis. radiated BSP colony females (IF), irradiated BSP colony males (IM), feral BSP+ females (UF) and RESULTS AND DISCUSSION feral BSP+ males (UM), respectively: Treatment A = 0:0:1:1 (control), Treatment B = 0:5:1:1, Treat- Effects of Gamma Radiation on DBM ment C = 0:10:1:1 and Treatment D = 5:5:1:1. Cages were checked every two days and eggs de- The results of our study on the effect of 200 Gy posited on the cabbage were tagged and counted. on percent emergence, percentage of adults with- The population increase in each field-cage was de- out deformities and longevity of DBM are pre- termined by counting the number of eggs, pupae sented in Table 1. In general, DBM females and adults for two generations. Three replications appear to be more sensitive to gamma radiation were completed. than males. When 6-day old pupae were treated with 200 Gy, 71.5% of the male pupae developed Combined Releases of Irradiated DBM as normal adults and 65.3% of these survived to and Cotesia plutellae in Field-Cages the 6th day, while the corresponding percentages Cotesia plutellae (CP) parasitoids from our col- for treated females were 70.6 and 46.5. Adult lon- ony (see above), one day-old 200 Gy treated colony gevity of both males and females was reduced by (BSP) DBM adults (I) and unirradiated wild about one-third as compared to controls. (BSP+) moths (U) were released into field-cages in Table 2 shows the results of measuring fecun- the following ratios: Treatment 1 = 0 CP: 0 I: 1 U dity, sterility, percent survival and sex ratio of DBM irradiated with 200 Gy and their F and F (control), Treatment 2 = 5 CP: 0 I: 1 U, Treatment 1 2 3 = 0 CP: 5 I: 1 U, and Treatment 4 = 2.5 CP: 2.5 progeny. Significant differences were found in all I: 1 U. In Trt. 4, the release of one day-old irradi- measured parameters when compared to controls. These significant effects persisted in the F ated moths occurred at day one and was followed 1 and F progeny. In the P generation, the mean ten days later with the release of one-day old par- 2 1 asitoid adults. Field-cages were checked every number of eggs laid by the controls was 154. This two days and the cabbage leaves on which eggs number decreased to an average of 84 eggs when had been deposited were tagged and eggs were the female was crossed to an irradiated male. counted. The size of the DBM population inside Further, the number of eggs produced by untreated females mated to F males was reduced to the field-cages was determined by counting DBM 1 eggs, pupae and adults. The size of the parasitoid about one-fifth the number laid by the controls. However, when F males were mated to untreated population was determined by counting the num- 2 ber of parasitoid cocoons present in each cage. females, the number of eggs produced per female was similar to the number produced when irradi- Statistical Analysis ated males were mated to normal females. The sterility of treated males was 62.1%, while that of

Data were subjected to either the pooled t-test F1 males was 94.8% and that of F1 females was

or to analysis of variance. Differences between 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 emergence1 without deformities1 to day 62 (days)3

Control Male 94.1 ± 3.1a 89.3 ± 2.7a 84.2 ± 5.3a 12.3 ± 2.4a Female 95.2 ± 1.8a 91.1 ± 5.8a 85.1 ± 4.6a 11.9 ± 1.4a 200 Gy Male 85.4 ± 6.3a 71.5 ± 7.2a 65.3 ± 7.4b 8.9 ± 0.8a Female 83.2 ± 4.1a 70.6 ± 7.8a 46.5 ± 8.5a 7.9 ± 1.0a

Means followed by the same letter within a column for each treatment are not signiﬁcantly different (P > 0.05; pooled t-test). 1Average of 200 pupae, 3 replications. 2Moths, which did not survive up to the sixth day usually, were not able to mate. 3Average of 100 moths, 3 replications. 202 Florida Entomologist 84(2) June 2001

TABLE 2. FECUNDITY, STERILITY, PERCENT SURVIVAL AND SEX RATIO OF PLUTELLA XYLOSTELLA IRRADIATED WITH 200

GY AS 6-DAY-OLD PUPAE AND THEIR F1 AND F2 PROGENY.

Mean ± SD

Sterility Fecundity (% of eggs that Survival Ratio of Mating type (total # of eggs)1 failed to hatch)1 (%)2 males/females

P1 generation UM × UF 156 ± 10a 12.7 ± 7.8a 78.5 ± 8.9b 1.01 ± 0.01a IM × UF 84 ± 3b 62.1 ± 6.8b 57.8 ± 7.6a 1.98 ± 0.07b

F1 generation UM × UF 145 ± 8b 10.3 ± 0.6a 85.1 ± 2.2c 0.97 ± 0.05a A × ± a ± b ± a ± b F1M UF 33 4 94.8 1.3 11.1 3.7 1.78 0.20 B × ± a ± b ± b ± b F1F UM 28 2 91.1 2.0 32.1 10.5 1.63 0.17

F2 generation UM × UF 140 ± 7b 11.4 ± 3.4a 75.7 ± 78a 1.03 ± 0.14a × ± a ± b ± a ± a F2AM UF 84 3 63.5 7.1 64.7 9.7 1.18 0.11 × ± a ± b ± a ± b F2AF UM 80 7 61.8 8.2 65.3 13.3 1.42 0.13 × ± a ± b ± a ± ab F2BM UF 87 6 64.8 11.8 62.9 12.3 1.25 0.12 × ± a ± b ± a ± b F2BF UM 86 3 62.4 5.6 70.1 10.6 1.34 0.10

I = irradiated, U = unirradiated. Means followed by the same letter within a column for each treatment are not signiﬁcantly different (P = 0.05; Tukey’s Honest Signiﬁcant Difference). 1Average of 20 pairs of moths, 3 replications. 2Percent survival of 100 neonate larvae to adult, 3 replications. A, B Male and female progeny from each F1 cross were followed separately.

61.8% to 64.8%. Survival to adulthood in progeny but might be explained by differences in DBM of untreated moths varied between 75-85%, while strain, rearing methods and dose calibration. it was 57.8% in the F1 generation, from 11.1% to

32.1% in the F2 and from 62.9% to 70.1% in the F3. Chromosomal Aberrations The sex ratio was biased in favor of males among

F1 and F2 progeny. Cytological examination of chromosomal aber- These results differ from those reported by Su- rations in the primary spermatocytes in 4th-instar trisno et al. (1993) and Sutrisno & Hoedaya larvae (Table 3) showed abnormal chromosomes (1993). We report higher levels of sterility than evident in 86.9%, 21.5% and 9.7% of larvae from those reported by these authors. In addition, the the F1, F2 and F3 generations respectively, while fecundity of untreated females mated to irradi- none were evident in the untreated controls. ated, F1 and F2 males was lower than for un- DBM have 31 pairs of chromosomes, individually treated controls in our experiments, while recognizable during metaphase-I. Reciprocal Sutrisno et al. (1993) and Sutrisno & Hoedaya translocations in the form of rings or chains, in- (1993) report that fecundity in these crosses was volving four or six chromosomes were observed in equal to that observed in the untreated controls. the F1 generation. These particular aberrations The reasons for these differences remain unclear, 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

F3 MALE PROGENY.

No. with chromosomal Frequency of aberrations Generation No. of nuclei examined1 aberrations (%)

F1 92 80 86.9

F2 79 17 21.5

F3 72 7 9.7 Control 77 0 0.0

1Testes 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. Nguyen Thi & Nguyen Thanh: F1 sterility in DBM suppression 203

Carpenter (1991) and Zhang et al. (1993) re- In Trichoplusia ni Holt & North (1970) report port that incidence of visible chromosomal aber- that the ability of fully sterile males to transfer

rations in F1 and F2 larvae of Helicoverpa zea and sperm was considerably lower than in unirradi- Ostrinia furnacalis is dose dependent, and that ated males. They found that when sperm were de- aberrations occur most frequently in the F1 and posited into the bursa copulatrix, the eupyrene progressively less frequently in subsequent gen- sperm lacked motility, while the apyrene sperm erations. North (1975) reported that male prog- were able to migrate to the spermatheca. In con- eny of H. zea treated with 200 Gy showed at least trast, treating DBM pupae with a substerilizing one translocation. Our observations on DBM dose of radiation (200 Gy) resulted in males able agree with results reported by these authors for to transfer normal ratios of eupyrene:apyrene other Lepidoptera. sperm. Our results for DBM are similar to those reported by El-Naggar et al. (1984) on Agrotis yp- silon, Carpenter et al. (1987) on Helicoverpa zea, Sperm Transfer and Sallam & Ibrahim (1993) on Spodoptera littoralis. Table 4 shows the percentage of matings on each of five consecutive days by irradiated (200 Gy) and unirradiated DBM males. This percent- Effect of Mating Status and Type age diminished for both groups with each succes- and Ratio of Sperm on Oviposition sive mating. Lepidopteran males produce apyrene and eupyrene sperm that is transferred North & Holt (1971) reported that when T. ni to the female during mating. Eupyrene sperm are females mated with an irradiated male they often nucleate and therefore capable of fertilization, failed to deposit a normal number of eggs. They while apyrene sperm are smaller and anucleate. suggested that this might be caused by inade- Both types are present in the spermatophore and quate sperm or accessory gland fluid transfer to migrate to the spermathecae in the female after the female during mating. Table 5 shows the ef- copulation is complete (North & Holt 1971). The fect of mating status and type and ratio of sperm percentage of treated and untreated DBM males on oviposition in DBM. Females that received a that transferred sperm in each of five consecutive normal sperm complement laid significantly matings is given in Table 4. No reduction in (twice as many) more eggs than those receiving sperm transfer was evident among the first, sec- an abnormal sperm ratio. It appears that type ond and third matings for either male group. The and quantity of sperm transferred significantly percentage of males that transferred a normal ra- influences fecundity in DBM females. tio of eupyrene to apyrene sperm remained fairly Holt & North (1970) reported that early in cop- high during the first three consecutive matings ulation the spermatophore in Trichoplusia ni for both groups. 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 tested1 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

1Irradiated and unirradiated males were allowed to mate with unirradiated virgin females on each of five consecutive days. Mating pairs were removed 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. 2The 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). 204 Florida Entomologist 84(2) June 2001

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:apyrene1 77 234 ± 26d Abnormal eupyrene:apyrene1 55 112 ± 16c No sperm1 32 73 ± 14b None (virgin females) 42 47 ± 11a

Means followed by a different letter within a column are significantly different (P = 0.05; Tukey’s Honest Significant Difference). 1Unirradiated 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 flooding ratio with unirradiated moths, there was laying of a significant number of eggs. This obser- a significant reduction in the number of F1 and F2 vation appears to hold true for DBM. In our ex- adults emerging in the field-cages as compared to periments mated females that received no sperm the control (Trt. A). A 50-60% reduction in the F1 laid significantly more eggs than virgin females, and 59-68% in the F2 generation were observed which suggests that fecundity in DBM is also for field-cages receiving Trt. B and C, respectively. influenced by the secretions from the accessory When irradiated females and males were re- gland transferred during mating. leased at a 5:5:1:1 overflooding ratio (Trt. D), the

decrease in Fl adult emergence was not signiﬁ- BSP Mutant cantly different than for the control (<3%). How-

ever, adult emergence in the F2 generation was Reciprocal crosses made between BSP and/or reduced by almost 90%. This degree of suppres- BSP+ virgin females and males confirm that the sion was significantly greater than that achieved gene responsible for the mutation black stripe in cages where only irradiated males had been re- pupa (BSP) is dominant and most likely located leased. Nonetheless, 1.4 times more neonates on an autosome. were produced by Treatment D in the F1 generation as compared to the control, and these neonates caused greater host plant damage. Evaluation of F1 Sterility for Suppression of DBM in Field-Cages Several investigators have formulated hypotheses concerning the role that released irradiated Evaluating the results of inherited sterility as females might play in the suppression of popula- a method for population suppression is particu- tions subjected to the sterile insect technique larly complex. Released partially sterile individu- (Whitten & Taylor 1970, Allam & Galun 1976). If als mate with released untreated individuals to partially sterile females are released they could produce partially sterile progeny. The progeny contribute a significant fraction of the progeny continue to do the same until the experiment is produced in the target population. This increase

stopped and effects are ascertained. Several in the number of F1 progeny may benefit a release marking techniques (such as radioisotopes, exter- program when the released females carry lethal nally applied fluorescent powders and internal oil chromosomal changes that will be transferred to soluble dyes) have been used to identify released the target population. However, the major sup- Lepidopteran insects. Unfortunately, these mark- pression of the target population is deferred to ers do not allow the descendants of a released (but greatly enhanced in) the second generation. insect to be tracked through subsequent genera- North & Holt (1971) suggested that the maximum tions. Bartlett (1967) suggested that mutations economic efficiency in the use of inherited steril- could be used as biological markers. Further- ity for the suppression of lepidopteran popula- more, he suggested that dominant and co-domi- tions requires the release of both sexes of nant mutants are most useful, as they can partially sterile moths. Our results support the identify not only the released individuals but also above analyses and findings. their progeny and descendants in subsequent In our field-cage studies BSP individuals were generations. The presence of the BSP mutant in identified among the progeny in all cages except + our DBM colony made it possible for us to exam- the control. The ratio of BSP to BSP in the F1 gen- ine the mating interactions of the irradiated DBM eration was 2.21, 3.41 and 8.94 in Treatments B, through several generations. C, and D, respectively. The ratio of BSP to BSP+ in

The results of our ﬁeld-cage experiments are the F2 generation was 0.11, 0.09 and 0.07 in Trts. shown in Table 6. When treated males (Trts. B B, C, and D, respectively (Table 6). All individuals and C) were released at either a 5:1 or a 10:1 over- expressing the black stripe pupal mutant were Nguyen Thi & Nguyen Thanh: F1 sterility in DBM suppression 205

TABLE 6. INFLUENCE OF RELEASING IRRADIATED (200 GY) AND UNIRRADIATED BSP PLUTELLA XYLOSTELLA ADULTS AT

DIFFERENT RATIOS ON THE DEMOGRAPHIC PARAMETERS OF THE F1 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

F1 generation A-control 3243 ± 767a 3037 ± 64c 1015 ± 167b 921 ± 5b 0 (0:0:50:50) B 2653 ± 667a 2238 ± 338b 542 ± 124a 459 ± 87a 2.21 (0:250:50:50) (50.16%) C 2397 ± 645a 1490 ± 166a 415 ± 90a 362 ± 51a 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 ± 198d 2345 ± 160c 2154 ± 145c 0 B 4246 ± 361a 3166 ± 323c 923 ± 74b 879 ± 82b 0.11 (59.19%) C 3619 ± 212a 1523 ± 105b 729 ± 70ab 684 ± 68b 0.09 (68.25%) D 4370 ± 466a 688 ± 270a 648 ± 47a 266 ± 43a 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). 1Values 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. 2BSP+ = wild moth and BSP = mutant moth. Three replications were completed.

descendants of released DBM, and it was these because of the lower fertility in the F1 as compared individuals that transmitted the sterility factors to the P1 generation. However, the lower fertility into subsequent generations. In fact, the ratios of in the F1 generation would call into question the BSP to BSP+ in the various ﬁeld-cages are the economic feasibility of this option.

ratios of substerile to fertile DBM in the F1 and F2 generations. The presence of BSP-marked indi- Combined Releases of Irradiated DBM viduals is the most compelling evidence of the and Cotesia plutellae in Field-Cages mating interaction between released substerile and wild moths. The usefulness of mutant mark- Knipling (1979) suggested that combining par- ers as a tool to monitor the outcome of sexual asitoid releases with sterile insect release might interactions between irradiated (BSP) and unir- yield both additive and synergistic effects. Al- radiated (BSP+) DBM has been demonstrated in though the modes of action of both tactics are dif- these experiments. ferent, the effectiveness of the sterile insect Even though our results would suggest that technique increases the ratio of adult parasitoids male only releases should be considered for DBM, to adult hosts, while the action of parasitoids in- no efﬁcient technique is currently available to sep- creases the ratio of sterile to fertile insects. Even arate large numbers of male and female pupae. As greater suppression could be expected if parasi- a consequence, simultaneous release of treated toids are combined with the releases of partially males and females is unavoidable at this time. In sterile insects. Carpenter (1993) demonstrated

our experiments, population suppression in the F2 that the economic beneﬁts of combining inherited generation for bisexual releases was signiﬁcantly sterility and parasitoids would be greatest when higher than that observed in the F2 for male only the ratio of irradiated to unirradiated moths is releases, even at a ratio of 10:1. However a higher ≤10:1 and the ratio of parasitoid to hosts is ≤5:1.

amount of crop damage by F1 larvae was observed Our objective was to investigate the potential when both irradiated substerile male and female of using combined releases of partially sterile DBM were released into the ﬁeld-cages. One po- DBM with releases of Cotesia plutellae, a speciﬁc tential solution to avoid excessive crop damage larval parasitoid, to suppress wild DBM popula-

would be to rear the DBM F1 generation in the lab- tions. The results are summarized in Table 7. In oratory and release F1 adults instead of their irra- cages receiving partially sterile DBM (Trts. 3 and

diated parents. This would reduce crop damage 4), the number of F1 eggs was signiﬁcantly (1.5 206 Florida Entomologist 84(2) June 2001

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 F1 AND F2 GENERATIONS IN FIELD-CAGES.

Mean ± SD Treatment1 CP:I: U No. eggs No. neonates No. pupae No. adults3

F1 Generation Treatment 1 298 ± 67a 262 ± 42a 127 ± 33b 108 ± 25a (0:0:10) Treatment 2 279 ± 40a 252 ± 34a 87 ± 10ab 78 ± 17a (50:0:10) (27.78%) Treatment 3 417 ± 21b 255 ± 41a 115 ± 25ab 104 ± 16a (0:50:10) (0%) Treatment 42 432 ± 20b 262 ± 41a 66 ± 15a 63 ± 15a (25:25:10) (41.67%)

F2 Generation Treatment 1 2796 ± 415b 2534 ± 204c 1572 ± 297d 1431 ± 2214d Treatment 2 2267 ± 308b 2018 ± 195b 1108 ± 109c 1002 ± 86c (29.98%) Treatment 3 1432 ± 289a 288 ± 56a 261 ± 48b 257 ± 51b (82.04%) Treatment 42 843 ± 152a 195 ± 8a 52 ± 4a 40 ± 5a (96.58%)

Means followed by the same letter within a column are not significantly different (P = 0.05; Tukey’s Honest Significant Difference). 1CP = Cotesia plutellae; I = irradiated BSP moth; U = unirradiated BSP+ moth. 2Irradiated DBM were released on day 1 and were followed 10 days later with the release of Cotesia plutellae. 3Values 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 ing irradiated DBM than in those receiving mutations in the irradiated moths killed many parasitoids. embryos during development, no significant dif- The numbers of parasitoids, BSP, and BSP+ ference was found in the number of neonates marked moths recovered from each of the treat- among treatments. Thus, the level of host damage ments are summarized in Table 8. In Treatment 2 caused by larvae in all four populations was al- (parasitoids only), the population of C. plutellae most identical. The number of adult moths was decreased by about 20% in the Fl generation and lowest in Treatment 4, although differences increased by about 75% in the F2 generation. among treatments were not significant. However, However, in Trt. 4 (irradiated DBM + parasitoids) in the F2 generation the number of eggs in Trts. 3 the size of the parasitoid population increased by

and 4 was signiﬁcantly lower than the number of about 50% in F1 generation and declined by about

eggs in Trts. 1 and 2. Treatment 4 had the lowest 15% in the F2. These observations might suggest number of eggs at 843. The number of neonates that C. plutellae may have had a higher survival

for each of the treatments was signiﬁcantly lower rate on irradiated F1 (BSP) larvae than on the than the number of neonates in the control. Fur- wild type (BSP+) larvae. However the greater

thermore, the number of neonates in Trt. 3 (288) number of C. plutellae in the F2 generation in Trt. and Trt. 4 (195) was signiﬁcantly lower than the 2 is largely due to lower number of DBM larvae in number of neonates in Trt. 2 (2018). As a result, Trt. 4 (195 neonates). the level of damage on host plants was the lowest Our data suggest that the use of both tactics, in cages treated with both irradiated males and inherited sterility and releases of the parasitoid, females and parasitoids. C. plutellae, may be feasible for managing early- Each of the treatments signiﬁcantly sup- season populations of DBM. Partially sterile pressed population growth as compared to the DBM adults could be released to produce large

control. However, population suppression was numbers of F1 sterile larvae on early-season an-

significantly greater in cages receiving a combi- nual hosts. These F1 larvae could in turn serve as nation of irradiated (200 Gy) DBM males and fe- hosts for C. plutellae and other parasitoids males followed by a single release of C. plutellae. present in the field. In this way the next genera- When only one tactic was used, population sup- tion of parasitoids would be increased, and any pression was significantly higher in cages receiv- surviving partially sterile larvae would become Nguyen Thi & Nguyen Thanh: F1 sterility in DBM suppression 207

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 ± SD1

Population (CP:I:U)1 Parasitoids BSP BSP+

F1 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

1BSP+ = wild moth; BSP = mutant moth; I = irradiated BSP moth; U = unirradiated BSP+ moth; CP = Cotesia plutellae. Three replications were conducted. partially sterile adults (Carpenter et al. 1996). CARPENTER, J. E., C. M. MANNION, AND HIDRAYANI. Further studies are needed to measure the in- 1996. Potential of combining inherited sterility and ﬁeld effects of these combined tactics on the early- parasitoids for managing lepidopteran pests, pp. season population dynamics in DBM. 273-287. In Working Material First FAO/IAEA Re- search Coordination Meeting on Evaluation of population suppression by irradiated Lepidoptera and ACKNOWLEDGMENTS their progeny. 24-28 April 1995. Jakarta, Indonesia. EL-NAGGAR, S., M. M. MEGAHED, H. A. SALLAM, AND We are grateful to Waldemar Klassen, Bill S. M. IBRAHIM. 1984. Inherited sterility among Agro- Butt, Leo LaChance, James Carpenter and Jorge tis ipsilon laboratory populations exposed to gamma Hendrichs for documents and recommendations. irradiation. Insect Sci. Appl. 56: 501-3. This work was supported by the International HOLT, G. G., AND D. T. NORTH. 1970. Effects of gamma Atomic Energy Agency under Research Contract irradiation on the mechanism of sperm transfer in Trichoplusia ni. J. Insect Physiol. 16: 2211-2222. No. 7174/RB and the Dalat Nuclear Research In- KNIPLING, E. F. 1970. Suppression of pest Lepidoptera stitute under Research Contract VH 10/92. by releasing partially sterile males: a theoretical ap- praisal. Bioscience. 20: 465-470. REFERENCES CITED KNIPLING, E. F. 1979. The basic principles of insect population suppression and management. USDA, Agri- ALLAM, G., AND R. GALLUN. 1976. Optimal sex ratio for cultural Handbook No 512. the control of insects by the sterility method. Ann. LACHANCE, L. E. 1985. Genetic methods for the control Entomol. Soc. Amer. 60: 41-3. of Lepidopteran species: status and potential. Rep. BARTLETT, A. C. 1967. Genetic markers in the boll wee- ARS 28, USDA, Washington DC. vil. J. Hered. 58: 159-63. NORTH, D. T., AND G. G. HOLT. 1971. Radiation studies BARTLETT, A. C., AND J. R. RAULSTON, JR. 1982. The of sperm transfer in relation to competitiveness and identiﬁcation and use of genetic marker for popula- oviposition in the cabbage looper and corn earworm, tion dynamics and control studies in Heliothis, pp. pp. 87-111. In Application of induced sterility for 15-20. In Proc. International Workshop on Heliothis control of Lepidopterous populations. IAEA, Vienna. Management. 15-20 November 1981. Patancheru, NORTH, D. T. 1975. Inherited sterility in Lepidoptera. AP, India. Annu. Rev. Entomol. 20: 167-82. CARPENTER, J. E., J. R. YOUNG, A. N. SPARKS, H. L. OMAR, D., AND M. MANSOR. 1993. Effect of substerilizing CROMROY, AND M. A. CHOWDHURY. 1987. Corn ear- doses of radiation on the biology of diamondback

worm (Lepidoptera: Noctuidae): Effects of substeril- moth, pp. 3-10. In Radiation induced F1 sterility in izing doses of radiation and inherited sterility on Lepidoptera for area-wide control. IAEA, Vienna. reproduction. J. Econ. Entomol. 80: 483-489. SALLAM, H. A., AND S. M. IBRAHIM. 1993. Inherited ste- CARPENTER, J. E. 1991. Effect of radiation dose on the rility in progeny of gamma irradiated male cotton incidence of visible chromosomal aberrations in leafworm, Spodoptera littoralis (Boisd), pp. 81-100.

Helicoverpa zea (Lepidoptera: Noctuidae). Environ. In Radiation induced F1 sterility in Lepidoptera for Entomol. 20: 1457-1459. area-wide control. IAEA, Vienna.

CARPENTER, J. E. 1993. Radiation induced F1 sterility in SUTRISNO, S., M.S. HOEDAYA, D. SUTARDI, AND A. RAH-

Helicoverpa zea (Boddie): Potential for Area-Wide AYU. 1993. Radiation induced F1 sterility in dia-

Control, pp. 37-48. In Radiation induced F1 sterility in mondback moth, Plutella xylostella L., and tropical Lepidoptera for Area-Wide Control. IAEA, Vienna. armyworm, Spodoptera litura F., pp. 23-36. In Radi- 208 Florida Entomologist 84(2) June 2001

ation induced F1 sterility in Lepidoptera for area- WALDER, J. 1988. Genetic method for separation of wide control. IAEA, Vienna. males and females of the medﬂy on the basis of pupal SUTRISNO, S., AND M. HOEDAYA. 1993. The sterile insect colour, pp. 205-210. In Modern insect control: Nuc- technique and transmission of inherited sterility to lear techniques and biotechnology. IAEA, Vienna. control the diamondback moth, Plutella xylostella WHITTEN, M. J., AND W. C. TAYLOR. 1970. A role for ster- (L.) and the cabbage webworm Crocidolomia binota- ile females in insect control. J. Econ. Entomol. 63: lis Zell, pp. 395-403. In Management of Insect Pests: 269-72. Nuclear and related molecular techniques. Symp. ZHANG, H. R., Y. Y. LI, H. S. WANG, AND W. ZHANG. Proc. 19-23 October 1992. IAEA, Vienna. 1993. Studies on chromosomal aberrations and inher- TALEKAR, N. S. 1992. Management of diamondback ited sterility in Asian corn borer, Ostrinia furnacalis

moth and other Crucifer pests. In Proc. Second Inter- (Guence), pp. 117-123. In Radiation induced F1 steril- national Workshop. Taiwan, AVRDC. 603 pp. ity in Lepidoptera for area-wide control. IAEA, Vienna.

Henne &Johnson: Parasitism of Scapteriscus in Louisiana 209

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, Acridomyia

RESUMEN

Los grillos topo del genero Scapteriscus fueron introducidos accidentalmente en el sur de los Estados Unidos hace un siglo y se consideran ser parásitos importantes económicamente en regiones sureñas de EE.UU. Los grillos topo fueron muestreados usando trampas acústicas en Baton Rouge y New Orleáns, Louisiana en el otoño de 1998, la primavera y otoño de 1999, y la primavera del 2000. Scapteriscus borelli tiene un periodo de vuelo estacional comenzando tarde en febrero y continuando hasta junio. Un periodo de vuelo mas corto ocurre en el otoño, comenzando a mediados de septiembre y continuando hasta noviembre. S. vicinus fue capturado solo entre ﬁnes de febrero y ﬁnes de abril. Determinamos que S. borellii estaba siendo parasitado dentro de las trampas acústicas por la mosca tachinida Ormia ochracea y que S. vicinus fue parasitado por la mosca esp. Acridomyia.

Three species of mole crickets, Scapteriscus America, where it attacks mole crickets of the ge- vicinus Scudder, S. borellii Giglio-Tos and S. ab- nus Scapteriscus (Fowler 1987). It has been re- breviatus Scudder, were accidentally introduced leased at many sites in Florida and is established into the southern United States from South there (Frank et al. 1996). America from around 1899 to 1926 (Walker & Nickle 1981). Scapteriscus vicinus and S. borellii MATERIALS AND METHODS have since spread throughout the coastal plain from southeastern Texas to southeastern North Acoustic traps that use artificially produced Carolina (Parkman et al. 1996), with isolated pop- calling songs of mole crickets capture large num- ulations of S. borellii reported from Arizona bers of these insects, and they are used for popu- (Nickle & Frank 1988) and California (Frank lation surveys and behavioral studies (Walker 1994). Scapteriscus spp. mole crickets, especially 1988). Traps in this study were constructed ac- S. vicinus, are considered to be serious pests of cording to details given in Parkman and Frank turf and pasture grasses throughout the south- (1992) with some modifications. Our traps con- eastern U.S. (Hudson et al. 1988). sisted of 1 m diameter sheet metal funnels sus- Using traps with artificial calling songs of S. pended from metal yokes. The yokes were borellii and S. vicinus, surveys of these two spe- supported by 10 cm × 10 cm × 100 cm wooden cies were conducted in Baton Rouge and New Or- posts buried into the ground to a depth of 30 cm. leans, Louisiana, in the fall of 1998, the spring A single 20 l plastic bucket was attached to the and fall of 1999, and the spring of 2000. This was bottom of each funnel to hold captured insects. done in order to ascertain the population levels in Buckets were suspended above ground level to these areas and to determine the flight periods of minimize predation of captured mole crickets by introduced mole crickets in southeastern Louisi- red imported fire ants (Solenopsis invicta Buren). ana prior to release of Ormia depleta (Wiede- Holes were drilled through the bottom of the mann) (Diptera: Tachinidae) in these cities for buckets to allow rainwater to drain. Two sound biological control. This fly is native to South traps were deployed at Burden Research Station

210 Florida Entomologist 84(2) June 2001 in Baton Rouge (31°24.46 N, 91°06.74 W) during mole crickets captured in our sound traps were fall 1998, the spring and fall of 1999 and spring much lower than those reported in Florida. 2000. A second set of traps was also deployed in Trap abundance cycles of 7-12 days were ob- City Park, New Orleans (29°59.70 N, 90°05.33 W) served during the fall 1998 and 1999, and spring during fall 1999 and spring 2000. A different song 1999 and 2000 trap data (Figs. 1 and 2). Ngo and was played over each of the traps; one synthesized Beck (1982) also observed trap abundance cycles the song of S. borellii males and the other the lasting ca. 9 days for S. borellii in Florida and at- song of S. vicinus males. The traps were sepa- tributed this phenomenon to egg laying cycles of rated by at least 2 m as suggested by Walker S. borellii females. Walker and Nation (1982) re- (1982). Artificial crickets were obtained from ported that some S. borellii males will call during Night Caller Artificial Crickets (Eco-Sim, Gaines- the fall months and females can respond in signif- ville, FL), and they were powered by 12V, 7.0 icant numbers. They found that some S. borellii amp-hour, lead acid gel-cell rechargeable batter- females do mate during the fall, since 7 out of 25 ies (Power Sonic PS-1270). Batteries were re- females attracted to synthetic calling song in Oc- charged once per week. tober to December had sperm in their spermathe- Traps were serviced at least every other day. cae. It is not generally believed, however, that egg Captured adults of Scapteriscus spp. were placed laying occurs during the fall. in zippered plastic bags which were placed in a cooler for transport to the laboratory. Adults were Parasitism of S. borellii by O. ochracea held individually in 15 dram snap-lid, plastic vials (WWR Scientific, West Chester, PA). Each vial Ormia ochracea adults were commonly col- contained at least 4 cm (~35 ml) of moist sand. lected in the S. borellii traps but were never col- The mole crickets were checked each day for par- lected from the S. vicinus traps during fall 1998 asitism. Dead mole crickets were removed from and 1999. O. ochracea were often observed resting vials and the sand searched for fly puparia. Lar- inside trap buckets and caller housings. No O. vae of O. ochracea Bigot require approximately ochracea were reared from ca. 200 Scapteriscus one week to complete development in the labora- spp. collected during each spring of 1999 and 2000. tory at 23-25°C (Wineriter & Walker 1990). After This was expected because O. ochracea adults three weeks, all puparia were removed from the were never observed at our traps during spring sand and placed inside 15 dram snap-lid vials 1999 and 2000. Of 88 S. borellii captured at New onto 3 cm × 3 cm moist paper towels until emer- Orleans and Baton Rouge between 7 October and gence of adults. Adult flies were identified using 18 October 1999, 18 were parasitized by O. ochra- keys in Sabrosky (1953). Voucher specimens of O. cea, yielding 34 puparia (range 1-4 puparia per ochracea, G. rubens Scudder, S. borellii and S. host), (Table 1). A total of 14 adults of O. ochracea vicinus were deposited in the Louisiana State eclosed from the puparia. Flies (n = ca. 5-10) were University Arthropod Museum. Larvae of Acri- regularly observed resting inside the caller hous- domyia were identified using keys to immature ing during the day. When the S. borellii trap buck- Diptera in Teskey et al. (1991). ets were opened to inspect the contents, several dozen flies would escape. We were often able to collect flies, usually 10-20, from inside the trap buck- RESULTS AND DISCUSSION ets that were either dead or too weak to fly. Walker (1993) found that calls of S. borellii and S. vicinus Seasonal Distribution attracted few and zero O. ochracea, respectively. Tachinid flies of the tribe Ormiini are orthop- Numbers of S. borellii and S. vicinus captured teran parasitoids specializing on crickets and katy- in fall 1998, spring and fall 1999, and spring 2000 dids (Walker 1986, 1989). They are known to occur are shown in Figures 1 and 2. The flight period for throughout the southeastern United States from S. borellii in southeastern Louisiana begins in Florida to Texas (Walker 1989; Robert & Hoy late February and lasts into June. A lesser flight 1994). Females of the phonotactic tachinid fly period occurs in the fall, starting in mid-Septem- O. ochracea are larviporous (Frank 1994), are ber and lasting into November. For S. vicinus, the attracted to artificially produced songs of its host, flight period in southeastern Louisiana also begins Gryllus integer and larviposit in the vicinity of in late February and continues into late April. No calling males (Cade 1975; Walker 1989). Walker S. vicinus were captured at either of the two trap- (1986, 1989) captured large numbers of O. ochra- ping locations during fall of the years 1998 or cea at traps using artificially produced Gryllus 1999. Flight periods of S. borellii and S. vicinus in rubens Scudder songs. Mangold (1978) found that southeastern Louisiana are similar to those re- O. ochracea also was attracted to synthesized ported by Walker et al. (1983) for S. borellii and S. songs of S. borellii males and was able to success- vicinus at Gainesville, Florida (29°40’N), except fully rear O. ochracea to adulthood on 1 of 5 that we never captured S. vicinus in our traps S. borellii and on one G. rubens artificially in- during the fall months. In addition, numbers of fested. Wineriter and Walker (1990) also reared Henne &Johnson: Parasitism of Scapteriscus in Louisiana 211

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. 212 Florida Entomologist 84(2) June 2001

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

O. ochracea from S. borellii. According to Hudson with S. borellii for several days in our traps in- et al. (1988), larvae of O. ochracea have been col- variably resulted in a portion of the mole crickets lected in the field only from crickets of the genus being parasitized. Hundreds of first-instar larvae Gryllus. As Mangold (1978) points out, the rear- of O. ochracea also were collected from the trap ing of O. ochracea on S. borellii does not indicate buckets, suggesting that larvipositing by O. that this is a natural host. Instances of parasit- ochracea females occurred inside the trap buck- ism of S. borellii by O. ochracea are probably an ets. Our results are in contrast to data reported in artifact of the trapping technique (Walker & Win- Frank et al. (1996), where only one out of tens of eriter 1991). Confinement of O. ochracea females thousands of Scapteriscus mole crickets captured Henne &Johnson: Parasitism of Scapteriscus in Louisiana 213

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

*1 = numbers of O. ochracea puparia reared from S. borellii in parentheses. in a survey was parasitized, presumably by O. of S. vicinus (~130 s-1) is much faster than both S. ochracea. The authors did not state what species borellii and G. rubens. In addition, the carrier fre- of Scapteriscus was parasitized. quencies of S. borellii and S. vicinus are much Three female G. rubens were collected in the lower than G. rubens. As Walker (1993) points out, S. borellii sound trap at Baton Rouge between 14- our knowledge of cricket communication, and that 18 October 1999. Mangold (1978) also reported of their acoustically attracted parasitoids, is far that several Gryllus spp. were attracted to artifi- from complete and future studies should examine cial songs of S. borellii in Florida. Of particular in- other components of sound that may be important. terest is the attraction of O. ochracea and G. rubens to artificial songs of S. borellii but not of S. Parasitism of S. vicinus by Acridomyia sp. vicinus. The calling songs of S. borellii and G. rubens are similar (Burk 1982). Males of In late March 2000, two S. vicinus that were S. borellii broadcast songs at a carrier frequency captured in an acoustic trap in New Orleans were of 2.7 kHz, with a pulse rate of ~50 pulses s-1 found to have been parasitized by Acridomyia sp. (Walker 1982). Males of G. rubens broadcast songs (Diptera: Anthomyiidae). One of the parasitized at a carrier frequency of 4.8 kHz, also with ~50 S. vicinus contained six maggots, the other con- pulses s-1. (Walker 1986). In addition, calling songs tained only one. Unfortunately, no adult flies were of S. vicinus have a carrier frequency that falls in obtained. Larvae of flies in this genus are para- between that of S. borellii and G. rubens (3.3 kHz), sitoids of grasshoppers (Acrididae) (Dahlem & but have a much faster pulse rate of ~130 s-1 Thompson, 1991). Little else is known about mem- (Walker 1993). Robert et al. (1992) found that the bers of this genus. Are Acridomyia sp. attracted to hearing organ of O. ochracea females is most sen- the calling songs of S. vicinus or were these two sitive to frequencies in the range of 4 to 6 kHz. mole crickets accidentally parasitized? There is, however, no reason to suggest that O. ochracea females would not respond to carrier frequencies outside these ranges. In fact, Walker ACKNOWLEDGMENTS (1993) demonstrated that O. ochracea females are The authors would like to thank Daniel J. Gill, attracted to carrier frequencies between 2.4 and County Agent, Orleans Parish for his assistance 6.8 kHz. According to Chapman (1982) there is no in arranging for the deployment of sound traps on evidence that frequency discrimination alone is City Park property in New Orleans. Thanks are especially important to the insect. The pulse repe- also extended to Drs. Abner Hammond and Gregg tition frequency of insect songs is probably a more Henderson (Louisiana State University) for their important factor in their recognition because im- helpful comments and criticisms of an earlier pulses occur in the auditory nerves in synchrony draft of this manuscript. Approved for publication with sound pulses. This may explain why O. by the Director, Louisiana Agricultural Experi- ochracea and G. rubens were attracted to artificial ment Station as Manuscript number 00-17-0632. calling songs of S. borellii at our trapping stations but not to those of S. vicinus. In Walker (1986), O. ochracea females were attracted to synthesized REFERENCES CITED calls of G. rubens but not G. firmus. The carrier CADE, W. 1975. Acoustically orienting parasitoids: fly frequencies of these two species fall within the phonotaxis to cricket song. Science 190: 1312-1313. most sensitive range of O. ochracea females (4-6 BURK, T. 1982. Sticky panel catches of the tachinid Eu- kHz), but songs of G. firmus have a much slower phasiopteryx ochracea. Florida Entomol. 65(2): 291- pulse rate of ~17 s-1 (Walker 1986). The pulse rate 292. 214 Florida Entomologist 84(2) June 2001

CHAPMAN, R. F. 1982. Insects: Structure and Function. ROBERT, D., J. AMOROSO, AND R. R. HOY. 1992. The evo- Third Edition. Harvard University Press. Cam- lutionary convergence of hearing in a parasitoid fly bridge, MA. 919 pp. and its cricket host. Science 258: 1135-1137. DAHLEM, G. A. AND F. C. THOMPSON. 1991. Anthomyi- ROBERT, D., AND R. R. HOY. 1994. Overhearing cricket idae (Muscoidea). Chapter 37: Diptera, pp. 853-857. love songs. Natural History 103(6): 49-50. In Immature Insects, Volume II. F. W. Stehr (ed.). SABROSKY, C. W. 1953. Taxonomy and host relations of Kendall/Hunt Publishing Company. Dubuque, IA. the tribe Ormiini in the western hemisphere. Proc. 975 pp. Entomol. Soc. Washington 55: 167-183. FOWLER, H. G. 1987. Field behavior of Euphasiopteryx TESKEY, H. J., B. A. FOOTE AND F. W. STEHR. 1991. Key depleta (Diptera: Tachinidae): Phonotactically ori- to families of larvae. Chapter 37: Diptera, pp. 708- enting parasitoids of mole crickets (Orthoptera: 730. In Immature Insects, Volume II. F.W. Stehr Gryllotalpidae: Scapteriscus). J. New York Entomol. (ed.). Kendall/Hunt Publishing Company. Dubuque, Soc. 95(4): 474-480. IA. 975 pp. FRANK, J. H. 1994. Biological control of pest mole crick- WALKER, T. J. 1982. Sound traps for sampling mole ets, pp. 343-352. In Pest Management in the Sub- cricket flights (Orthoptera: Gryllotalpidae: Scap- tropics: Biological Control a Florida Perspective. D. teriscus). Florida Entomol. 65(1): 105-110. Rosen, F. D. Bennett and J. L. Capinera (eds.). Inter- WALKER, T. J. 1986. Monitoring the flights of field crick- cept Ltd. Andover, U.K. 717 pp. ets (Gryllus spp.) and a tachinid fly (Euphasiopteryx FRANK, J. H., T. J. WALKER, AND J. P. PARKMAN. 1996. ochracea) in north Florida. Florida Entomol. 69(4): The introduction, establishment, and spread of 678-685. Ormia depleta in Florida. Biological control 6: 368- WALKER, T. J. 1988. Acoustic traps for agriculturally im- 377. portant insects. Florida Entomol. 71(4): 484-492. HUDSON, W. G., J. H. FRANK, AND J. L. CASTNER. 1988. WALKER, T. J. 1989. A live trap for monitoring Eu- Biological control of Scapteriscus spp. mole crickets phasiopteryx and tests with E. ochracea (Diptera: (Orthoptera: Gryllotalpidae) in Florida. Bull. Ento- Tachinidae). Florida Entomol. 72(2): 314-319. mol. Soc. Am. 34: 192-198. WALKER, T. J. 1993. Phonotaxis in female Ormia ochra- MANGOLD, J. R. 1978. Attraction of Euphasiopteryx cea (Diptera: Tachinidae), a parasitoid of field crick- ochracea, Corethrella sp. and Gryllids to broadcast ets. J. Insect. Behav. 6(3): 389-410. songs of the Southern Mole Cricket. Florida Ento- WALKER, T. J. AND D. A. NICKLE. 1981. Introduction and mol. 61(2): 57-61. spread of pest mole crickets: Scapteriscus vicinus NGO DONG AND H. W. BECK. 1982. Mark-release of and S. acletus reexamined. Ann. Entomol. Soc. Am. sound attracted mole crickets: flight behavior and 74(2): 158-163. implications for control. Florida Entomol. 65(4): 531- WALKER, T. J. AND J. L. NATION. 1982. Sperm storage in 538. mole crickets: fall matings fertilize spring eggs in NICKLE, D. A., AND W. FRANK. 1988. Pest mole crickets, Scapteriscus acletus. Florida Entomol. 65(2): 283- Scapteriscus acletus (Orthoptera: Gryllotalpidae), 285. reported established in Arizona. Florida Entomol. WALKER, T. J., J. A. REINERT, AND D. J. SCHUSTER. 71(1): 90-91. 1983. Geographical variation in flights of the mole PARKMAN, J. P., AND J. H. FRANK. 1993. Use of a sound cricket, Scapteriscus spp. (Orthoptera: Gryllotalpi- trap to inoculate Steinernema scapterisci (Rhabdita: dae). Ann. Ent. Soc. Am. 76(3): 507-517. Steinernematidae) into pest mole cricket popula- WALKER, T. J. AND WINERITER, S. A. 1991. Hosts of a tions (Orthoptera: Gryllotalpidae). Florida Entomol. phonotactic parasitoid and levels of parasitism 76(1): 75-82. (Diptera: Tachinidae: Ormia ochracea). Florida En- PARKMAN, J. P., J. H. FRANK, T. J. WALKER, AND D. J. tomol. 74(4): 554-559. SCHUSTER. 1996. Classical biological control of Scap- WINERITER, S. A., AND T. J. WALKER. 1990. Rearing pho- teriscus spp. (Orthoptera: Gryllotalpidae) in Florida. notactic parasitoid flies (Diptera: Tachinidae: Ormia Environ. Entomol. 25(6): 1415-1420. spp.). Entomophaga 35: 621-632.

Ellis et al.: Evaluation of Serangium 215

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

DONNA ELLIS1, RICHARD MCAVOY1, LUMA ABU AYYASH1, MELISA FLANAGAN1, 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 poinsettia with biological agents has been unreliable. Serangium parcesetosum Sicard, a coccinellid 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. parcesetosum 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 predator 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

RESUMEN El control de la mosca blanca (Bemisia argentifolii Bellows & Perring) en poinsetias de in- vernadero con agentes biológicos ha sito errático. Serangium parcesetosum Sicard, un predador coccinélido, parece tener gran potencial para el control de B. argentifolii. En nuestro estudio, cambios dinámicos en poblaciones de B. argentifolii en poinsetias enjauladas en respuesta a S. parcesetosum fueron observados. Las moscas blancas fueron liberadas en poinsetias enjauladas de 1 a 10 adultos por planta y 6 semanas después S. parcesetosum fueron liberados de 0,2, o 4 adultos por planta. Dentro de 2 semanas desde la introducción de Se- rangium la mortalidad de la mosca blanca incremento dramáticamente, y por las próximas 10 semanas los niveles de moscas permanecieron en o cerca de aquellos observados al mo- mento de introducción del predador. Larvas de escarabajos fueron observadas de 2 a 10 semanas después de la liberación 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- mentación continua de escarabajos individuales. Nuestros datos sugieren que Serangium pudiera servir bien en un programa de control de especies múltiples de moscas blancas en poinsettia. Sin embargo, mas investigación es necesaria sobre las interacciones de especies múltiples dentro la especie (plaga / planta), y en estrategias de control de liberación.

Silverleaf whitefly, Bemisia argentifolii Bel- annual wholesale value (>$220 million dollars) lows & Perring (Homoptera: Aleyrodidae, also (USDA 1997). It appears to be a good candidate- known as the sweetpotato whitefly, B. tabaci crop for biological control because it is produced (Gennadius) Biotype B), is the most important ar- as a monoculture and has few serious pest prob- thropod pest on greenhouse grown poinsettias lems other than Bemisia argentifolii (Parrella et (Ecke et al. 1990). Poinsettia cuttings often arrive al. 1991). In practice however, economic biological infested with whitefly nymphs at levels well be- control systems capable of suppressing B. argen- low economic thresholds (Helgesen & Tauber 1977; tifolii to the low thresholds required for ornamen- Hoddle et al. 1999) but whitefly populations rap- tal crops have been elusive (Parrella et al. 1991). idly increase to exceed economic thresholds in the For example, Hoddle et al. (1997a) reported that absence of effective controls. when Encarsia formosa Gahan (Hymenoptera: Poinsettia is the single largest potted flower- Aphelinidae) was used as the control agent, 23 to ing greenhouse crop grown in the U.S. in terms of 70% of poinsettia plants were infested with im- both number of pots produced (>59 million) and mature whitefly at the end of the season. By com-

216 Florida Entomologist 84(2) June 2001

parison they observed 30% of poinsettias in MATERIALS AND METHODS commercial retail outlets were infested with immature whitefly. With the E. formosa Beltsville Four rooted poinsettia (Euphorbia pulcher- strain, end-of-season whitefly infestation ranged rima Willd. Ex. Klotzsch. cv. ‘Freedom Red’) cut- from 77 to 100% of plants under various release tings were transplanted into individual 30 cm rates compared to 28% of plants observed in com- pots on 16 July 1997 in the University of Connect- mercial retail outlets (Hoddle et al. 1997b). Heinz icut research greenhouse range. Each exclusion & Parrella (1994) observed satisfactory whitefly cage was constructed of a white organdy sleeve control with a combination of Encarsia luteola supported by 75 cm bamboo plant stakes and Howard and Delphastus pusillus LeConte (Co- placed around each pot. Cages were sealed above leoptera: Coccinellidae) but at a cost 5-times the four poinsettia plants and below the lip of higher than insecticide-based control. With each pot. Velcro strips were used on two vertical weekly releases of Eretmocerus eremicus n. sp. seams, one on each side of the cage, to facilitate Rose & Zolnerowich (Hymenoptera: Aphelinidae), access to the plants. Hoddle et al. (1998) reported that 73 and 83% of After plants were established in the pots, sil- plants were infested with immature whitefly at verleaf whiteflies were introduced on the caged the end of the season compared to only 28% of poinsettia plants on 23 Aug. 1997 at a rate of ei- plants in commercial retail outlets. These studies ther 1 or 10 adults per plant (equivalent to 4 or 40 underscore the need for continued evaluation of adult whiteflies per cage). On 3 Oct., Serangium promising new biological control agents. parcesetosum were introduced into cages at 0, 2 Serangium parcesetosum Sicard is a coccinellid and 4 adults per plant (equivalent to 0, 8 and 16 predator that has demonstrated potential for the Serangium per cage, respectively). The result was biological control of silverleaf whitefly (Legaspi a 2 × 3 factorial design with initial levels of either et al. 1996). This species was originally collected 1 or 10 whiteflies per plant and 0, 2, or 4 Seran- from India in 1929 for release as a biological con- gium per plant. Treatments were arranged in a trol agent of citrus whitefly, Dialeurodes citri randomized complete block with five replications. Ashmead (Aleyrodidae), in the Union of Soviet Poinsettias in separate cages, but without either Socialist Republics (Kapur 1954; Timofeyeva & prey or Serangium, were used to evaluate the ef- Nhuan 1979). As a result of the success in that fects of whitefly on plant growth. Prey and Seran- biological control program, and because of its re- gium were shipped overnight from the USDA discovery during foreign exploration in Podumbu, APHIS Mission Plant Protection Center (Mission, India, S. parcesetosum is currently being re- TX) in insulated containers with ice packs. A searched as a predator of silverleaf whitefly. small Hibiscus plant infested with whitefly pupae Serangium parcesetosum (herein referred to as was shipped just prior to emergence of the adults. Serangium) has been known from the available Whiteflies were held in a controlled temperature literature to feed mainly on citrus whitefly, al- chamber until adults emerged within 48 h. Newly though in field trials in the U.S. it has also at- emerged whitefly adults were aspirated and then tacked silverleaf whitefly (M. C., unpublished). transferred into the treatment cages. Serangium All of the known coccinellids belonging to the tribe were shipped as adults in paper cartons with or- Serangiini are obligate predators of whiteflies, or ganza lids for ventilation. Each container held 25 in a few cases, scale insects (Gordon 1985). adults. A Hibiscus leaf with whiteflies was in- Laboratory studies to date show that both lar- cluded in each container for Serangium feeding in vae and adults of Serangium are voracious feed- transit. Immediately upon arrival, Serangium bee- ers, capable of consuming large numbers of tles were introduced into treatment cages using a immature silverleaf whiteflies in short periods of fine camelhair paintbrush. time. Legaspi et al. (1996) showed adults consumed approximately 400 whitefly nymphs in a Monitoring Silverleaf Whitefly 24h period. Serangium larvae consumed 25 to 50 and Serangium Populations whitefly eggs or nymphs in 24 h, depending on the larval stage (M. C., unpublished). Furthermore, Two leaves per plant (8 leaves per cage) were Legaspi et al. (1996) determined the cumulative harvested weekly from 3 Oct. to 5 Dec. 1997. lifetime predation rate to be approximately 5,000 Leaves were selected from the strata of the plant whitefly nymphs per adult Serangium. These canopy with the greatest number of late instar data suggest that Serangium may have the poten- whitefly nymphs; the sample strata was deter- tial to control silverleaf whitefly at moderate to mined each week just prior to leaf harvest. A 25 high levels. However, low whitefly infestation lev- cm2 section of each leaf was examined under a dis- els may not be adequate to sustain Serangium re- secting microscope and the number of whitefly production or even adult survival. eggs and the number of live and dead nymphs and In this study we investigate the effects of Se- pupae were recorded. Immature whitefly were rangium release rates on the population dynam- judged dead when they appeared discolored or ics of B. argentifolii on caged poinsettia. desiccated, or when the empty integument

Ellis et al.: Evaluation of Serangium 217 showed evidence of Serangium feeding. As leaf to monitor temperature. Light intensity was mon- samples were harvested they were visually itored in the greenhouse immediately above the checked for Serangium, and all larvae and adults exclusion cages, and also above the plant canopy were returned to their respective cages. within the cages. Air temperature was monitored At the conclusion of the study, 8 Dec. 1997, a within the cages in the plant canopy and above stratified leaf sample was collected from each the canopy, and directly outside of the cages. For cage and whitefly population and percent mortal- the duration of the study, daily photosynthetic ity were determined. The stratified sample con- photon flux (±SE) averaged 9.1 ± 0.5 mol·m-2·day-1 sisted of two leaves per cage from each of four in the greenhouse above the cage and 8.0 ± 0.5 locations on the plant: bracts, upper canopy, mid- mol·m-2·day-1 above the plant canopy in the cage. dle canopy and lower canopy. The two leaves per Air temperature in the cage and in the plant can- stratum were collected randomly and examined opy (in the cage) averaged 21.3 ± 0.1°C and 21.4 ± for whitefly life stages with a dissecting micro- 0.2°C (respectively) and the ambient air tempera- scope. The poinsettia plants were then destruc- ture in the greenhouse (outside the cage) aver- tively harvested and total laminar surface area aged 22.4 ± 0.2°C. (leaves plus bracts) was determined for all plants in each treatment using a LI-COR 1600 leaf area RESULTS meter (LI-COR, Inc. Lincoln, NE). Plants were separated into bracts, leaves and stems and dried Initial silverleaf whitefly release rates greatly in a forced-air oven at 70°C for one week. Plant affected final population densities of all whitefly dry mass was then recorded. 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; Data Analysis P = 0.031). This effect was most evident when whitefly populations were left uncontrolled. For The mean density of whitefly eggs, nymphs, example, the final density of nymphs was approx- and pupae (per 25 cm2) was calculated for each imately 10-times higher in cages with initial re- treatment. Percent whitefly mortality was calcu- lease rates of 10 whitefly adults per plant than in lated as follows [1]: cages with initial release rates of 1 whitefly adult per plant (Fig. 1b). A similar pattern was observed [(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-normal data for whitefly and Serangium counts and for plant measurements were transformed using the formula: log10 (x + 1), with x representing dependent variables. Non-normal data for whitefly mortality were transformed using the formula: Arcsine (x/100)½, with x representing percent whitefly mortality. Analysis of variance was performed 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 (laminar surface area and shoot dry mass) were subjected to a two-way analysis of variance with seven treatments and five replicates. The treatments included the six whitefly-Serangium combinations 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- × Fig. 1a-c. Changes in silverleaf whitefly life stages on bell Scientific 21 Datalogger (Campbell Scientific, poinsettia plants over time in the absence of a biological Logan, UT) at 1-minute intervals. LI-COR 190- control agent. Plants were initially inoculated with ei- SA quantum sensors (LI-COR, Inc. Lincoln, NE) ther 1 or 10 whitefly adults per plant on 23 August. were used to monitor photosynthetic photon flux Stages include; a) eggs, b) nymphs, and c) pupae. Verti- and copper-constantan thermocouples were used cal bars denote standard error of the mean.

218 Florida Entomologist 84(2) June 2001

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 (respectively) 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 approximately 55% at final harvest. Over the 10 week experimental 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 treatments (Fig. 3a). For example, mortality was only 10 and 38% for the 2 and 4 Serangium treatments (respectively) on 16 Oct. but reached approximately 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 period, 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 Fig. 2a-c. Changes in silverleaf whitefly life stages on contrast, mortality in cages without Serangium poinsettia plants over time in response to different initial Serangium parcesetosum release rates. Poinsettia 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 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. 1a) and pupae (Fig. 1c) 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 introduction (Fig. 2). For example, nymphal prey densities 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- Fig. 3a-b. Changes in silverleaf whitefly mortality (%) ulations remained nearly constant when exposed on poinsettia plants over time in response to Serangium to either the 2 or 4 beetles per plant release rates parcesetosum release rates of 2 or 4 adults per plant (a), (Fig. 2b), while nymphal densities increased up to or in the absence of biological control agents (b). Poinset- 70 fold in the ensuing 10 week period without Se- tia plants were initially inoculated with either 1 or 10 rangium (Fig. 1b). Within both the 1 and 10 adult whiteflies per plant on 23 Aug., and 6 weeks later S. parcesetosum was introduced at 0, 2, or 4 adults per whitefly treatments, similar final prey densities plant. Percent whitefly mortality was calculated using were observed for the high and low Serangium the equation: [(dead nymphs + dead pupae)/(live nymphs treatments (Fig. 2). + live pupae + dead nymphs + dead pupae)]*100. Arrows A dramatic increase in prey mortality was ob- denote Serangium introduction on 3 October. Vertical served within 14 days (16 Oct.) of Serangium re- bars denote standard error of the mean. Ellis et al.: Evaluation of Serangium 219 averaged 4.2% (±0.6% SE) in the 10 whitefly 0.11) and shoot dry mass (84.7 ± 1.9 g/cage, df = 6, cages and 6.8% (±2.3% SE) in the 1 whitefly cages 23; F = 0.86; P = 0.54) were similar for plants in during the final 10 weeks of the study (Fig. 3b). all treatments. At final harvest, live immature whiteflies were observed throughout the plant canopy (Table 1). DISCUSSION In cages without Serangium, approximately 70% of whiteflies were located in the upper leaf strata Consumers have a low tolerance for insect and on the red-colored poinsettia bracts. With pests on greenhouse ornamentals like poinsettia. Serangium present prey were more uniformly Consequently, high standards must be set when distributed in the leaf/bract strata in the 1 white- evaluating the effectiveness of natural enemies fly cages than in the 10 whitefly cages. This may and their management. Inundative releases of indicate that Serangium needed to move more Encarsia formosa can produce satisfactory re- quickly to the upper canopy to find prey when sults if introduced in sufficient numbers before prey populations were low but not when popula- whitefly populations begin to build (Hoddle et al. tions were high. In the 1 whitefly treatment cages 1997a,b). However, in instances where Encarsia with Serangium, the average live immature fail to control whiteflies, alternative measures are whitefly counts observed throughout the canopy required in order to maintain a salable plant were about 16% of those without predators (Table (Parrella et al. 1991; Heinz & Parrella 1994). 1). In the 10 whitefly treatment cages with Seran- In our study, silverleaf whitefly reached dam- gium, the average live immature prey counts aging populations 6-8 weeks after introduction were about 10% of those without predators. Aver- when left uncontrolled even when initial popula- age live immature prey counts were similar for tions were low (1 adult per plant). Heinz & Par- both the 2 and 4 Serangium treatments in all four rella (1994) observed a dramatic increase in strata of the plant canopy with an initial prey whitefly populations on greenhouse grown plants release of 1 per plant (Table 1). Only small differ- (both inside and outside of exclusion cages) after ences in the number of live immature whiteflies nine weeks exposure to whiteflies even in the in the middle and lower leaf strata were observed presence of weekly releases of E. luteola. However, between the 2 and 4 Serangium treatments with a series of three weekly releases of the predatory an initial whitefly release of 10 per plant. beetle Delphastus pusillus (1 beetle per plant per At final harvest, the highest prey mortality week) effectively checked whitefly population was observed in the lower half of the plant canopy growth until the study was ended 3 weeks after (middle and lower leaf strata v. upper leaf and the final release. In our study, a single release of 2 bract strata) (Table 1). Throughout the plant can- Serangium per plant effectively checked further opy, the highest mortality consistently occurred increases in prey population for up to 10 weeks. in cages with Serangium and in cages with high Heinz & Parrella (1994) recovered several initial whitefly release rates (Table 1). adult D. pusillus 3 weeks after the last release, Very few Serangium were recovered from but no evidence of successful predator reproduc- cages at time of final harvest and the total num- tion was reported. Hoelmer et.al. (1993) reported ber (larvae + adults) of Serangium recovered did that D. pusillus required 100-150 whitefly eggs not vary with treatment (P≤0.05). In cages with per day to initiate and sustain oviposition. In our an initial inoculation rate of 1 prey, only adult Se- study, Serangium larvae were first observed 2 rangium were recovered and at an average den- weeks after adults were released in cages with sity of 0.2 and 1.2 per cage for the 2 and 4 high initial whitefly levels (data not shown) but Serangium release rate treatments, respectively. not in cages with low initial whitefly levels. In In cages with an initial inoculation rate of 10 cages with high initial prey levels, Serangium lar- prey, 2 and 2.4 Serangium per cage were recov- vae were recovered as late as 10 weeks after ered from the 2 and 4 Serangium release rate adults were introduced. Cohen & Brummett’s treatments (respectively) and Serangium larva (1997) data suggest that Serangium could con- (1.2 per cage) were only observed in the 2 Se- sume a sufficient number of whitefly immatures rangium release rate treatment. During the final during a daily 10 hour feeding to meet the mini- weeks of this study, whitefly mortality rates de- mum methionine requirement for normal growth clined in all Serangium treatments (Fig. 3a) and and development. This calculation assumes an simultaneously whitefly populations increased average handling time of 1 minute per prey item, (Fig. 2). These data suggest that low Serangium and as with D. pusillus (Hoelmer et al. 1993), it counts at final harvest were not indicative of the appears from our study that Serangium can only predator levels that prevailed during the first reproduce (without nutritional augmentation) eight weeks following Serangium release when under high prey populations. maximum prey control was observed (Fig. 2). Legaspi et al. (1996) reported that the average Poinsettia growth was unaffected by whitefly life-span of Serangium ranged from 75 days at populations in this study. Total laminar surface 20°C to 25 days at 30°C. In our study, typical com- area (1.6 ± 0.03 m2/cage, df = 6, 23; F = 1.99; P = mercial cropping practices were used, poinsettia 220 Florida Entomologist 84(2) June 2001 . 11.5 9.6 9.8 10 1.6 SE) ± ± ± ± ± ± 5.8 34.7 17.0 (0.004) (0.014) F-value HARVEST (<0.001) (% (P-value)

Mortality FINAL

1.0 64.8 1.5 69.7 0.4 31.4 4.8 1.8 0 10.0 AT SE)

± ± ± ± ± 24.7 4.0 ± ± 5.7 47.9 16.0 (0.002) (0.014) F-value (<0.001) Whiteﬂy (P-value) (No. (No. CANOPY

4.1 168 17.9 1.3 10.9 1.9 3.3 7.4 SE) ± ± ± ± 11.4 8.4 2.0 4.7 ± 7.1 2.2 ± ± 11.3 POINSETTIA (0.007) (0.028) (0.145) F-value

(% (P-value) Mortality THE

6.3 66 5.2 8.3 0.9 79 3.0 49.5 1.7 24.2 SE) ± ± ± ± ± 30.6 17.1 ± ± 7.0 0.8 29.4 (0.007) (0.006) (0.456) LEVELS F-value

Whiteﬂy (P-value) (No. (No. FOUR

12.7 16.8 8.9 6.6 11.4 3.5 3.5 2.9 3.3 13.5 SE) ± ± ± ± ± 4.5 123 ± 4.7 7.1 5.2 ± (0.02) (0.025) (0.056) F-value (% (P-value) Mortality MORTALITY

Statistical effects 18.5 38.2 25.5 52.8 18.5 6.7 2.0 37.1 1.6 5.7 SE) ± ± ± ± ± 100 14 ± ± 32.4 14.6 22.42 (0.005) F-value (<0.001) (<0.001) WHITEFLY Whiteﬂy (P-value)

(No. (No. OF

9.9 37.2 4.0 33.7 5.0 2.8 4.9 3.5 ± SE) ± ± ± ± 1.2 1.3 0.4 INCIDENCE (0.334) (0.308) (0.704)

F-value (% (P-value) Mortality AND

a 4.5 6.4 10.2 16.2 1.1 5.0 1.0 5.8 4.0 0 49.3 SE) ± ± ± ± ± 48 0 477 ± ± (0.014) (0.009) (0.426) WHITEFLY F-value

(P-value) Whiteﬂy (No. (No. of leaf surface. 2 SILVERLEAF

df 2 0.9 IMMATURE

LIVE

(No. per plant) (No. ENSITY 1 4 1.8 1 2 1.5 1 0 8.2 Serangium 10 4 16.7 1010 0 2 104 18.8 × Initial release rate treatments Sample strata in poinsettia canopy Whitefly Serangium Bracts Upper leaves Middle leaves Lower leaves 1. D Silverleaf whitefly counts are expressed per 25cm a ABLE Source of variation WhiteflySerangiumWhitefly interaction 1 2 17.2 6.5 T Ellis et al.: Evaluation of Serangium 221

canopy temperature averaged 21.6°C and the GORDON, R. D. 1985. The Coccinellidae (Coleoptera) of crop matured in a normal time period. Legaspi America North of Mexico. J. New York Entomol. Soc. et.al. (1996) reported a mean life-time cumulative 93(1): 1-912. predation of 4909 whitefly (eggs and immature HEINZ, K. M. AND M. P. PARRELLA. 1994. Biological con- stages) for Serangium at a mean temperature of trol of Bemisia argentifolii (Homoptera: Aleyrodi- ° dae) infesting Euphorbia pulcherrima: Evaluations 20 C. Even without reproductive success, the sin- of releases of Encarsia luteola (Hymenoptera: Aphe- gle Serangium release in our study effectively linidae) and Delphastus pusillus (Coleoptera: Coc- prevented prey populations from increasing over cinellidae). Environ. Entomol. 23: 1346-1353. a 10-week period (Fig. 1). It appears that this suc- HEINZ, K. M. AND J. M. NELSON. 1996. Interspecific in- cess was largely due to the prolonged survival and teractions among natural enemies of Bemisia in an continuous feeding of individual adult beetles. inundative biological control program. Biol. Control Due to the relatively high number of whiteflies 6: 384-393. needed to sustain Serangium reproduction and HELGESEN, R. G. AND M. J. TAUBER. 1977. The whitefly- the extremely low pest levels tolerated on orna- Encarsia system: A model for biological control on short-term greenhouse crops. U.S. Agricultural Re- mental crops, it is unlikely that Serangium could search Service Northeast Region. 85: 71-73. function effectively as the sole biological control HODDLE, M., R. VAN DRIESCHE, AND J. SANDERSON. agent on a crop like poinsettia. However, Seran- 1997a. Biological control of Bemisia argentifolii (Ho- gium would be especially useful for suppressing moptera: Aleyrodidae) on poinsettia with inundative localized pest population increases or ‘hot spots’ releases of Encarsia formosa (Hymenoptera: Aphe- in the greenhouse, or as the primary biological linidae): Are higher release rates necessarily better? control agent on crops such as greenhouse tomato Biol. Control 10: 166-179. where pest population tolerance levels are higher HODDLE, M. S., R. G. VAN DRIESCHE, AND J.P. SANDER- than for ornamental crops. Based on our data it SON. 1997b. Biological control of Bemisia argentifolii (Homoptera: Aleyrodidae) on poinsettia with inun- appears that Serangium might be best suited for dative releases of Encarsia formosa Beltsville strain inclusion in a multiple species biological control (Hymenoptera: Aphelinidae): Can parasitoid repro- approach to silverleaf whitefly management on duction augment inundative releases? J. Econ. Ento- ornamental crops. As an obligate whitefly preda- mol. 90: 910-924. tor with a voracious feeding potential, Serangium HODDLE, M. S., R. G. VAN DRIESCHE, J. P. SANDERSON, is capable of checking rapid increases in whitefly AND O. P. J. M. MINKENBERG. 1998. Biological con- populations (based on the caged studies herein), trol of Bemisia argentifolii (Hemiptera: Aleyrodidae) thus potentially enabling whitefly parasitoid spe- on poinsettia with inundative releases of Eret- cies such as Eretmocerus or Encarsia to suppress mocerus eremicus Hymenoptera: Aphelinidae): do release rates affect parasitism? Bull. Entomol. Res. 88: whiteflies to acceptable thresholds. Heinz & Nel- 47-58. son (1996) found that D. pusillus provided the HODDLE, M. S., R. G. VAN DRIESCHE, AND J. P. SANDER- greatest suppression of silverleaf whitefly when SON. 1999. Biology and use of the whitefly parasitoid used in conjunction with one or more species of Encarsia formosa. Annu. Rev. Entomol. 43: 645-669. Encarsia. In order to determine if Serangium HOELMER, K. A., L. S. OSBORNE AND R. K. YOKOMI. would be effective in such a role, interspecific in- 1993. Reproduction and feeding behavior of Delphas- teractions between predator and parasite within tus pusillus (Coleoptera: Coccinellidae), a predator the host species (pest/plant), as well as release of Bemisia tabaci (Homoptera: Aleyrodidae). J. Econ. management strategies, must be investigated. Entomol. 86: 322-329. KAPUR, A. P. 1954. A new species of Coccinellidae (Co- leoptera) predacious on the citrus white-fly in India. ACKNOWLEDGMENTS Rec. of the Ind. Mus. 52: 189-193. LEGASPI, J. C., B. C. LEGASPI, R. L. MEAGHER, JR. AND Storrs Agricultural Experiment Station scien- M. A. CIOMPERLIK. 1996. Evaluation of Serangium tific contribution #1906. This research was parcesetosum (Coleoptera: Coccinellidae) as a biolog- funded, in part, by the University of Connecticut ical control agent of the silverleaf whitefly (Homop- Storrs Agricultural Experiment Station and tera: Aleyrodidae). Environ. Entomol. 25: 1421-1427. PARRELLA, M. P., T. D. PAINE, J. A. BETHKE, K. L. ROBB USDA APHIS. AND J. HALL. 1991. Evaluation of Encarsia formosa (Hymenoptera: Aphelinidae) for biological control of REFERENCES CITED sweetpotato whitefly (Homoptera: Aleyrodidae) on poinsettia. Environ. Entomol. 20: 713-719. BARTLETT, M. S. 1937. Some examples of statistical SAS INSTITUTE. 1995. SAS/STAT version 6. SAS Insti- methods of research in agriculture and applied biol- tute, Cary, NC. ogy. J. Royal Statist. Soc. Suppl. 4: 137-170. TIMOFEYEVA, T. V. AND H. D. NHUAN. 1979. Morphology COHEN, A. C. AND D. L. BRUMMETT. 1997. The non- and biology of the Indian ladybird Serangium abundant nutrient (NAN) concept as a determinant parcesetosum Sicard (Coleoptera: Coccinellidae) pre- of predator-prey fitness. Entomophaga 42: 85-91. dacious on the citrus whitefly in Adzharia. Entomol. ECKE, P. J., O. A. MATKIN, AND D. E. HARTLEY. 1990. In- Rev. 57: 210-214. sects and other pests, pp. 139-166. In Ecke, Matkin, USDA 1997. Floriculture Crops. 1997 Summary. USDA, and Hartley (eds.). The Poinsettia Manual. Published National Agricultural Statistics Service, Washing- by Paul Ecke. Poinsettias, Encinitas, CA. 267 pp. ton, D.C.

222 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 investigated 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

RESUMEN

Una inspeccion de termitas se llevo a cabo en el bosque del Delta Experimental Forest al centro-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 especies de termitas subterráneas en la familia Rhinotermitidae: Reticulitermes flavipes (Kollar) y Reticu- litermes virginicus (Banks). R. flavipes fue la especie mas común y constituyo el 81.3% de las ocurrencias de termitas. De los 685 pedazos de madera inspeccionados, 16.5% tenían termitas. El porcentaje de las dos especies de termitas varió entre lotes. El porcentaje de materiales de madera con señas de actividad de termitas o la presencia de termitas forrajeras fue correlacionada positivamente con el diámetro de los materiales de madera (R = 0.85). La probabilidad de un tronco, rama o tocón ser atacado por estas termitas incremento un 1.3% al incrementar el diámetro de estos materiales por 1 cm. El porcentaje de madera muerta con señas de actividad de termitas vario entre 11.6 a 67.2% entre los lotes muestreados. Las termitas fueron significativamente menos abundante en el Lote 3, lo cual puede corresponder a una elevación mas baja y una humedad de suelo mas alta.

Termites play an important role in forest eco- conducted a survey of termites in an experimental systems, especially in tropical rain forests, where forest. In this study, we tried to determine the spe- they occur in extremely dense populations (Har- cies composition and their foraging activities as- ris 1966; Gray 1972; Matsumoto 1976). Termites sociated with dead wood in a forest environment. promote organic matter decomposition, alter soil The objectives of this study were to quantitatively properties, and provide food to other animals describe termite distribution patterns and feeding (Wood & Sands 1978; La Fage & Nutting 1978). activities in different forest ecosystems. Although their ecological importance is well known, termites have rarely been quantitatively studied in forests of the United States (Gentry & MATERIALS AND METHODS Whitford 1982; Howard et al. 1982). Like any other insects, the abundance of ter- Study Sites mites is influenced by the distribution and quality of their food materials. However, there is very little This study was carried out at the Delta Exper- information regarding the relationship between imental Forest near Stoneville, Mississippi. The termites and size or distribution of wood materials study site is mostly level with gentle slopes (<2°) (Gentry & Whitford 1982; Jones et al. 1995). at some sections. The soil type is fine-textured In Mississippi, Reticulitermes flavipes (Kollar) Sharkey clay soil with clay particle content varies and Reticulitermes virginicus (Banks) appear to from 60 to 85% (Krinard & Johnson 1985). It ex- be the most common termite species. In 1998, we pands considerably when wet in the winter and

Wang & Powell: Survey of termites 223

cracks profusely when dry in the summer. The station, http://www.deltaweather.msstate.edu/). At study sites were sometimes under water in the each plot, three 200-m long, 6-m wide transects in wet winter months (December-February). Aver- north-south direction were delineated. Diameter age air temperature in January and July from at the large end of all logs, branches, and stumps 30-year data is 5.1°C and 27.6°C, respectively >3 cm in diameter and partially or entirely within (Boykin 1995). The average annual precipitation each transect was measured. Each was examined is 1326 mm. for the presence of termites or signs of termite ac- Three 200 ha experimental plots were delin- tivity. Termite galleries, fecal debris, and/or body eated for the survey based on differences in tree parts were noted as signs of termite activity. De- stand. termining whether termite galleries exist in wood Plot 1 is a sweetgum (Liquidambar styraciflua was usually not difficult. Galleries made by other L.) plantation established in 1964-65 at 3 × 3-m arthropods had a more regular shape and not con- spacing. The diameter at breast height is 23.4 ± nected to each other into great lengths or width. 1.2 (mean ± standard error) cm. Some sugarber- Soil particles often exist in termite galleries or on ries (Celtis laevigata Willdenow) have grown into surface of the wood. There were occasions when small to large trees. Common shrubs were: sugar- the wood materials were too decayed to determine berry, hawthorn (Crataegus spp.), willow oak whether or not termites had existed before. These (Quercus phellos L.), Chinese privet (Ligustrum wood materials were not included in the analysis. sinense Loureiro), and blackberry (Rhubus sp.). Results were recorded as three categories: I) with Plot 2 is a naturally regenerated tree stand. live termites, II) with sign of termite activity, or Major trees species were: red oaks (Quercus spp.), III) without termites and no sign of termite activ- sweetgum, sugarberry, overcup oak (Quercus ity. When live termites were found, soldiers were lyrata Walter), green ash (Fraxius pennsylvanica collected for species determination. Host trees Marshall), and elms (Ulmus spp.). Common un- were not identified. Termite soldiers were exam- derstory shrubs were: Chinse privet, swamp dog- ined under an Olympus SZX12 dissecting scope. wood (Cornus stricta Lamarck), Eastern Typically, two or three termite soldiers which are swampprivet (Forestiera acuminata (Michaux) representative of the soldiers in size and mor- Poiret), American snowball (Styrax americana phology were examined. More soldiers were ex- Lamarck), green ash, elms, deciduous holly (Ilex amined if necessary to determine the species. decidua Walter), and blackberry. Termites were identified to species using the key Plot 3 is an even-aged stand of natural regen- provided by Scheffrahn & Su (1994), and identifi- eration resulting from a clearcut in 1937. Major cations of representative specimens were verified tree species were: green ash, cottonwood (Populus by Rudolf Scheffrahn. Voucher specimens have deltoides Marshall), sugarberry, sweetgum, red been deposited in collections of Stoneville Re- oaks, black willow, elms, and white oaks. Under- search Quarantine Facility, USDA Agricultural story shrubs were similar to Plot 2 but at a much Research Service, Stoneville, MS. lower density. This plot is at a slightly lower elevation than plots 1 and 2 and is more likely to be Statistical Analysis flooded in wet winter months. Soil water content of the plots was measured The presence or absence of live termites or on September 15, 1999. The precipitation during signs of termite activity in dead wood materials one month prior to the sampling date was 35.3 were recorded as 1 or 0, respectively. The wood mm. The precipitation during the same period of materials were grouped into 6 categories accord- 1998 was 68.1 mm. Nine soil samples were taken ing to diameter. The actual diameter of the 6 cat- from the three transects of each plot. The soil was egories ranged from 3-5.9, 6-8.9, 9-11.9, 12-14.9, taken from 0-9 cm of the “A” layer. Then they 15-17.9, and ≥18 cm, respectively. Analysis of were dried in an oven at 105°C for 48 hrs. Soil Variance (ANOVA) was performed to test for dif- moisture content was determined by the differ- ferences between diameter groups and plots us- ence between fresh soil weight and dry soil weight ing PROC MIXED of the SAS software (SAS over fresh soil weight. Institute 1999). Then the data were further analyzed for the trend between diameter of the wood Survey materials and presence of termites or signs of termite activity. R2 of the regression equation was A survey of termites was conducted from Octo- determined by the ratio of the sum of squares for ber 20 through November 25, 1998 in plots 1, 2, diameter groups as a trend with 1 degree of free- and 3, in that order. Weather data in the three dom divided by the sum of squares for diameter plots during the survey were: average maximum groups with 5 degrees of freedom. Soil moisture air temperature 22.9, 29.5, and 17.6°C, respec- content data of the plots were compared by tively; average minimum air temperature 8.6, Tukey’s Studentized Range Test after ANOVA. 13.6, and 8.1°C, respectively; average daily rainfall All analyses were performed by SAS software 0.3, 1.5, 6.4 mm, respectively (Stoneville weather (SAS Institute 1999).

224 Florida Entomologist 84(2) June 2001

RESULTS AND DISCUSSION with termites in Plot 3 was significantly lower than that in plots 1 and 2 (Fig. 1) (F = 23.04; df = Species Composition and Distribution of Termites 2, 674; P < 0.0001). This pattern is same as the result on the percentage of wood with signs of ter- Among the 685 branches, logs, and stumps ex- mite activity. The lower temperatures during the amined, 114 (16.5%) had live termites. Two ter- survey might have an impact on the low number mite species were found, Reticulitermes flavipes of termite occurrences in Plot 3. However, the dif- (Kollar) and R. virginicus (Banks). Species were ference between percentage of wood with termites not identified in 7 samples because no soldiers and that with signs of termite damage in Plot 3 is were collected. Among the 107 identified termite proportionally smaller than those in plots 1 and 2 samples, R. flavipes and R. virginicus were found (Fig. 1). This suggests that the influence of tem- in 12.7% and 2.9% of the samples, respectively perature on termite occurrences in Plot 3 was not (Table 1). Reticulitermes flavipes was most fre- significantly higher than that in plots 1 and 2. quently encountered and occurred in 81.3% of the termite samples. This is similar to observations Relationship between Termite Activity by Howard et al. (1982) in southern Mississippi. and Size of Wood Materials The relative abundance of R. virginicus in plots 1- 3 was 7.7%, 21.4%, and 53.8%, respectively. The Most of the wood samples in the three plots high percentage in Plot 3 was not precise because were <12 cm in diameter. The percentage of wood termites in 7 of the 20 samples were not identi- samples with diameter ≥12 cm in the plots 1-3 fied. However, the percentage of R. virginicus in was 1.8%, 5.1%, and 19.7%, respectively. A cause- Plot 3 was at least 35.0%, which was still higher effect relationship existed between percentage of than the other plots. This higher percentage samples with signs of termite activity and diame- might relate with higher soil moisture. Soil mois- ter of the wood (F = 20.34; df = 1, 508; P < 0.0001; ture in Plot 3 (21.4 ± 0.8%) was significantly R2 = 0.73) (Fig. 2). The chance of a sample being higher than that in Plot 1 (19.8 ± 0.8%) measured infested by termites increased 1.3 ± 0.3% as the on September 15, 1999 (F = 12.90; df = 2, 24; P = diameter of the wood increased 1 cm. Perhaps this 0.0002). As reported by Howard et al. (1982), R. is because larger wood materials tend to maintain flavipes tends to occupy higher, more arid places moisture, which is important for termite survival in southern Mississippi. and foraging. According to the regression equa- The percentage of samples with signs of ter- tion, Percentage = 0.327 + 0.013 diameter, even a mite activity in plots 1-3 was 60.0%, 67.2%, and very small dead branch such as 1 cm in diameter 11.6%, respectively. The percentage in plots 1 and will have a 34% chance of having been attacked 2 was significantly higher than Plot 3 (F = 132.76; by termites. df = 2, 508; P < 0.0001) (Fig. 1). The much lower The percentage of samples with termites termite activity that observed in Plot 3 might in- present was also positively correlated with diame- dicate unsuitable conditions for termites, espe- ter of the wood material (F = 30.82; df = 1, 674; P cially for R. flavipes. Plot 3 tended to be flooded for < 0.0001; R2 = 0.72) (Fig. 3). The chance of a sample a longer period of time and more frequently than with termites present increased 1.3 ± 0.2% as the plots 1 and 2 during the winter months. This diameter of the wood materials increased 1 cm. might have created an unsuitable condition for In conclusion, we found two species of termites termites to survive in the winter. in the Delta Experimental Forest near Stoneville, Live termites were found in 24.8%, 22.2%, and Mississippi. Reticulitermes flavipes was the most 7.0% of the logs, branches, and stumps in plots 1- common termite species. The occurrence of ter- 3, respectively. The percentage of wood materials 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

Wang & Powell: Survey of termites 225

Fig. 3. Relationship between diameter of wood sam- Fig. 1. Percentage of dead wood samples with ter- ples and the percentage with termites present (Percent- mites or with signs of termite activity in Delta Experi- age = 0.044 + 0.013 × diameter, R2 = 0.72, n = 678). mental Forest, Mississippi. wood materials by the two termite species was actual length of the 4 categories ranges from <3, 3- positively correlated with the diameter of the 5.9, 6-8.9, and ≥9 m, respectively. Although the wood materials. This has practical significance length of wood had significant effect on the perfor termite control and baiting studies. It provides centage of wood with live termites (F = 11.23; df = an estimate of the probability of termite attack on 3, 666; P < 0.0001), it did not have significant effect wood materials. on the percentage of wood with signs of termite ac- Gentry & Whitford (1982) sampled lowland tivity (F = 0.67; df = 3, 502; P = 0.57). A long hardwood forests and pine plantations in South branch with small diameter may be less likely to Carolina (similar latitude as in our study location), be attacked by termites than a short branch with and found that the occurrences of R. flavipes and large diameter. Therefore, length alone is not a R. virginicus varied among different habitats. Reti- good predictor for estimating the probability of ter- culitermes flavipes represented 26.6-60% of the termite attack on the wood materials. mites, which was lower than what we observed on Jones et al. (1995) found a positive relation- the 3 sites on the Delta Experimental Forest. In ship (R2 = 0.50) between volume of wood and dry their study, nearly all the wood greater than 2 cm wood termite colony size on Mona Island, Puerto diameter had signs of termite usage. The termite Rico. In a laboratory experiment, Hedlund and population or activity in their study location was Henderson (1999) found termite consumption much higher than in our study location. rate increased as the wood volume increased. Ter- We measured the length of every piece of wood mites foraged less actively when larger wood ex- in this survey. The wood materials were grouped isted. These findings support our conclusion that into 4 categories according to their length. The larger wood tends to be more attractive for termites 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 grateful to Deborah Boykin (USDA Agricultural Re- search Service) for help in the statistical analysis, and Rudolf Scheffrahn, University of Florida for verifying identiﬁcations. Steve Meadows from USDA Forest Service provided the vegetation information of the study plots. The manuscript was Fig. 2. Relationship between diameter of wood sam- improved by E. W. Riddick, J. A. Morales-Ramos, ples and the percentage with signs of termite activity W. D. Woodson, T. E. Nebeker, and three anony- (Percentage = 0.327 + 0.013 × diameter, R2 = 0.73, n = mous reviewers. This research was funded 512). through USDA Agricultural Research Service. 226 Florida Entomologist 84(2) June 2001

REFERENCES CITED termites (Isoptera: Kalotermitidae) of Mona Island. Florida Entomol. 78: 307-313. BOYKIN D. L. 1995. Weather data summary for 1964-1993 KRINARD, R. M. AND R. L. JOHNSON. 1985. Eighteen- at Stoneville, Mississippi. Mississippi Agricultural year development of sweetgum (Liquidambar & Forestry Experiment Station Tech. Bull. 201. 49 pp. styraciflua L.) plantings on two sites. Tree Planters’ GENTRY, J. B. AND W. G. WHITFORD. 1982. The relation- Notes. 36: 6-8. ship between wood litter infall and relative abun- LA FAGE, J. P. AND W. L. NUTTING. 1978. Nutrient dy- dance and feeding activity of subterranean termites namics of termites, pp. 165-232. In M. V. Brian (ed.). Reticulitermes spp. in three southeastern coastal Production ecology of ants and termites. Cambridge plain habitats. Oecologia. 54: 63-67. University Press, Cambridge. GRAY, B. 1972. Economic tropical forest entomology. MATSUMOTO, T. 1976. The role of termites in an equato- Ann. Rev. Entomol. 17: 313-354. rial rain forest ecosystem of west Malaysia. I. Popu- HARRIS, W. V. 1966. The role of termites in tropical for- lation density, biomass, carbon, nitrogen and estry. Insectes Soc. 8: 255-266. calorific content and respiration rate. Oecologia HEDLUND, J. C. AND G. HENDERSON. 1999. Effect of (Berl.) 22: 153-178. available food size on search tunnel formation by the SAS INSTITUTE. 1999. SAS OnlineDoc™, Version 7-1. Formosan subterranean termite (Isoptera: Rhinoter- SAS Institute, INC. Cary, NC. mitidae). J. Econ. Entomol. 92: 610-616. SCHEFFRAHN, R. H. AND N.-Y. SU. 1994. Keys to soldier HOWARD, R. W., S. C. JONES, J. K. MAULDIN, AND R. H. and winged termites (Isoptera) of Florida. Florida BEAL. 1982. Abundance, distribution, and colony Entomol. 77: 460-474. size estimates for Reticulitermes spp. (Isoptera: Rhi- WOOD, T. G. AND W. A. SANDS. 1978. The role of ter- notermitidae) in southern Mississippi. Environ. En- mites in ecosystems, pp. 245-292. In M. V. Brian tomol. 11: 1290-1293. (ed.). Production ecology of ants and termites. Cam- JONES, S. C., C. A. NALEPA, E. A. MCMAHAN, AND J. A. bridge University Press, Cambridge. TORRES. 1995. Survey and ecological studies of the

McFarland & Hoy: Survival of D. citri and parasitoids 227

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 tolerances compared to these parasitoid populations. Adult survival was evaluated under relative 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 survived 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, biological control

RESUMEN

La habilidad de la plaga exótica del cítrico, Diaphorina citri Kuwayama, y sus dos parasitoides, Tamarixia radiata (Waterson) y Diaphorencyrtus aligarhensis (Shafee, Alam & Aga- rwal) de sobrevivir bajo diferentes humedades relativas (7%, 33%, 53%, 75% y 97%) a 25 y 30°C fueron comparadas. Los datos obtenidos pueden ayudar a predecir las limitaciones po- tenciales climáticas a su establecimiento en la Florida e indican si la plaga tiene diferentes tolerancias climáticas en comparación a estas poblaciones parasitoides. La supervivencia de adultos fue evaluada bajo humedades relativas mantenidas por soluciones de sal saturada. D. citri sobrevivió 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- blación T. radiata (Taiwán) demostró el mayor requerimiento de humedad en todas las temperaturas y humedades relativas probadas. La población T. radiata (Vietnam) sobrevivió mas que la de Taiwán, sugiriendo que las dos poblaciones pueden ejecutar de manera dis- tinta en diferentes regiones geográﬁcas. D. aligarhensis y T. radiata (Vietnam) sobrevivieron duraciones de tiempo similares, excepto bajo humedades relativas altas, indicando que los requerimientos de humedad para estas dos poblaciones son comparables.

The Asian citrus psylla, Diaphorina citri Ku- ever, the worst threat is that D. citri is an efﬁcient wayama, was found for the ﬁrst time in June 1998 vector of the bacterium, Liberobacter asiaticum, by personnel in the Division of Plant Industry, which causes greening disease. This is the most Florida Department of Agriculture and Consumer serious disease of citrus in the world, causing re- Services (Hoy & Nguyen 1998; Halbert 1998; duced production and eventual death of the trees Mead 1977). It has successfully established in (McClean & Schwarz 1970). Florida and it will probably colonize all citrus- No effective eradication efforts are known for growing areas in Florida and may spread to Lou- D. citri, but classical biological control of the psyl- isiana, Texas, Arizona and California citrus. lid vector should contribute to suppression of D. citri damages citrus by depleting sap from their populations. Two parasitoids have been im- the plant and, because its saliva is toxic, growing ported (Hoy et al. 1999; Hoy & Nguyen 2000). shoots are distorted and may die. It also causes Tamarixia radiata (Waterston) is an ectoparasi- damage by excreting honeydew, which allows the toid imported from Taiwan and Vietnam, while growth of sooty mold (Chien & Chu 1996). How- Diaphorencyrtus aligarhensis (Shafee, Alam &

228 Florida Entomologist 84(2) June 2001

Agarwal) is an endoparasitoid of D. citri obtained day, the psyllids, T. radiata and D. aligarhensis from Taiwan. Approximately 12,000 T. radiata were set up 15 days, 12 days and 18 days, respec- were released into Florida between 15 July and 1 tively, before the start of the experiments. The December 1999. D. aligarhensis was approved for day before starting an experiment, all old psyllids release on 15 March 2000 and 5000 were released and parasitoids were collected to ensure that only during the 2000 growing season (Hoy, unpublished). new individuals would be present in the cages. The success of classical biological control pro- D. citri is collected by aspirating adults off the grams may be determined by the use of the appro- plant, while T. radiata can be collected off the top priate natural enemy ecotypes. Moisture require- of the mesh cage. D. aligarhensis is aspirated ments may be especially important in determining most often from the bottom of the cage. Individual suitability of an imported biological control agent adults were placed in groups of five into 28.35 g for release and establishment in a new geographic condiment cups (Plastics, Inc. St. Paul, MN). The area. Only a few experiments have been conducted lid of the cup was covered with mesh glued to it to compare the water balance or the temperature- with clear nail polish. All containers were cleaned relative humidity relations in a pest and its para- with 70% EtOH before being reused. sitoids. Yoder & Hoy (1998) found the citrus leafminer and two populations of its endoparasitoid Pretreatment Conditions Ageniaspis citricola Logvinovskaya both require a After collection, all individuals were pre- moisture-rich environment although the citrus treated for 21 hours by placing them into bell jars leafminer is less dependent on high relative hu- placed in an incubator (Percival I-30B) pro- midities than its parasitoid, A. citricola. grammed with a photoperiod of 18:6 (L:D) at The objective of this study is to compare, under 17°C. RH within the bell jars was maintained by laboratory conditions, the effect of temperature a saturated salt solution, Mg(NO ) 6H 0, which and relative humidity on the survival of an exotic 3 2 2 yields a 56% RH (Winston & Bates 1960; O’Brien citrus pest, D. citri and its two parasitoids T. radi- 1948; Carr & Harris 1949). Specimens were given ata and D. aligarhensis. This information will no water or honey. Pretreatment was expected to help in mass rearing these parasitoids and may remove adsorbed water from the cuticular surface help to predict potential climatic limitations to and to ensure a uniform physiological state. Pre- their establishment in Florida. treatment minimizes the effects of ingestion, reproduction, excretion and defecation (Arlian & MATERIALS AND METHODS Ekstrand 1975). The survival rate should reflect Rearing the relative changes in water balance of the insects before desiccation (Wharton 1985). D. citri was reared on Murraya paniculata (Chien et al. 1989), ornamental orange jasmine, Experimental Conditions in the quarantine facility at the University of At 2 PM specimens were transferred into Florida, Gainesville (Skelley & Hoy, unpublished). stainless steel chambers (30.5 cm × 30.5 cm × 30.5 The plants were pruned, fertilized and held under cm) (Fisher Scientific, Pittsburgh, PA) held a photoperiod of 16:8 (L:D) at 21 to 24°C to stim- within a single incubator (Percival I-35 series) at ulate the growth of new flushes because adult either 25 or 30°C with a photoperiod of 18:6 (L:D). psyllids lay eggs only on newly sprouted leaf Plastic cups containing five adults were placed buds. Trees that had aphid or scale infestations into a chamber with either 7, 33, 53, 75 or 97% were sprayed with an application of oil or pyre- RH maintained by saturated salt solutions. Re- thrum (Prentiss, Inc., Sandersville, GA). agent grade salts (Sigma, St. Louis, MO) dis- D. citri was reared in quarantine under a pho- solved in distilled water were used: 200 ml of toperiod of 18:6 (L:D) at 26°C and 60% relative saturated salt solution was placed into chambers humidity (RH). T. radiata colonies from Taiwan with the three higher RH, and 100 ml of salt solu- and Vietnam and D. aligarhensis from Taiwan tion was used in the two lower RH because they were reared in separate cages and rooms in quar- are hygroscopic substances and should be formu- antine to reduce the risk of contamination. Both lated as a paste to maintain the desired RH. parasitoid species were reared at 27°C and 60% These salts were chosen because they maintain RH under a photoperiod of 18:6 (L:D) in cages (76 the same RH at 25 and 30°C (Winston & Bates cm × 46 cm × 66 cm) constructed with 1.27 cm 1960). The chemicals used were sodium hydroxide diameter polyvinyl chloride pipe and covered with (7% RH), magnesium chloride hexahydrate (33% a fine nylon mesh. RH), magnesium nitrate hexahydrate (53% RH), Specimen Collection sodium chloride (75% RH) and potassium sulfate (97% RH). RH in each chamber was confirmed by Adults used for experiments were collected at a hygrometer (Thomas Scientific, Swedesboro, 5 PM on the day of their emergence. To ensure NJ), as well as the salt crystal method outlined by that all four populations emerged on the same Winston & Bates (1960), which utilizes the fact

McFarland & Hoy: Survival of D. citri and parasitoids 229 that a salt crystal will pick up moisture and deli- D. aligarhensis and T. radiata (Vietnam) sur- quesce in relative humidities which are at or vived similar lengths of time under all RH at 25 above that maintained by its saturated solution. and 30°C, so the moisture requirements for these A very small crystal is introduced into the closed two populations appear comparable (Figs. 1, 2). chamber and observed under magniﬁcation; if the LT 10 and LT90 values can be found easily from RH is below that for the salt, there will be no the ﬁgures, but the differences are small and not change. If the RH is very close, the deliquescence many inferences about the populations can be made will be only partial; if just above there will be a at the ends of the curves. The LT50 values of the narrow rim of liquid around the mass. Experi- pest D. citri were always higher than the LT50 val- mental units were placed randomly into each RH ues of its parasitoid populations at 25°C (Fig. 1). ° chamber. LT 50 values at 25 C for D. aligarhensis are dif-

ferent from LT50 values for the T. radiata (Taiwan) Survivability Tests population (P < 0.05) (Table 1). D. aligarhensis

and T. radiata (Vietnam) had different LT50 val- Survival of individuals was recorded every ues, except at 53 and 97% RH (z = 0.10, P > 0.05;

three hours. RH chambers were sampled sepa- z = 0.75, P > 0.05) (Table 1). The LT50 values of the rately and specimens were out of the chamber for two T. radiata populations are not different at 7 a maximum of ten minutes. Insects found on their and 33% RH (z = 1.55, P > 0.05); z = 0.10, P > 0.05) dorsal surface that failed to respond to probing (Table 1). were considered dead. All dead specimens were All parasitoids held at 25°C were dead by 40.5 sexed. hours, whereas a few psyllids survived up to 94.5 hours (Fig. 1). There were no differences between Statistics survival of males and females of the D. citri and T. radiata populations, perhaps because there is no Four replicates were tested at 25°C under each apparent difference in size. The D. aligarhensis RH on separate dates, while three replicates were population is made up of females only. ° ° run at 30 C and each RH condition. At 25 C, a to- The LT50 values of the pest were equal to or

tal of 400 individuals (100 per experiment) were greater than the LT50 values of its parasitoids at tested for D. citri and the two T. radiata popula- 30°C (Table 1). As the RH increased to 97%, the

tions. Only 300 individuals (75 per experiment) of LT50 of the D. citri population was different from the D. aligarhensis population were tested at that of D. aligarhensis and the T. radiata (Viet- ° ° 25 C. At 30 C, a total of 300 individuals (100 per nam) population (P < 0.05) (Table 1). The LT50 val- experiment) were tested for all four populations. ues of the T. radiata (Taiwan) population were Mean percentage survival (±SEM) was calculated different (P < 0.05) from the D. citri, D. aligarhen- for all four populations at each temperature-RH sis and T. radiata (Vietnam) populations at all

combination. LT50 values were calculated by add- RH tested (Table 1). Again, there were no differing the time it took to get 50% mortality under ences in survival rates of males and females. All each condition on each date. A grand mean and parasitoids held at 30°C were dead by approxi- SEM was determined for each population at each mately 40.5 hours, but a few psyllids managed to

temperature-RH combination. LT50 values were survive up to 52.5 hours (Fig. 2). compared statistically by z test statistics. The data suggest that, especially at 75 and 97% RH at 25°C and 97% RH at 30°C, some RESULTS AND DISCUSSION D. citri will have up to two times longer to ﬁnd food and water in the ﬁeld compared to its parasi- The survival of D. citri, D. aligarhensis and toids (Figs. 1D, 1E, 2E). D. citri also survived well T. radiata populations increased with increasing at the lower RH of 7 and 33%, suggesting that it RH (Figs. 1, 2). When the temperature was in- could disperse and survive in the more arid condi- creased from 25 to 30°C, adult survival of all pop- tions found in western citrus-growing regions in ulations decreased, as expected (Figs. 1, 2). At 75 the United States (Figs. 1A, 1B, 2A, 2B). and 97% RH at both temperatures, D. citri sur- The RH responses of the Vietnam population vived longer than the parasitoid populations, sug- of T. radiata were different from those of the Tai- gesting it has a lower net water loss rate (Figs. wan population, especially at 30°C, suggesting 1D, 1E, 2D, 2E). that the two populations of T. radiata may be The T. radiata (Taiwan) population showed the ecotypes (populations varying in their ability to greatest moisture requirement at all tempera- survive in different climates). tures and RH tested. The T. radiata (Vietnam) This information is helpful in rearing these population survived longer than the Taiwan pop- parasitoids for release in biological control pro- ulation at 25°C and 53, 75 and 97% RH as well as grams because it indicates there are no large dif- all RH tested at 30°C (Figs. 1C, 1D, 1E, 2). This ferences in pest and natural enemy responses to suggests that the two populations may perform the RH and temperatures likely to be encoun- differently in different geographical regions. tered in the rearing facility. The ability of D. citri 230 Florida Entomologist 84(2) June 2001

Fig. 1. Survival curves of the Asian citrus psylla, Diaphorina citri, and its two parasitoids, Diaphorencyrtus aligarhensis 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 of D. citri and parasitoids 231

Fig. 2. Survival curves of the Asian citrus psylla, Diaphorina citri, and its two parasitoids, Diaphorencyrtus aligarhensis 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). 232 Florida Entomologist 84(2) June 2001

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 50 values (SEM) in hours at relative humidity (%) of

Species 7 33537597

------at 25°C ------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 30°C ------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 (RAPD-PCR) and DNA sequence analyses of two is interesting and difficult to interpret (Figs. 1E, Actin genes led to the conclusion that the two pop- 2E). It could mean that D. citri is better adapted ulations actually are cryptic species, even though to very high RH than its natural enemies. The these two populations of A. citricola can not be data obtained compare the intrinsic water loss separated on the basis of morphological charac- rates of these four populations under extreme teristics (Hoy et al. 2000). Additional analysis of conditions of no food or water, and the ability of the two populations of T. radiata could establish D. citri, D. aligarhensis and T. radiata popula- whether they are cryptic species. tions to disperse to more optimal sites was pre- cluded. In citrus groves D. citri can feed on host ACKNOWLEDGMENTS plants to obtain water and food. Likewise, the parasitoids can host feed for moisture and nutri- We thank John Capinera and James Nation for ents, feed on honeydew, drink droplets of water providing advice on the manuscript, Lucy Skelley and disperse to find microhabitats more suitable for her assistance in rearing Murraya paniculata, for survival. psyllids and parasitoids, and Ramon Littell for Yoder & Hoy (1998) found two populations of his help with the statistical analyses. This work A. citricola and their host, the citrus leafminer, was funded in part by the Davies, Fischer and both require a moisture-rich environment al- Eckes Endowment. This is University of Florida though the citrus leafminer is less dependent on Agricultural Experiment Station Journal Series high RH than its parasitoids. The results of this No. R-07835. study were similar, with D. citri and its parasitoids requiring a moisture-rich environment, al- REFERENCES CITED though D. citri does not depend on high RH as much as its parasitoids, especially the T. radiata ARLIAN, L. G., AND I. A. EKSTRAND. 1975. Water balance (Taiwan) population. D. citri survived longer at in Drosophilia pseudoobscura, and its ecological im- higher temperatures and lower RH than the T. ra- plications. Ann. Entomol. Soc. America 68: 827-837. diata (Taiwan) and the D. aligarhensis popula- CARR, D. S., AND B. L. HARRIS. 1949. Solutions for con- tions (Figs. 2A, 2B). stant humidity. Indust. Eng. Chem. 41: 2014-2015. CHIEN, C. C., AND Y. I. CHU. 1996. Biological control of Hoy et al. (2000) found slight physiological dif- citrus psyllid, Diaphorina citri in Taiwan. Inter. J. ferences in the Taiwan and Australian ecotypes of Pest Mgt. 34: 93-105. A. citricola, which indicated genetic differences CHIEN, C. C., S. C. CHIU, AND S. C. KU. 1989. Biological exist. Subsequent analysis of Random-Amplified- control of Diaphorina citri in Taiwan. Fruits 44: 401- Polymorphic-DNA Polymerase-Chain-Reaction 407. McFarland & Hoy: Survival of D. citri and parasitoids 233

HALBERT, S. E. 1998. Asian citrus psyllid-A serious po- MEAD, F. W. 1977. The Asiatic citrus psyllid, Diaphorina tential exotic pest of Florida citrus. http://www.ifas. citri Kuwayama (Homoptera: Psyllidae). Florida Dept. ufl.edu/~entweb/DCITRI.htm. of Agriculture and Consumer Services, Division of HOY, M. A., AND R. NGUYEN. 1998. Citrus psylla: Here Plant Industry. Entomol. Circular 180. Reprinted in in Florida—An action plan-updated. http://extlab1. 1998 as Featured creature-Asian citrus psyllid. entnem.ufl.edu/PestAlert/hoy-0615.htm. http://gnv.ifas.ufl.edu/~insect/CITRUS/acpsyllid.htm. HOY, M. A., AND R. NGUYEN. 2000. Classical biological O’BRIEN, F. E. M. 1948. The control of humidity by sat- control of brown citrus aphid. Release of Lipolexis urated salt solutions. J. Sci. Instruments 25: 73-76. scutellaris. Citrus Industry 8(12): 24-26. WHARTON, G. W. 1985. Water balance of insects, pp. 565- HOY, M. A., R. NGUYEN, AND A. JEYAPRAKASH. 1999. 601. In G. A. Kerkut and L. I. Gilbert (eds.). Compre- Classical biological control of the Asian citrus psylla- hensive insect physiology biochemistry and pharma- release of Tamarixia radiata. Citrus Industry 80(9): cology. Oxford: Pergamon Press. 20-22. WINSTON, P. W., AND P. S. BATES. 1960. Saturated salt HOY, M. A., A. JEYAPRAKASH, R. MORAKOTE, P. K. C. LO, solutions for the control of humidity in biological re- AND R. NGUYEN. 2000. Genomic analyses of two pop- search. Ecology 41: 232-237. ulations of Ageniaspis citricola (Hymenoptera: En- YODER, J. A., AND M. A. HOY. 1998. Differences in water cyrtidae) suggest that a cryptic species may exist. relations among the citrus leafminer and two different Biol. Cont. 17: 1-10. populations of its parasitoid inhabiting the same ap- MCCLEAN, A. P. D., AND R. E. SCHWARZ. 1970. Greening parent microhabitat. Entomol. Exp. Appl. 89: 169-173. of blotchy-mottle disease in citrus. Phytophylactica 2: 177-194.

234 Florida Entomologist 84(2) June 2001

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 vanillae (L.), Lepidoptera: Nymphalidae) which accounted for 58% of the insects counted; the highest rate was 3,162/h, with an estimate of total season migration 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 vanillae, migration, dispersal

RESUMEN

Se describe la migración de 5 especies Lepidóptera y 4 especies Odonata en el otoño de 1999 hacia el norte a lo largo de la península de St. Joseph, condado del Golfo, en la región noroeste de la Florida. Las cuentas mas altas fueron de la mariposa Agraulis vanillae (L.) (Lepidóp- tera: Nymphalidae), la cual constituyo el 58% de los insectos contados; la cuenta mas alta fue de 3,162/h, con un estimado de migración estacional total de mas de 250,000 individuos. La libélula Anax junis (Drury 1773) (Odonata: aeshnidae) le sigue en numero con una cuenta total de 3,297/h. El medio y periodo cumbre para estas dos especies fue la primera semana de Octubre. El patrón de vuelo observado puede demostrar una aversión a cruzar el agua.

Due to its southern location, Florida may be (Fig. 1). The count location was on an elevated, 50 either a migration pathway, stopover, or desti- m long boardwalk bisecting the peninsula, at Ea- nation for migrant insects. Migrating butterflies gle Harbor (29°45.98’N, 85°24.29’W) in St. Joseph usually fly close enough to the ground and often Peninsula State Park. At the boardwalk, the penin sufficient numbers to attract public attention. insula has a width of 200 m and is 11 km from the Large scale fall movements of the monarch but- north tip (Fig. 1). terfly (Danaus plexippus (L.), Lepidoptera: There are only a few small freshwater wet- Nymphalidae) have been documented westward lands on the peninsula that support small breed- along the Gulf Coast (Urquhart & Urquhart 1978, ing populations (<50 pair) of Odonata including Van Hook & Hermann 1999). Additionally, 5 spe- A. junius, black saddlebags (Tramea lacerata cies of butterflies have been reported migrating in Hagen 1861, Odonata: Libellulidae) and Carolina a generally southward direction in peninsular saddlebags (Tramea carolina (L.), Odonata: Libel- Florida (Walker 1979; Walker 1991). Localized lulidae). There were low numbers of breeding but- fall movements of massive numbers of the com- terflies including D. plexippus and Gulf fritillary mon green darner dragonfly (Anax junius (Drury butterfly (Agraulis vanillae (L.), Lepidoptera: 1773) Odonata: Aeshnidae) have been docu- Nymphalidae) on the peninsula. mented on the Atlantic coast of Florida (Russell et Counts were made in 2, 5-minute periods per al. 1998). This paper presents fall migration hour, 1-2 days per week, from 3 to 11 hours each observations for both Odonata and Lepidoptera at day. All species with ≥3 cm wing span were counted. a location along the Gulf Coast in the Florida Flight direction, estimated height, predation, Panhandle. perching, tandem flights, and copulation, were recorded. Weather conditions, particularly frontal MATERIALS AND METHODS boundary, and wind direction were recorded. Hourly flight rates (in any flight direction) were Both butterflies (Lepidoptera) and dragonflies calculated from the average of the 2, 5-minute (Odonata) were counted on St. Joseph Peninsula, counts in an hour. Highest observed hourly migra- Gulf County, Florida from 14 Aug through 7 Dec, tion rates for the fall for each species were re- 1999. The peninsula is 24 km long, oriented ported. Linear interpolation between the observed north-south, roughly parallel to the mainland hourly flight rates, first between hours in a day,

Sprandel: Migration at St. Joseph Peninsula 235

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).

236 Florida Entomologist 84(2) June 2001

RESULTS AND DISCUSSION necessarily reflect the direction of a potentially larger migration on the mainland. A total of 12,616 insects were counted in 222, There have been observations from boats (e.g., 5-minute count intervals on 19 days from 14 Aug Lowery 1946) and oil platforms (Baust et al. 1981, to 7 Dec. Most (98.7%) were heading north along Russell 1999) of some dragonflies and butterflies the peninsula (Fig. 1), with the remainder either flying over the Gulf. My observations of monarch patrolling (Odonata) or heading south (both Odo- migration match previously recorded western nata and Lepidoptera). The most common mi- movement of eastern populations (Urquhart & grant was the Gulf fritillary butterfly, A. vanillae, Urquhart 1978; Van Hook & Hermann 1999); and followed by the common green darner dragonfly, destinations may be either the Gulf Coast states, A. junius (Table 1). All insects appeared to use or Mexico. These data do not suggest a peninsular powered flight rather than soaring. Although Florida destination for all Gulf fritillary (A. vanil- there were peak flights during the first week of lae) and cloudless sulphur butterflies (Phoebis October (Fig. 2), there was a steady and persis- sennae (L) Lepidoptera: Pieridae), as suggested tent flight on each clear day. by other studies (Walker 1979; Walker 1991). Pre- One possible flight pattern is that the insects vious reports of the long-tailed skipper (Urbanus head south down the continent, fly west when proteus L., Lepidoptera: Hesperiidae) at Carra- reaching the Gulf, and are then funneled north belle Beach (80 km east), indicated flight direc- via the peninsula. The northeast prevailing wind tions in both coastal directions (roughly east and direction in the fall (Winsberg 1990) would pro- west, Walker and Littell 1994). Destination and vide a further push from the mainland onto the migration patterns of Odonates are generally un- peninsula (Fig. 1). Local sea breezes, would com- known (May 1992) though northward movement plicate this general pattern. The insects may ex- of A. junius in summer and scarcity of adult hibit some risk avoidance by not crossing the open A. junius and T. lacerata in winter in Florida have Gulf. This migration route has been documented been suggested (Paulson 1966; Dunkle 1989). for 3-5,000 hawks annually, with the heaviest The peak day on 2 Oct (Figs. 2, 3) was 3 days flights 3-4 days after the passage of northern cold after the passage of a cold front. Some studies fronts when the wind is from the northeast (Sted- have reported the passage of cold fronts as an im- man 1984). After reaching the north tip of the petus for migration (Russell et al. 1998), but the peninsula, it is unknown whether the insects fly following weekend also had high numbers, de- the 2-km distance to the mainland or head south- spite no frontal activity. During drizzle the flight west over the Gulf (to uncertain landfall). The low dropped off to zero, but on the 2 overcast days (18 number of south-flying insects counted suggests Sep, 27 Sep) the high A. vanillae rates were still that they are not back-tracking down the penin- 48 and 2,472/h; while for A. junius the high rates sula. Observations from the peninsula may not 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 rate1 50% period3 Species (date and hour) % total2 (median) Est. total4

Odonata Anax junius 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

1Average rate from 2 5-minute counts/h, and date and hour of the count in parenthesis. 2Percentage of total count of all insects for this species. 3The 50% period is the period between 25% and 75% completion of seasonal migration. 4Estimated total is based on linear interpolation between observed hourly rates both between hours and days.

Sprandel: Migration at St. Joseph Peninsula 237

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 pattern, 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 represent an evening descent from above the height of direct observation (Corbet 1999). Behaviors other than flight included 51 instances of predation by A. junius on A. vanillae, stable fly (Stomoxys calcitrans (L), Diptera: Mus- cidae, see Wright 1945; Fye et al. 1980), mosquitoes (Diptera: Cilicidae), long-tailed skipper Fig. 2. Estimated daily survey totals for Gulf fritil- (U. proteus), buckeye butterfly J. coenia, and lary butterfly (Agraulis vanillae) and common green black saddlebags dragonfly T. lacerata. The only darner (Anax junius) from 14 Aug-7 Dec 1999 on the St. Joseph Peninsula, Gulf County, Florida. other instances of predation were twice by T. lacerata on stable flies, and once by a wandering glider dragonfly (P. flavescens) on the buckeye For both the Lepidoptera and Odonata the pe- butterfly (J. coenia). No predation on dragonflies riod when the middle 50% of the seasonal flight was observed despite abundance of potentially occurred varied by species (Table 1) with the ear- predatory birds including merlin (Falco colum- liest being P. sennae and the latest being the barius), American kestrel (Falco sparverius) (e.g., buckeye, Junonia coenia (L., Lepidoptera: Walter 1996) barn swallow (Hirundo rustica), Nymphalidae). The whole extent of the flight pe- tree swallow (Tachycineta bicolor), and logger- riod was much larger. For example, the flights head shrike (Lanius ludovicianus). Only dragon- ranged from 14 Aug to 28 Nov for the dragonflies flies were observed preying on Lepidoptera, as A. junius and the wandering glider Pantala flave- described above. There was some mortality from scens (F., Odonata: Libellulidae). Flights began on vehicles on the peninsula road of both butterflies 5 Sep for all butterflies and extended to 28 Nov for and dragonflies. Some butterflies paused within D. plexippus and J. coenia. By 7 Dec, A. vanillae 100 m north of the boardwalk on flowering plants. was the only species observed. There were 28 pairs of A. junius flying in tandem (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 indicating copulation (20 Nov). There was one instance 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 variability within the day (e.g., Fig. 3) and sharp peaks between days (Fig. 2), indicate that the linear interpolation method should not be viewed as an absolute number, but rather as an index of the relative abundance of the migrants. These migration levels are less than the millions in massive migratory swarms (May 1995; Russell et al. 1998) of Odonates, and less than the estimated 30 million seasonally migrating Lepidoptera in peninsular Florida (Walker 1991). This study does Fig. 3. Hourly flight rates by hour of the day (07:00- indicate a slow and steady migration for both 19:00) on 2 Oct 1999 for migrating Gulf fritillary butterfly dragonflies and butterflies at this location. Al- (Agraulis vanillae) and common green darner (Anax though none of these species are considered junius) on the St. Joseph Peninsula, Gulf County, Florida. threatened (Logan 1997) or rare (Deyrup & Franz 238 Florida Entomologist 84(2) June 2001

1994), their migrations may be endangered LOWERY, G. H. JR. 1946. Evidence of trans-Gulf migra- (Brower & Malcom 1991). Further studies along tion. Auk 63: 175-211. obvious migration corridors, and observations MAY, M. L. 1992. Migrating dragonflies in North Amer- from the tip of St. Joseph Peninsula, would help ica. Argia 4(2): 4-8 piece together flight patterns. More intensive sur- MAY, M. L. 1995. Dependence of flight behavior and heat production on air temperature in the green darner veys over multiple years would help determine if dragonfly Anax junius (Odonata: Aeshnidae). J. Exp. there were synoptic or local weather correlates to Bio. 198: 2385-2392. migration. Protection along migration paths PAULSON, D. R. 1966. Dragonflies (Odonata: Anisop- might include avoiding use of insecticides, and tera) of South Florida. Ph.D. Dissertation, Univ. of not mowing nectar plants. The impact of coastal Miami, 603 pp. development on these routes is unknown. RUSSELL. R. W., M. L. MAY, K. L. SOLTESZ, AND J. W. FITZPATRICK. 1998. Massive swarm migrations of dragonflies (Odonata) in Eastern North America. ACKNOWLEDGMENTS American Midland Nat. 140: 325-342. Michael May alerted me to the general interest RUSSELL. R. W. 1999. Migration Over the Gulf. Louisiana Museum of Natural Science. http://transgulf. org/. in these phenomena. Wendy Brill, Jerrell J. Daigle, STEDMAN, S. J. 1984. St. Joseph Peninsula hawk migra- Michael May, Tom Walker, and George Wallace tion. Dep. Nat. Res., Tallahassee, FL. reviewed earlier drafts of this manuscript. URQUHART, F. A., AND N. R. URQUHART. 1978. Autum- nal migration routes of the eastern population of the REFERENCES CITED monarch butterfly (Danaus p. plexippus L.; Dan- aidae; Lepidoptera) in North America to the over- BAUST, J. G., A. H. BENTON, AND G. D. AUMANN. 1981. wintering site in the Neovolcanic Plateau of Mexico. The influence of off-shore platforms on insect disperals Canadian J. Zool. 56: 1759-1764. and migration. Bull. Entomol. Soc. America 27:23-25. VAN HOOK, T. V., AND S. HERMANN. 1999. Monarch fall BROWER, L. P., AND S. B. MALCOM. 1991. Animal migra- migratory population monitoring at St. Marks Na- tions: endangered phenomena. American Zool. tional Wildlife Refuge: 1985-86, 1988-89, 1991-92, 31:265-276. 1997-98. Prelim. rept. to Florida Fish and Wild. CORBET, P. S. 1999. Dragonflies: behavior and ecology of Cons. Comm., Tallahassee. Odonata, Cornell University Press, Ithaca, NY. WALKER, T. J. 1979. Migrating lepidoptera: are butter- DEYRUP, M., AND R. FRANZ (eds.). 1994. Rare and en- flies better than moths? Florida Entomol. 63: 79-98. dangered biota of Florida. Vol. IV—invertebrates. WALKER, T. J. 1991. Butterfly migration from and to University Press of Florida, Gainesville. peninsular Florida. Ecol. Entomol. 16:241-252. DUNKLE, S.W. 1989. Dragonflies of the Florida penin- WALKER, T. J. AND R. C. LITTELL. 1994. Orientation of sula, Bermuda, and the Bahamas, Sci. Pub. Nature fall-migrating butterflies in North Peninsular Flor- Guide No. 1, Gainesville, FL. ida and source areas. Ethology 98: 60-84. FYE, R. L., J. BROWN, J. RUFF, AND L. BUSCHMAN. 1980. WALTER, S. 1996. Fort Tilden dragonfly migration A survey of northwest Florida for potential Stable fly watch. Argia 8(1): 8-13. breeding. Florida Entomol. 63(2): 246-251. WINSBERG, M. D. 1990. Florida weather. University of LOGAN, T. H. 1997. Florida’s endangered species, threat- Central Florida Press, Orlando. ened species, and species of special concern: official WRIGHT, M., 1945, Dragonflies predaceous on the lists. Florida Game and Fresh Water Fish Commis- Stablefly, Stomoxys calcitrans (L.), Florida Entomol. sion, Tallahassee. 28: 11-13.

Liu & Yue: Effects of Temperature on Lipaphis erysimi 239

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 YUE1 Vegetable IPM Laboratory, Texas Agricultural Experiment Station, Texas A&M University 2415 East Highway 83, Weslaco, TX 78596-8399

1Permanent 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 temperatures 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 apterous 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, respectively, 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, respectively, compared with 52.5, 90.8, 83, and 29.7 nymphs per apterous adult at the same temperature regimes.

Key Words: Lipaphis erysimi, turnip aphid, alate aphid, apterous aphid, development, reproduction, cabbage, vegetable

RESÚMEN

Bajo temperaturas constantes en lanratorio, se estudió el desarrollo, la longevidad, la supervivencia y la fecundidad en col de las formas alada y áptera del áfido del nabo Lipaphis erysimi (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 ápteras fue de 13.9, 6.8, 6.1 y 5.0 dias. Las ninfas aladas se desarrolaron de 1.9-3.0 dias más rápido a 15, 20 y 25°C que las ninfas ápteras, pero el tiempo de desarrollo entre las formas alada y áptera no fue significativamente diferente a 30°C. La longevidad de los adultos 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 áfidos ápteros bajo los régimenes correspondientes de temperatura. La fecundidad también fue perceptiblemente menor para los adultos alados que para los adultos áptreos. Los adultos alados producen 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 áptero bajo el mismo régimen de temperaturas.

The turnip aphid, Lipaphis erysimi (Kalten- The life history parameters, such as develop- bach), is a worldwide pest on Brassica crops ment, longevity, survivorship and fecundity of the (Begum 1995; Liu et al. 1997; Prasad 1988; Yue & alate form of L. erysimi were not well documented, Liu 2000). The biology of apterous L. erysimi on and these biological characteristics and parame- several Brassica vegetables were well documented ters are essential for effective aphid management (Ahlawat & Chenulu 1982, Amjad and Peters (Halbert et al. 1981). In this paper, we report the 1992; Chander & Phadke 1994; Castle et al. 1992; effects of four different constant temperature re- Prasad & Phadke 1984; Edelson et al. 1993; Set- gimes on development, survivorship, longevity okuchi & Muma 1993; Singh et al. 1983; Singh & and fecundity of both the alate and apterous forms Sachan 1995). The nymphs and adults suck the of L. erysimi on cabbage in the laboratory. sap from leaves, young shoots, inﬂorescence and young pods, resulting in chlorophyll reduction or MATERIAL AND METHODS even plant death. Additionally, alate L. erysimi can transmit some important plant virus diseases, Host Plants such as sugar cane mosaic virus, cucumber mosaic virus and bean yellow mosaic potyvirus (Ahlawat Cabbage, Brassica oleracea var. capitata L. & Chenulu 1982; Castle et al. 1992). (Grand Slam Hybrid), was seeded in styrofoam

240 Florida Entomologist 84(2) June 2001

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 leaving 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 cabbage in a greenhouse for >1 year. Both alate and apterous adult aphids were collected from the greenhouse colony, and were transferred onto potted cabbage plants in an air-controlled insectary at 25 ± 2°C 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 bottom 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 water-saturated paper tissue in each petri dish. At the time of the experiment, neonate nymphs (<24 h old 1st instars) were collected from the laboratory colony using a small camel hair brush (#000) Fig. 1. Development, longevity and fecundity of both and placed into each rearing arena. Thirty to 40 alate and apterous alate Lipaphis erysimi on cabbage aphids were used for each treatment. Four con- under four constant temperatures in the laboratory. stant temperatures, 15, 20, 25, and 30°C, were maintained in growth chambers (Percival, Boone, IA) with a photoperiod of 14:10 (L:D) h and 50- At the four temperatures, alate nymphs devel- 75% RH. Development, molting, survival and oped significantly faster at higher temperatures number of newborn nymphs were recorded daily than at lower temperatures (F = 18.6; df = 3, 102; until the females died. Newborn nymphs were re- P = 0.0001). The developmental duration was moved after the daily recording until the death of shortest at 30°C (5.4 d), followed by at 25°C (8.0 the adult. Leaf disks were replaced at the first d), at 20°C (9.5 d), and the longest at 15°C (15.8 sign of deterioration, normally at 4-5 d intervals. d). Similarly, apterous nymphs also developed faster at higher temperatures than at lower tem- Data Analysis peratures (F = 9.3; df = 3, 101; P = 0.0002). The developmental duration was shortest at 30°C (5.0 Aphids were excluded from the data set if they d), followed by at 25°C (6.1 d), at 20°C (6.8 d), and died within 24 h or they were never observed the longest at 15°C (13.9 d). Alate nymphs devel- feeding. Developmental duration, longevity and oped significantly longer than apterous nymphs reproduction of both alate and apterous L. ery- at 15, 20 and 25°C (F = 11.2- 23.14; df = 1, 102; P simi under the four constant temperatures were = 0.0025-0.0001). Those under 30°C did not show analyzed using a two-way analysis of variance × significant differences in developmental dura- (ANOVA) (temperatures aphid forms), and the tions (F = 1.13; df = 1, 102; P = 0.5741). means were separated using the least significant difference (LSD) test at P = 0.05 upon a signifi- Survivorship and Longevity cant F-test (SAS Institute 1996). All nymphs in both alate and apterous forms survived to adulthood (Figs. 1 and 2). The longev- RESULTS AND DISCUSSION ities of both alate and apterous adults were signif- Nymph Development icantly affected by temperature. The longevity of alate adults was significantly shorter at 15°C Temperature significantly affected the develop- than those at the other three temperatures (F = ment for both alate and apterous nymphs (Fig. 1). 7.13; df = 3, 104; P = 0.0471). In contrast, the lon-

Liu & Yue: Effects of Temperature on Lipaphis erysimi 241

most favorable conditions. The nymphs developed fastest at 30°C, and slightly slower at 25 and 20°C. DeLoach (1974) found that L. erysimi on turnip could continue to reproduce at 30°C, but failed to do so at 35°C. Similarly, Liu (1991) found the most favorable temperature for L. erysimi was 26°C. At 8.3 and 35°C, few nymphs developed to adults, but none produced any offspring. At 11.3 and 32.8°C, few adults successfully reproduced. Therefore, the lower and upper threshold temperature for L. erysimi should be higher than 8°C and lower than 33°C, with the most favorable temperature between 25 and 27°C. 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 25°C (39.0 nymphs/adult) and 20°C (37.9 nymphs/adult), followed by these (11.9 nymphs/adult) at 30°C, and the fewest at 15°C (7.9 nymphs/adult). Apterous adults produced most nymphs at 20°C (90.8 nymphs/adult), followed by 83.0 nymphs/adult at 25°C, 52.5 Fig. 2. Survival rates and natality of both alate and nymphs/adult at 15°C, and the fewest, 29.7 apterous Lipaphis erysimi on cabbage at different tem- nymphs/adult at 30°C. Between the two forms, peratures in the laboratory. apterous adults produced 6.6-, 2.4-, 2.1- and 2.5- fold more nymphs per adult than alate adults of the corresponding temperature. gevities of apterous adults declined as the tem- Both alate and apterous adults started to pro- peratures increased from 15°C to 30°C. The duce nymphs 1 or 2 d after the last molting, so the longest longevity (25.3 d) was obtained at 15°C, adults from last molting to the first reproduction followed by 21.3 d at 20°C, 17.5 d at 25°C, and the were only 1-2 d old (Fig. 2). Both fecundity and re- shortest, 11.7 d at 30°C. Between the two forms, productive period varied at the four temperature alate adults lived a significantly shorter period regimes. Generally, the alate aphids had lower than apterous adults (F = 9.87-12.23; df = 1, 102; daily reproductive rates and shorter period of re- P = 0.0012-0.0001) at 15, 20 and 30°C except for production than the apterous forms at the same those at 25°C (F = 0.97; df = 1, 102; P = 0.8754). temperature. At 15°C, reproductive periods lasted The largest difference in longevity between the 12 d at 15°C, compared with 24 d at 20 and 25°C, two forms was found at 15°C at which the apter- respectively, and only 11 d at 30°C. Daily fecundity ous adults lived ≈2-fold longer than that of alate at the peak reproduction period was 3-4 nymphs adults (25.3 d vs. 12.6 d respectively). At 20°C, the at 20 and 25°C, <2 nymphs at 30°C and 15°C. apterous adults lived 3.6 d longer than the alate The reproductive periods of apterous lasted 34 adults. The longevities of both forms were almost d at 15°C, compared with 24 d at 20°C, 25 d at the same at 25°C. In contrast, the apterous adults 25°C, and only 11 d at 30°C. An aphid could pro- lived 5.5 d shorter than the alate adults (17.2 d vs. duce 8-9 nymphs per d at the peak reproduction 11.7 d) at 30°C. period at 20 and 25°C, 6-7 nymphs at 30°C, and 2- Similar effects were also reported by DeLoach 3 nymphs at 15°C. (1974) and Liu (1991). Aphid development and re- Our results clearly indicate that many biologi- production typically increase from zero at a low cal parameters, including nymph development, temperature threshold, reach a maximum at the adult longevity and fecundity differed between most favorable temperature, then decrease rap- alate and apterous forms. Similar results were re- idly to zero at a lethal threshold. Like other ported by Takaoka (1973) who reviewed that aphids in general, L. erysimi reared at tempera- longer nymph period, longer adult longevity and tures above the upper or below the lower thresh- lower fecundity of alate virginoparae were found olds develop more slowly than those under the in many species of aphids compared to those of 242 Florida Entomologist 84(2) June 2001

their apterous form. Information from this study DELOACH, C. J. 1974. Rate of increase of populations of will aid in predicting the population dynamics of cabbage, green peach and turnip aphids at constant L. erysimi on cole crops in south Texas. The sus- temperatures. Ann. Entomol. Soc. Am. 67: 332-340. ceptibility to low (<10°C) and high temperature EDELSON, J. V., J. J. MAGARO, AND H. BROWNING. 1993. (>35°C) may present a partial explanation for the Control insect pests on broccoli in southern Texas: A comparison between synthetic organic insecticides and low field populations of L. erysimi in the summer biorational treatments. J. Entomol. Sci. 28: 191-196. and in the winter months when temperatures are HALBERT, S., E. IRWIN, AND R. M. GOODMAN. 1981. Alate often >30 and <10°C, respectively. Meanwhile, aphid species and their relative importance as field the information on L. erysimi obtained under con- vectors of soybean mosaic virus. Comp. Biochem. stant temperatures may not be directly applicable Physiol. 97: 1-9. for field populations that may be affected by fluc- LIU, S. S. 1991. The influence of temperature on the pop- tuating temperatures (Liu & Meng, 1990) and ulation increase of Myzus persicae and Lipaphis ery- other biotic and abiotic factors. simi. Acta Entomol. Sinica 34: 189-197. LIU, S. S., AND X. D. MENG. 1990. The relationships between temperature and development rates of Myzus ACKNOWLEDGMENTS persicae and Lipaphis erysimi. Acta Phytophyl. Sin- ica 17: 169-175 We thank B. Legaspi (USDA-ARS, Weslaco, LIU, S. S., X. G. WANG, X. J. WU, Z. H. SHI, Q. H. CHEN, TX) for reviewing the early version of this manu- AND H. X. HU. 1997. Population fluctuation of aphids script, and M. Morales, J. Martinez, M. De Leon on crucifer vegetables in Hangzhou suburbs. Chi- and J. Martinez, Jr. for their technical assistance. nese J. Appl. Ecol. 8: 510-514. Publication of this paper was approved by the PRASAD, S. K., AND K. G. PHADKE. 1984. Yield loss in Center Director of Texas Agricultural Research some improved varieties of rapeseed and mustard by Center at Weslaco, and the Head of the Depart- aphid, Lipaphis erysimi Kalt. Indian J. Entomol. 46: 250-253. ment of Entomology, Texas A&M University at PRASAD, S. K. 1988. Screening of germplasm of mustard College Station. (Brassica juncea) for resistance to Lipaphis erysimi (Kalt.). Indian J. Entomol. 48: 227-230. REFERENCES CITED SAS INSTITUTE. 1996. The SAS system for Windows, Re- lease 6.11. SAS Institute, Cary, NC. AHLAWAT, Y. S., AND V. V. CHENULU. 1982. Studies on SETOKUCHI, O., AND S. T. MUMA. 1993. Ecology of the transmission of radish mosaic virus by the aphid, aphids on sugarcane iii. Relationship between Lipaphis erysimi. Indian Phytopathol. 35: 633-638. alighting of aphid vectors of sugarcane mosaic virus AMJAD, M., AND D. C. PETERS. 1992. Survival, develop- and infecting in fields. Japanese J. Appl. Entomol. ment, and reproduction of turnip aphids (Ho- Zool. 37: 11-16. moptera: Aphididae) on oilseed Brassica. J. Econ. SINGH, B., R. SINGH, AND M. S. MAHAL. 1983. Assess- Entomol. 85: 2003-2007. ment of loss in yield of Brassica juncea by Lipaphis BEGUM S. 1995. Observations on the economic threshold erysimi (Kalt.). II. Economics of aphid control. In- level of the mustard aphid Lipaphis erysimi (Kalten- dian J. Ecol. 10: 279-284. bach) on mustard in Bangladesh. Bangladesh J. SINGH, C. P., AND G. C. SACHAN. 1995. Estimation of Zool. 23: 13-16. losses in yield of rapeseed, Brassica campestris, by CASTLE, S. J., T. M. PERRING, C. A. FARRAR, AND A. N. the mustard aphid, Lipaphis erysimi (Kalt.) in Tarai, KISHABA. 1992. Field and laboratory transmission of India. Insect Sci. Applic. 16: 283-286. watermelon mosaic virus 2 and zucchini yellow mo- TAKAOKA, I. 1973. Review on the emergence of alate vi- saic virus by various aphid species. Phytopathology viparous females of Aphididae. Bull. Okayama To- 82: 235-240 bacco Exp. Stat. 32: 101-135. CHANDER S., AND K. G. PHADKE. 1994. Economic injury YUE, B., AND T.-X. LIU. 2000. Host selection, develop- levels of rapeseed aphids (Lipaphis erysimi) deter- ment, survival and reproduction of turnip aphid mined on natural infestations and after different insec- (Homoptera: Aphididae) on green and red cabbage ticide treatments. Intern. J. Pest Manag. 40: 107-110. varieties. J. Econ. Entomol. 93: 1308-1314.

Landolt: Cabbage looper orientation to potato plants 243

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 Hübner, responded in a ﬂight tunnel to potted potato plants (Solanum tuberosum). Percentages of moths attracted to uninjured 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

RESUMEN

Polillas de repollo Trichoplusia ni Hübner, sin experiencia previa, respondieron en un túnel de vuela a plantas de papa en tiestos (Solanum tuberosum). Los porcentajes de polillas atraí- das a plantas de papa sin daño, plantas de papa con daño mecánico, y plantas de papa tratadas con regurgitante de larvas del escarabajo de papa de Colorado, Leptinotarsa decemlineata Say, fueron similares, sindicando la falta de efecto al tratamiento de la planta. La atracción de hembras de polilla a plantas de papa fue incrementada siguiendo contacto previo (experiencia) de la polilla con la planta de papa. Este incremento en respuesta a plantas de papa con polillas con experiencia ocurrió aun si las plantas no tenían daño, tenían daño mecánico, o tratadas con regurgitante larval del escarabajo de papa de Colorado. Poli- llas preacondicionadas a plantas tratadas con regurgitante exhibieron tiempos similares de atracción a plantas mecánicamente dañadas y a plantas tratadas con regurgitante. Sin embargo, polillas preacondicionadas a plantas mecánicamente dañadas respondieron mas a plantas mecánicamente dañadas en comparación con plantas tratadas con regurgitante.

The cabbage looper, Trichoplusia ni (Hübner) do not know if cabbage looper moths are more is a polyphagous herbivore that can feed on a di- strongly attracted to injured plants because of versity of plant taxa (Sutherland & Greene 1984). constitutive or induced plant odors, or how cab- The cabbage looper moth exhibits weak attraction bage looper moth learning of host plant odor responses to a wide range of host plants, a behav- (Landolt & Molina 1996) might be affected by ior that may bring it to a host habitat (Landolt these changes in host plant chemistry. 1989). This response is enhanced by conspeciﬁc The cabbage looper larvae are found on potato larval feeding or mechanical damage to the plant plants and complete development on potato foliage (Landolt 1993). Attraction of cabbage looper (Sutherland & Greene 1984; Landolt, unpublished moths to plants is also enhanced following contact data), qualifying potato as a host plant, although it with a host plant (experience), indicating learn- is not considered to be a common pest of potato. Po- ing of host plant odors (Landolt & Molina 1996). tato plants respond to mechanical injury, feeding Cabbage looper moths previously exposed to cel- by Colorado potato beetle, Leptinotarsa decemlin- ery (Apium graveolens) or cotton (Gossypium hir- eata (Say), and applications of Colorado potato sutum) were more likely to orient to that same beetle regurgitant, in part by increasing the re- species of plant rather than to the other species of lease of volatile chemicals (Bolter et al. 1997). host plant (Landolt and Molina 1996). These pre- Treatment of potato foliage with regurgitant from vious studies did not consider possible differen- larvae of the Colorado potato beetle or from cab- tial roles of constitutive (stored and released) and bage looper larvae makes potato plants more at- induced (produced de novo) odor chemistry pro- tractive to Colorado potato beetle females (Landolt duced by plants in response to injury. That is, we et al. 1999). The Colorado potato beetle is a spe-

244 Florida Entomologist 84(2) June 2001

cialist on species of Solanum and might be ex- together in screened cages (45 × 45 × 45 cm) at the pected to be adapted to the defensive chemicals, beginning of the scotophase. Moths were provided including volatiles, produced by induced potato sugar water and water as in the emergence cages. plants. Karban and Baldwin (1997) suggest that Females were removed from mating cages near specialist herbivores might respond positively and the end of the subsequent photophase and were generalist herbivores might respond negatively to placed in another cage. Assays were conducted induced host plant kairomones. It would be of in- during the first 3 h of the following scotophase. terest then to determine how a polyphagous herbi- For each set of assays, a subset of female moths vore such as the cabbage looper responds to plants were recaptured and were dissected to verify that following such treatments. they mated, evidenced by the presence of a sper- The objectives of these studies were to deter- matophore in the bursa copulatrix. mine if cabbage looper moths are attracted to po- In all experiments, mated female moth re- tato plants, if they are attracted more strongly to sponses to potted plants were evaluated in a flight potato plants that are mechanically injured (to re- tunnel that was similar to that described by lease constitutive odorants), and if they are more Landolt and Molina (1996). Charcoal-impreg- weakly attracted to potato plants that are treated nated fiberglass filters were used at both ends of to produce and release induced odorants. Also, ef- the tunnel to minimize contamination of the tun- fects of previous exposure or experience with nel and experimental room with plant and other plants on cabbage looper moth attraction to unin- odors. The experimental room was equipped with jured and injured potato plants was examined, overhead red incandescent lamps to facilitate ob- with the expectation that moth attraction re- servation of moths. Flight tunnel room conditions sponses might be enhanced following experience were 22-24°C and 50-70% RH. Plants tested for with undamaged and mechanically-damaged attractiveness to moths were placed at the center plants, but might not be enhanced following expe- of the upwind end of the flight tunnel. Moths were rience with induced plants. released from open 30 ml polystyrene vials near the center of the downwind end of the flight tunnel and were observed for two minutes. Released MATERIALS AND METHODS moths were scored for upwind oriented flight (zig- General Methods zagging upwind flights within the probable boundaries of the odor plume) and for contact Russet-Burbank potato plants were used in all with the plant. A replicate consisted of a series of experiments. Plants were grown in a glass green- 5-10 moths tested per plant. No plant was used house from September through April in Yakima, more than once either in a conditioning treatment Washington, but with supplemental lighting from or in an assay. sodium lamps and 400 watt metal halide lamps Four experiments were conducted to evaluate providing a 14:10 L:D photoperiod. Plants were moth attraction to potato plants. started with potato eyes in soil in 15 cm diam pots. Soil was a mix of sand, peat moss and fertilizer. Naive Moths to Uninjured Potato Plants Plants were used in experiments when 4 to 6 weeks old (30-35 cm in height) and before blooming. The first experiment tested the hypothesis Three to five days before experiments, plants that naive moths are attracted to uninjured po- were placed in a controlled environment room at tato plants. Naive mated female moths were as- 25°C and 50% RH. Lighting was supplied with sayed for attraction to an uninjured potato plant overhead 400 watt metal halide lamps on a light or to a pot of soil (as an experimental control). Ten cycle coinciding with the light cycle for the cab- replicates were conducted, with 80 moths tested bage looper moths. to plants and 80 moths tested to pots of soil. Cabbage looper moths were obtained from a colony established in 1997 at the Yakima Agricul- Naive Moths to Injured Potato Plants tural Research Laboratory, with stock that originated from northern Florida in 1987. Pupae were The second experiment evaluated the effects of sorted by sex and placed in cages with dishes of plant injury on attraction of naive moths to potato sugar water on cotton balls and with water dis- plants. Two non-competitive tests were conducted pensers on the cage tops. Pupae were moved daily using naive female moths. These two tests were to new cages to provide cohorts of moths of dis- 1) a comparison of moth responses to uninjured po- crete ages. All pupae and adult moths (including tato plants and to potato plants that were damaged mating cages, see below) were kept in an environ- mechanically and 2) a comparison of moth re- mentally controlled room with reversed light cy- sponses to uninjured potato plants and potato cle (lights off at 0900 h and lights on at 1700 h), plants treated with regurgitant from larvae of the 24°C, and 60% RH. Colorado potato beetle. To incur mechanical dam- To obtain mated females, groups of 30 males age to the potato plants, three leaves on a potato (3-6 d old) and 25 females (2-3 d old) were placed plant were cut with scissors, lengthwise about 3 cm

Landolt: Cabbage looper orientation to potato plants 245 along the main leaf axis. This was done one hour Discrimination of Uninjured vs Injured Potato before assays were started. Treatment of plants by Experienced Moths with beetle regurgitant were made to three leaves of each plant. Leaves were first scraped with a ra- The fourth experiment evaluated the ability of zor blade (about one cm2). A fourth or fifth instar experienced moths to discriminate between me- beetle larva was gently squeezed until it produced chanically-damaged plants and plants that had a droplet of regurgitant at the mouthparts which been treated with Colorado potato beetle regur- was then applied to the scraped area of leaf. This gitant. The objective of this experiment was to was done 24 h before assays were started. determine if moths preconditioned on one type of Nine bioassay replicates were conducted, with injured potato plant (mechanically-damaged or 90 moths tested per treatment and 90 per control. regurgitant-treated) would respond better to the In both studies, the sequence of plants tested type of plant they were preconditioned with, com- (treatment and control) was reversed daily for the pared to the other type of plant. There were 4 9 days. treatment regimes: 1) moths that were placed with mechanically-damaged plants in one scoto- Experienced Moths to Uninjured phase and were then tested for responses to and Injured Potato Plants mechanically-damaged plants in the following scotophase, 2) moths that were placed with The third experiment evaluated the effects of mechanically-damaged plants in one scotophase prior contact (experience) with potato plants on and were then tested for responses to regur- moth attraction to potato plants. Three tests were gitant-treated plants in the following scotophase, conducted to compare the responses of naive ver- 3) moths that were placed with regurgitant- sus experienced mated female moths to potato treated plants in one scotophase and were then plants that were either uninjured, were mechani- tested for responses to mechanically-damaged cally-damaged, or were treated with Colorado po- plants in the following scotophase, and 4) moths tato beetle regurgitant. For each assay, mated that were placed with regurgitant-treated plants females were divided into 2 equal groups when in one scotophase and were then tested for re- separated from males in the mating cages. One sponses to regurgitant-treated plants in the fol- group was placed in a larger screened cage (45 × lowing scotophase. 45 × 45 cm) with a potted potato plant, at the on- Mated females were subdivided into 2 equal set of the scotophase (experienced moth group). groups when initially separated from males in the The other group was placed in a similar cage with mating cages, near the end of a photophase. These no plant (naive moth group). Both cages were female moths were placed either in a cage with a kept in the controlled environment room used to mechanically-damaged plant (cut with scissors as hold plants for bioassays, on a 14:10 L:D light cy- before) or in a cage with a regurgitant-treated cle, 50% RH and 25°C for 24 h. Female moths plant (Colorado potato beetle larval regurgitant were then removed from these cages and held in applied to scrapes on 3 leaves 24 h before). After 30 × 30 × 30 cm screened cages in the controlled 24 h, during the last 2 h of the photophase, these environment room holding moths and no plants, moths were then removed from the cages contain- with water and sugar water, until used in bio- ing plants and were placed in 2 clean cages and assays in the following scotophase. A comparison held another 2-4 h until they were used in bio- was first made of naive versus experienced moths assays. For the bioassay, a mechanically-damaged to an undamaged plant. Naive moths were tested plant was placed in the flight tunnel and 5 moths for attraction to and contact with an uninjured that had been preconditioned with a mechani- potato plant, followed by the testing of experi- cally-damaged plant and 5 moths that had been enced moths tested to the same plant. The follow- preconditioned with a regurgitant-treated plant ing day, using a new plant, a set of experienced were then tested alternately for responses to the moths were tested for responses to an uninjured same plant. A regurgitant-treated plant was then plant, followed by the testing of naive moths. This placed in the flight tunnel and 5 moths that had was continued for 12 assay sets, with 80 naive been preconditioned with a mechanically-dam- and 80 experienced moths tested to uninjured aged plant and 5 moths that had been precondi- potato plants. This protocol was then followed to tioned with a regurgitant-treated plant were compare naive versus experienced moth re- tested alternately for responses to this plant. This sponses to a mechanically damaged plant, with a protocol was followed on 10 different days, provid- total of 80 naive and 80 experienced moths tested ing a total of 50 moths tested for each treatment to mechanically damaged plants in 16 bioassay category. sets. A comparison was then made of naive versus For experiments 1, 2 and 3, percentage re- experienced moth responses to regurgitant treated sponse data for treatment pairs were analyzed by potato plants, with 60 naive and 60 experienced a paired t test to determine if responses differed moths tested to regurgitant treated potato plants between treatments or between treatment and in 10 bioassay sets. control, with a significance level of P ≤ 0.05. Per-

246 Florida Entomologist 84(2) June 2001 centage response data for the treatment regimes than percentages of naive moths landing on those of experiment 4 were compared using Tukey’s same plants. Test following an ANOVA. Discrimination of Uninjured vs Injured Potato RESULTS by Experienced Moths

Naive Moths to Uninjured Potato Plants Greatest attraction response rates were seen for moths preconditioned on mechanically-dam- The percentages of naive moths that flew up- aged plants and were assayed to other mechani- wind towards uninjured Russet Burbank potato cally-damaged plants (Fig. 1). The attraction of plants were significantly greater than the per- moths that were preconditioned on regurgitant- centages of moths responding to a pot of soil (Ta- treated plants to either mechanically-damaged or ble 1). A significant percentage of moths tested regurgitant-treated plants was similar. These re- also landed on uninjured potato plants and none sponses, both attraction and landing on plants, landed on pots of soil (Table 1). were also similar to those of moths preconditioned on mechanically-damaged plants and as- Moths to Injured Potato Plants sayed to regurgitant-treated plants (Fig. 1).

Percentages of naive moths attracted to me- DISCUSSION chanically-damaged and uninjured plants were not significantly different (Table 1). Percentages Results indicate significant but weak attrac- of naive moths attracted to uninjured plants were tion of naive cabbage looper moths to Russet Bur- not significantly different from those attracted to bank potato plants. This finding is similar to the regurgitant-treated plants. There were also no weak attraction responses of naive cabbage significant differences between percentages of looper moths to cabbage (Brassica oleracea), cel- moths landing on plants, for either of the treat- ery, tomato (Lycopersicon esculentum), and soy- ment comparisons (Table 1). bean (Glycine max) plants (Landolt 1989). This low level attraction to a wide variety of plants Experienced Moths to Uninjured (Landolt 1989) may be a host-habitat finding and Injured Potato Plants strategy rather than a host-finding behavior, as was suggested previously (Landolt 1993). Moths preconditioned (experienced) on potato There was no significant enhancement of at- plants were attracted significantly more often to traction of cabbage looper moths to potato plants potato plants, than were naive moths (Table 2). cut with scissors to produce mechanical damage. Increased attraction of preconditioned moths to Because mechanical damage causes short term potato plants was observed when those plants increased emission of volatile chemicals from po- were uninjured, mechanically-damaged, or tato (Bolter et al. 1997), a heightened response by treated with Colorado potato beetle regurgitant cabbage looper moths was expected. Similar me- (Table 2). Additionally, increased landing was ob- chanical damage to foliage of cotton plants (Gos- served in moths that were preconditioned either sypium hirsutum) increased their attractiveness on uninjured plants or on plants with mechanical to cabbage looper moths (Landolt 1993) and simi- damage (Table 2). Percentages of preconditioned lar damage to cabbage plants increased their at- moths landing on regurgitant-treated plants tractiveness to Mamestra brassicae (L.) moths were numerically but not statistically greater (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.

Landolt: Cabbage looper orientation to potato plants 247

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- tive to female Colorado potato beetle (Landolt et damaged cabbage plants (Landolt 1993). It is not al. 1999). In previous studies, conspecific larval yet evident why orientation to mechanically-dam- damage made cotton plants more attractive to aged plants may be enhanced in some cases and cabbage looper moths (Landolt 1993) and made not in others. cabbage plants more attractive to M. brassicae There was also no enhancement of moth at- moths (Rojas 1999). However, such damage to traction to potato plants treated with regurgitant cabbage plants made them less attractive to cab- of larvae of the Colorado potato beetle. This treat- bage looper moths (Landolt 1993) and such dam- ment is known to stimulate the production and age to chrysanthemum plants made them less prolonged release of odorants from potato (Bolter attractive to M. brassicae moths (Rojas 1999). As et al. 1997) and makes potato plants more attrac- with the varying responses of moths to mechanically-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 positive effect of cabbage looper moth experience with potato plants prior to testing moths in flight tunnel assays. This enhancement of attraction responses occurred regardless of whether the plant was uninjured, mechanically-damaged or regurgitant-treated. Similarly, greater numbers of moths contacted the plants if they had prior experience 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 learning, as described by Papaj and Prokopy (1989). Landolt and Molina (1996) reported strong en- Fig. 1. Mean (±SE) percentages of mated female cab- hancement of host attraction responses by mated bage loopers attracted to (solid bars) and contacting female cabbage looper moths following prior expe- (open bars) Russet Burbank potato plants in a flight rience with host plants, using cotton, celery, and tunnel, after preconditioning contact with other potato soybean plants. In those studies it was shown plants during the previous scotophase. The first treat- that brief contact with the plant was all that was ment letter indicates the type of plant the moths were required to strongly increase the subsequent re- preconditioned with and the second letter indicates the type of plant the moths were presented with in the flight sponse of the moth to that plant species. Evidence tunnel assays. Plants for preconditioning and for flight of learning of host plant odor chemistry also ex- tunnel assays were either mechanically-damaged (C) or ists for the moths Helicoverpa armigera (Hübner) were regurgitant-treated (R). Bars with the same letter (Cunningham et al. 1998), Heliothis virescens are not significantly different by Tukey’s Test (P ≤ 0.05). (Hartlieb 1996) and Spodoptera littoralis (Boisdu- 248 Florida Entomologist 84(2) June 2001 val) (Fan et al. 1997) and for the phytophagous looper (Landolt 1993). Additional work is needed beetles L. decemlineata (Say) (Visser & Thiery to determine the chemical bases for the response 1986) and Diaprepes abbreviatus (L.) (Harari & differences observed among plants. Prior contact Landolt 1999). In contrast, Rojas and Wyatt with a plant increases the subsequent responsive- (1999) noted decreased responsiveness to host ness of the moth to the same type of plant, indi- plants by Mamestra brassicae L. following earlier cating associative learning of host plant odor contact with host plants. when contact is made with plant foliage. The in- In previous studies (Landolt and Molina 1996), ability of the moth to selectively respond to potato it was shown that cabbage looper moth learning plants with different types of injury still leaves of host plant odor was specific to the plant species open the question of the relative significance of used in preconditioning. When moths had prior constitutive versus induced odorants to cabbage contact with cotton plants, they were more looper host finding with other species of plants. strongly attracted to other cotton plants and not to celery plants. Similarly, when moths had prior ACKNOWLEDGMENTS contact with celery plants, they were more strongly attracted to other celery plants and not Technical assistance was provided by L. Bid- to cotton plants. In the studies described herein, dick, P. Chapman, R. S. Hofstetter, and D. Larson. this discrimination between plants did not occur L. Finch, J. Jewett, and C. Albano provided cab- when moths had prior contact with mechanically- bage looper moths for use in these experiments. damaged potato plants or regurgitant-treated This work was supported in part by funding from potato plants and were tested for attraction to the Washington State Potato Commission. Cab- both types of potato plants. There was some en- bage looper eggs were kindly provided by Ken hancement of response to those plants following Haynes, Dept. Entomology, Lexington, KY, for es- moth preconditioning on mechanically-damaged tablishment of the colony at the Yakima Agricul- plants, but moths responded similarly to the two tural Research Laboratory. Helpful comments to types of injury. Apparently, cotton and celery improve the manuscript were made by C. O. plants have strongly divergent odor chemistries Calkins, N. Epsky, H. C. Reed, D. C. Robacker, and with little, if any, qualitative overlap, making it M. Zlotina. more likely that an insect could learn and discriminate separate odors. However, the odor REFERENCES CITED chemistries of mechanically-damaged (to release BOLTER, C. J., M. DICKE, J. J. A. VAN LOOP, J. H. VIS- constitutive odors) and regurgitant-treated (to re- SER, AND M. A. POSTHUMUS. 1997. Attraction of Col- lease induced odors) potato plants overlap exten- orado potato beetle to herbivore-damaged plants sively (Bolter et al. 1997) and moths that are during herbivory and after its termination. J. Chem. preconditioned on potato plants with one type of Ecol. 23: 1003-1023. injury may be unable to discriminate between CUNNINGHAM, J. P. M. F. A. MALLOW, D. J. WRIGHT, plants with either type of injury. AND M. P. ZALUCKI. 1998. Learning in host selection It was hypothesized that the cabbage looper in Helicoverpa armigera (Hubner) (Lepidoptera: moth, as a generalist herbivore, might show en- Noctuidae). Animal Behav. 55: 227-234. FAN, R., P. ANDERSON, AND B. S. HANSSON. 1997. Be- hanced attraction to mechanically-damaged havioral analysis of olfactory conditioning in the plants because of increased emission of volatiles moth Spodoptera littoralis (Boisd.) (Lepidoptera: and might show decreased attraction to Colorado Noctuidae). J. Exp. Biol. 200: 2969-2976. potato beetle regurgitant-treated plants because HARARI, A. R., AND P. J. LANDOLT. 1999. Feeding expe- of the induction of defensive chemistry associated rience enhances attraction of female Diaprepes ab- with that treatment to potato (Bolter et al 1997) breviatus (L.) (Coleoptera: Curculionidae) to food and similar treatments with lepidopterous larvae plant odors. J. Insect Behav. 12: 415-422. to other plants (Tumlinson et al. 1992; Turlings et HARTLIEB, E. 1996. Olfactory conditioning in the moth al. 1995). In the case of cabbage looper orientation Heliothis virescens. Naturwissenshaften 83: 87-88. KARBAN, R., AND I. T. BALDWIN. 1997. Induced re- to potato odor, those expectations appear incor- sponses to herbivory. University of Chicago Press, rect because observed response rates were similar Chicago. 319 pp. to uninjured and injured potato, both by naive LANDOLT, P. J. 1989. Attraction of the cabbage looper to and by experienced moths. host plants and host plant odor in the laboratory. En- Host-finding by the cabbage looper moth ap- tomol. Exp. Appl. 53: 117-124. pears to involve several behavioral strategies, LANDOLT, P. J. 1993. Effects of host plant leaf damage on host-habitat location, responses to some plant in- cabbage looper moth attraction and oviposition. En- jury volatiles, and learning of host plant odor. Ev- tomol. Exp. Appl. 67:79-85. idence indicates a weak response to a variety of LANDOLT, P. J., AND O. MOLINA. 1996. Host-finding by cabbage looper moths (Lepidoptera: Noctuidae): plant species that may bring them into the vicin- Learning of host odor upon contact with host foliage. ity of possible hosts (Landolt 1989). Injury to J. Insect Behav. 9: 899-908. plants may (cotton) or may not (cabbage, potato) LANDOLT, P. J., D. LARSON, J. H. TUMLINSON, AND H. increase a plant’s attractiveness to cabbage ALBORN. 1999. Attraction of Colorado potato beetle Landolt: Cabbage looper orientation to potato plants 249

(Coleoptera: Chrysomelidae) to damaged and chemi- ment of Cabbage Looper Populations. USDA Tech. cally induced potato plants. Environ. Entomol. 28: Bull. 1684. 973-978. TUMLINSON, J. H., T. C. J. TURLINGS, AND W. J. LEWIS. PAPAJ, D. R., AND R. J. PROKOPY. 1989. Ecological and 1992. The semiochemical complexes that mediate evolutionary aspects of learning in polyphagous in- insect parasitoid foraging. Agric. Zool. Reviews. 5: sects. Annual Review of Entomology. 34: 1281-1288. 221-252. ROJAS, J. C. 1999. Influence of host plant damage on the TURLINGS, T. C. J., J. H. LOUGHRIN, P. J. MCCALL, U. S. host-finding behavior of Mamestra brassicae (Lepi- R. ROSE, W. J. LEWIS, AND J. H. TUMLINSON. 1995. doptera: Noctuidae). Environ. Entomol. 28: 588-593. How caterpillar-damaged plants protect themselves ROJAS, J. C., AND T. D. WYATT. 1999. The role of pre- by attracting parasitic wasps. Proc. National Acad. and post-imaginal experience in the host-finding and Sciences of the USA. 92: 4169-4172. oviposition behavior of the cabbage moth. Physiol. VISSER, J. H., AND D. THIERY. 1986. Effects of feeding Entomol. 24. experience on the odour-conditioned anemotaxes SUTHERLAND, D. W. S., AND G. L. GREENE. 1984. Culti- of Colorado potato beetles. Entomol. Exp. Appl. 42: vated and wild host plants, pp. 1-13. In P. D. Lingren 198-200. and G. L. Greene (eds.). Suppression and Manage-

250 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. ALLEN1, 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 loggerhead (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 moisture 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 potential impacts of S. invicta on turtle hatchlings. Over 70% of hatchlings were killed by S. invicta 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 nelsoni, Solenopsis invicta, turtles

RESUMEN

Se han observado hormigas bravas (Solenopsis invicta Buren) cada vez mas en nidos de tortugas marinas (Caretta caretta L.) y (Chelonia mydas L.) en la Florida, y en los nidos de tortugas de agua dulce. Estas hormigas pueden ser atraídas al disturbio, mucosidad y humedad asociada con anidaje de tortugas y establecen túneles de forraje hacia nidos de tortuga poco después de la puesta de huevos, así incrementando la vulnerabilidad de los recién nacidos a predación por la hormiga brava. Llevamos a cabo experimentos con una tortuga de agua dulce (Pseudemys nelsoni Carr) para determinar el impacto potencial de S. invicta en tortugas re- cién nacidas. Mas del 70% de los recién nacidos fueron muertos por S. invicta durante el pro- ceso de salir del cascaron o poco después de salir. Para determinar la extensión de infestación de S. invicta en playas donde anidan tortugas marina, muestreamos playas conocidas por tener nidos por todo el estado de la Florida. Exámenes de playas indicaron que S. invicta esta presente y mucha la mayoría de las playas y dunas a lo largo de la costa Floridana.

There has been considerable concern and de- Red imported fire ants were observed feeding on bate over the potential impact of fire ants on nest- pipped eggs, and stinging, killing and subse- ing sea turtles, but little quantitative evidence quently feeding on turtle hatchlings (Wilmers et exits. Information on the impact of Solenopsis in- al. 1996). Moulis (1997) documented a 15% de- victa (Buren) on hatchling turtles has been largely crease in hatchling release rate for loggerhead sea incidental and anecdotal. Fire ants may impact turtles emerging from nests infested with fire ants turtle populations directly by preying on hatch- as compared to uninfested nests. The ultimate ef- lings and/or indirectly by stinging hatchlings, fect of this predation on sea turtle populations and resulting in reduced weight gain and survival. the magnitude of the problem else-where is un- Wilmers et al. (1996) documented an increase known. The nesting period (May-August) for log- in the presence of fire ants in green (Chelonia my- gerheads in the eastern United States (Johnson et das L.) and loggerhead (Caretta caretta L.) turtle al. 1996) corresponds with a concentrated period nests on undeveloped island beaches off Florida. of brood production in S. invicta. During that

Allen et al.: Fire ant impacts on turtles 251

time, protein needs for fire ants are maximal (So- Commission incubation facilities (Gainesville, rensen et al. 1983). For secure turtle populations, FL) with food supplied ad libitum, and measured high juvenile mortality may not affect population weekly for 6 weeks to determine if differences in size (Congdon & Gibbons 1990), but for small pop- weight gain between treated and untreated ulations, decreases in annual cohort size may af- groups existed (Allen et al. 1997). fect population viability (Heppell et al. 1996). To determine the presence of S. invicta on sea- Our objectives were to assess the potential im- turtle nesting beaches, we sampled for fire ants at pacts of the invasive non-indigenous ant, S. in- 18 known sea turtle nesting beaches throughout victa, on hatching turtles by using the eggs of a Florida. Collection localities are given in Table 1. freshwater species (Florida red-bellied turtle, Transects consisting of approximately 20 samples Pseudemys nelsoni Carr) in a controlled experi- at 10-m intervals were established along dune mental setting. Pseudomys nelsoni often lays its lines. Multiple transects (2 to 7) were sampled at eggs in alligator nests, approximately 20% of which each locality. Baits on all transects consisted of are infested with fire ants in central Florida (Allen ground beef, except for those in Duval, St. Johns et al. 1997). In addition, we determined the geo- and Volusia counties, which consisted of a sugar- graphic extent of S. invicta occurrence on sea turtle based bait attractive to a variety of ants, newly nesting beaches throughout the state of Florida by formulated by the U.S.D.A.-A.R.S. Baits were left sampling beaches with baits attractive to ants. in the field for approximately 1 h before being collected and transported to U.S.D.A.-A.R.S. facilities in Gainesville, Florida, for sorting and MATERIALS AND METHODS identification of ant species. To assess the potential for S. invicta impact on the eggs and hatchlings of turtles we conducted a RESULTS laboratory experiment using P. nelsoni eggs. The eggs of this freshwater turtle species are elliptical Pseudemys nelsoni clutch size varied from 6-16 and approximately 2.5 cm long, occur in clutches eggs, but only 2-11 of the eggs in a given clutch of about 15, and are found regularly in American ultimately hatched. In control groups, 59% of the alligator nests in Florida. Although the shape of eggs did not hatch, and in those exposed to fire eggs in this species is different from the generally ants 37% did not hatch. The cause or causes of round shape of sea turtle eggs, and egg and clutch inviability were not determined but may be at- size vary among turtle species, we have observed tributed to flooding or crushing by the attendant no differences in attractiveness among eggs of female alligator prior to collection. During and several different species regardless of size or after hatching, 100% of hatching turtles in con- shape. Pseudemys nelsoni and many other turtle trol groups (17) survived, and were eventually species, including sea turtles, share the trait of released. The proportion successfully hatching in emerging from the nest only after most or all of clutches exposed to fire ants (10 of 35) was signif- the clutch has hatched. icantly less (median = 33%, range 0-55%; Mann- Ten clutches of P. nelsoni eggs were collected Whitney Rank Sum Test t = 40.0, df = 8, P = (1996) from Lake Apopka in central Florida and 0.008). Approximately half of the mortalities oc- transferred to ten 61 × 36 × 13 cm enclosures at curred while the hatchlings were still in the egg, the U.S.D.A.-A.R.S. Imported Fire Ant Labora- while most others died less than 1 h after emer- tory, in Gainesville, Florida. Five clutches served gence. Fire ants did not breach turtle eggs, but as controls. For both control and treated clutches, entered the eggs as soon as a pipped hole was eggs were placed in sphagnum nesting material present. Too few individuals exposed to fire ants adjacent to a shallow pan of water, which allowed survived to assess differences in weight gain individual hatchlings immediate access to water between the two groups. upon emergence. Treated clutches were main- We collected 734 ant bait samples and a total tained identically to control clutches, but were ex- of 31,392 ants from Florida sea turtle nesting posed to a field-collected mound of S. invicta at beaches. About 40% of the collected ants (12,658) the opposing end of each enclosure. This con- were S. invicta. Fire ants were detected foraging trolled situation simulated the natural conditions along dune lines on sea turtle nesting beaches in for the many P. nelsoni clutches which share alli- all regions of the state, and were detected on 13 of gator nests with fire ant mounds (Allen et al. 18 specific sites (Table 1). Within those 18 sites, 1997). The enclosures allowed fire ants to forage fire ant occurrence on baits varied from 0 to 63%, among the clutch as may occur within natural and represented from 0 to 97% of the individuals turtle nests (Wilmers et al. 1996; Allen et al. collected. Fire ant occurrence followed no obvious 1997). Fire ants were provided with honey as a geographic pattern. Ants were abundant at some food source. Eggs were observed twice daily as very remote locations (e.g., Boca Grande Key near they approached hatching and constantly as pip- Key West) but were uncommon on beaches at some ping commenced. Surviving turtles were translocations that have undergone extensive human ferred to the Florida Game and Fresh Water Fish disturbance (e.g., northeastern Florida beaches).

252 Florida Entomologist 84(2) June 2001

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

% baits No. No. with No. other % Region Site baits S. invicta S. invicta ants S. invicta

East Coast North Crescent Beach 30 0 0 612 0 Daytona Beach 30 7 52 90 37 Appollo State Park 30 0 0 658 0 Panhandle Cape San Blas 40 18 867 1,732 33 Southwest Clam Pass Park South 20 60 869 98 90 Vanderbilt Beach 40 18 691 1,103 39 Park Shore Beach 39 21 142 30 83 Collier Co., Gullivan Key 69 1 3 1675 0.1 Collier Co., Turtle Key 21 0 0 1163 0 Collier Co., “B” Key 30 63 4,480 127 97 West Panther Key 20 0 0 987 0 Westcentral Pinellas Co., Sand Key 60 35 3,570 1,143 76 Pinellas Co., Passe Grille 60 7 188 2,546 7 Southeast Indian River Co., Sebastian Inlet 60 5 153 798 16 Palm Beach Co., J. D. MacArthur State Park 60 0 0 1,174 0 Keys Boca Grande 50 26 1,613 2,392 40 Marquesas 50 4 29 582 5 Matecumbe 25 4 1 1,914 0.5

Totals 734 15 12,658 18,824 29

DISCUSSION aprons of gopher tortoises as colony sites (personal observation). Nearly half of the P. nelsoni killed by Our surveys found S. invicta present on most S. invicta successfully exited from their eggs and beaches, at both the wrack and dune lines. Our reached the water before succumbing to the effects and other observations indicate that fire ants often of envenomization. In a natural setting, these in- are present in sea turtle nest cavities (e.g., Wilm- dividuals would have been considered to have ers et al. 1996; Parris et al. 2001). The egg-laying hatched successfully, thus under-estimating fire process may initially attract fire ants because it ant-induced mortality by about 50%. Our labora- represents a local disturbance and food source. tory research only documented direct mortality of Mucous associated with the egg laying process is hatchlings. However, other research has shown se- an attractive food for fire ants and sea turtle nest rious indirect effects on individual animals (Amer- cavities provide a desirable micro-climate for fire ican alligators, Alligator mississippiensis Daudin, ants. It appears that fire ants cannot breach intact Allen et al. 1997; northern bobwhite, Colinus vir- sea or freshwater turtle eggs (Wojcik & Allen, un- ginianus L., Giuliano et al. 1996) stung non-le- published data). However, once fire ants build sub- thally by S. invicta. These effects included the loss terranean foraging trails to a site that has of digits and appendages, and reduced weight provided food, such as turtle nest cavities, they gain, both likely to affect survival in the wild. maintain those foraging tunnels. Additionally, This work indicates that hatching turtles are post-laying disturbances caused by predators such very vulnerable to predation by S. invicta and as raccoons (Procyon lotor L.) or ghost crabs (Ocy- that S. invicta is now a common component of the pode sp.) that fracture some eggs may attract fire ant community of sea turtle nesting beaches. ants to nest cavities. Thus, fire ants may maintain However, population-level impacts are unknown. a presence in the nest cavity until hatching. Moulis (1997) documented a 15% decrease in Our experiments with P. nelsoni eggs indicate hatchling release rate for sea turtles (C. caretta) that turtle hatchlings are both highly attractive emerging from nests infested with fire ants as and vulnerable to fire ants. Presumably, endan- compared to uninfested nests, but predation by gered species such as sea turtles or gopher tor- vertebrate predators (e.g., raccoons) can vary be- toises attempting to hatch from nests with tween 5 to 90% (Ratnaswamy et al. 1997). established fire ant foraging tunnels are equally Fire ant populations are increasing in terms of as vulnerable. Fire ants often use the burrow both the spatial extent of infestation (Cokendol-

Allen et al.: Fire ant impacts on turtles 253

pher & Phillips 1989; Callcott & Collins 1996) REFERENCES CITED and population densities of sites already infested (Wojcik 1994). Due to the sensitivity of using sea ALLEN, C. R., K. G. RICE, D. P. WOJCIK, AND H. F. PER- turtle hatchlings as experimental units, this re- CIVAL. 1997. Effect of red imported fire ant envenom- search did not document the extent of S. invicta ization on neonatal American alligators. J. Herpetol. utilization of sea turtle nesting cavities or inter- 31: 318-321. actions between sea turtles and fire ants in natu- CALLCOTT, A. A., AND H. L. COLLINS. 1996. Invasion and range expansion of the imported fire ants (Hy- ral settings. However, our experiment with P. menoptera: Formicidae) in North America from nelsoni was conservative because hatchlings had 1918-1995. Florida Entomol. 79: 240-251. immediate access to water upon emergence, COKENDOLPHER, J. C., AND S. A. PHILLIPS, JR. 1989. whereas in the wild, hatchlings of many species Rate of spread of the red imported fire ant, Solenop- may spend several hours in the nest cavity before sis invicta (Hymenoptera: Formicidae) in Texas. emergence. We conclude that there is a very real Southwest. Nat. 34: 443-449. potential for negative impacts by S. invicta on CONGDON, J. D., AND J. W. GIBBONS. 1990. The evolu- hatchling turtles. tion of turtle life histories, pp. 45-54. In J. W. Gibbons Additional investigation of the effect of S. in- (ed.). Life History and Ecology of the Slider Turtle. Smithsonian Institution Press, Washington, D.C. victa on hatching sea turtles is vital because fire GIULIANO, W. M., C. R. ALLEN, R. S. LUTZ, AND S. DEMA- ants occur throughout sea turtle nesting habitat RAIS. 1996. Effects of imported fire ants on northern in the United States, they are increasing in abun- bobwhite chick survival and body mass. J. Wildl. dance, and they clearly have the potential to neg- Manage. 60: 309-313. atively impact hatchlings of sea turtles, other HEPPELL, S. S., L. B. CROWDER, AND D. T. CROUSE. turtle species, and many other species in terres- 1996. Models to evaluate headstarting as a manage- trial ecosystems. In the case of sea turtles, further ment tool for long-lived turtles. Ecol. Appl. 6: 556-565. research is also needed to determine why certain JOHNSON, K. A. BJORNDAL, AND A. B. BOLTON. 1996. Ef- beaches possessed higher densities of S. invicta, fects of organized turtle watches on loggerhead (Caretta caretta) nesting behavior and hatchling pro- and what beach management activities (e.g., duction in Florida. Cons. Biol. 10: 570-577. beach renourishment, degree of disturbance, hu- MOULIS, R. A. 1997. Predation by the imported fire ant man activity levels) may influence S. invicta in- (Solenopsis invicta) on loggerhead sea turtle festation and spread. (Caretta caretta) nests on Wassaw National Wildlife Refuge, Georgia. Chelonian Cons. Biol. 2: 433-436. ACKNOWLEDGMENTS PARRIS, L. N., M. M. LAMONT, AND R. R. CARTHY. 2001. Observations of predation by red imported fire ants We wish to extend thanks to J. P. Hosford, M. (Solenopsis invicta) on hatching loggerhead sea tur- Lamont, and A. Quistorff for aiding in the collec- tles (Caretta caretta). Herp. Rev. (in press). tion of beach samples. We thank J. Sullenger and RATNASWAMY, M. J., R. J. WARREN, M. T. KRAMER, AND B. Mayfield for collecting, processing and identify- M. D. ADAM. 1997. Comparisons of lethal and nonle- ing the samples from beaches. We thank T. Wilm- thal techniques to reduce raccoon depredation of sea ers for providing access to the Marquesas, and to turtle nests. J. Wildl. Manage. 61: 368-376. SORENSEN, A. A., T. M. BUSCH, AND S. B. VINSON. 1983. T. Hopkins for providing access to beaches and Behavior of worker subcastes in the fire ant, Sole- housing during sampling in southwest Florida. nopsis invicta, in response to proteinaceous food. We thank A. Garmestani for providing access to Physiol. Entomol. 8: 83-92. beaches in the Ten Thousand Islands. We wish to WILMERS, T. J., E. S. WILMERS, M. MILLER, AND P. thank R. Carthy, D. Epperson, and R. Moulis for WELLS. 1996. Imported fire ants (Solenopsis invicta): reviewing an earlier draft of this manuscript. Our a growing menace to sea turtle nests in Key West laboratory work with turtles was approved by the National Wildlife Refuge, pp. 341-343. In J. A. Kein- University of Florida Animal Care and Use Com- ath, D. E. Barnard, J. A. Musick, and B. A. Bell (eds.). mittee. The South Carolina Cooperative Fish and Proc. Fifteenth Annual Workshop on Sea Turtle Biol- ogy and Conservation. Wildlife Research Unit is jointly supported by a WOJCIK, D. P. 1994. Impact of the red imported fire ant cooperative agreement among the USGS/BRD, on native ant species in Florida, pp. 269-281. In D. F. the South Carolina Department of Natural Re- Williams (ed.). Exotic Ants: Biology, Impact, and sources, Clemson University, and the Wildlife Control of Introduced Species. Westview Press, Boul- Management Institute. der, CO.

254 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. 1 Natural Sciences Collegium, Eckerd College, St. Petersburg, FL 33711

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

3 U.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-indigenous 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 conducted 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 partially 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 aristodemus, Solenopsis invicta

RESUMEN

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 hábitat de este bosque tropical esta fragmentado por calles y desarrollo humano que puede alterar el microhabitat dentro de los bosques e incrementar la probabilidad de inva- sión por predadores y competidores exóticos. Una especie no indígena 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 reducción en S. invicta como resultado de la aplicación de trampas químicas de hormigas. En adición, llevamos a cabo experimentos de laboratorio para determinar que tan vulnerable son las etapas de vida de P. aristodemus a predación por S. invicta. Encontramos S. invicta en un 50% de lotes en los bosques, hasta 40 m del borde del bosque. Tratamientos químicos fueron solo parcialmente efectivos para disminuir la abundancia de S. invicta, y el efecto fue de corta duración. Todas las etapas de vida de P. aristodemus fueron vulnerables a predación por S. invicta. La restauración del hábitat que re- ducen la abundancia de S. invicta puede ser el método mas efectivo en costo y a largo plazo para reducir los impactos 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 255

Dade Counties, Florida, including torchwood, eggs, larvae and pupae are readily discovered and Amyris elemifera (L.), and wild lime, Zanthoxy- consumed by red imported fire ants, and 3) ex- lum fagara (L.) Sarg., (Emmel 1986, 1995). Both plore the effectiveness of fire ant control in areas of these trees are relatively small and tend to pro- important to Schaus swallowtail reproduction. duce suckers around the base. It appears that leaves on these suckers may be the preferred ovi- MATERIALS AND METHODS position locations for Schaus (Bagget 1982; Em- mel 1995). Young torchwood and wild lime leaves Red Imported Fire Ant Foraging are the primary food of most Schaus caterpillars. Like other butterflies, the Schaus is an important To determine fire ant abundance and ability to pollinator of native plants, serves as a food source forage into closed canopy tropical hardwood ham- for insectivorous species, and contributes to the mock, we established bait transects on North Key biological diversity of the Florida Keys. Largo, Florida. North Key Largo is a long and The Schaus swallowtail was listed as Federally narrow (<1 km) island at the northernmost por- endangered in 1984 because of population de- tion of the Florida Keys. There are several resi- clines caused by the destruction of its tropical dential developments, but most of North Key hardwood hammock habitat, mosquito control Largo is federally and state owned. In the center practices, and over-harvesting by collectors. Rein- of the key, a well-traveled highway (SR 905) bi- troductions occurred between 1995 and 1997 (U.S. sects the hardwood hammock. Recently, Schaus Fish and Wildlife Service 1999) and in 1998 the swallowtails have been successfully reintroduced Schaus swallowtail was documented on 13 areas in this area (U.S. Fish and Wildlife Service 1999). on the mainland and the Upper and Middle Keys. Twenty transects were placed perpendicular to Currently, efforts are underway to protect the SR 905 into the Key Largo hammock. Ten of the remaining hardwood hammocks in south Florida transects were on the federally-owned north side from commercial and residential development as of SR 905 and the other 10 were directly across well as from pesticide spraying (U.S. Fish and the road on the state-owned south side of SR 905. Wildlife Service 1999). Even if these measures Habitat on both sides of SR 905 was similar. are successful, much of the existing habitat is al- Transects were separated by 100 m and consisted ready fragmented by roads and human develop- of 10 sampling stations spaced 5 m apart begin- ment that may alter the microhabitat within the ning at known fire ant infested areas (the road- hammocks (Saunders et al. 1991) and increase side) and continuing perpendicular into the intact the probability of invasion by non-native preda- hardwood hammock. tors and competitors (Usher 1988). At each of the 10 sampling stations along all 20 An invasive non-native species that may have transects, we placed two terrestrial and two arbo- a negative impact on the Schaus swallowtail is real baits, with members of each pair separated the red imported fire ant (Solenopsis invicta Bu- by >1 m. The paired terrestrial baits (one honey, ren). Red imported fire ants were first recorded in one hamburger meat) were placed directly on the the upper Florida Keys in 1976 (Callcott & Col- ground on pieces of aluminum foil. The paired lins 1996), but were considered to be restricted to arboreal baits were placed in plastic condiment disturbed areas (Deyrup et al. 1988; Porter 1992). cups with 5-10, 3-6 mm holes punched throughout During a Keys-wide ant survey in 1996, S. invicta the cup. Both arboreal cups were placed 1-1.5 m was identified on 10 of the 14 major keys (Forys et from the ground in a tree as close to the terres- al. 1999). Solenopsis invicta was found in every trial bait as was possible. major habitat type including hardwood hammocks, pinelands, salt and freshwater marshes, Predation on Swallowtails and disturbed areas (e.g., roadsides, parking lots) (Forys et al. 1999). While little research has been To determine the attractiveness and vulnera- conducted on red imported fire ant predation on bility of Schaus swallowtails to red imported fire Lepidoptera, the presence of S. invicta in the trop- ants we conducted an experiment using eggs, lar- ical hardwood hammocks is of particular concern vae, and pupae of the giant swallowtail (P. cre- for the Schaus swallowtail because red imported sphontes) Cramer as a surrogate species. The fire ants are known to prey on a wide range of giant swallowtail is a common Key’s resident that other invertebrates (Porter & Savignano 1990). is similar to the Schaus swallowtail in its distri- Schaus swallowtail eggs, larvae, and pupae may bution and natural history. Both species of swal- be vulnerable because they occur on tree species lowtails occur in the Florida Keys, utilize species (e.g., torchwood, wild lime) that generally occupy of Rutaceae as host plants, and lay eggs singly on habitat edge where red imported fire ant infesta- leaves. The larvae of both eat new leaves, do not tions tend to be the highest. use nests, pupae of both hibernate and are similar The objective of this study was to 1) measure in structure and appearance (Scott 1986). The gi- the abundance of red imported fire ants in Schaus ant swallowtail reproduces more frequently (has swallowtail habitat, 2) determine if swallowtail multiple broods in one year) and adults can be

256 Florida Entomologist 84(2) June 2001 found throughout the year, while the Schaus by placing hamburger and honey baits every 5 m swallowtail has only one set of brood a year and along 50-m transects extending into the ham- adults are found mid March through mid Septem- mock at the midpoint of each treatment and con- ber (Emmel 1995). trol areas before and after treatment. These Ten eggs, larvae (third and fourth instar), and transects were surveyed twice before treatment pupae were purchased from a commercial butter- in March and July 1997, and three times after fly farm (Robert Brown, Butterfly Paradise, 19940 treatment in October and December 1997 and Adams Rd., Ft. Myers, FL). Each egg, larva, and March 1998. We compared the number of red im- pupa was individually attached 1 m off the ground ported fire ants collected at each hammock on a wild lime tree placed in enclosures with ac- transect in the treated and untreated areas for tive red imported fire ant colony. For comparison each survey using a Mann-Whitney U Test be- we also placed 10 balls of hamburger meat of cause the data was not normally distributed and/ equal size to the larvae and pupae one meter off or variances were not equal. the ground on a wild lime tree in an additional 10 enclosures with an active red imported fire ant RESULTS colonies. The eggs had been laid on wild lime leaves and we attached these leaves to the wild Red Imported Fire Ant Foraging lime tree using gardeners wire. The pupae were placed in small porous cups and were attached to In the North Key Largo hammock, red im- the wild lime tree. The larvae were directly placed ported fire ants were identified on 8 of the 10 on leaves. The enclosures were monitored for transects from the south side of SR905 and 2 of three hours and both the length of time for the fire the 10 transects from the north side of SR905. ants to discover (make first contact) and entirely Most of the baits with red imported fire ants were consume the hamburger balls and swallowtail near the road. The maximum foraging distance eggs, larvae, and pupae were recorded. A t-test into the hammock was 40 m. Red imported fire was used to compare the time to discovery for each ants were detected arboreally at 3 transects on swallowtail life stage and the hamburger meat. the south side of the road, up to a distance of 25 m No statistical comparisons were made between into the hammock. time to consumption for the life stages and hamburger meat because the life stage differed in Predation on Swallowtails shape and consistency from the meat. Red imported fire ants predated all of the im- Effectiveness of Fire Ant Control mature swallowtail life stages. Fire ants discovered the butterfly stages faster than the Five, 50-m sections of the shoulder of the main hamburger meat although this difference was sig- road that bisects the Key Largo hammock nificant only for the larval life stage (t = 4.66, d.f. (SR905) were treated in July, 1997, using a fire = 18, P < 0.0001) (Table 1). The larval stage was ant bait, Amdro®, broadcast from the back of a the first to be discovered by fire ants and was con- four-wheeler and leaving 5, 50-m untreated areas sumed the fastest (Table 1). However, three larvae as controls. Areas >100 m were left between treat- escaped predation during the three h experiment ment and control areas. The active ingredient in by moving to higher branches of the wild lime tree Amdro® is hydramethylnon, a metabolic inhibi- after being initially detected by a fire ant. All of tor that usually kills queens, workers and the col- the pupae and eggs were discovered and con- ony within 2-4 weeks (Williams 1994). sumed by fire ants. It took ants the longest to con- To measure the effectiveness of this treatment, sume the pupal life stage because the fire ants red imported fire ant abundance was monitored 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.

Forys et al.: Fire ant impacts on swallowtails 257

Effectiveness of Fire Ant Control forded some protection. The high rate of predation was surprising due to the suite of anti- Before treating with Amdro®, the average num- predator behaviors that swallowtails exhibit such ber of red imported fire ants in the proposed treat- as laying one egg per leaf, secretive behavior of ment areas and untreated areas did not differ larvae, and the production of foul-smelling scents significantly (March 1997: T = 92.5, P = 0.36, n1 = from the osmeteria when larvae are disturbed 10, n2 = 10; July 1997: T = 101.0, P = 0.78, n1 = 10, (Rutkowski 1971). The ability of the red imported n2 = 10). In October, 1997, three months after the fire ant to penetrate the pupae was particularly July treatment, there were no red imported fire disturbing. While these results were found in a ants at any of the bait transects in the treated area laboratory experiment on a related species, it is and this was significantly fewer than in the un- probable that red imported fire ants impact treated areas (T = 75.0, P = 0.03, n1 = 10, n2 = 10). Schaus swallowtails in nature. A manipulative However, five and eight months after the treat- field experiment with red imported fire ant popu- ment, the number of red imported fire ants was not lation reductions and careful monitoring of significantly lower in the treated areas (December Schaus swallowtail life stages and populations 1997: T = 92.0, P = 0.34, n1 = 10, n2 = 10; March would provide definitive evidence. 1998: T = 109.5, P = 0.76, n1 = 10, n2 = 10). Overall, Recently, Schaus swallowtail reintroductions the number of red imported fire ants declined in occurred in state and federally owned hammocks both treated and untreated areas after the July on northern Key Largo (Emmel 1995; U.S. Fish 1997 treatment, probably due to the unusually dry and Wildlife Service 1999). We found that red im- conditions during that time period (Fig. 1). ported fire ants were abundant on both the edges of the north Key Largo hammock and up to 40m DISCUSSION into the interior. Fire ants were more abundant terrestrially than arboreally, but fire ants did for- The lab experiment we conducted provides ev- age arboreally. Because of the abundance of red idence of the potential for red imported fire ant imported fire ants and the results of the predation predation on the Giant swallowtail, and suggests experiment, it is possible that fire ants are a that other species of butterflies that occur in the threat to the long-term success of Schaus swal- southeastern United States also may be vulnera- lowtail reintroductions. ble, including Schaus swallowtail. Fire ants pre- To further increase the chance of survival of dated on all terrestrial life history stages of the the reintroduced Schaus swallowtail populations, swallowtail, although the mobility of larvae af- red imported fire ant populations should be reduced 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 affected 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 abandoned 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 densities. 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- Fig. 1. The average number of red imported fire ants ported fire ants on the south and north sides of (Solenopsis invicta) on baits at the 10 transects treated with Amdro® to kill fire ants and the 10 left untreated. the road. The July 1997 sampling date occurred immediately Previous studies in this area have indicated before the Amdro® treatment. An “*” indicates that the that roads bisecting the hammock are especially average number of ants significantly differed (p < 0.05) attractive colonization sites for fire ants (Forys et between the treated and untreated transects. al. 1999). The removal and restoration of aban-

258 Florida Entomologist 84(2) June 2001

doned roads and access paths, and limiting dis- CALLCOTT, A. M. A., AND H. L. COLLINS. 1996. Invasion turbance of road shoulders, will probably lower and range expansion of red imported fire ant (Hy- fire ant populations in the area. Reducing the menoptera: Formicidae) in North America from abundance of red imported fire ants in the Key 1918-1995. Florida Entomol. 79: 240-251. Largo hammock would be beneficial to the DEYRUP, M. A., N. CARLIN, J. TRAGER, AND G. UMPHREY. 1988. A review of the ants of the Florida keys. Flor- Schaus, as well as a suite of other rare inverte- ida Entomol. 71: 163-176. brate and vertebrate species that may be suscep- EMMEL, R. C. 1995. Habitat requirements and Status of tible to predation (e.g., Key Largo cotton mouse, the Endemic Schaus Swallowtail in the Florida Keys. Peromyscus gossypinus; Key Largo woodrat, Florida Game and Fresh Water Fish Commission, Neotoma floridana smalli; Florida tree snails, Tallahassee, FL. Final Report GFC-86-023. 202 pp. Liguus fasciatus). EMMEL, R. C. 1986. Status survey and habitat requirements of Florida’s endemic Schaus swallowtail butterfly. Florida Game and Fresh Water Fish ACKNOWLEDGMENTS Commission, Tallahassee, FL. Final Report GFC-84- 028. 22 pp. This project was funded by the Florida Game FORYS, E. A., C. R. ALLEN, AND D. P. WOJCIK. 1999. The and Fresh Water Fish Commission’s Nongame potential for negative impacts by red imported fire Wildlife Program (NG95-018). B. Stieglitz and R. ants (Solenopsis invicta) on listed herpetofauna, Skinner helped us by providing access and timely mammals, and invertebrates in the Florida Keys. permits to refuge and state owned land. P. Frank Florida Game and Fresh Water Fish Commission, and S. Klett provided input on study design. J. Tallahassee, FL. Final Report NG95-018. Sullenger and B. Mayfield sorted and identified PORTER, S. D. 1992. Frequency and distribution of po- all of the ants. D. P. Wojcik provided ultimate ant lygyne fire ants (Hymenoptera: Formicidae) in Flor- identification and helped in the field. H. Collins ida. Florida Entomol. 75: 248-257. PORTER, S. D., AND D. A. SAVIGNANO. 1990. Invasion of and associates helped design the bait treatment. polygyne fire ants decimates native ants and dis- J. Hosford and M. Pratt assisted with data collec- rupts arthropod community. Ecology 71: 2095-2106. tion and entry. An earlier version of this paper RUTKOWSKI, F. 1971. Observations on Papilio aristode- was improved by incorporating comments from D. mus ponceanus (Papilionidae). J. Lep. Soc. 25: 126- Cook, J. Isely, and J. Zettler. The South Carolina 136. Cooperative Fish and Wildlife Research Unit is SAUNDERS, D. A., R. J. HOBBS, AND C. R. MARGULES. jointly supported by a cooperative agreement 1991. Biological consequences of ecosystem fragmen- among the USGS/BRD, the South Carolina De- tation: a review. Cons. Biol. 5: 18-32. partment of Natural Resources, Clemson Univer- SCOTT, J. A. 1986. Butterflies of North America. Stan- ford University Press, Stanford, CA. 583 pp. sity, and the Wildlife Management Institute. U.S. FISH AND WILDLIFE SERVICE. 1999. South Florida multi-species recovery plan. Atlanta, GA. 2172 pp. REFERENCES CITED USHER, M. B. 1988. Biological invasions of nature re- serves: a search for generalizations. Biol. Cons. 44: ALLEN, C. R., R. S. LUTZ, AND S. DEMARAIS. 1995. Red 119-135. imported fire ant impacts on northern bobwhite pop- WILLIAMS, D. F. 1994. Control of the Introduced Pest So- ulations. Ecol. App. 5: 632-638. lenopsis invicta in the United States, pp. 282-292. In BROWN, C. H. 1976. A colony of Papilio aristodemus D. F. Williams (ed.). Exotic ants: Biology, impact, and ponceanus (Lepidoptera: Papilionidae) in the upper control of introduced species. Westview Press, Boul- Florida Keys. J. Georgia Entomol. Soc. 11: 117-118. der, CO.

Mao et al: Sweeptotato weevil host plant resistance 259

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 effect 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 different from Beauregard and Centennial. Roots of the same genotype grown in different locations 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

RESUMEN

Fue investigado el efecto de genotipo del camote, la duración de almacenamiento y el lugar de producción sobre la alimentación y oviposición de Cylas formicarius (Fab.). El genotipo del camote tuvo un efecto significativo sobre la alimentación y velocidad de oviposición dadas las opciones de raíz y no raíz. Beauregard y Centennial fueron igualmente susceptibles entre los genotipos evaluados. W-250 tuvo un menor numero de agujeros y huevos a 7 y 25 días des- pués del cosechado. A 85 días después de cosechado, W-244 tuvo menos agujeros y huevos, mientras que W-250 no tuvo diferencias significativas con Beauregard y Centennial. Raíces del mismo genotipo sembradas en diferentes localidades tuvieron diferente numero de agujeros y huevos. Los resultados sugieren que antixenosis es responsable 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 producción, pero los resultados finales de- penden mas en los genotipos.

Sweetpotato weevil (SPW), Cylas formicarius tance in the field (Rolston et al. 1979; Mullen et (Fab.), is a major constraint to sweetpotato [Ipo- al. 1980b, 1981, 1982, 1985; Taleker 1987b) and moea batatas (L.) Lam.] production worldwide laboratory (Mullen et al. 1980a; Barlow & Rolston (Chalfant et al. 1990; Jansson & Raman 1991). It 1981; Nottingham et al. 1987, 1989; Ratnayake attacks sweetpotato both in the field and during 1995; Story et al. 1996, 1999a, b, c). However, lit- storage. Adults make feeding and oviposition tle success has been realized in the development punctures on the root surface that can reduce root of resistant cultivars, partly because of inconsis- quality and market value. Larval tunneling in tencies in the performance of selected breeding roots induces terpenoid production that renders lines (Talekar 1987a, b) and a lack of understand- even slightly damaged roots unfit for human and ing of the resistance mechanisms. animal consumption (Cockerham et al. 1954; Uri- The expression of insect resistance can be in- tani et al. 1975). Due to the concealed nature of fluenced by many environmental factors (Smith the feeding habit, control of SPW is difficult. The 1989). Identification of these factors would help to use of resistant sweetpotato cultivars is a poten- explain the inconsistent performance of resistant tially viable option that could be an economical genotypes. Such information would also be useful component in the integrated management of SPW in facilitating the development of resistant culti- (Martin & Jones 1986; Collins et al. 1991). vars and understanding the underlying mecha- Many studies have been conducted on SPW re- nisms of resistance. In temperate growing areas sistance in sweetpotato indicating variable resis- like the United States, storage roots are cured by

260 Florida Entomologist 84(2) June 2001 keeping them in a specially designed facility total darkness to eliminate light as a variable. maintained at about 30°C and 85% to 90% RH for Cores from only one genotype were presented to 4 to 7 days. Cured roots are often evaluated over a the weevils in no-choice tests. In choice tests, one period of several months in sweetpotato breeding core from each genotype was randomly arranged programs. Thus curing and storage time may af- on the plate and presented to the insects. fect the outcome of SPW resistance evaluations Four sweetpotato genotypes were chosen ac- because physical and chemical changes occur in cording to their performance in no-choice whole- the roots (Bouwkamp 1985). In addition, sweetpo- root laboratory evaluations (Story et al. 1996). tato is grown throughout a wide geographic W-244 and W-250 were breeding lines shown to be range. Wide variations in SPW resistance be- resistant to SPW. Beauregard and Centennial tween production sites have been observed in field were two susceptible cultivars. To determine the plots (Talekar 1987b). However, until this study, effect of curing and storage time, bioassays were no comparative study has been done under con- conducted with roots of the following groups: non- trolled laboratory conditions to determine the ef- cured 7 days after harvest (DAH), cured 25 DAH, fect of storage and production site on SPW feeding and cured 85 DAH. Storage roots were produced and oviposition. We evaluated 4 sweetpotato gen- using standard practices at Burden Research otypes (“Beauregard”, “Centennial”, “W-244”, and Plantation, Baton Rouge, Louisiana. Slips were “W-250”) to determine the effects of storage and planted on July 5, 1996 with 0.3 m spacing in 20- production site on SPW feeding and oviposition. plant plots with rows separated by 1.2 m. Storage roots were harvested on November 1, 1996, cured (30°C, 90% RH for 7 days), and stored at 15 ± 2°C. MATERIALS AND METHODS At each test date, both no-choice and choice tests were conducted with complete randomized exper- Insect rearing imental designs and 8 replications (8 US#1 roots A SPW colony was established from a field col- for each genotype). lected population (about 500 insects) and main- Three sweetpotato growing regions were cho- tained in the laboratory on storage roots of sen to evaluate the effect of production site on the Beauregard in plastic containers (5.6 L) with expression of SPW resistance. They were Baton screen covers at 28 ± 2°C and 85 ± 10% RH. In Rouge, Louisiana (LA), Edisto, South Carolina preparing experimental insects, 5 fresh storage (SC), and Pontotoc, Mississippi (MS). Storage roots (US #1) were exposed to about 1000 adults roots were produced at each site using similar (male and female) for 5 days, then were removed production practices. Non-cured 7 DAH roots of and kept under the conditions described above. all 4 genotypes from LA and MS and cured 25 Emerging adults (male and female) were col- DAH roots of 3 genotypes (Beauregard, Centen- lected weekly and held with fresh storage roots. nial and W-250) from LA and SC were used. No- Female adults 3-4 weeks old were used in the bio- choice tests were conducted with 8 replications. assays to ensure adequate egg-laying capability (Wilson et al. 1988). Data Analysis All data (average number of feeding punctures Bioassay or number of eggs per root core) were analyzed The assay technique was an adaptation of one with the PC SAS General Linear Model (GLM) pro- previously described by Mullen et al. (1980a) and cedure (SAS Version 6.12 1990), followed by Tukey has been used in several SPW feeding and oviposi- multiple range tests for mean separations. The ef- tion studies (Nottingham et al. 1987; Wilson et al. fect of storage time was tested as a fixed block ef- 1988). It consisted of a 24-well tissue culture plate fect by pooling data from all 3 age groups. Curing (12.5 × 8.5 × 2.0 cm; Falcon®) placed in a rectangu- effect was tested using a contrast statement. Pro- lar clear plastic container (17 × 12 × 6 cm). Cores duction site effect was analyzed as a fixed block ef- were cut from selected roots with a cork borer (1.6 fect in a randomized complete block design. In all α cm diameter) and inserted into the wells so that tests the significance level was = 0.05. only the surface of the root periderm was exposed. The cores had the same diameter as the wells, pro- RESULTS viding a close fit. Female adults were kept without food for 3 hours before being introduced into the Genotype, Curing and Storage Time Effects arena at the rate of 2 weevils per root core. A moist cotton ball was placed in the container to maintain Significant differences in both choice and no- 90-100% RH and prevent desiccation of the root choice tests in feeding and oviposition were found cores. After 24 hours the number of feeding punc- among the 4 genotypes. In both no-choice and tures on each core was recorded, and after 48 choice tests, W-250 had the lowest number of feed- hours the number of eggs was counted. All tests ing punctures and eggs at 7 and 25 DAH (Table 1). were conducted at 28 ± 5°C, 85 ± 10% RH under At 85 DAH, W-244 had the least numbers of feed-

Mao et al: Sweeptotato weevil host plant resistance 261

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 puncture1 Eggs1 Feeding puncture1 Eggs1

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

1Means 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- vars. Beauregard and Centennial were uniformly nificantly different from Beauregard and susceptible across the three root age groups. Centennial (Table 1). The curing process did not have a significant effect on feeding and oviposi- Production Site Effects tion in both choice and no-choice tests. Storage time had a significant effect on the number of Non-Cured Roots. A significant production site eggs deposited in no-choice tests (F = 61.52, df = effect was found for the number of feeding punc- 2,84, P = .0001), but not in choice tests. Storage tures (F = 5.72, df = 1,56, P = 0.0202) on the non- time did not have an effect on feeding punctures. cured roots from Louisiana and Mississippi where Cultivar and storage time interaction effect was Mississippi roots received higher number of feed- significant in all cases. W-250 had some resis- ing punctures than Louisiana roots (Table 2). tance relative to the susceptible cultivars when However, the number of eggs deposited was not the roots were non-cured 7 DAH and cured 25 significantly different (F = 0.05, df = 1,56, P = DAH, but the resistance factors were diminished 0.8915). The interaction effect of genotype and in cured 85 DAH roots (Table 1). The opposite production site was highly significant for both trend was found with W-244, in which significant feeding and oviposition (F = 4.63, df = 3,56, P = differences in the number of punctures and eggs 0.0058, F = 6.33, df = 3,56, P = 0.0009, respec- were detected only with cured 85 DAH roots when tively), indicating that the feeding and oviposi- they were compared with the susceptible culti- 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

1The tests were conducted using non-cured roots 7 days after harvest. 2Means followed by the same letter within production site are not significantly different (p > 0.05, Tukey). 262 Florida Entomologist 84(2) June 2001 between these two sites (Table 2). For Louisiana nayake 1995; Story et al. 1996) and Centennial grown roots, all 4 genotypes were significantly (Mullen et al. 1980b; Nottingham et al. 1989; different from each other in number of feeding Rolston et al. 1979) in both field and laboratory punctures, while no significant difference was tests. W-244 and W-250 are two breeding lines found between Beauregard and Centennial in the with resistance to SPW (Ratnayake 1995; Story et number of eggs laid. Centennial had the most al. 1999a). The lower numbers of feeding punc- feeding punctures and eggs. For Mississippi tures and eggs on the roots of these two lines sug- grown roots, significant differences were found gests that antixenosis was responsible for at least between the two susceptible cultivars in both part of SPW resistance and that the resistant fac- number of feeding punctures and eggs. Signifi- tor(s) may have a broad spectrum. Talekar (1987a) cant differences were not found between W-244 argued against the feasibility of nonpreference in and W-250 but they were different from suscepti- sweetpotato. He pointed out that it had little value ble cultivars. Beauregard was the preferred geno- because weevils lack choices among sweetpotato type for both feeding and oviposition. genotypes in commercial plantings. We found that Cured Roots. A significant production site ef- SPW exhibited feeding and oviposition differences fect was found between the number of eggs depos- among sweetpotato genotypes under no-choice ited (F = 4.38, df = 1,42, P = 0.0424) in Louisiana conditions, suggesting the possibility of utilizing and South Carolina cured roots where South antixenosis in SPW management. Carolina roots had higher number of eggs (Table No-choice and choice are the two experimental 3). The number of feeding punctures was not sig- settings for evaluating plant resistance to insects. nificantly affected by production site (F = 1.90, df Sometimes, results from these two kinds of tests = 1,42, P = 0.1723). Although no statistically sig- appear to be contradictory. Resistant genotypes nificant production site and genotype interaction identified under choice conditions can receive effects were found, there was a trend toward dif- more feeding damage than susceptible genotypes ferent performance of W-250 from these two loca- when insects are forced to feed on only one geno- tions. Roots grown in Louisiana had significantly type (Tingey 1986). Usually, under choice condi- fewer punctures and eggs for W-250 when com- tions, susceptible plants receive higher levels of pared to Beauregard and Centennial. However, damage than resistant plants when compared to no differences were detected among the 3 geno- the results of no-choice conditions. This results in types grown in South Carolina. a larger variance among genotypes under the choice conditions. We found no significant differ-

DISCUSSION ences in number of feeding punctures (Fs = 1.2513,

df1 = df2 = 95, P = 0.13818) and eggs (Fs = 1.2781,

Plant resistance to insects may be due to anti- df1 = df2 = 95, P = 0.11679) between choice and no- biosis, antixenosis (nonpreference), tolerance, or choice tests when testing for equal variance as de- escape. All these types have been reported in scribed by Sokal and Rohlf (1981). sweetpotato resistance to SPW (Waddill & Curing and storage are common postharvest Conover 1978; Barlow & Rolston 1981; Mullen et procedures for sweetpotato in temperate growing al. 1981; Talekar 1987b; Ratnayake 1995). This areas. During these processes many physical and study evaluated antixenosis effects (plants lack chemical changes may occur in the roots. For ex- the characteristics that attract insects and are ample, curing promotes wound periderm forma- avoided by insects) on feeding and oviposition by tion on injured surfaces, thus reducing decay and female adult of SPW. We found that Beauregard water loss (Bouwkamp 1985). Storage has been and Centennial were preferred by SPW with re- reported to induce changes in carbohydrate com- spect to both feeding and oviposition. These re- position, enzyme activities and cell wall compo- sults are consistent with previous reports that nents (Takahata et al. 1995; Walter & Palma have shown the susceptibility of Beauregard (Rat- 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

1The tests were conducted using cured roots 25 days after harvest. 2Means followed by the same letter within production site are not significantly different (p > 0.05, Tukey). Mao et al: Sweeptotato weevil host plant resistance 263

on SPW feeding and oviposition behaviors. How- COLLINS, W., A. JONES, M. A. MULLEN, N. S. TALEKAR, ever, as storage time lengthened, SPW feeding AND F. W. MARTIN. 1991. Breeding sweet potato for and oviposition rates changed among genotypes. insect resistance: A global overview, pp. 379-397. In This suggests that storage time may influence the Jansson & Raman (eds.). Sweet Potato Pest Manage- expression of SPW resistance, but the effect dif- ment: A Global Perspective,. Westview Press, Boul- der, USA. fers with each genotype. JANSSON R. K., AND K. V. RAMAN 1991. Sweet potato The importance of environmental factors in pest management: a global overview, pp. 2-12. In the expression of SPW resistance has been noted Jansson & Raman (eds.). Sweet Potato Pest Manage- by Talekar (1987 b). Our study also had some sig- ment: A Global Perspective. Westview Press, Inc. nificant production site effects and the interac- MARTIN, F. W., AND A. JONES. 1986. Breeding sweet po- tions of genotype and production site on SPW tatoes. In Janich (ed.). Plant Breeding Reviews IV, feeding and oviposition. Previous studies have re- pp. 313-345. AVI, Westport, CT, USA. lated SPW resistance to the presence and concen- MULLEN, M. A., A. JONES, R. DAVIS, AND G. C. PEAR- tration of a pentacyclic triterpene, boehmeryl MAN. 1980a. Rapid selection of sweet potato lines resistant to the sweetpotato weevil. HortScience 15: acetate, in the periderm tissues of sweetpotato 70-71. roots. This chemical has been identified as a SPW MULLEN, M. A., A. JONES, R. T. ARBOGAST, J. M. oviposition stimulant (Son 1989; Wilson et al. SCHALK, D. R. PATERSON, T. E. BOSWELL, AND D. R. 1989). Our study suggests the possibility of the EARHART. 1980b. Field selection of sweet potato lines presence of deterrent(s) or repellent(s) in the re- and cultivars for resistance to the sweetpotato wee- sistant genotypes or a reduction of boehmeryl ac- vil. J. Econ. Entomol. 73: 288-290. etate. Environmental factors very likely influence MULLEN, M. A., A. JONES, AND R. T. ABROGAST. 1981. such phytochemicals and hence alter the level of Resistance of sweet potato lines to infestations of resistance. sweet potato weevil, Cylas formicarius elegantulus (Summers). HortScience 16:539-540. In conclusion, SPW exhibited different feeding MULLEN, M. A., A. JONES, D. R. PATERSON, AND T. E. and oviposition preferences among sweetpotato BOSWELL. 1982. Resistance of sweet potato lines to genotypes. Curing, storage time, and production the sweet potato weevil. HortScience 17: 931-932. site influenced SPW feeding and oviposition be- MULLEN, M. A., A. JONES, D. R. PATERSON, AND T. E. havior. When screening for SPW resistance, all BOSWELL. 1985. Resistance in sweet potatoes to the conditions associated with testing materials sweetpotato weevil, Cylas formicarius elegantulus (storage roots) and environmental conditions (Summers). J. Entomol. Sci. 20(3): 345-350. should be kept as consistent as possible. Poten- NOTTINGHAM, S. F., D. D. WILSON, R. F. SEVERSON, AND tially resistant lines should be evaluated under S. J. KAYS. 1987. Feeding and oviposition preferences of the sweet potato weevil, Cylas formicarius multiple sets of environmental conditions over a elegantulus, on the outer periderm and exposed in- period of several years. ner core of storage roots of selected sweet potato cultivars. Entomol. Exp. Appl. 45: 271-275. NOTTINGHAM, S. F., K.-C. SON, D. D. WILSON, R. F. SEV- ACKNOWLEDGMENTS ERSON, AND S. J. KAYS. 1989. Feeding and oviposi- The authors wish to thank Dr. Paul Thompson tion preferences of sweet potato weevil, Cylas of the Pontotoc Research and Extension Center, formicarius elegantulus (Summer), on storage roots of sweet potato cultivars with differing surface Mississippi State University, and Dr. Janice Bo- chemistries. J. Chem. Ecol. 15: 895-903. hac of the U.S. Vegetable Laboratory, Charleston, RATNAYAKE, B. M. 1995. Determination of categories of South Carolina for providing storage roots for host plant resistance to the sweetpotato weevil, Cy- testing. Jeff Murray is thanked for his assistance las formicarium elegantulus, in various genotypes of in conducting these studies. Approved for publica- sweet potato (Ipomoea batatas). MS Thesis, Missis- tion by the Director of the Louisiana Agricultural sippi State University. Experiment Station as manuscript number 00- ROLSTON, L. H., T. BARLOW, T. HERNANDEZ, AND S. S. 17-0223. NILAKHE. 1979. Field evaluation of breeding lines and cultivars of sweet potato for resistance to the sweet potato weevil. HortScience 14: 634-635. REFERENCES CITED SAS INSTITUTE 1990. SAS/STAT user’s guide. Release 6.12. SAS Institute, Cary, NC. BARLOW, T. AND L. H. ROLSTON. 1981. Types of host SMITH, C. M. 1989. Plant resistance to insects: A funda- plant resistance to the sweetpotato weevil found in mental approach. John Wiley & Sons. Inc. sweet potato roots. J. Kansas Entomol. Soc. 54: 649- SOKAL, R. R., AND F. J. ROHLF. 1981. Biometry. 2nd ed. 657. Freeman, New York. BOUWKAMP, J. C. 1985. Sweet potato products: a natural SON, K.-C. 1989. Phytochemistry of the sweet potato, Ip- resource for the tropics. CRC Press, Boca Raton, FL. omoea batatas (L.) Lam., storage root in relation to CHALFANT, R. B., R. K. JANSSON, D. R. SEAL, AND J. M. susceptibility to the sweet potato weevil, Cylas form- SCHALK. 1990. Ecology and management of sweetpo- micarius elegantulus (Summers). Ph.D. Disserta- tato insects. Ann. Rev. Entomol. 35: 157-180. tion, University of Georgia, Athens. COCKERHAM, K. L., O. T. DEAN, M. B. CHRISTIAN, AND STORY, R. N., A. HAMMOND, J. MURRAY, L. H. ROLSTON, L. D. NEWSOM. 1954. The biology of the sweetpotato AND D. LABONTE. 1996. selection for host plant resis- weevil. La. Agric. Exp. Stn. Tech. Bull. No. 483. tance in sweetpotatoes to the sweetpotato weevil. 264 Florida Entomologist 84(2) June 2001

1995, pp. 71-79. In Sweetpotato Research 1996. activities of sweetpotato lines during storage. J. Ag- LAES Mimeo series No. 117. ric. Food Chem. 43: 1923-1928. STORY, R. N., A. HAMMOND, D. LABONTE, P. THOMPSON, TINGEY, W. M. 1986. Techniques for evaluating plant re- AND J. BOHAC. 1999a. Evaluation of sweetpotato ger- sistance to insects, pp. 251-284. In J. R. Miller and T. mplasm for resistance to sweetpotato weevil, 1996. A. Miller [eds.], Insect-plant interactions. Springer, Arthropod Management Tests 24: 436-437. New York. STORY, R. N., A. HAMMOND, D. LABONTE, P. THOMPSON, URITANI, I., T. SAITO, H. HONDA, AND W. K. KIM. 1975. AND J. BOHAC. 1999b. Evaluation of sweetpotato ger- Induction of furanoterpenoids in sweet potato roots mplasm for resistance to sweetpotato weevil, 1997. by the larval components of the sweetpotato weevils. Arthropod Management Tests 24: 437-438. Agric. Biol. Chem. 37: 1857-1862. STORY, R. N., A. HAMMOND, D. LABONTE, P. THOMPSON, WADDILL, V. H., AND R. A. CONOVER. 1978. Resistance of AND J. BOHAC. 1999c. Evaluation of sweetpotato ger- white-ﬂeshed sweet potato cultivars to the sweet po- mplasm for resistance to sweetpotato weevil, 1998. tato weevil. HortScience 13: 476-477. Arthropod Management Tests 24: 438-439. WALTER, W. M., JR., AND C. S. PALMA. 1996. Effect of TALEKAR, N. S. 1987a. Feasibility of the use of resistant long-term storage on cell wall neutral sugars and ga- cultivar in sweet potato weevil control. Insect Sci. lacturonic acid of two sweetpotato cultivars. J. Agric. Applic. 8: 815-817. Food Chem. 44: 278-281. TALEKAR, N. S. 1987b. Resistance in sweetpotato to WILSON, D. D., R. F. SEVERSON, K.-C. SON, AND S. J. sweetpotato weevil. Insect Sci. Applic. 8: 819-823. KAYS. 1988. Oviposition stimulant in sweet potato TAKAHATA, YASUHIRO, TAKAHIRO NADA, AND TATSUO periderm for the sweetpotato weevil, Cylas formicar- SATO. 1995. Changes in carbohydrates and enzyme ius elegantulus. Environ. Entomol. 17: 691-693.

Yanoviak: Treehole macroinvertebrates 265

CONTAINER COLOR AND LOCATION AFFECT MACROINVERTEBRATE COMMUNITY STRUCTURE IN ARTIFICIAL TREEHOLES IN PANAMA

STEPHEN P. YANOVIAK Department of Zoology, University of Oklahoma, Norman, OK 73019-0235 USA

Current address: Evergreen State College, Lab 1, Olympia, WA 98505

ABSTRACT

I investigated the effects of habitat color and location on community structure in artificial water-filled treeholes in the forest of Barro Colorado Island, Panama. The macroinvertebrate fauna of 9 replications (5 in understory, 4 in tree-fall gaps) of black, blue, red, and green 650 ml plastic cups were censused weekly for 7 wks. Macroinvertebrate abundance and species richness were greater in understory cups than in gap cups. Seven species colonized black cups exclusively. Black cups in the understory, and red and black cups in gaps, attracted more species on average than other colors. Species richness and abundance were consistently lowest in green cups. Species were more broadly distributed among cup colors in the understory, suggesting that diffuse light conditions influenced color perception. There was no overlap in species composition between water in the artificial treeholes and water held by red Heliconia bracts or green tank bromeliads.

Key Words: color, microcosm, mosquitoes, phytotelmata, tree-fall gaps, tropics

RESUMEN

Investigué los efectos del color de hábitat y localidad en la estructura de la comunidad en huecos de árboles artificiales llenos de agua en el bosque de Isla Barro Colorado, Panamá. La fauna macro invertebrada de 9 replicaciones (5 en el “understory”, 4 en espacios abiertos de árboles caídos) de vasos plásticos de 650 ml de color negro, azul, rojo, y verde fueron observadas semanalmente por 7 semanas. La abundancia macro invertebrada y la riqueza de especies fueron mayor en vasos del “understory” que en los vasos de espacios abiertos. Siete especies colonizaron vasos negros exclusivamente. Los vasos negros en el “understory”, y los vasos rojos y negros en los espacios, atrajeron un promedio mayor de especies que otros colores. Riqueza y abundancia de especies fueron mas bajas consistentemente en vasos verdes. Las especies fueron distribuidas mas ampliamente entre colores de vaso en el “understory”, sugiriendo que condiciones de luz difusa influyen percepción de color. No hubo área común en composición de especies entre agua en los huecos de árbol artificiales y agua retenida por brácteas rojas de Heliconia o bromelias de tanque verde.

Container-breeding mosquitoes use a variety and rain water) are often used for field-based pop- of physical and chemical cues when selecting ovi- ulation and community-level studies. These con- position substrates (Bates 1949; Bentley & Day tainers provide suitable mimics of the treehole 1989), and many species show preferences for habitat, and typically attract the same fauna specific habitat colors (Frank 1985, 1986, and ref- found in the natural setting (e.g., Fincke et al. erences therein). Most investigations of mosquito 1997; Yanoviak, in press). Artificial treeholes are response to habitat color have been lab-based, usually clear (Pimm & Kitching 1987; Srivastava and color preferences have not been studied for & Lawton 1998), black or brown (Fincke et al. other taxa that typically coexist with mosquitoes 1997; Yanoviak 1999a), or blue (O. M. Fincke, in the field. Although mosquitoes generally domi- Dept. Zoology, Univ. Oklahoma, pers. comm.). nate the macrofauna of tropical phytotelmata, How such color differences may affect community several other insect taxa are also common in structure in these experimental systems has these habitats (e.g., Fish 1983). The effects of hab- never been investigated. itat color on colonization by non-mosquito aquatic The potential importance of habitat color ex- macroinvertebrates in tropical phytotelmata are tends beyond artificial treeholes; there is consid- not known. erable variation in color among natural phyto- Water-filled treeholes are common phytotel- telmata. Common phytotelmata in the lowland mata in many temperate and tropical forests forests of Panama include water-filled treeholes, (Snow 1949; Kitching 1971), and occur in both tank bromeliads (e.g., Vriesia spp., Guzmania tree-fall gaps and forest understory. Artificial spp.), fallen palm petioles and spathes (e.g., of As- treeholes (plastic containers containing leaf litter trocaryum standleyanum Bailey), and exocarps of

266 Florida Entomologist 84(2) June 2001 fallen Tontelea ovalifolia (Miers) A. C. Smith ginning 26 July 1997. I occasionally collected sub- fruits. Treehole interiors typically appear black or samples of pupae and late-instar larvae to confirm brown; bromeliads are generally green; palm field identifications. Water temperatures were spathes and T. ovalifolia husks are initially measured at irregular intervals with a Corning® cream-colored, but gradually become orange, red- modular electronic probe. The experiment was dish, and eventually dark brown as they age terminated after 7 wks because detritus accumu- (pers. obs.). In addition, the red and green bracts lations and fungal or algal growth changed the in- of Heliconia spp. often contain sufficient water to terior color of some cups to dark brown. support aquatic macroinvertebrate assemblages In addition to the fauna of artificial treeholes, (e.g., Seifert & Seifert 1979; Naeem 1988; Louni- I qualitatively sampled macroinvertebrates living bos & Machado-Allison 1993). in nearby water held by 14 tank bromeliads, 20 T. This study examined the effects of container ovalifolia exocarps, 12 fallen palm spathes, and color and location (tree-fall gap or forest under- 12 Heliconia latispatha Benth. bracts (from 12 story) on colonization of artificial treeholes by different plants) for comparison with the species macroinvertebrates under field conditions. Spe- composition of communities in the plastic cups. cifically, I hypothesized that container location Taxa that could not be identified in the field were and color would affect macroinvertebrate species collected and reared in the lab. composition, species richness, and abundance. Differences in macroinvertebrate species rich- Earlier work on this system showed that highly ness and abundance in the artificial tree holes exposed treeholes (in forest canopy) contained were analyzed with 2-way repeated-measures fewer species than holes in the understory ANOVAs using location (gap or understory) and (Yanoviak 1999b). Thus, I predicted that macroin- color as main effects. When significantly differ- vertebrate species richness and abundance would ent, means were compared with Ryan-Einot-Gab- be lower in gap holes of this study. Based on field riel-Welsch multiple range tests (SAS 1989). observations and other studies showing oviposi- Abundance data were log(x+1) transformed prior tion preference for dark containers (e.g., to analysis to correct variance heterogeneity McDaniel et al. 1976), I predicted that black cups (Sokal & Rohlf 1981), but all means presented in would attract more species than other colors. Fi- results were calculated from untransformed data. nally, because color is a potentially important cue Sørensen’s (1948) coefficient of similarity [Cs = 2j for species colonizing bromeliads (Frank 1985, · (a + b)-1, where j = number of species common to 1986), I expected that green cups would attract two treatments, and a and b = the number of spe- some taxa (e.g., Wyeomyia spp. mosquitoes) that cies in each treatment] was used to quantify over- normally inhabit tank bromeliads in Panama. lap in species composition (all replicates pooled within gap or understory locations) among the four cup colors. MATERIALS AND METHODS Reflectance data for the red, blue, and green In mid-July 1997, I established 9 replications cups were obtained with an Ocean Optics® S2000 of four colored plastic cups (black, blue, green, spectrophotometer. Measurements were taken and red; 650 ml, 8.5 cm diameter × 12 cm height - under dim illumination to reduce the variance at- Churchill Container Corp., Shawnee, KS, USA) in tributable to environmental lighting. The probe the seasonally wet tropical forest of Barro Colo- was dark-spectrum calibrated by placing it into a rado Island (BCI), Panama (see Leigh et al. 1996 black plastic bag, and a reference spectrum was for a site description). Four replicates were lo- obtained by measuring an Ocean Optics reflec- cated in tree-fall gaps and 5 in forest understory. tance standard (a white piece of plastic). Three I secured each cup to a pole-sized (10-20 cm diam- separate recordings, each resulting in 1570 data eter) tree approximately 1 m above the ground (as points between 360 and 860 nm, were taken from described in Yanoviak 1999a). Cups initially con- each cup. Reflectance spectra for the red, blue, tained ca. 400 ml rain water and a strip of balsa and green cups are shown in Fig. 1. wood (0.2 × 4 × 16 cm) as an oviposition site for insect colonists (Novak & Peloquin 1981). The balsa RESULTS strip was small enough relative to the cup that it had little effect on general color appearance of the Artificial treeholes located in the understory habitat. Holes drilled in the cup rims maintained were colonized by more individuals and more spe- water levels ca. 100 ml below capacity. Thus, any cies than cups in tree-fall gaps (Tables 1 and 2; insect entering to oviposit was exposed to the cup compare Figs. 2 and 3). Conditions in the cups dif- color from all sides and from below. Cups within a fered dramatically between locations. Tempera- replication were separated by > 5 m and the dis- tures in gap cups exceeded 40°C on sunny days, tance between replicates was > 100 m. I allowed and all gap cups contained abundant filamentous rain water and detritus to accumulate naturally. algae by Week 5. In understory cups, tempera- The macroinvertebrate fauna (organisms > 0.5 tures never exceeded 32°C and algae were virtu- mm) of all cups was censused weekly for 7 wks be- ally nonexistent. There were no significant inter-

Yanoviak: Treehole macroinvertebrates 267

Black cups were colonized by the most taxa overall, whereas green cups attracted the fewest species (Table 2). Seven species, including four non-mosquito taxa, occurred exclusively in black cups. Overlap in species composition among different colors was marginally greater in the understory than in gaps (Wilcoxon 2-sample test using Sørensen’s coefficients obtained for each location, P = 0.077), and assemblages in understory black cups showed higher similarity to assemblages in red and blue cups than to those in green cups (Ta- ble 3). Several species that avoided blue and/or green cups in gaps colonized one or both of those Fig. 1. Reflectance spectra for the red, blue, and colors in the understory (Table 2). green cups used in this study. Each point is the mean of There was no overlap between the species com- 30 data associated with 10 wavelength measurements position of water held by bromeliads or H. latis- × (3 replicates 10 adjacent wavelengths), and each mean patha bracts on BCI and the cups used in this was plotted against its median wavelength value. Vari- experiment. However, several treehole species ance was very low; error bars (±1 SD) are shown only for a subset of the means. were found in water held by palm spathes and T. ovalifolia exocarps; both were dominated by the mosquitoes Trichoprosopon digitatum (Rondani) actions between treehole color and location for and Limatus spp. both macroinvertebrate species richness and abundance (Table 1), so effects of color were tested DISCUSSION separately for gap and understory locations. Average species richness and abundance dif- My results show that the location and color of fered among cup colors in both gaps and under- artificial treeholes can influence species richness, story (Table 1), but significant effects did not abundance, and composition in container commu- occur in either location until Week 4 (based on nities. As predicted, macroinvertebrate species Ryan post-hoc tests). Species richness was great- richness and abundance were lower in artificial est in black cups in the understory (Fig. 2a) and treeholes located in gaps. This result is likely due black and red cups in gaps (Fig. 3a) after 7 wks of to oviposition preferences for shaded vs. exposed colonization. Macroinvertebrate abundance was sites. Prior work on this system showed that some significantly lower in green cups in forest under- species are more common in shaded holes story, but did not differ among black, blue, and (Yanoviak 1999c) and that highly exposed canopy red cups on Weeks 6 and 7 (Fig. 2b). Black and red tree holes contain fewer species than understory cups in gaps contained more individuals than did holes (Yanoviak 1999b). It is also probable that green cups by the end of the study (Fig. 3b). The extremely high water temperatures in gap cups significant time*treatment interaction for under- depressed richness and abundance through larval story species richness, but not for abundance (Ta- mortality. Larvae of several mosquito species can- ble 1), reflects the early successional status of the not survive temperatures above 40°C for more artificial treeholes (Yanoviak, in press). Species than a few minutes (Bates 1949). Finally, abun- richness in understory cups increased over time dant algae in gap cups may have deterred ovipo- whereas abundance remained relatively constant sition by chemical or other means, and occa- (Fig. 2). The lack of a similar time interaction in sionally caused larval mortality by restricting gaps is attributed to high variance (Fig. 3). their movements within the cups (pers. obs.).

TABLE 1. REPEATED-MEASURES ANOVA OUTPUT. SS = TYPE III SUM OF SQUARES. TIME = P-VALUE FOR UNIVARIATE TEST OF TIME*TREATMENT INTERACTION. * = P < 0.05, ** = P < 0.005.

Abundance Richness

Treatment SS df F Time SS df F Time

Location 10.45 1,28 16.83** 0.71 31.74 1,28 26.07** 0.02 Color 16.21 3,28 8.70** 0.07 66.94 3,28 18.32** <0.01 Location*Color 0.36 3,28 0.20 0.68 4.21 3,28 1.15 0.13 Color, Gap 5.64 3,12 7.72** 0.19 17.21 3,12 11.61** 0.40 Color, Underst. 11.59 3,16 4.27* 0.35 58.54 3,16 11.08** <0.01

268 Florida Entomologist 84(2) June 2001

TABLE 2. DISTRIBUTION OF INVERTEBRATE TAXA AMONG DIFFERENT COLORED CUPS IN FOREST UNDERSTORY AND TREE- FALL GAP LOCATIONS. VALUES ARE MAXIMUM NUMBER OF INDIVIDUALS OBSERVED IN A CUP WITHIN A TREAT- MENT. MINIMUM ABUNDANCE WAS ZERO IN MOST CASES. MEAN ABUNDANCE AND MEAN RICHNESS ARE THE AV- ERAGE (±1 SD) NUMBER OF INDIVIDUALS AND SPECIES WITHIN A TREATMENT, ALL CENSUS DATES COMBINED.

Understory Gap

Taxon Black Blue Green Red Black Blue Green Red

Annelida: Naididae Dero sp. 115 7 0 0 0 0 0 0 Odonata: Pseudostigmatidae Mecistogaster spp. 1000 1000 Coleoptera: Scirtidae Prionocyphon sp. 16000 4000 Diptera: Ceratopogonidae Bezzia snowi Lane 5 2 0 5 4 0 0 0 Forcipomyia spp. 26 0 0 1 3 0 37 0 Diptera: Chironomidae Chironomus sp. 0000 1000 Diptera: Culicidae Aedes terrens (Walker) complex 35 1 2 35 9 0 0 15 Anopheles eiseni Coq. 4 0 0 0 0 0 0 0 Culex conservator D. & K. 8 5 0 0 5 0 0 0 C. corrigani D. & K. 33 0 0 18 2 0 0 0 C. urichii (Coq.) 21 59 10 58 12 2 0 44 Haemagogus equinus Theobald 7 6 1 9 8 4 0 23 H. leucotaeniatus (Komp) 0 0 0 1 0 0 0 0 H. lucifer (Howard, Dyar & Knab) 0 0 0 15 0 9 0 0 Limatus assuleptus (Theobald) 16 15 15 38 6 5 1 32 Orthopodomyia fascipes (Coq.) 0 0 0 0 1 0 0 0 Toxorhynchites theobaldi (D. & K.) 1 0 0 0 1 0 0 0 Diptera: Psychodidae Telmatoscopus spp. 0000 8030 Diptera: Syrphidae Copestylum rafaelanum (Townsend) 69 0 0 0 0 0 0 0 Diptera: Tipulidae Sigmatomera spp. 0000 0050

Total no. of taxa represented 14 7 4 9 14 4 4 4 Mean abundance 22.3 10.5 3.0 19.1 4.8 1.3 2.1 11.3 (31.20) (16.18) (4.87) (14.89) (4.80) (2.10) (6.95) (13.41) Mean richness 2.5 1.2 0.7 2.0 1.3 0.4 0.2 0.9 (1.71) (1.10) (0.77) (1.17) (1.00) (0.56) (0.55) (0.80)

Cup color signiﬁcantly affected macroinverte- Differences in distributions of species among brate species richness and abundance in both lo- cup colors between gap and understory locations cations and, as predicted, black cups attracted the most likely reﬂect differences in how colors are most species overall. Although many taxa colo- perceived in these light environments. (One ex- nized more than one color of cup, the general dis- ception among the taxa found in this study is the tribution of species among colors suggests that annelid Dero sp., which is primarily dispersed by most prefer to oviposit in dark containers or avoid phoresy.) Most insects are unable to see red green oviposition sites. This pattern is well docu- (Chapman 1998), thus red containers probably mented for several mosquito species (e.g., Will- appear dark gray to potential colonists. Wave- iams 1962; Wilton 1968; McDaniel et al. 1976; lengths of incident light in forest understory are Hilburn et al. 1983; Beehler et al. 1992; Jones & shifted toward the blue/green end of the spectrum Schreiber 1994; also reviewed by Frank 1985), by the surrounding vegetation and toward red un- and at least partly explains why the green cups of der overcast conditions (Endler 1993), thus blue this study contained few species and individuals. and green cups in forest understory may also Yanoviak: Treehole macroinvertebrates 269

Fig. 2. Mean (±SE) macroorganism species richness Fig. 3. Mean (±SE) macroorganism species richness (A) and abundance (B) in different colored cups located (A) and abundance (B) in different colored cups located in forest understory. N = 5 for each mean. Within a in tree fall gaps. N = 4 for each mean. Within a week, week, means followed by the same letter do not differ means followed by the same letter do not differ based on based on post-hoc tests (only noted where differences oc- post-hoc tests (only noted where differences occurred; a curred; a single letter was used for overlapping means). single letter was used for overlapping means). Some er- Some error bars were omitted for clarity. ror bars were omitted for clarity. have been perceived as gray or reddish. Results of this study support such a conclusion. Blue and eni Coquillett only in black cups probably reflects red cups attracted more species overall in the un- a color preference; several other Anopheles spe- derstory than in gaps, and species that colonized cies prefer to oviposit in dark containers (re- three or more colors tended to be more abundant viewed by Frank 1985). Mecistogaster spp. and in red and black. The mosquitoes Culex urichii Toxorhynchites theobaldi (Dyar & Knab) are top (Coquillett) and Limatus assuleptus (Theobald) predators in this system, and typically exist in are exceptions to the latter, but both are also hab- low abundance due to competition and cannibal- itat generalists and will colonize water in almost ism (Fincke 1999). Treeholes are a limiting repro- any container (pers. obs.). ductive resource for pseudostigmatids (Fincke Five of the 7 species found only in black cups 1992a), and the species occurring on BCI avoid occurred in very low abundance. The midge Chi- other phytotelmata, including bromeliads (Fincke ronomus sp. and the mosquito Orthopodomyia 1992b). Lab studies showed that some Toxorhyn- fascipes (Coquillett) are normally more abundant chites species prefer to oviposit in black contain- in artificial treeholes than was observed here ers (Hilburn et al. 1983; Jones & Schreiber 1994). (Yanoviak, in press), so their distribution in this Therefore, it is reasonable to conclude that the study may not reflect color preferences. Despite presence of these two taxa only in black cups re- its low abundance, the presence of Anopheles eis- flects a habitat color preference.

TABLE 3. SØRENSEN’S SIMILARITY COEFFICIENTS FOR MACROINVERTEBRATE ASSEMBLAGES OCCURRING AMONG THE FOUR CUP COLORS IN UNDERSTORY AND TREE-FALL GAP LOCATIONS.

Understory Gap

Black Blue Red Black Blue Red

Blue 0.667 — — 0.333 — — Red 0.609 0.625 — 0.444 0.750 — Green 0.444 0.727 0.615 0.333 0.250 0.250 270 Florida Entomologist 84(2) June 2001

The lack of species overlap between the colored FINCKE, O. M. 1992b. Behavioural ecology of the giant cups and similarly colored phytotelmata on BCI damselflies of Barro Colorado Island, Panama, pp. can be explained by physical attributes of these 102-113. In D. Quintero and A. Aiello (eds.). Insects systems other than color. First, the general form of Panama and Mesoamerica. Oxford Univ. Press, of a container may influence colonization. For ex- Oxford. FINCKE, O. M. 1999. Organization of predator assem- ample, Frank (1986) used artificial bromeliads to blages in Neotropical tree holes: effects of abiotic fac- show that ovipositing mosquitoes are sensitive to tors and priority. Ecol. Entomol. 24: 13-23. the general shape of plant containers. Second, FINCKE, O. M., S. P. YANOVIAK, AND R. D. HANSCHU. macroinvertebrate colonization is influenced by 1997. Predation by odonates depresses mosquito the vertical position of phytotelmata. Fallen fruit abundance in water-filled tree holes in Panama. husks and palm spathes occur at ground level, Oecologia 112: 244-253. whereas all of the containers used in this study FISH, D. 1983. Phytotelmata: flora and fauna, pp. 1-27. were ca. 1 m above the ground. Some mosquito In J. H. Frank and L. P. Lounibos (eds.). Phytotel- species in the BCI forest (e.g., Limatus spp., T. mata: terrestrial plants as hosts for aquatic insect communities. Plexus, Medford, NJ. digitatum) are sensitive to this difference in FRANK, J. H. 1985. Use of an artificial bromeliad to show height (Yanoviak 1999b, in press). Finally, many the importance of color value in restricting the coloni- species respond to chemical cues when selecting zation of bromeliads by Aedes aegypti and Culex quin- oviposition sites (Bentley & Day 1989), and these quefasciatus. J. Am. Mosq. Control Assoc. 1: 28-32. cues may complement or exceed the effects of FRANK, J. H. 1986. Bromeliads as ovipositional sites for color (Lounibos & Machado-Allison 1993). Wyeomyia mosquitoes: form and color influence behavior. Florida Entomol. 69: 728-742. HILBURN, L. R., N. L. WILLIS, AND J. A. SEAWRIGHT. CONCLUSIONS 1983. An analysis of preference in the color of oviposition sites exhibited by female Toxorhynchites r. My results suggest that differential coloniza- rutilus in the laboratory. Mosq. News 43: 302-306. tion and harsh environmental conditions in tree- JONES, C. J., AND E. T. SCHREIBER. 1994. Color and fall gaps tend to reduce the abundance and diver- height affects oviposition site preferences of Toxo- sity of macroinvertebrates in artificial tree holes. rhynchites splendens and Toxorhynchites rutilus Container color appears to play a role in habitat rutilus (Diptera: Culicidae) in the laboratory. Envi- selection for several species in this system, but ron. Entomol. 23: 130-135. perception of color may be confounded by variable KITCHING, R. L. 1971. An ecological study of water-filled light conditions in forest understory. Additional tree-holes and their position in the woodland ecosys- studies under controlled laboratory conditions tem. J. Anim. Ecol. 40: 281-302. LEIGH, E. G., JR., A. S. RAND, AND D. M. WINDSOR (eds.). are needed to determine oviposition preferences 1996. The ecology of a tropical forest. 2nd ed. Smith- of the many non-mosquito colonists of tropical sonian, Washington, DC. phytotelmata. LOUNIBOS, L. P., AND C. E. MACHADO-ALLISON. 1993. Field test of mosquito ovipositional cues from Vene- ACKNOWLEDGMENTS zuelan phytotelmata. Florida Entomol. 76: 593-599. MCDANIEL, I. N., M. D. BENTLEY, H.-P. LEE, AND M. A. Almanza assisted in the field and R. Fuller YATAGAI. 1976. Effects of color and larval-produced provided reflectance data for the cups. Comments oviposition attractants on oviposition of Aedes trise- from L. P. Lounibos, D. Srivastava, and an anony- riatus. Environ. Entomol. 5: 553-556. mous reviewer improved the manuscript. This work NAEEM, S. 1988. Predator-prey interactions and community structure: chironomids, mosquitoes and was supported by funds from the IIE/Fulbright copepods in Heliconia imbricata (Musaceae). Oecolo- Foundation and the University of Oklahoma. gia 77: 202-209. NOVAK, R. J., AND J. J. PELOQUIN. 1981. A substrate LITERATURE CITED modification for the oviposition trap used for detect- ing the presence of Aedes triseriatus. Mosq. News 41: BATES, M. 1949. The natural history of mosquitoes. 180-181. MacMillan, Gloucester, MA. PIMM, S. L., AND R. L. KITCHING. 1987. The determi- BEEHLER, J., S. LOHR, AND G. DEFOLIART. 1992. Factors nants of food chain lengths. Oikos 50: 302-307. influencing oviposition in Aedes triseriatus (Diptera: SAS. 1989. SAS/STAT user’s guide, volume 2, GLM- Culicidae). Great Lakes Entomol. 25: 259-264. VARCOMP. SAS Institute Inc., Cary, NC. BENTLEY, M. D., AND J. F. DAY. 1989. Chemical ecology SEIFERT, R. P., AND F. H. SEIFERT. 1979. A Heliconia in- and behavioral aspects of mosquito oviposition. sect community in a Venezuelan cloud forest. Ecol- Annu. Rev. Entomol. 34: 401-421. ogy 60: 462-467. CHAPMAN, R. F. 1998. The insects: structure and func- SNOW, W. E. 1949. The arthropoda of wet tree holes. tion. 4th ed. Cambridge Univ. Press, Cambridge. Ph.D. Dissertation, Univ. of Illinois, Urbana. 235 p. ENDLER, J. A. 1993. The color of light in forests and its SOKAL, R. R., AND F. J. ROHLF. 1981. Biometry. W. H. implications. Ecol. Monogr. 63: 1-27. Freeman & Co., New York. FINCKE, O. M. 1992a. Interspecific competition for tree SØRENSEN, T. 1948. A method of establishing groups of holes: consequences for mating systems and coexist- equal amplitude in plant sociology based on similar- ence in neotropical damselflies. American Nat. 139: ity of species content. K. Danske Vidensk. Selsk. 80-101. Biol. Skrift 5: 1-34. Yanoviak: Treehole macroinvertebrates 271

SRIVASTAVA, D. S., AND J. H. LAWTON. 1998. Why more quito yield in Neotropical tree hole microcosms. productive sites have more species: experimental Oecologia 120: 147-155. test of theory using tree-hole communities. Ameri- YANOVIAK, S. P. 1999b. Community structure in water- can Nat. 152: 510-529. ﬁlled tree holes of Panama: effects of hole height and WILLIAMS, R. E. 1962. Effect of coloring oviposition me- size. Selbyana 20: 106-115. dia with regard to the mosquito Aedes triseriatus YANOVIAK, S. P. 1999c. Distribution and abundance of (Say). J. Parasit. 48: 919-925. Microvelia cavicola Polhemus (Heteroptera: Veli- WILTON, D. P. 1968. Oviposition site selection by the idae) on Barro Colorado Island, Panama. J. New tree-hole mosquito, Aedes triseriatus (Say). J. Med. York Entomol. Soc. 107: 38-45. Entomol. 5: 189-194. YANOVIAK, S. P. 2001. The macrofauna of water-filled YANOVIAK, S. P. l999a. Effects of leaf litter species on tree holes on Barro Colorado Island, Panama. Bio- macroinvertebrate community properties and mos- tropica 33: (in press).

272 Florida Entomologist 84(2) June 2001

PUPATION BIOLOGY OF FRANKLINOTHRIPS ORIZABENSIS (THYSANOPTERA: AEOLOTHRIPIDAE) AND HARVESTING AND SHIPPING OF THIS PREDATOR

MARK S. HODDLE, KIKUO OISHI, AND DAVID MORGAN Department of Entomology, University of California, Riverside, CA 92521, USA

ABSTRACT In the laboratory, 82-99% of late second instar Franklinothrips orizabensis Johansen larvae abandoned avocado branches and artificial branches constructed of wooden dowels and were recovered below branches trapped on tangle foot coated plastic sheets, suggesting a preference by this life stage for selection of pupation sites beneath host plants. Of three media tested (coarse and fine vermiculite, and parafilm cones) for harvesting F. orizabensis pupae in cocoons, parafilm cones were most easily harvestable from colonies, and 44% of deployed late second stage larvae that were recovered used parafilm cones for pupation in experimental cages. Harvesting and shipping trials using aspirated adult F. orizabensis or pupae in parafilm cones showed significant differences in survivorship when held in the laboratory or shipped round trip from Riverside, California to Amherst, Massachusetts. Survivorship of aspirated adults was reduced on average by 41% following shipping, and mortality was highest for adult males. Transit survivorship was increased by 53% if F. orizabensis were shipped as cocoons in parafilm cones. Inclusion of ice packs in polystyrene boxes did not significantly increase survivorship rates for F. orizabensis adults or pupae that were either retained in the laboratory or shipped. This result may have been an artifact resulting from the time of year (i.e., May and temperatures were moderately cool) when shipping trials were conducted.

Key Words: Franklinothrips, pupation biology, harvesting, shipping

RESUMEN En el laboratorio, 82-99% de las larvas en el segundo instar de Franklinothrips orizabensis abandonaron las ramas de aguacate y las ramas artificiales construidas de clavijas de madera y fueron recuperadas bajo ramas atrapadas en hojas plásticas con capa pegajosa “tangle foot”, sugiriendo una preferencia de selección de lugares de pupación debajo de plantas huéspedes por esta etapa de vida. De les tres medios probados (vermiculita fina y ordinaria, y conos de parafina) para cosechar pupas de Franklinothrips orizabensis en capullos, conos de parafina fueron los mas fáciles de cosechar de las colonias, y el 44% de larvas de segunda etapa que fueron recuperadas usaron conos de parafina para pupación en jaulas experimen- tales. Ensayos de cosecha y embarque usando adultos aspirados de Franklinothrips orizabensis o pupas en conos de parafina demostraron diferencias significativas en supervivencia al ser llevadas a cabo en el laboratorio o embarcadas ida y vuelta desde Riverside, California hasta Amherst, Massachussets. Supervivencia de adultos aspirados fue reducida un promedio de 41% después de embarque, y la mortalidad fue mayor entre machos adultos. Supervi- vencia de transporte fue incrementada por 53% si Franklinothrips orizabensis eran embarcadas como capullos en conos de parafina. La inclusión de bolsas de hielo en cajas de poliestireno no incrementó significativamente las cantidades de supervivencia para adultos o pupas de Franklinothrips orizabensis que fueron retenidas en el laboratorio o embarcadas. Este resultado puede haber sido un artefacto resultando por el tiempo del año (es decir, Mayo y las temperaturas eran moderadamente frías) cuando los ensayos de embarque fueron llevados a cabo.

Inoculative, augmentative, and inundative bi- facilitate rapid pest outbreaks as thrips natural ological control programs targeting pestiferous enemy densities have declined because of lack of thrips in perennial outdoor crops have seldom prey. (3) Incompatibility of thrips natural ene- been successful (Parker & Skinner 1997). The ma- mies with broad-spectrum insecticides used to jor limiting factors that have been identiﬁed as control other crop pests. (4) The high cost of insec- constraints on successful thrips biological control tary-reared natural enemies make large-scale programs for outdoor crops are: (1) lack of effec- ﬁeld releases for thrips control into outdoor crops tive resident natural enemies that respond in a of low value unfeasible economically (Grafton- rapid density dependent manner to increasing Cardwell & Ouyang 1995a; Parker & Skinner thrips densities. (2) Thrips phenology and life cy- 1997; Parrella & Lewis 1997). Natural enemies cle characteristics can result in long periods of that have been commonly observed with phytoph- low thrips densities. In some instances this may agous thrips in perennial tree crops are phy-

Hoddle et al.: Franklinothrips pupation biology 273

toseiid mites and predatory thrips (Parker & ments and diets for mass rearing this predator Skinner 1997, Parrella & Lewis 1997). have been evaluated (Hoddle et al. 2000, 2001) In some instances, thrips biological control by and limited field trials evaluating releases of Type IV phytoseiid mites (i.e., specialized pollen F. orizabensis against S. perseae on avocados have feeders that exhibit generalist predatory activity been conducted (Silvers 2000). [McMurtry & Croft 1997]) can be enhanced by Augmentative field releases of mass-reared F. pollen bearing wind break trees (Grout & Rich- orizabensis onto avocado trees failed to signifi- ards 1990, 1992a,b) or cover crops in orchards cantly suppress S. perseae populations in Califor- (Grafton-Cardwell et al. 1999a). Crop manage- nia. This may have been due more to the poor ment practices such as pruning that enhances quality of adults which suffered high mortality succulent leaf material can also promote in- (>50%) after shipping from an insectary in Europe creased densities of Type IV phytoseiids (Grafton- rather than the inherent ineffectiveness of Cardwell & Ouyang 1995b; Grafton-Cardwell F. orizabensis (Silvers, 2000). In order to fully ex- 1997). Augmentative releases of insectary-reared plore the potential of augmentative releases of phytoseiids for thrips control have been shown to F. orizabensis for controlling of S. perseae in Cali- significantly reduce densities of Scirtothrips citri fornia avocado orchards, low impact techniques for Moulton (Thysanoptera: Thripidae) on citrus (Cit- harvesting and distribution that minimize transit rus spp.), but, at present, this technology is not mortality of this predator are required. One possi- cost effective (Grafton-Cardwell & Ouyang ble approach would be to collect and transport F. 1995a; Grafton-Cardwell et al. 1999b). orizabensis when larvae are pupating within pro- Scirtothrips perseae Nakahara (Thysanoptera: tective silk cocoons as opposed to aspirating and Thripidae) was first discovered damaging avocado shipping adults. The purpose of this study was to foliage and fruit in southern California orchards investigate the pupation biology and behavior of F. in 1996, and at time of discovery was a species orizabensis, and to develop techniques based on an new to science (Nakahara 1997). In 1998, crop understanding of pupation behavior for collecting losses due to down-graded fruit and increased and shipping pupating larvae and compare survi- production costs due to S. perseae feeding damage vorship rates to currently employed methods for were estimated to have cost California growers collecting and shipping adult predators. (US) $7-$13 million (Hoddle et al. 1998, 1999). Foreign exploration efforts to determine the na- MATERIALS AND METHODS tive range of S. perseae indicate that this pest is of Central American origin (Hoddle et al. 1999) and Franklinothrips orizabensis Colony associated natural enemies collected concurrently from avocados in Latin America have included Franklinothrips orizabensis colonies were Franklinothrips spp. (Hoddle, unpublished). maintained in cages in a temperature controlled Surveys in southern California avocado or- room (25°C, 60% RH, L:D 14:10) on lima beans chards for indigenous natural enemies associated (Phaseolus lunatus Linnaeus variety “Baby Ford- with S. perseae have revealed that an undescribed hook” [plants are needed for oviposition of eggs by species of Franklinothrips orizabensis Johanson female F. orizabensis]) at the University of Cali- (Thysanoptera: Aeolothripidae) is the dominant fornia Riverside, California, USA. Colony reared predator where this pest has attained high densi- F. orizabensis were fed irradiated Ephestia kueh- ties (>10 S. perseae larvae per leaf) (Hoddle, un- niella (Zeller) (Lepidoptera: Pyralidae) eggs (sup- published). Female Franklinothrips spp. lay eggs plied by Beneficial Insectaries, Oak Run, CA, directly into plant tissue. Developing larvae pass USA) which were liberally deposited on upper through two instars (second instars are distin- surfaces of horizontal bean leaves. Our colony guished from firsts by red hypodermal pigments), was initiated with adult F. orizabensis collected before pupating within silk cocoons which are from an avocado orchard infested with S. perseae spun from secretions produced from the anal rein Fallbrook, California, USA. Adult progeny pro- gion (Arakaki & Okajima 1998). duced by field collected F. orizabensis used to ini- Unlike Euseius tularensis Congdon (Acari: tiate the colony were deposited with the Phytoseiidae), which in some instances can regu- Systematic Entomology Laboratory, USDA-ARS, late citrus thrips, S. citri (Moulton) (Grafton- Beltsville, Maryland, USA and identified as Cardwell & Ouyang 1995b), E. hibisci (Chant) a F. orizabensis by Dr. S. Nakahara. common Type IV phytoseiid in southern Califor- nia avocado orchards, has not been observed to re- Source of Second Instar Franklinothrips orizabensis spond in a significant density dependent manner Larvae for Experiments to increasing S. perseae populations (Hoddle unpublished). Because of industry interest, the po- Undersides of mature ‘Hass’ avocado leaves tential for using F. orizabensis for augmentative collected from the Biological Control Grove (F. releases against S. perseae in avocado orchards is orizabensis has not been collected here) at the being investigated. Optimal temperature require- University of California, Riverside were pre-

274 Florida Entomologist 84(2) June 2001

sented to colony-reared female F. orizabensis for clamp. A second versatile clamp was used to hold oviposition (Hoddle et al. 2000). Adult females a clear sheet of plastic (100cm × 30 cm) covered were confined with male F. orizabensis within with tanglefoot under the artificial branch. Prox- modified Munger cells (Munger 1942, Morse et al. imal ends of artificial branches were coated with 1986), fed irradiated E. kuehniella eggs, and left tanglefoot to prevent larval escape. Outlines of to oviposit in temperature cabinets (25°C, L:D artificial branches were drawn in tanglefoot. Two 14:10 h). types of artificial branches were used: (1) wooden At the end of the 24 h oviposition period, the leaf dowels without pupation refugia, and (2) wooden area enclosed by the Munger cell which was ex- dowels with pupation refugia. Refugia consisted posed to ovipositing females was excised from the of four 3mm diameter holes drilled equidistant avocado leaf. Trimmed leaves were labeled, placed around the dowel’s circumference at 20 cm inter- on water-saturated foam pads in stainless steel vals from the distal end. Artificial pupation sites trays, and incubated at 25°C. Leaves were exam- were provided to determine the propensity of ined daily for emerged F. orizabensis larvae. F. orizabensis larvae to stay on branches to pu- Emerged first instar larvae were collected with a pate when abundant sites were available in com- fine camel hair brush, placed individually in 1 dram parison to artificial branches without refugia, or glass shell vials with irradiated E. kuehniella eggs naturally occurring pupation sites on avocado as food, and vials were sealed with parafilm® branches. Ten late second instar F. orizabensis (American National Can, WI). Larvae were reared larvae of known age were placed on avocado leaf in temperature controlled cabinets (25°C, L:D 14:10 bouquets attached to artificial branches and sup- h) in shell vials until second instars which were plied with irradiated E. kuehniella eggs as food. then used for experiments. Late second instar lar- Larvae were left for three days and numbers of vae select pupation sites and spin pupation cocoons larvae on tanglefoot covered plastic sheets were within which propupal and pupal stages occur. recorded. The perpendicular distance jumped from branches was determined by measuring dis- Pupation Behavior tances from larvae to drawn branch outlines in tanglefoot. Artificial branches were examined un- Pupation behavior experiments were con- der a dissecting microscope and numbers of lar- ducted in the laboratory at 25°C and ambient vae pupating on wooden dowels were recorded. light to determine the proportion of F. orizabensis Experiments were replicated 10 times for artifi- larvae that pupate on avocado branches and arti- cial branches with and without refugia. ficial wooden branches. Pupation on avocado branches. Individual Pupal Harvesting Techniques branches were selected on small (1.5-2 m tall) Hass avocado trees planted in 20 liter plastic pots. The pupation behavior study outlined above in- Tanglefoot was applied at the base of branches at dicated that >80% of late second instar F. oriza- trunk attachment to prevent larvae leaving ex- bensis larvae would fall or jump from natural and perimental branches. Clear plastic sheets (60 cm artificial branches to seek pupation sites below × 40 cm) covered with tanglefoot were placed un- branches (see Results Section). Since late second der each experimental branch and the outline of instar larvae spin silk cocoons we sought to iden- the overhanging branch was drawn in the tangle- tify a media within which larvae would pupate foot. Ten late second instar F. orizabensis larvae of that could allow easy harvesting of F. orizabensis known age were placed on avocado leaves and in cocoons. Evaluations of pupation media were supplied with irradiated E. kuehniella eggs as conducted in temperature controlled cabinets food. Larvae were left for three days and then (25°C, L:D 14:10). Five late second instar F. oriza- numbers recovered from tanglefoot covered plas- bensis larvae were placed with pupation media in tic sheets were recorded, and the perpendicular glass Petri dishes (9.5 cm diameter) with lids distance jumped from branches was determined which were sealed with parafilm and left for three by measuring distances from larvae to drawn days in temperature controlled cabinets. Cocoons branch outlines in tanglefoot. Avocado branches were then located in Petri dishes and numbers of were removed from trees and examined under a adult F. orizabensis that successfully emerged fol- dissecting microscope and numbers of larvae pu- lowing harvesting were recorded daily. pating on branches were recorded. This Vermiculite. Vermiculite (Therm-o-Rock West experiment was replicated 10 times. Inc., Chandler, AZ) was sieved into two grades Pupation on artificial branches. Artificial coarse (all granules that were not retained by 20 branches were constructed of wooden dowels mesh USA Standard Testing Sieve) and fine (all 90cm long and 0.75 cm wide and bouquets of avo- granules not retained by 35 mesh USA Standard cado leaves wrapped in moist paper towels were Testing Sieve). Five ml of each material was attached to distal ends of dowels with parafilm. placed into glass Petri dishes and the ability to Proximal ends of artificial branches were at- harvest cocoons spun onto vermiculite granules tached to a 1.2 m ringstand with a versatile was assessed after three days. Each vermiculite Hoddle et al.: Franklinothrips pupation biology 275 treatment was replicated 10 times. Collection of Harvesting of Franklinothrips orizabensis Pupae cocoons was attempted by sieving vermiculite from Laboratory Colonies with a 20 mesh sieve based on the assumption that cocoons encased with vermiculite would be Parafilm cones were readily used as pupation too large to pass through and would be retained. sites by F. orizabensis larvae in glass Petri dishes Parafilm cones. Parafilm cones (diameter 3 mm, and small rearing cages (see Results Section). The height 5 mm) (Fig. 1) were constructed by wrap- efficacy of using parafilm cones to harvest F. oriz- ping strips of parafilm (4 mm wide, 15 mm long) abensis pupae was determined in laboratory colo- around the tapered end of a camel hair brush. nies. F. orizabensis colonies used for this Five parafilm cones were placed in each Petri experiment were fed irradiated E. kuehniella dish, and this treatment was replicated 10 times. eggs and maintained in cages (75 cm × 40 cm × 45 Cone utilization for pupation by F. orizabensis cm) with 9-12 lima bean plants. Plants were larvae was visually assessed after three days. raised 3 cm from the cage floor on metal pipes Parafilm cones and fine vermiculite. Five para- fixed to cage walls. Under these metal supports a film cones and 5 mls of fine vermiculite were com- clear plastic sheet (70 cm × 30 cm) was positioned bined in a glass Petri dish. This treatment was holding 100 parafilm cones (Fig. 2). Parafilm replicated 6 times, and utilization of either cones cones were left beneath plants for three days be- or vermiculite for pupation was assessed after fore the plastic sheet with cones was removed. three days. Cones containing pupae were counted, held in Petri dishes in a temperature controlled cabinet ° Selection of Pupation Sites in Small Rearing Cages (25 C, L:D 14:10), and number and sex of emerging adult F. orizabensis was recorded daily. This Thirty individually-reared late second instar trial was replicated 10 times and approximately F. orizabensis were hand placed on four lima bean 300 adult F. orizabensis were present in mass- plants contained within rearing cages (30 cm × 30 rearing cages each time. cm × 37.5 cm), fed irradiated E. kuehniella eggs which were liberally deposited on upper surfaces Shipping Adult and Pupal Franklinothrips orizabensis of horizontal bean leaves, and maintained in a temperature controlled room (25°C, 60% RH, L:D Thirty individually-reared adult F. orizabensis 14:10) for three days. Cages were supplied with (15 male and 15 female; all were 1-2 days of age) 50 parafilm cones which were distributed on pot- were aspirated into three dram glass shell vials, ting media and cage floors. After three days, num- supplied with irradiated E. kuehniella eggs as bers of F. orizabensis cocoons in parafilm cones, in food, and sealed with a wad of cotton wool. Thirty cage corners and sleeves, on bean plants, and in F. orizabensis pupae (1-2 days of age) in parafilm vermiculite in which bean plants were growing cones were placed in three dram shell vials and (plants were destructively sampled) were deter- sealed with cotton wool. Aspirated adults and pu- mined. After inspection for cocoons, experimental pae in parafilm cones were each subjected to one cages were examined every two days for 10 days of the following treatments: for adults that emerged from cocoons that were Aspirated adults and cocoons retained in the not detected during initial cage inspection. This laboratory. The purpose of this experiment was to treatment was replicated 10 times. quantify predator mortality in the absence of shipping stress. Aspirated adults and pupae in parafilm cones were placed in sealed polystyrene foam boxes (20.5 cm × 16.5 cm × 15.5 cm) with or without ice packs. Temperatures and humidities in boxes were recorded every 10 mins with Hobo data loggers (Onset Computer Corp., Pocasset, MA). Survivorship of aspirated adults was recorded after 48 h and pupae in parafilm cones were removed from boxes after 48 h and reared in a temperature controlled cabinet (25°C, L:D 14:10) to determine emergence rates and sex. This treatment was replicated five times. Aspirated adults and cocoons shipped to Mas- sachusetts. The purpose of this experiment was to quantify predator mortality due to shipping stress. Adult and pupal F. orizabensis were collected, prepared, and shipped in polystyrene foam boxes with or without ice-packs with Hobo data Fig. 1. Schematic of a parafilm cone used for harvest- loggers as described for the laboratory retention ing Franklinothrips orizabensis pupae. study. Boxes were shipped Federal Express prior- 276 Florida Entomologist 84(2) June 2001

Fig. 2. Parafilm cones on a clear plastic sheet that is placed beneath elevated bean plants in a Franklinothrips orizabensis rearing cage. Franklinothrips orizabensis pupae are harvested from the laboratory colony in parafilm cones. ity overnight to Amherst, Massachusetts and star F. orizabensis larvae were recovered (Table 1). then returned immediately the next day by Fed- On avocado branches and artificial branches with eral Express priority overnight to Riverside, Cal- refugia <20% of recovered larvae pupated on ifornia. Upon receipt in Riverside, proportion of branches, and the majority were recovered from adults surviving was determined, and pupae in tanglefoot coated plastic below branches. This re- parafilm cones were removed from boxes and sult indicated that late second instar F. orizaben- reared in a temperature controlled cabinet (25°C, sis larvae may prefer to pupate below trees, or L:D 14:10) to determine emergence rates and sex. suitable sites for pupation on branches were ei- Round trip transit time from California to River- ther not available or located. Artificial branches side was approximately 48 h. This treatment was without pupation refugia resulted in 100% branch replicated five times. abandonment by larvae (Table 1). Of larvae recov- Analysis of collecting and shipping data. Anal- ered from plastic sheets, 80-98% were found ysis of adult and cocoon survivorship data was within 0-2.5 cm of branches, indicating that larvae performed on logit transformed data (ln live/ fall rather than actively jump away from branches dead) after weighing for sample size. The effects when searching for pupation sites below host of shipping, cooling, life stage, and sex on survi- plants (Table 1). High rates of branch abandon- vorship of F. orizabensis were tested for signifi- ment may make it possible to collect F. orizabensis cance using Chi-square analysis and pair-wise T- below host plants if suitable pupation sites for co- test comparisons (0.025 level of significance). coon construction can be provided that allow for Analysis of temperature and humidity data. easy harvesting of this life stage. Temperature and humidity recorded for each day of the two day period for both laboratory held and Pupal Harvesting Techniques shipped polystyrene foam boxes was analyzed using ANOVA and Tukey’s Studentized range test In coarse vermiculite, 80% of deployed late sec- for means separation (0.05 level of significance). ond instar F. orizabensis larvae pupated successfully (Table 2). Use of this media for harvesting RESULTS cocoons was unsuitable as larvae spun silk cocoons between vermiculite particles and the bot- Pupation Behavior toms and sides of Petri dishes, or crawled into cracks in vermiculite particles making detection In all three treatments (i.e., natural avocado difficult. Consequently, a technique for harvest- branches and artificial branches with and without ing pupal F. orizabensis based on sieving for pupation refugia) >90% of deployed late second in- cocoons encased in vermiculite would not be prac- Hoddle et al.: Franklinothrips pupation biology 277

TABLE 1. MEAN PERCENTAGE RECOVERY (± SE) OF FRANKLINOTHRIPS ORIZABENSIS LARVAE RELEASED ONTO AVOCADO BRANCHES, ARTIFICIAL BRANCHES LACKING PUPATION SITES, AND ARTIFICIAL BRANCHES WITH PUPATION SITES.

Artiﬁcial branches Artiﬁcial without branches with Avocado pupation pupation branches refugia refugia

% Total larval recovery 93.00 ± 2.13 98.00 ± 1.33 95.00 ± 1.67 % Larvae pupating on branch 1.11 ± 0.01 0.00 ± 0.00 17.51 ± 0.01 % Larvae recovered from sticky traps 0-2.5cm from branch 97.78 ± 0.01 90.89 ± 0.01 80.38 ± 0.01 % Larvae recovered from sticky traps 2.5-5.0 cm from branch 1.11 ± 0.01 9.11 ± 0.07 2.11 ± 0.01 % Larvae recovered from sticky traps >5.0 cm from branch 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 tical. The use of fine vermiculite resulted in 100% vae (Table 4). Greater numbers of females were mortality of larvae as this grade probably acted as harvested using parafilm cones, and 0-67% (mean an abrasive desiccant (Table 2). 35% ± 6.50 [SE]) of cones would have more than In Petri dishes, parafilm cones were utilized as one pupating larva. Adult emergence rates from pupation sites by 80% of deployed late second in- parafilm cones placed in colony cages was high, star F. orizabensis larvae of which 72% emerged >97% (Table 4). successfully. All recovered larvae had spun cocoons inside parafilm cones (Table 2). Parafilm Shipping Adult and Pupal Franklinothrips orizabensis cones combined with fine vermiculite resulted in 67% successful pupation and all recovered pupae Handling and shipping. Survivorship of adult were found inside parafilm cones. Provision of F. orizabensis was significantly affected by ship- parafilm cocoons mitigated the adverse effects of ping (χ2 = 44.39, df = 1, p < 0.0005) with survivor- fine vermiculite on pupation success rates (Table ship rates of adults being reduced on average by 2). Parafilm cones were the most successful media 41% in comparison to F. orizabensis that were tested for harvesting F. orizabensis pupae. held in the laboratory and not subjected to shipping stress (Fig. 3). Adult male F. orizabensis suf- Selection of Pupation Sites in Small Rearing Cages fered significantly higher mortality rates than females (χ2 = 34.18, df = 1, p < 0.0005) with sur- Of the 300 second instar F. orizabensis larvae vival being reduced on average by 24% (Fig. 3). released into small rearing cages 58% were recov- Survivorship of F. orizabensis was significantly ered. The highest numbers of recovered pupae increased when predators were shipped as co- were found inside parafilm cones (Table 3). No pu- coons (χ2 = 185.10, df = 1, p < 0.0005) and transit pating larvae were found on bean leaves or stems. survivorship was increased on average by 53%. Approximately 10% of pupae were found inside (Fig. 3). Inclusion of icepacks in polystyrene foam seed coats attached to cotyledons, and a similar boxes that were either retained in the laboratory percentage of cocoons were found in vermiculite or shipped roundtrip from California to Massa- potting mix down to a depth of 2 cm (Table 3). chusetts did not significantly alter survivorship rates in comparison to the same treatments with- Harvesting of Franklinothrips orizabensis Pupae out icepacks (χ2 = 0.62, df = 1, p = 0.43). from Laboratory Colonies Temperature and humidity. Significant differences in temperature (F = 2627, df = 3, 1448, p < Over a three day period, 48% of parafilm cones 0.0005) (Fig. 4A) and humidity (F = 173, df = 3, in cages were used by pupating F. orizabensis lar- 1448, p < 0.0005) (Fig. 4B) existed between poly-

TABLE 2. MEAN PERCENTAGE (± SE) OF FRANKLINOTHRIPS ORIZABENSIS IN EACH OF FOUR PUPATION MEDIA CON- TAINED IN PETRI DISHES THAT EITHER PUPATED SUCCESSFULLY AND EMERGED AS ADULTS, DIED AS LARVAE OR PUPAE, OR WERE NOT RECOVERED.

Pupation Media % Emerged % Dead % Unrecovered

Coarse vermiculite 80.00 ± 5.96 6.00 ± 3.06 14.00 ± 6.70 Fine vermiculite 0.00 ± 0.00 100 ± 0.00 0.00 ± 0.00 Parafilm cones 72.00 ± 4.42 8.00 ± 4.42 20.00 ± 5.16 Parafilm cones & fine vermiculite 66.67 ± 4.22 16.67 ± 6.15 16.67 ± 6.15 278 Florida Entomologist 84(2) June 2001

TABLE 3. MEAN PERCENTAGE (± SE) RECOVERY OF FRANKLINOTHRIPS ORIZABENSIS COCOONS FROM DIFFERENT LOCA- TIONS IN SMALL REARING CAGES.

Location of Cocoons in Cage % Recovered

Cage sleeves 10.40 ± 4.34 Cage corners 7.01 ± 1.49 Paraﬁlm cones on potting media & cage ﬂoor 44.02 ± 6.86 Seed coat attached to cotyledons 9.61 ± 5.53 Potting soil 0-1cm deep 7.22 ± 3.52 1-2cm deep 3.89 ± 1.24 >2 cm deep 0.00 ± 0.00 Dead larvae & pupae 3.51 ± 1.24 Adults emerged into cages after inspection for cocoons 13.58 ± 3.10

styrene foam boxes that were retained in the lab- been inferred from analysis of leaf duff samples oratory. Boxes with ice-packs were significantly from commercial avocado orchards in southern cooler and had higher humidity rates, particu- California where F. orizabensis specimens have larly on the second day, presumably resulting been recovered to a depth of 2.5 cm into the soil from condensation on ice packs. The mean tem- (Hoddle et al. 1998). perature also increased in boxes with ice packs on Host plant abandonment rates by F. orizaben- the second day (Fig. 4A). Significant differences in sis larvae prior to pupation in avocado orchards temperature (F = 1422, df = 3, 2672, p < 0.0005) have not been quantified. Our estimates of larval (Fig. 4C) and humidity (F = 513, df = 3, 2672, p < abandonment on young avocado trees in the labo- 0.005) (Fig. 4D) existed between polystyrene foam ratory may have been over-estimated as young boxes that were shipped round trip from Califor- trees have fewer bark fissures that can be used as nia to Massachusetts. Boxes with ice-packs were pupation refugia in comparison to mature trees in significantly cooler, especially on the first day, and orchards. This suggestion is supported by higher had higher humidity rates, particularly on the recovery rates of F. orizabensis pupae on artificial second day, probably from condensation on ice branches with pupation refugia. In comparison to packs. Humidity in boxes without ice packs was phytophagous thrips, there is little published in- constant during the transit period (Fig. 4B). formation on pupation behavior and site selection by predatory thrips in field situations and this DISCUSSION area would benefit from more research. Coarse and fine vermiculite were unsuitable In the laboratory, the majority (82-99%) of re- pupation substrates for harvesting F. orizabensis covered late second instar F. orizabensis larvae pupae. In Petri dishes with coarse vermiculite, actively abandoned natural and artificial wooden pupating larvae constructed cocoons by attaching dowel branches to search for pupation sites below silk to walls and floors of dishes, and coarse ver- host plants. Predatory and phytophagous thrips miculite particles were then attached to cocoon have been captured emerging from the ground be- surfaces not adhered to the Petri dish. Pupating neath citrus trees (Childers et al. 1994), and pu- F. orizabensis could not be harvested in this me- pation beneath host plants is common for dia as cocoons were destroyed (i.e., cocoons were pestiferous phytophagous thrips (Grout et al. ripped open exposing pupae) when removal of 1986; Harrison 1963; Okada 1981; Reed & Rich vermiculite granules was attempted. Fine ver- 1975; Schweizer & Morse 1996, 1997; Tsuchida miculite was an unsuitable pupation substrate as 1997). Host plant abandonment in the field has it resulted in mortality of 100% of second instar

TABLE 4. UTILIZATION OF PARAFILM CONES BY FRANKLINOTHRIPS ORIZABENSIS IN MASS REARING CAGES.

Measured Variable Mean ± SE

Paraﬁlm cones utilized by pupating larvae 48.33% ± 3.43 Male Franklinothrips orizabensis emerging from cones 39.49% ± 1.79 Female Franklinothrips orizabensis emerging from cones 60.51% ± 1.79 No. adult Franklinothrips orizabensis emerging per cone 1.31 ± 0.07 Pupal mortality in paraﬁlm cones 2.35% ± 0.35 Hoddle et al.: Franklinothrips pupation biology 279

Fig. 3. Survivorship of aspirated adult male and female Franklinothrips orizabensis and pupae in parafilm cones either retained in the laboratory at ambient temperatures or cooled with ice packs, or shipped roundtrip from Riv- erside, California to Amherst, Massachusetts either with or without ice packs. Mean treatment proportions surviving with the same letters are not significantly different (0.025 level of significance).

Fig. 4. Mean temperatures (A) and humidities (B) in polystyrene foam boxes with and without ice packs when retained in the laboratory for 48 h, and mean temperatures (C) and humidities (D) when polystyrene boxes were shipped round trip from Riverside, California to Amherst, Massachusetts with or without ice packs. Mean temperatures and humidities followed by the same letters are not signiﬁcantly different (0.05 level of signiﬁcance). 280 Florida Entomologist 84(2) June 2001

F. orizabensis larvae. Placing parafilm cones in ACKNOWLEDGMENTS Petri dishes with fine vermiculite significantly increased larval and pupal survivorship. This work was supported in part by the Cali- Parafilm cones were utilized by late second in- fornia Avocado Commission. Dr. Roy Van Drie- star F. orizabensis larvae as pupation sites in sche, University of Massachusetts at Amherst Petri dishes, small rearing cages and laboratory kindly assisted with the shipping aspects of this colonies. In small rearing cages where known trial. We thank Dr. Miwa Takano-Lee for critically numbers of second instar larvae were deployed, evaluating this work. 44% of recovered F. orizabensis pupae were found in parafilm cones. Parafilm cones may have been desirable pupation sites because of the conical de- REFERENCES CITED sign (i.e., the tapering nature of the unit and its horizontal position) resulted in larvae being able ARAKAKI, N., AND S. OKAJIMA. 1998. Notes on the biol- to find a desirable wall width within the cone to ogy and morphology of a predatory thrips, Frankli- adhere silk strands during cocoon construction. In nothrips vespiformis (Crawford) (Thysanoptera: commercial mass rearing operations, the use of Aeolothripidae): First record from Japan. Entomol. parafilm cones or some other artificial pupation Sci. 1: 359-363. site under host plants (e.g., small, mass produced CHILDERS, C. C., S. NAKAHARA, AND R. J. BESHEAR. clear plastic cones) that are easily harvestable 1994. Relative abundance of Frankliniella bispinosa may be cost effective for rapidly collecting and and other species of Thysanoptera emerging from shipping F. orizabensis pupae. The use of elec- soil beneath “Navel” orange trees in Florida during spring flowering. J. Entomol. Sci. 29: 318-328. tronic scanning devices could conceivably assist in GRAFTON-CARDWELL, E. E., AND Y. OUYANG. 1995a. sorting cones with and without pupae as cones Augmentation of Euseius tularensis (Acari: Phy- with cocoons turn a blackish color as adults ma- toseiidae) in Citrus. Environ. Entomol. 24: 738-747. ture. This color change could aid electronic detec- GRAFTON-CARDWELL, E. E., AND Y. OUYANG. 1995b. Ma- tion and mechanical sorting (Petitt et al. 1996; nipulation of the predacious mite, Euseius tularensis Smittle et al. 1986; Whitten 1969; Wolf et al. (Acari: Phytoseiidae), with pruning for citrus thrips 1972). Alternatively, weight differences between control, pp. 251-254. In Thrips Biology and Manage- cones with and without pupae may be amenable ment. B. L. Parker, M. Skinner and T. Lewis (eds.). to air stream separation (Jackson et al. 1996). Me- Plenum Press, New York. GRAFTON-CARDWELL, E. 1997. Manipulation of E. tula- chanical harvesting and sorting of F. orizabensis rensis for improved thrips control. Citrograph 82: 8. pupae in artificial pupation media could signifi- GRAFTON-CARDWELL, E. E., Y. OUYANG, AND R. L. cantly reduce production costs for this predator. BUGG. 1999a. Leguminous cover crops to enhance F. orizabensis larvae that pupate in sites other population development of Euseius tularensis (Acari: than cones could be a source of adults for sustain- Phytoseiidae) in citrus. Biol. Control 16: 73-80. ing cage colonies and may mitigate the need to set GRAFTON-CARDWELL, E. E., Y. OUYANG, AND R. A. aside portions of the harvested product for colony STRIGGOW. 1999b. Predacious mites for control of cit- maintenance. It is possible that harvesting pupae rus thrips, Scirtothrips citri (Thysanoptera: Thripi- in cones and not returning individuals to colonies dae) in nursery crops. Biol. Control 14: 29-36. GROUT, T. G., J. G. MORSE, AND O. L. BRAWNER. 1986. that exhibit a preference for cones could select for Location of citrus thrips (Thysanoptera: Thripidae) individuals that do not utilize harvestable media pupation: tree or ground? J. Econ. Entomol. 79: 59-61. for pupation. GROUT, T. G., AND G. I. RICHARDS. 1990. The influence of Survivorship of F. orizabensis during shipping windbreak species on citrus thrips (Thysanoptera: was greatly enhanced if predators were shipped as Thripidae) populations and their damage to South pupae in parafilm cocoons. Adult F. orizabensis, es- African citrus orchards. J. Entomol. Soc. South Af- pecially males, were extremely sensitive to aspira- rica 53: 151-157. tion and transit stress, and high mortality resulted GROUT, T. G., AND G. I. RICHARDS. 1992a. The dietary ef- when adult males and females were shipped long fects of windbreak pollens on longevity and fecundity of a predacious mite Euseius addoensis addoensis distances. Transit mortality of adult and pupal F. (Acari: Phytoseiidae) found in citrus orchards in orizabensis was not significantly reduced by inclu- South Africa. Bull. Entomol. Res. 82: 317-320. sion of ice packs in polystyrene foam boxes. This GROUT, T. G., AND G. I. RICHARDS. 1992b. Euseius ad- may have been an artifact resulting from the time doensis addoensis, an effective predator of citrus of year this shipping trial was conducted as ship- thrips, Scirtothrips aurantii, in the eastern Cape ments were made during May when weather con- Province of South Africa. Experimental and Applied ditions were cool. Inclusion of ice packs with Acarology 15: 1-13. shipments of F. orizabensis pupae would be recom- HARRISON, J. O. 1963. Notes on the biology of banana mended, especially during summer. flower thrips, Frankliniella parvula, in the Domini- can Republic (Thysanoptera; Thripidae). Ann. Ento- Work is currently underway with commercial mol. Soc. America 56: 664-666. insectaries in California to implement mass rear- HODDLE, M. S., J. G. MORSE, P. PHILLIPS, AND B. FABER. ing of F. orizabensis for field trials against S. per- 1998. Progress on the management of avocado thrips. seae in avocado orchards. California Avocado Society Yearbook 82: 87-100. Hoddle et al.: Franklinothrips pupation biology 281

HODDLE, M. S., J. G. MORSE, P. PHILLIPS, B. FABER, Y. PARRELLA, M. P., AND T. LEWIS. 1997. Integrated Pest WEE, AND S. PEIRCE. 1999. Avocado thrips update. Management (IPM) in Field Crops, pp. 595-614. In Citrograph 84: 13-14. Thrips as Crop Pests. T. Lewis (ed.). CAB Interna- HODDLE, M. S., L. ROBINSON, K. DRESCHER, AND J. tional, Wallingford. JONES. 2000. Developmental and reproductive biol- PETITT, F. L., D. S. SCHUMAN, D. O. WIETLISBACH, AND ogy of a predatory Franklinothrips orizabensis (Thys- J. A. COFFELT. 1996. An automated system for collec- anoptera: Aeolothripidae). Biol. Control 18: 27-38. tion and counting of parasitized leafminer (Diptera: HODDLE, M. S. J. JONES, K. OISHI, D. MORGAN, AND L. Agromyzidae) larvae. Florida Entomol. 79: 450-454. ROBINSON. 2001. Evaluation of diets for the develop- REED, D. K., AND J. R. RICH. 1975. A new survey tech- ment and reproduction of Franklinothrips orizaben- nique for citrus thrips. J. Econ. Entomol. 68: 739-742. sis (Thysanoptera: Aeolothripidae). Bull. Entomol. SCHWEIZER, H., AND J. G. MORSE. 1996. Pupation sites Res. (in press). of Scirtothrips citri (Thysanoptera: Thripidae) and po- JACKSON C. G., H. T. CHAN JR., M. H. TANIGUCHI, D. B. tential management through increasing mortality of CHURCHILL, AND D. M. BILSLAND. 1996. Pneumatic instars on the ground. J. Econ. Entomol. 89: 1438-1445. air separation for the sorting of parasitized and un- SCHWEIZER, H., AND J. G. MORSE. 1997. Factors influ- parasitized fruit fly (Diptera; Tephritidae) puparia. encing survival of citrus thrips (Thysanoptera: J. Econ. Entomol. 89: 353-358. Thripidae) propupae and pupae on the ground. J. MCMURTRY, J. A., AND B. A. CROFT. 1997. Life-styles of Econ. Entomol. 90: 435-443. phytoseiid mites and their roles in biological control. SILVERS, C. 2000. Biological control of Scirtothrips per- Ann. Rev. of Entomol. 42: 291-321. seae Nakahara in California avocados: assessment of MORSE, J. G., T. S. BELLOWS JR., AND Y. IWATA. 1986. two generalist predators. MS Thesis, University of Technique for evaluating residual toxicity of pesti- California at Riverside. 103 pp. cides to motile insects. J. Econ. Entomol. 79: 281-283. SMITTLE, J., J. A. SEAWRIGHT, AND B. K. BIRKY. 1986. MUNGER, F. 1942. A method for rearing citrus thrips in Use of a commercial color sorter to separate sexes of the laboratory. J. Econ. Entomol. 35: 373-375. stable fly (Diptera: Muscidae) pupae from a genetic NAKAHARA, S. 1997. Scirtothrips perseae (Thysan- sexing strain. J. Econ. Entomol. 79: 877-878. optera: Thripidae), a new species infesting avocado TSUCHIDA, K. 1997. Adult emerging sites of Thrips in southern California. Insecta Mundi 11: 189-191. palmi (Thysanoptera; Thripidae) in an egg plant OKADA, T. 1981. Searching behavior for pupation place field with mulching sheet. Applied Entomol. and by late second instar larvae of Scirtothrips dorsalis Zool. 32: 246-249. Hood (Thysanoptera: Thripidae). Japanese Soc. Ap- WHITTEN, M. J. 1969. Automated sexing of pupae and plied Entomol. and Zool. 25: 10-16. its usefulness in control by sterile insects. J. Econ. PARKER, B. L., AND M. SKINNER. 1997. Integrated Pest Entomol. 62: 272-273. Management (IPM) in Tree Crops, pp. 615-638. In WOLF, W. W, M. W. STIMMAN, AND G. K. PARKER. 1972. Thrips as Crop Pests. T. Lewis (ed.). CAB Interna- A mechanism to aid sorting of male and female cab- tional, Wallingford. bage looper pupae. J. Econ. Entomol. 65: 1044-1047.

282 Florida Entomologist 84(2) June 2001

EFFECTS OF VEGETATION CONTROL ON PARASITOIDS OF THE NANTUCKET PINE TIP MOTH, RHYACIONIA FRUSTRANA (LEPIDOPTERA: TORTRICIDAE)

KENNETH W. MCCRAVY1, AND C. WAYNE BERISFORD Department of Entomology, University of Georgia, Athens, GA 30602

1Current Address: Department of Biology, Valdosta State University, Valdosta, GA 31698

ABSTRACT The Nantucket pine tip moth (Rhyacionia frustrana (Comstock)) is host to numerous parasitoid species that can cause substantial moth mortality. Little is known regarding the effects of forest management practices on these parasitoids. Abundance of parasitoids and parasitism rates, based on rearing of adult moths and parasitoids, were compared in herbicide-treated and untreated loblolly pine, Pinus taeda L., stands in the southeastern Georgia coastal plain. Three parasitoids, Lixophaga mediocris Aldrich, Eurytoma pini Bugbee, and Hyssopus rhyacioniae Gahan accounted for over 70% of total parasitism. Parasitism rates did not differ signiﬁcantly between treated and untreated plots. Tip moth damage was higher in the untreated plots. Malaise trapping of parasitoids yielded no signiﬁcant difference in numbers of tip moth parasitoids or total parasitoids captured in treated and untreated plots, suggesting a functional response of parasitoids to tip moth densities. Higher levels of naturally occurring vegetation did not improve tip moth control through increased parasitism rates.

Key Words: Rhyacionia frustrana, parasitoids, vegetation control, Pinus taeda, parasitism

RESUMEN La polilla de pino Nantucket (Rhyacionia frustrana (Comstock)) es huésped a numerosas especies parasitoides que pueden causar mortalidad substancial de polilla. Poco se sabe en re- lación con los efectos de practicas de control forestal en estos parasitoides. La abundancia de parasitoides y las incidencias de parasitismo, basado en el cultivo de polillas adultas y parasitoides, fueron comparadas en áreas de pinos Pinus taeda L., sin tratar y tratados con her- bicida, en el plano costeño del sureste de Georgia. Tres parasitoides, Lixophaga mediocris Aldrich, Eurytoma pini Bugbee, y Hyssopus rhyacioniae Gahan resultaron en mas del 70% del parasitismo total. Incidencias de parasitismo no difirieron significativamente entre terrenos tratados y no tratados. Daño de polilla de pino fue mayor en los terrenos no tratados. Capturas de parasitoides no produjeron diferencias significativas en números de parasitoides de polilla o en el total de parasitoides capturados en terrenos tratados y no tratados, sugiriendo una respuesta funcional de parasitoides a densidades de polilla de pino. Niveles mas elevados de presencia de vegetación natural no mejoro el control de polilla de pino a tra- vés de cantidades más altas de parasitismo.

The Nantucket pine tip moth, Rhyacionia frus- moth population ﬂuctuations have been associ- trana (Comstock), is a common insect pest of ated with intensive forest regeneration practices, southern pines, attacking seedlings and saplings including chemical control of competing vegeta- of loblolly (Pinus taeda L.), shortleaf (P. echinata tion to increase seedling growth and survival Miller), and Virginia (P. virginiana Miller) pines (Miller & Stephen 1983; Nowak & Berisford (Berisford 1988). Eggs are laid on needles and 2000). Tip moth populations may be constrained shoots. Early instar larvae mine needles and fas- to some degree by natural agents, including para- cicle sheaths. Later instars feed on the meristem- sitoids and predators (Eikenbary & Fox 1968a,b; atic tissue of shoots and buds, causing shoot Freeman & Berisford 1979; Wallis et al. 1980; death and losses in form, wood quality and vol- Gargiullo & Berisford 1983; Warren 1985; Mc- ume growth (Cade & Hedden 1987). The moth has Cravy & Berisford 1998, 2000). Approximately 60 2 to 5 generations annually (depending on cli- species of parasitoids have been associated with matic conditions) and overwinters as a pupa in- the Nantucket pine tip moth (Yates & Beal 1962; side of the dead shoot or bud (Berisford 1988). Frank & Foltz 1997). However, potential effects of Damage caused by the Nantucket pine tip intensive forest management practices on tip moth is highly variable, and may be negligible or moth parasitoids have received little attention. high enough to cause tree mortality. Increased tip Herbaceous vegetation can be an important

McCravy & Berisford: Vegetation and tip moth parasitoids 283 source of food for adult parasitoids (Leius 1961, wintering generation were done in 2 stages. Half 1963; Syme 1975), and can also be important as of the shoots were collected in late September, refugia and in maintaining suitable microclimatic 1996 and half were collected in mid-January, conditions (Reed et al. 1970; Powell 1986). Pimen- 1997, in order to sample parasitoids that might tel’s enemy impact hypothesis (Pimentel 1961) emerge before the onset of winter as well as those suggests that natural enemies of herbivorous in- emerging in late winter and early spring. Spring sects are more effective in diverse systems than in (1st) generation collections were done in early simple ones. In a review of tests of this hypothe- May, 1997. Because of low tip moth infestations in sis, however, Russell (1989) found the results to this generation, 1,000 shoots were collected from be inconclusive for parasitism. each plot. Shoots were returned to the laboratory We initiated a study to determine if chemical and placed in ventilated rearing containers (Ber- control of competing vegetation affects parasitism isford et al. 1971), separated by site and treat- of the Nantucket pine tip moth. The objective of ment. Emerging adult moths and parasitoids this study was to compare rates of parasitism and were collected every 2-3 days. Moths and parasi- abundance of parasitoids of the tip moth in herbi- toids were counted and parasitoids were identi- cide-treated and untreated loblolly pine stands. fied to species using Yates’ (1967) key, and placed in 70% EtOH. At the end of emergence for each MATERIALS AND METHODS collection, shoots were removed from the rearing cans, dissected, and examined for the presence of The study was conducted in 1996-97 at 2 one-yr- tip moths, parasitoids, or cocoons. Series of each old P. taeda plantations in Burke County, in the apparent species were sent to appropriate taxo- Georgia coastal plain. Site 1 was 70 ha and Site 2 nomic authorities for positive determinations. was 47 ha. Tree density at each plantation was ap- Tip moth parasitism rates were calculated prox. 1500 trees/ha. In spring, 1996, each site was based on relative numbers of emerged tip moth divided into 2 equally sized plots, with 1 randomly and parasitoid adults. Two parasitoids, Hyssopus selected plot receiving treatment with the herbi- rhyacioniae Gahan and Pteromalus Swederus sp., cides hexazinone (Velpar®, DuPont, Wilmington, were determined to be gregarious based on dis- DE) and sulfometuron methyl (Oust®, DuPont, section of individual tip moth-infested shoots, av- Wilmington, DE) at rates of 2.34 and 0.22 liters/ha, eraging 12.2 ± 1.20 (mean ± SE, n = 15) and 2.92 respectively. The remaining halves were left as un- ± 0.45 (n = 12) individuals per brood, respectively. treated plots. The treatment plots at each site were Total numbers of individuals reared for these 2 treated again in fall, 1996 with imazapyr (Arse- species were divided by these means to provide a nal®, American Cyanamid, Princeton, NJ) and more accurate estimate of the number of parasit- glyphosate (Accord®, Monsanto, St. Louis, MO) at ism events. 0.58 and 2.38 liters/ha, respectively. All herbicides At site 1, parasitoids were collected using mal- were applied by helicopter. There was no replica- aise traps to estimate the relative abundance of tion within sites. Rather, sites were treated as rep- parasitoids in the herbicide-treated and un- licates to decrease the potential for confounding treated plots. Two traps, 1 in each treatment plot, effects due to parasitoid immigration into small were operated for 8, 5-day trapping periods bi- plots. In spring, 1996, 50 pines were randomly cho- weekly from mid-June to early October 1996. sen in each plot of each study site to serve as per- Traps were randomly relocated for each trapping manent study trees. Tip moth infestations were period. Insects were collected in 70% EtOH. At the evaluated on these trees by counting numbers of end of each trapping period, samples were taken infested shoots in each generation, when tip moths to the laboratory and parasitoids, defined as Par- were in the late larval and pupal stages and dam- asitica (Huber 1993) plus tachinids (Diptera), age was most obvious. Non-pine vegetation was were removed and counted. Recognizable tip moth quantified and characterized for each plot as well. parasitoids were also separated and counted. Twenty randomly located 1 m2 quadrats were sam- Mean percentage ground cover and mean num- pled in each plot during each of the 4 tip moth gen- ber of flowering plants per plot were compared be- erations. For each quadrat, a visual estimate of tween treatments using paired t-tests. Two-way percentage ground cover was taken, and numbers repeated measures analysis of variance, with gen- of flowering plants were counted. eration as the repeated variable, was used to com- Collections of infested shoots for rearing of tip pare mean numbers of infested shoots per tree moths and parasitoids were made during the late between treatments and sites, to compare overall larval/pupal stages of each tip moth generation. parasitism rates between treatments and sites, For summer (2nd), fall (3rd), and winter (4th) and to analyze species-specific parasitism by the generations, 2,000 infested shoots were randomly most common parasitoid species in relation to collected from non-permanent study trees in each treatment. Percent parasitism data were arcsine plot. Summer generation collections were done in transformed prior to analysis. Paired t-tests were early July and fall generation collections in mid- used to compare numbers of parasitoids captured August, 1996. Collections of shoots for the over- in malaise traps between treatments. It was noted

284 Florida Entomologist 84(2) June 2001 that larger parasitoids appeared to be relatively Overall, 7,198 parasitoids were reared, includ- more common in samples from the untreated ing 17 species in 9 families representing 2950 plots. Therefore, chi-square tests were used to an- parasitism events. Three species, Lixophaga me- alyze ratios of ichneumonoids and tachinids to mi- diocris Aldrich, Eurytoma pini Bugbee, and Hys- crohymenoptera reared and captured in treated sopus rhyacioniae Gahan accounted for over 70% and untreated plots. All analyses were done using of total parasitism. Mean percent parasitism was SigmaStat Version 2.0 software package (Jandel 47.47 ± 7.94 in the treated plots and 46.65 ± 7.47

Scientiﬁc Software 1995). in the untreated plots (F(1,3) = 0.01, P = 0.92). Dif- ference in parasitism rates between sites was not

RESULTS signiﬁcant (F(1,3) = 5.56, P = 0.10), and there was

no significant interaction (F(1,3) = 0.24, P = 0.66). The most common plant species found and Parasitism was highest in spring (73.93 ± 2.94) months in which flowering individuals were and lowest in winter (22.36 ± 1.33). present are shown in Table 1. Mean percent There was no relationship between treatment ground cover (±SE) was significantly greater in and contribution to total parasitism among the 3

the untreated plots than in the treated plots (t(7) = most common parasitoid species (F(2,6) = 0.06, P = 8.48, P < 0.001), indicating that herbicide treat- 0.94). However, as a group, ichneumonoids comments were effective (Fig. 1). Mean number of prised a greater proportion of rearings, relative to ﬂowering plants was also greatest in the un- microhymenoptera, in the untreated than the

treated plots (t(7) = 2.82, P = 0.03) (Fig. 1). herbicide-treated plots at Site 1 (14.27% vs. 2 Tip moth infestation levels, based on numbers 8.02%; X (1) = 13.53, P < 0.001) and Site 2 (17.39% 2 of damaged shoots per tree, were higher in the un- vs. 11.57%; X (1) = 8.57, P = 0.003). treated plots than the treated plots (10.89 ± 1.41 Malaise trapping resulted in 2,981 total para- ± vs. 2.64 0.49; F(1,3) = 33.31; P = 0.01). Infestation sitoid captures. Mean numbers of captures per levels ranged from 7.96 ± 2.50 in the fall genera- trapping period (±SE) were 189.88 ± 49.15 in the tion to 15.41 ± 4.15 in the winter for the untreated herbicide-treated plot and 182.75 ± 36.90 in the plots, and from 1.22 ± 0.18 in spring to 3.79 ± 0.21 untreated plot, and were not signiﬁcantly differ-

in summer for the treated plots (Fig. 2). There ent (t(7) = 0.11, P = 0.916; Fig. 3). Ichneumonoids

was no signiﬁcant difference between sites (F(1,3) = accounted for 9.09% of total captures and 11.77%

1.26, P = 0.34), and no signiﬁcant interaction (F(1,3) of total parasitic hymenoptera, while microhy- = 1.04, P = 0.38). menoptera accounted for 68.13% and 88.23% of

TABLE 1. COMMON PLANT SPECIES FOUND IN QUADRAT SAMPLING AND MONTHS IN WHICH FLOWERING INDIVIDUALS WERE FOUND

Collection Date

Family and Species Jun 96 Aug 96 Oct 96 Apr 97

Asteraceae Eupatorium capillifolium (Lamarck) Small + Gnaphalium obtusifolium L. + + Solidago sp. L. + Caryophyllaceae Cerastium glomeratum Thuillier + Cyperaceae Cyperus retrorsus Chapman + Fabaceae Stylosanthes biflora Britton, Sterns, and Poggenburg + + Tephrosia spicata (Walter) Torrey and Gray + Hypericaceae Hypericum denticulatum Humboldt, Bonpland, and Kunth + Liliaceae Smilax sp. L. + Passifloraceae Passiflora incarnata L. + Vitaceae Vitis rotundifolia Michaux +

McCravy & Berisford: Vegetation and tip moth parasitoids 285

Fig. 1. Mean percentage ground cover (±SE) and mean number of ﬂowering plants (±SE) per 1 m2 quadrat in herbicide-treated and untreated plots. Observa- Fig. 3. Mean numbers of parasitoids captured (±SE) tions were averages of 20 quadrats for each study site in malaise traps per 5-day trapping period in herbicide- and tip moth generation (n = 8, each mean). treated and untreated plots. Between-plot differences were non-signiﬁcant (P > 0.20, each comparison).

these totals, respectively. Tachinids comprised DISCUSSION 22.78% of total captures. There were no signiﬁ- cant between-treatment differences in captures of Most studies of tip moth damage in relation to ichneumonoids (t(7) = 1.037, P = 0.334), microhy- forest regeneration practices have found an in-

menoptera (t(7) = 0.409, P = 0.695), or tachinids verse relationship between damage and amount

(t(7) = 0.328, P = 0.753). However, there was a sig- of competing vegetation (Berisford & Kulman nificantly greater proportion of ichneumonoids 1967; White et al. 1984; Hood et al. 1988; Ross et and tachinids captured, relative to microhy- al. 1990). However, Miller & Stephen (1983) and menoptera, in the untreated (35.29%) than the Nowak & Berisford (2000) found no differences in 2 treated (28.57%) plot (X (1) = 15.199, P < 0.001). A damage levels between herbicide-treated and un- total of 136 known tip moth parasitoids were cap- treated plots, but greater temporal variation in tured. Mean numbers captured/trap period (±SE) tip moth damage levels in the treated plots. We were 9.50 ± 2.84 in the herbicide-treated plots also found greater temporal variation in damage ± and 7.50 1.84 in the untreated plots (t(7) = 0.528, levels in treated plots, at least on a relative scale, P = 0.613). Lixophaga mediocris (72% of total) with a roughly 3-fold difference between peak and and E. pini (14% of total) were the most common lowest infestation rates in the treated plots, but species captured. only a 2-fold difference in the untreated plots (Fig. 2). Nowak & Berisford (2000) also found that infestation levels were generally lower in treated than untreated plots at low tip moth densities, as was the case in our study. There was little difference in variation in parasitism rates between treatments in our study, suggesting that the greater relative fluctuations in tip moth damage in the treated plots, and greater overall damage in the untreated plots, were not due to disruption of parasitoid populations. The overall tip moth parasitism rate of 47% generally agrees with that of 42% found by Free- man & Berisford (1979) in the Georgia piedmont and 41% found by Eikenbary & Fox (1965) in the South Carolina coastal plain, but is higher than the 26% rate obtained by the latter authors in the South Carolina piedmont. Tip moth parasitism rates were highest in the spring generation and lowest in the winter in our study. Gargiullo & Fig. 2. Mean numbers of infested shoots per tree Berisford (1983) found a similar pattern for tip (±SE) in herbicide-treated and untreated plots for 4 tip moth parasitism in the Georgia piedmont. If tip moth generations (P = 0.004, all generations). moth parasitoids attack specific life stages, the 286 Florida Entomologist 84(2) June 2001 increased asynchrony of tip moth development in R. Hedden (Clemson University), J. Meeker (Flor- later generations (Fettig et al. 2000) could make it ida Dept. of Agriculture and Consumer Services) more difficult for the parasitoids to find suitable J. Nowak (University of Georgia), and S. Salom hosts, since fewer tip moths in the required life (Virginia Polytechnic Institute and State Univer- stage(s) would be present at any given time. sity) provided helpful comments on earlier ver- Tip moth populations were approx. 2 to 5 times sions of the manuscript, as did the anonymous higher in the untreated plots, based on damage reviewers. Thanks also to R. Weseloh (The Con- estimates, but there was no difference in parasit- necticut Agricultural Experiment Station) for ism rates between treatments, meaning that suggesting use of repeated measures ANOVA. L. greater total parasitism occurred in the un- Coote (Royal Ontario Museum), C. Darling (Royal treated plots. Malaise trapping showed no differ- Ontario Museum), G. Gibson (Agriculture and ence in parasitoid abundance between plots, Agri-Food Canada), E. Grissell (USDA, System- suggesting a greater number of moths parasitized atic Entomology Laboratory), S. Heydon (Univer- per parasitoid in the untreated plots, a functional sity of California-Davis), J. Luhman (Minnesota response perhaps resulting from greater parasi- Dept. of Agriculture), A. Menke (USDA, System- toid fecundity or longevity, or less searching time atic Entomology Laboratory), M. Schauff (USDA, due to higher host densities. The ratio of ichneu- Systematic Entomology Laboratory), A. Sharkov monoids and tachinids to microhymenoptera (Ohio State University), J. Whitfield (University reared and captured in malaise traps was great- of Arkansas), M. Wood (Agriculture and Agri-Food est in the untreated plots, suggesting that vegeta- Canada), and G. Zolnerowich (Texas A&M Uni- tion control in pine plantations may have a versity) provided positive identifications of para- relatively greater impact on populations of the sitoids. This project was supported by the Pine larger parasitoids. Larger-bodied parasitoids may Tip Moth Research Consortium and the Georgia have greater metabolic requirements, which Traditional Industries Program. could affect the amount and type of food needed. Small wasps are also probably more affected by REFERENCES CITED wind currents that result from lack of vegetation to act as a windbreak. There is also evidence that BERISFORD, C. W. 1988. The Nantucket pine tip moth, some female ichneumonoids must feed at flowers pp. 141-161. In A. A. Berryman (ed.). Dynamics of to complete egg development (van Emden 1963). forest insect populations: patterns, causes, implica- Numerous studies (Syme 1975; Foster & Ruesink tions. Plenum Publishing Corporation, New York. 1984; Wäckers & Swaans 1993; Idris & Grafius BERISFORD, C. W., AND H. M. KULMAN. 1967. Infesta- tion rate and damage by the Nantucket pine tip 1995, 1996, 1997; Johanowicz & Mitchell 2000) moth in six loblolly pine stand categories. For. Sci. have documented the importance of flower avail- 13: 428-438. ability on fecundity, survival, and parasitism BERISFORD, C. W., H. M. KULMAN, AND H. J. rates by ichneumonoids. Detailed studies of spe- HEIKKENEN. 1971. Factors affecting distribution and cific plant associations of tip moth parasitoids, abundance of hymenopterous parasites of Ips spp. and effects of vegetation control practices on bark beetles in Virginia (Coleoptera: Scolytidae). Ca- these plants, are needed. nadian Entomol. 103: 235-239. In summary, we found that total tip moth par- CADE, S. C., AND R. L. HEDDEN. 1987. Growth impact of asitism was greater in untreated than in herbi- pine tip moth on loblolly pine plantations in the Oua- chita mountains of Arkansas. South. J. Appl. For. 11: cide-treated plots, but parasitism rates were 128-133. equal between plots. Tip moth damage was sub- EIKENBARY, R. D., AND R. C. FOX. 1965. The parasites of stantially higher in untreated than in herbicide- the Nantucket pine tip moth in South Carolina. treated stands, suggesting that vegetation man- South Carolina Agri. Expt. Sta. Tech. Bull. 1017, agement practices do not necessarily affect the Clemson, SC. role of parasitoids in tip moth population control. EIKENBARY, R. D., AND R. C. FOX. 1968a. Arthropod predators of the Nantucket pine tip moth Rhya- cionia frustrana. Ann. Entomol. Soc. Am. 61: 1218- ACKNOWLEDGMENTS 1221. EIKENBARY, R. D., AND R. C. FOX. 1968b. Responses of We thank M. Dalusky (University of Georgia) Nantucket pine tip moth parasites to tree level, ori- for technical advice, and N. LeCroy, R. Garland, J. entation and hosts per pine tip. Ann. Entomol. Soc. Smith, T. Jackson, B. Dorough, M. Morrow, and D. Am. 61: 1380-1384. Hart (University of Georgia) for invaluable tech- FETTIG, C. J., K. W. MCCRAVY, AND C. W. BERISFORD. nical assistance. B. Haynes and the late M. Moore 2000. Effects of Nantucket pine tip moth insecticide (University of Georgia) provided advice on vege- spray schedules on loblolly pine seedlings. South. J. tation sampling and assistance with plant identi- Appl. For. 24: 106-111. FOSTER, M. A., AND W. G. RUESINK. 1984. Influence of fications, respectively. J. Seckinger, S. Cameron, flowering weeds associated with reduced tillage in and F. Bevan (International Paper) were of great corn on a black cutworm (Lepidoptera: Noctuidae) help in providing study sites. A. Braccia (Univer- parasitoid, Meteorus rubens (Nees von Esenbeck). sity of Georgia), C. Fettig (University of Georgia), Environ. Entomol. 13: 664-668. 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FRANK, J. H., AND J. L. FOLTZ. 1997. Classical biological MCCRAVY, K. W., AND C. W. BERISFORD. 2000. Parasi- control of pest insects of trees in the southern United toids of the Nantucket pine tip moth (Lepidoptera: States: a review and recommendations. United Tortricidae) in the coastal plain of Georgia. J. Ento- States Dep. Agric. For. Serv. FHTET-96-20. mol. Sci. 35: 220-226. FREEMAN, B. L., AND C. W. BERISFORD. 1979. Abun- MILLER, F. D., JR., AND F. M. STEPHEN. 1983. Effects of dance and parasitic habits of some parasitoids of the competing vegetation on Nantucket pine tip moth Nantucket pine tip moth (Lepidoptera: Tortricidae). (Lepidoptera: Tortricidae) populations in loblolly Canadian Entomol. 111: 509-514. pine plantations in Arkansas. Environ. Entomol. 12: GARGIULLO, P. M., AND C. W. BERISFORD. 1983. Life ta- 101-105. bles for the Nantucket pine tip moth, Rhyacionia NOWAK, J. T., AND C. W. BERISFORD. 2000. Effects of in- frustrana (Comstock), and the pitch pine tip moth, tensive forest management practices on insect infes- Rhyacionia rigidana (Fernald) (Lepidoptera: Tortri- tation levels and loblolly pine growth. J. Econ. cidae). Environ. Entomol. 12: 1391-1402. Entomol. 93: 336-341. HOOD, W. M., R. L. HEDDEN, AND C. W. BERISFORD. PIMENTEL, D. 1961. Species diversity and insect popula- 1988. Hazard rating forest sites for pine tip moth, tion outbreaks. Ann. Entomol. Soc. Am. 54: 76-86. Rhyacionia spp., in the Upper Piedmont Plateau. POWELL, W. 1986. Enhancing parasitoid activity in For. Sci. 34: 1083-1093. crops. In J. Waage and D. Greathead (eds.). Insect HUBER, J. T. 1993. Introduction, pp. 1-3. In H. Goulet parasitoids. Academic Press, New York. and J. T. Huber (eds.). Hymenoptera of the world: an REED, D. K., W. G. HART, AND S. J. INGLE. 1970. Influ- identification guide to families. Center for Land and ence of windbreaks on distribution and abundance of Biol. Res. Res., Ottawa, Ontario. brown scale in citrus groves. Ann. Entomol. Soc. Am. IDRIS, A. B., AND E. GRAFIUS. 1995. Wildflowers as nec- 63: 792-794. tar sources for Diadegma insulare (Hymenoptera: ROSS, D. W., C. W. BERISFORD, AND J. F. GODBEE, JR. Ichneumonidae), a parasitoid of diamondback moth, 1990. Pine tip moth, Rhyacionia spp., response to Plutella xylostella L. (Lepidoptera: Plutellidae). J. herbaceous vegetation control in an intensively site- Econ. Entomol. 24: 1726-1735. prepared loblolly pine plantation. For. Sci. 36: 1105- IDRIS, A. B., AND E. GRAFIUS. 1996. Effects of wild and 1118. cultivated host plants on oviposition, survival, and RUSSELL, E. P. 1989. Enemies hypothesis: a review of development of diamondback moth (Lepidoptera: the effect of vegetational diversity on predatory in- Plutellidae) and its parasitoid Diadegma insulare sects and parasitoids. Environ. Entomol. 18: 590- (Hymenoptera: Ichneumonidae). Environ. Entomol. 599. 25: 825-833. SYME, P. D. 1975. The effects of flowers on the longevity IDRIS, A. B., AND E. GRAFIUS. 1997. Nectar-collecting be- and fecundity of two native parasites of the Euro- havior of Diadegma insulare (Hymenoptera: Ichneu- pean pine shoot moth in Ontario. Environ. Entomol. monidae), a parasitoid of diamondback moth 4: 337-346. (Lepidoptera: Plutellidae). Environ. Entomol. 26: VAN EMDEN, H. F. 1963. Observations on the effect of 114-120. flowers on the activity of parasitic Hymenoptera. JANDEL SCIENTIFIC SOFTWARE. 1995. SigmaStat Statis- Entomol. Mon. Mag. 98: 265-270. tical Software User’s Manual, Version 2.0. Jandel WÄCKERS, F. L., AND C. P. M. SWAANS. 1993. Finding Corporation, San Rafael, CA. floral nectar and honeydew in Cotesia rubecula: ran- JOHANOWICZ, D. L., AND E. R. MITCHELL. 2000. Effects dom or directed? Proc. Exp. Appl. Entomol. 4: 67-72. of sweet alyssum flowers on the longevity of the par- WALLIS, G. W., F. M. STEPHEN, G. T. SCRIVEN, AND R. 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288 Florida Entomologist 84(2) June 2001

TRAPPING FALL ARMYWORM (LEPIDOPTERA: NOCTUIDAE) ADULTS IN TRAPS BAITED WITH PHEROMONE AND A SYNTHETIC FLORAL VOLATILE COMPOUND

ROBERT L. MEAGHER, JR. Center for Medical, Agricultural and Veterinary Entomology Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL 32604

ABSTRACT

Field experiments were conducted to determine the effectiveness of the ﬂoral compound phenylacetaldehyde in increasing capture of fall armyworm, Spodoptera frugiperda, males in pheromone-baited traps. Plastic Unitraps were placed in cotton and corn ﬁelds in north-central Florida and were baited with commercial sex pheromone and synthetic phenylacetaldehyde released from hollow polyethylene stoppers and glass microcapillary pipets. Addition of phenylacetaldehyde as a lure was not effective in collecting more moths and actually reduced numbers of moths captured compared to pheromone-baited traps. Nontarget Hy- menoptera were also collected in traps; more Sphecoidea were found in phenylacetaldehyde- baited traps compared to pheromone-baited traps.

Key Words: insect behavior, Spodoptera, phenylacetaldehyde, monitoring

RESUMEN

Experimentos de campo fueron llevados a cabo para determinar la efectividad del compuesto ﬂoral fenilacetaldehido en incrementar la captura de machos del gusano de otoño Spodop- tera frugiperda, en trampas con carnada de feromona. Trampas de plástico “Unitraps” fueron colocadas en campos de maíz y algodón en regiones del centro-norte de Florida y se les preparo con feromona de sexo comercial y fenilacetaldehido sintético soltados de tapones huecos de polietileno y de pipetas microcapilarias de cristal. La adición de fenilacetaldehido como atrayente no fue efectivo en la colección de mas polillas y en realidad redujo el numero de polillas capturadas en comparación con trampas con señuelo de feromona. Himenóptera en general también fueron colectadas en trampas; mas Sphecoidea fueron encontradas en trampas con fenilacetaldehido comparadas con trampas de feromona.

Populations of adult male Spodoptera fru- Advanced pest management techniques used giperda (J. E. Smith) (fall armyworm) are moni- to correlate adult populations with resulting lar- tored in agricultural systems with a multi- val densities, or with management strategies component sex pheromone as a lure in traps such as autodissemination of a pathogen, may re- (Mitchell et al. 1985; Tumlinson et al. 1986). How- quire improved attraction and trap capture of ever, chemicals other than sex pheromones have moths. My major objective for this research was to been isolated, identified, and bioassayed as moth test phenylacetaldehyde as an enhancement to attractants. Phenylacetaldehyde, along with sex pheromone in the collection of male fall army- other chemicals, has been isolated from many worm in field tests. Because of the collection of flowering plants and shrubs including Zea mays nontarget Hymenoptera in related studies L. (Poaceae) (Cantelo & Jacobson 1978), Araujia (Meagher & Mitchell 1999), a secondary objective sericofera Brothero (Asclepiadaceae) (Cantelo & was to compare collection of aculeate Hy- Jacobson 1979), Abelia grandiflora (André) (Ca- menoptera in pheromone- and phenylacetalde- prifoliaceae) (Haynes et al. 1991), Cestrum noc- hyde-baited traps. turnum (L.) (Solanaceae) (Heath et al. 1992), and Gaura spp. (Onagraceae) (Shaver et al. 1998). MATERIALS AND METHODS Field trapping with flowers or synthetic phenylacetaldehyde captured low numbers of S. fru- 1997 giperda (Cantelo & Jacobson 1979). Synthetic phenylacetaldehyde was tested in flight tunnel An experiment was conducted in northwestern bioassays with fall armyworm males and was Alachua County, Florida, from 21 July to 10 Octo- found to increase upwind flight (from 65.4% to ber. All-white Universal Moth Traps or Unitraps 85.2%) and contact with the source (from 43.8% to (Great Lakes IPM, Vestaburg, MI) were placed 69.3%) in combination with a sex pheromone lure along pivot roads and edges in an 80-ha field of (Meagher & Mitchell 1998). cotton, Gossypium hirsutum L. This field was sur-

Meagher: Trapping FAW with Floral Volatiles 289 rounded by ≈ 400 ha of cotton, separated by paved captured were sorted and numbers were com- and unpaved roads and forested strips. pared across treatments. Traps were baited with lures containing sex pheromone of the fall armyworm and phenylacet- Release Rates aldehyde. Three treatments were used: (1) Trécé® (Trécé, Inc., Salinas, CA) red septa lures contain- Release rates of phenylacetaldehyde in the ing components of the S. frugiperda pheromone, stoppers used in the 1997 experiment were deter- (2) phenylacetaldehyde (Aldrich Chemical Co., mined by the modified techniques of Heath & Milwaukee, WI) placed in hollow polyethylene Manukian (1992). Briefly this system consists of a stoppers (Kimble, Vineland, NJ, purchased through glass chamber (25.7 cm long and 7.6 cm ID) con- Thomas Scientific, Swedesboro, NJ, #9713-F28), structed of Pyrex glass with a glass frit inlet, a or (3) a combination of both lures. Pheromone ground-glass joint outlet, and a multiport collec- lures were attached to the bottom of a cork that tor base to which the collector traps were con- was placed in a hole in the canopy of the Unitrap. nected. Collector traps were made from a 4.0 cm The stopper with phenylacetaldehyde (0.5 ml per long by 4.0 mm ID piece of glass tubing and con- stopper) was hot-gun glued (Arrow Fastener Co., tained 50 mg of Super-Q® (Alltech Assoc., Deer- Saddle Brook, NJ) to the bottom of the cork, field, IL) as the adsorbent. Two stainless steel which was placed in the trap canopy. The combi- frits were used to contain the adsorbent. The col- nation lure was composed of a cork with attached lector traps were connected to stainless steel tub- stopper and the pheromone lure attached to the ing by 0.64 cm unions and 0.64 cm ID Teflon® side of the cork. All traps contained insecticide ferrules. These traps were cleaned by soxhlet ex- strips to kill moths that were captured (Hercon® traction with methylene chloride for 24 h and dried Vaportape II containing 10% 2, 2-dichlorovinyl in a fume hood prior to use. The airflow rate dimethyl phosphate, Hercon Environmental Co., through the volatile collection system was 1 L/min Emigsville, PA). Trap contents were removed during a 1-h collection period. Volatiles from the three times per week and pheromone and pheny- lures collected on the traps were eluted using 100 lacetaldehyde lures were replaced every two µl of high purity methylene chloride. Tetradecane weeks. The experiment was designed as a ran- was added as internal standard prior to analysis. domized complete block with three blocks of the Release rates were determined from three lures three treatments. The location of each trap within on days 3, 5, 7, and 14. The lures were held in a each block was changed weekly. hood without airflow between analyses. Gas chromatographic analyses were conducted 1998 with a Hewlett-Packard Model 5890A Series II gas chromatograph, equipped with a cool on-column In late March, fields in the same area in north- capillary injector (septum injector) and flame ion- western Alachua county were planted to silage ization detector. Helium was used as the carrier corn (Zea mays L.). An experiment was designed gas at a linear flow of 18 cm/sec. A combination of to compare capture of fall armyworm males in three fused silica columns connected in series by Unitraps that presented two lower release rates GlasSeal® connectors (Supelco Inc., Bellefonte, of phenylacetaldehyde in combination with Trécé PA) was used. A deactivated fused silica column, S. frugiperda pheromone lures. Phenylacetalde- 8.0 cm long by 0.5 mm ID, was connected between hyde was injected into disposable 100 µl glass mi- the injector and the retention gap column. This crocapillary pipets (Kimble, Vineland, NJ) that column permitted the use of 0.4 mm OD stainless had been flame-sealed at one end, and filled so steel needles with a septum injector for on-column that there would be a 10-mm length of the vapor- injections. The retention gap column used was 10 air column above the liquid. Holes were drilled m by 0.25 mm ID deactivated fused silica and the into corks that were placed in the canopy of the analytical column used for analysis was a 30 m by Unitrap, and microcapillaries were placed in the 0.25 mm ID (0.25 µm film) SE-30. corks. The double phenylacetaldehyde release Release rates of phenylacetaldehyde in the mi- rate contained two microcapillaries of the syn- crocapillaries used in the 1998 experiment were thetic compound. Therefore, the three treatments determined in a manner similar to that reported were: (1) Trécé S. frugiperda pheromone lures, (2) by Weatherston et al. (1985a, b), by which rates Trécé lures plus phenylacetaldehyde in one micro- are estimated mathematically based on size of mi- capillary pipet, or (3) Trécé lures plus phenyl- crocapillary, length of vapor-air column above the acetaldehyde in two pipets. Traps contained in- liquid, and volatility of the formulated compound. secticide strips to kill insects. The experiment was designed as a randomized complete block Statistics with four blocks of the three treatments, and trap location within a block was randomized weekly. For each experiment, insect numbers were Trapping began 27 April and ended 12 June. Ad- compared across treatments in a split block anal- ditionally, all aculeate Hymenoptera that were ysis of variance (ANOVA), where treatment was

290 Florida Entomologist 84(2) June 2001 the main plot and date was the subplot (Steel & Torrie 1980). To satisfy ANOVA assumptions, counts were log (x + 1) transformed before analysis. Treatment means or treatment combinations were separated by a LSD mean separation test or orthogonal comparisons (PROC GLM, CONTRAST statement, SAS Institute 1996). Untransformed means (±SE) are given in text and ﬁgures, whereas statistical results refer to transformed data.

RESULTS

1997

Fall armyworm males were collected from late Fig. 2. Release rate (µg/h) of phenylacetaldehyde July through early October, with a peak capture from hollow polyethylene stoppers over 14 days. of over 100 moths per night per trap in early Sep- tember (Fig. 1). Traps baited with pheromone alone consistently captured more moths than Several species of Hymenoptera were collected traps baited with both pheromone and phenylac- in relatively large numbers, including the bum- etaldehyde, or traps baited with phenylacetalde- blebee Bombus pennsylvanicus (De Geer), and the hyde alone (mean ± SE, moths per night: wasps Scolia n. nobilitata (F.), and Larra bicolor pheromone alone 21.6 ± 3.0, pheromone + pheny- F. (Table 1). More Sphecoidea were found in phe- lacetaldehyde 13.7 ± 1.6, phenylacetaldehyde nylacetaldehyde-baited traps than pheromone- alone 0.12 ± 0.03; F = 196.7; df = 2, 4; P < 0.0001). baited traps (Fig. 4) (contrast: F = 6.5; df = 1, 6; P The release rate for phenylacetaldehyde in the = 0.0436). Numbers of Apoidea, Scolioidea, Ves- stoppers over the 14-day period averaged 492.9 ± poidea, and Tiphioidea were similar among the 15.1 µg/h (Fig. 2). different treatments (P > 0.05). These results generally agree with those found in Meagher & 1998 Mitchell (1999).

Male fall armyworm numbers in traps were DISCUSSION low until late May (Fig. 3). More moths per night were collected from 29 May through 15 June in Previous research showed a numerical in- pheromone-baited traps (27.1 ± 5.0) than phero- crease in captures of males with traps baited with mone + phenylacetaldehyde-baited traps (12.5 ± both sex pheromone and phenylacetaldehyde 3.4) (F = 5.1; df = 2, 6; P = 0.0511). Capture in with ﬁve moth species (Noctuidae, Plusiinae) pheromone + double phenylacetaldehyde-baited (Creighton et al. 1973). In fact, most of the suc- traps (13.6 ± 2.4) was intermediate. The release cessful studies of attraction to phenylacetalde- rate for phenylacetaldehyde in the microcapillary hyde have been completed with species of tubes was initially 230.8 ng/h for the single tube Plusiinae (Smith et al. 1943; Creighton et al. and 461.6 ng/h for the double rate.

Fig. 3. Collection of male fall armyworm in Unitraps Fig. 1. Collection of male fall armyworm in Unitraps baited with sex pheromone only, or combinations with baited with sex pheromone, phenylacetaldehyde (PA two rates of phenylacetaldehyde (+PA and +2 PA), Ala- only), or a combination (+PA), Alachua Co., FL, 1997. chua Co., FL, 1998.

Meagher: Trapping FAW with Floral Volatiles 291

TABLE 1. SPECIES AND NUMBER OF HYMENOPTERA COLLECTED IN THE 1998 FLORAL LURE EXPERIMENTS, ALACHUA CO., FL.

Superfamily/family/tribe/genus/species Number collected

Apoidea Anthophoridae Anthophorinae Melissodes spp. 4 Xylocopinae Xylocopa virginica (L.) 2 Unidentiﬁed anthophorids 12 Apidae Bombinae Bombus bimaculatus Cresson 1 B. fraternus (Smith) 3 B. impatiens Cresson 7 B. pennsylvanicus (De Geer) 53 Apinae Apis mellifera L. 6 Halictidae Halictinae Agapostemon splendens (Lepeletier) 11 Scolioidea Scoliidae Campsomerinae Campsomeris (Dielis) plumipes fossulana (F.) 8 Scoliinae Scolia (Discolia) n. nobilitata (F.) 20 Sphecoidea Sphecidae Larrinae Larra bicolor F. 19 Philanthinae Cerceris bicornuta Guérin 11 Sphecinae Ammophila sp. 7 Bicyrtes quadrifasciata (Say) 3 Eremnophila aureonotata (Cameron) 1 Sceliphron caementarium (Drury) 1 Sphex sp. 1 Tiphioidea Tiphiidae Myzininae Myzinum sp. 7 Vespoidea Vespidae Polistinae Polistes carolina (L.) 2 Polistes sp. 13

1973; Cantelo & Jacobson 1979; Haynes et al. The disappointing results in these field experi- 1991; Landolt et al. 1991; Heath et al. 1992). ments showed that wild S. frugiperda moths were However, our research with laboratory-reared fall not attracted to phenylacetaldehyde-baited traps armyworm (Noctuidae, Amphipyrinae) in a flight alone, or in combination with sex pheromone. tunnel showed increased percentages of upwind Landolt et al. (1991) and Heath et al. (1992) re- flight and source contact in combination with a ported that cabbage looper, Trichoplusia ni (Hüb- sex pheromone lure (Meagher & Mitchell 1998). ner), moths responded to release rates ranging

292 Florida Entomologist 84(2) June 2001

that stimulate upwind flight in cabbage looper moths. J. Chem. Ecol. 17: 637-646. HEATH, R. R., AND A. MANUKIAN. 1992. Development and evaluation of systems to collect volatile semio- chemicals from insects and plants using charcoal-in- fused medium for air purification. J. Chem. Ecol. 18: 1209-1226. HEATH, R. R., P. J. LANDOLT, B. DUEBEN, AND B. LENC- ZEWSKI. 1992. Identification of floral compounds of night-blooming jessamine attractive to cabbage looper moths. Environ. Entomol. 21: 854-859. LANDOLT, P. J., B. LENCZEWSKI, AND R. R. HEATH. 1991. Lure and toxicant system for the cabbage looper (Lepidoptera: Noctuidae). J. Econ. Entomol. 84: 1344-1347. Fig. 4. Number of Hymenoptera captured per day in MEAGHER, JR., R. L., AND E. R. MITCHELL. 1998. Pheny- phenylacetaldehyde- (+PA) or double rate phenylacetal- lacetaldehyde enhances upwind flight of male fall ar- dehyde-baited (+2 PA) bucket traps in corn. All traps myworm (Lepidoptera: Noctuidae) to its sex contained Trécé S. frugiperda pheromone lures, Ala- pheromone. Florida Entomol. 81: 556-559. chua Co., FL, 1998. Means within Sphecoidea followed MEAGHER, JR., R. L., AND E. R. MITCHELL. 1999. Non- by the same letter are not significantly different. target Hymenoptera collected in pheromone- and synthetic floral volatile-baited traps. Environ. Ento- mol. 28: 367-371. from 50 ng/h to 4 µg/h in flight tunnel and screen- MITCHELL, E. R., J. H. TUMLINSON, AND J. N. MCNEIL. cage bioassays. Because counts were lower in the 1985. Field evaluation of commercial pheromone for- combined phenylacetaldehyde + pheromone- mulations and traps using a more effective sex pher- baited traps, it is assumed that fall armyworm omone blend for the fall armyworm (Lepidoptera: moths were able to perceive phenylacetaldehyde Noctuidae). J. Econ. Entomol. 78: 1364-1369. at the release rates tested (230 ng/h—500 µg/h), SAS INSTITUTE. 1996. SAS/STAT guide for personal computers, version 6.12 ed. SAS Institute, Cary, NC. but were repelled from those traps. SHAVER, T. N., P. D. LINGREN, J. R. RAULSTON, AND H. F. MARSHALL, JR. 1998. Plant chemicals as attracta- ACKNOWLEDGMENTS nts for Helicoverpa zea (Lepidoptera: Noctuidae) and other insect species. Southwest. Entomol. Suppl. No. Release rate determination of the phenylace- 21: 37-44. taldehyde was conducted in the laboratory of R. SMITH, C. E., N. ALLEN, AND O. A. NELSON. 1943. Some Heath by B. Dueben (USDA-ARS). Technical sup- chemotropic studies with Autographa spp. J. Econ. port in the field was provided by J. Brady and C. Entomol. 36: 619-621. Dillard. N. D. Epsky (USDA-ARS), D. L. Kline STEEL, R. G. D., AND J. H. TORRIE. 1980. Principles and procedures of statistics: a biometrical approach. (USDA-ARS) and J. Nation (Univ. of Florida) pro- McGraw-Hill, NY. vided helpful reviews of an earlier manuscript. TUMLINSON, J. H., E. R. MITCHELL, P. E. A. TEAL, R. R. HEATH, AND L. J. MENGELKOCH. 1986. Sex phero- REFERENCES CITED mone of fall armyworm, Spodoptera frugiperda (J. E. Smith): Identification of components critical to at- CANTELO, W. W., AND M. JACOBSON. 1978. Corn silk vol- traction in the field. J. Chem. Ecol. 12: 1909-1926. atiles attract many species of moths. J. Environ. Sci. WEATHERSTON, I., D. MILLER, AND L. DOHSE. 1985a. Health A14: 695-707. Capillaries as controlled release devices for insect CANTELO, W. W., AND M. JACOBSON. 1979. Phenylace- pheromones and other volatile substances—a re- taldehyde attracts moths to bladder flower and to evaluation: part I. Kinetics and development of a blacklight traps. Environ. Entomol. 8: 444-447. predictive model for glass capillaries. J. Chem. Ecol. CREIGHTON, C. S., T. L. MCFADDEN, AND E. R. CUTH- 11: 953-965. BERT. 1973. Supplementary data on phenylacetalde- WEATHERSTON, I., D. MILLER, AND J. LAVOE-DORNIK. hyde: an attractant for Lepidoptera. J. Econ. 1985b. Capillaries as controlled release devices for in- Entomol. 66: 114-115. sect pheromones and other volatile substances—a re- HAYNES, K. F., J. Z. ZHAO, AND A. LATIF. 1991. Identifi- evaluation: part II. Predicting release rates from celcon cation of floral compounds from Abelia grandiflora and teflon capillaries. J. Chem. Ecol. 11: 967-968.

Myartseva & Ruíz: Mexican Marietta 293

MEXICAN SPECIES OF PARASITOID WASPS OF THE GENUS MARIETTA (HYMENOPTERA: APHELINIDAE)

SVETLANA N. MYARTSEVA1,2 AND ENRIQUE RUÍZ-CANCINO1 1Centro de Investigación, UAMAC, Universidad Autónoma de Tamaulipas, 87149 Cd. Victoria, Tamaulipas, México

2National Institute of Deserts, Flora and Fauna, Ministry of Nature Use and Environment Protection of Turkmenistan, 744000 Ashgabat, Turkmenistan

ABSTRACT

A key to females and notes on 5 species of the Mexican fauna of the genus Marietta are given. Marietta montana n. sp. from the Biosphere Reserve “El Cielo”, State of Tamaulipas, México, is described and illustrated.

Key Words: Marietta n. sp., Tamaulipas, México

RESUMEN

Se presenta una clave y notas acerca de las 5 especies conocidas de la fauna mexicana del género Marietta. Se describe e ilustra Marietta montana n. sp. de la Reserva de la Biosfera “El Cielo” del Estado de Tamaulipas, México.

The world fauna of the genus Marietta in- Pinus trees, including P. patula, is described here cludes 19 species (Hayat 1998), seven of which are as new to science. known in the Nearctic region (Woolley 1997), in- Parasitoids were reared under laboratory con- cluding three in México. Species of the genus ditions from scale insects collected on young twigs Marietta are almost always hyperparasitoids of of Pinus trees in mountain landscapes. Some Homoptera, including Diaspididae, Coccidae and adult parasitoid individuals were mounted on other families. Beardsley & Tsuda (1990) noted card points, others were stored in 75% alcohol and Marietta pulchella Howard as a primary parasi- later some females and males were mounted on toid of Conchaspis angracei Cockerell (Fam. Con- slides in Canada balsam. The morphology of these chaspididae), which is common species of scale specimens was studied. The senior author illus- insect in México (Miller 1996). trated the morphological structures from slide The genus Marietta often referred to in earlier preparations using a stereoscope and drawing ap- literature as Perissopterus, a junior synonym, was paratus (by increasing × 280, × 400). first recorded from México by Howard (1895), who Some Marietta specimens were received for described M. mexicana from Guadalajara. In ad- study from Dr. V. A. Trjapitzin. The species iden- dition to the three species previously known from tification was carried out using the key of world México, we found M. graminicola Timberlake fauna of the genus Marietta (Hayat 1986). The 1925, collected in the State of Tamaulipas, and a original description of Marietta mexicana new species, M. montana Myartseva & Ruíz-Can- (Howard 1895) and other publications containing cino, reared from a diaspine scale in Tamaulipas. information about Mexican species of the genus Marietta were analyzed. MATERIALS AND METHODS An illustrated key to females and notes on five Mexican species of Marietta are provided, as well Specimens of the parasitoid genus Marietta as information on the genus Marietta. were collected by sweeping in several landscapes of México and reared from different hosts of scale insects (Homoptera: Coccoidea). One Marietta Genus: Marietta Motschulsky, 1863; species, reared in 1998 in La Perra, Reserve “El Type species: Marietta leopardina Motschulsky, 1863; Cielo”, Tamaulipas, México, from an unidentified Synonymy: Perissopterus Howard, 1895; Melanaspis species (Homoptera: Diaspididae) on Pseudaphelinus Brèthes, 1918.

KEY TO FEMALES OF THE KNOWN MEXICAN SPECIES OF MARIETTA

1. Antennal scape subtrapezoidal, less than 2× as long as wide, with irregular fuscous patch in the middle (Fig. 1). Fore wing with apical margin widely hyaline ...... M. graminicola Timberlake

294 Florida Entomologist 84(2) June 2001

—Antennal scape narrower, at least 2.5× as long as wide, with one or more narrow blackish longitudinal, transverse bands. Fore wing with apical margin not hyaline...... 2 2. Antennal scape with one blackish longitudinal band along the middle (Fig. 2). Fore wing with apical margin infuscated ...... M. pulchella (Howard) —Antennal scape with two blackish longitudinal or oblique transverse bands or with one transverse band (Figs. 3-5). Fore wing with fuscous band not touching apical margin ...... 3 3. Antennal scape more than 3× as long as wide, with two blackish longitudinal bands (Fig. 3). Clava with fuscous base and apex...... M. mexicana (Howard) —Antennal scape 3× or less as long as wide, with two blackish oblique transverse bands in the middle, or with one transverse band (Figs. 4-5) ...... 4 4. Antennal scape with two complete oblique transverse bands (Fig. 4). Clava uniformly fuscous ...... M. picta (André) —Antennal scape with one oblique transverse band, not touching its dorsal margin (Fig. 5). Clava with fuscous base and apex ...... M. montana n. sp.

Marietta montana Myartseva & Ruíz-Cancino, n. sp. whitish, with two elongate fuscous spots on each (Figs. 6-13) side; mesoscutum and scutellum yellowish or orange-yellowish; mesopleuron, metanotum and Description propodeum whitish-yellow. Fore wing whitish, or- namented with irregular fuscous pattern as in Female (Figs. 6-11). Fig. 10. Legs pale yellowish-white, with blackish bands: fore tibia with two bands, middle and hind Length: 0.80-0.93 mm, mean of 2 specimens in tibia each with three bands; basitarsus, tibial alcohol 0.93 mm and of 3 specimens on pins 0.80 spur, two last tarsal joints of middle leg fuscous, mm. basitarsus and two last tarsal joints of fore and Coloration: Head yellow, with slight orange hind legs fuscous. Gaster largely dusky dorsally, tinge; occiput with brown band around foramen, sides of tergites whitish-yellow, with black mar- darker on its sides; lower margin of face narrowly gins and two longitudinal spots on each. Ex- brownish; apices of mandibles dark brown to panded part of ovipositor brownish-black. black; maxillary and labial palpi whitish; setae on Structure: Head of the same width as mesos- frontovertex and face fuscous. Scape pale yellow- oma (in dried specimens wider than mesosoma), ish-white, with a short incomplete brown-blackish 1.5× wider than long, and approximately 1.5× oblique transverse band and small blackish spot wider than high. Frontovertex length slightly below on the ventral margin; pedicel pale yellow- more than width (Fig. 6). Ocelli in slightly obtuse ish-white except brown-blackish basal part, espe- triangle, distance from hind ocellus to eye and oc- cially dorsally, two funicle segments brownish; cipital margin approximately 1.5× the diameter of ﬁrst segment of clava in basal ⅓, second segment ocellus. Antennal (Fig. 7) radicle (R), scape (S), in basal half and on apex infuscated. Pronotum pedicel (P), two ﬂagellar segments (F1, F2) and two

Figs. 1-5. Antennal scape of Marietta species: 1-M. graminicola Timberlake, 2-M. pulchella (Howard), 3-M. mexicana (Howard), 4-M. picta (André) (by Jasnosh, 1966), 5-M. montana, sp. Nov.

Myartseva & Ruíz: Mexican Marietta 295

Figs. 6-13. Marietta montana, n. sp.: 6-frontovertex, 7-antenna, female, 8-mandible, 9-mesosoma, 10-fore wing, 11-ovipositor, 12-antenna, male, 13-male genitalia.

clava segments (F3, F4) with the following ratios of F4 with 2 and 2,2 linear sensillae in two rows, re- × length to width: R-6.5:6.5, S-45:15, P-25:10, F1-4:6, spectively. Clava 1.5 longer than scape. Mandi-

F2-4:8, F3-25:15, F4-42:16. Flagellar segments F3- bles with two teeth and short truncation (Fig. 8).

296 Florida Entomologist 84(2) June 2001

Maxillary and labial palpi 2- and 1-segmented, Holotype: female (card mounted). Paratypes: 2 respectively. Mesosoma (Fig. 9) with broad mesos- females and 1 male card mounted; 1 female, 2 cutum, 1.5× as wide as long, with 12-14 setae sit- males in Canada balsam; 2 females, 2 males in uated in 3 rows symmetrically, with reticulate 75% alcohol. México, Tamaulipas, Gómez Farías, sculpture; scutellum with 2 pairs of setae and Reserve “El Cielo”, La Perra (1900 m) 23-X-1998, similar sculpture to mesoscutum; the base of each S. N. Myartseva, all reared from diaspine scales setae on mesoscutum and scutellum surrounded Melanaspis sp. on Pinus spp. by small brownish spot; each side lobe and axilla with one seta. Fore wing (Fig. 10) 2.6× as long as Deposition wide; the longest marginal cilia more than 5.6× shorter than the maximum width of wing. Costal Female holotype and one male paratype (card cell short; submarginal vein with 3-4 setae; mar- mounted) are deposited in the National Museum ginal vein a little longer than submarginal vein, of Natural History, Washington, D.C., USA; one with 6-7 setae along anterior margin; stigmal female paratype (card mounted) is in the Depart- vein very short; basal part of wing glabrous. Disc ment of Zoology, Institute of Biology, National Au- of wing with two hyaline cells under stigmal vein tonomous University of México (UNAM), México in transverse row, apex with a triangular hyaline City; the remaining paratype specimens are de- cell. Metasoma slightly longer than mesosoma; posited in the Insect Museum-UAM Agronomía y ovipositor protruding from apex, 2.2× as long as Ciencias, Autonomous University of Tamaulipas middle tibia; valvulae II 1.8 times as long as val- (UAT), Ciudad Victoria, Tam., México. vulae III (Fig. 11). Etymology Male (Figs. 12-13). Marietta montana is named for its inhabita- Length: 0.66-0.93 mm, mean of 2 specimens in tion of the mountains of the Sierra Madre Orien- alcohol 0.93 mm, and one specimen on pin 0.66 tal, at La Perra, Gómez Farías (1900 m). mm. Coloration: Similar to female. NOTES ON OTHER MEXICAN SPECIES Structure: Differs from the female by rounded OF MARIETTA EXAMINED apex of gaster, by the 3—segmented ﬂagellum (clava undivided) (Fig. 12). Length: width ratios 1. M. graminicola Timberlake, 1925

of antennal segments R-F3 as follows: R-2,0, S-3,0, Material: México, Tamaulipas: Hidalgo, “El Ti-

P-2,2, F1-0,5, F2-1,6, F3-3,8; relative length of each gre”, VIII. 1992, 1 female (O. Escamilla); Gómez segment to F1 length: R-2,5, S-12,0, P-6,0, F1-1,0, Farías, Reserve “El Cielo”, Canindo, 4-X-1995, 1

F2-1,2, F3-16,2. Clava with 3,2,2 linear sensillae in female (V. Trjapitzin); Ciudad Victoria, UAT, X- three rows, respectively. Clava 1.4× longer than 1995, 1 female (V. Trjapitzin). scape. Genitalia (Fig. 13): phallobase 3.6× as long Using Hayat (1986) we determined that speci- as wide, digitus 1.5× as long as paramere; each mens collected in México belong to the species M. digitus with two teeth, each paramere with one graminicola. Literature: Coronado-Padilla & seta at apex. Sosa-Esquiliano, 1966:43 (México); Contreras- Coronado, 1972: 27-30 (México, ex Antonina Comments graminis Maskell); Rivera-Guillot, 1972:15 (Méx- ico); De Santis, 1979:317 (México); Hayat, Marietta montana n. sp. is similar in colora- 1986:10 (?México). tion and structure to Marietta picta (André) and especially to Marietta mexicana (Howard). M. Distribution: México, ?USA, Hawaii. montana can be distinguished from M. mexicana which has one brown transverse band on scape, 2. M. mexicana (Howard, 1895) the apical segment of scape clava more than 2× Material: México, Morelos, Cuernavaca, longer than ﬁrst segment, the disc of fore wing be- UAEM: 20-25-VIII-1995, 1 female (V. Trjapitzin); fore linea calva with two hyaline cells in the mid- 7-IV-1996, 1 female (E. Chouvakhina); 30-IV- dle; a longer marginal fringe; the body not so 1996, 1 female (G. Peña-Chora); all ex Parasaisse- extensively fuscous; the placoid sensillae on tia nigra (Nietner) (Homoptera: Coccidae), new scutellum situated anterior to level of the second host record for M. mexicana, on Ficus benjamina; pair of setae, which are closer to apex of scutel- San Luis Potosí, 11-XI-1999, 2 females (S. Myart- lum; and the propodeum with a distinctly ele- seva), ex white scale; 4 females and 2 males (S. vated median triangular area. Females and males Myartseva) ex Ceroplastes sp. of M. montana can be distinguished from M. picta Literature: Howard, 1895: 21-23 Guadalajara, which has one brown transverse band on scape, ex Ceroplastes sp., Pseudococcus agavis MacGre- infuscated clava on base and apex, and a different gor as juccae, Coccus hesperidum Linnaeus); fore wing color pattern. García-Martell, 1973: 19 (Morelos, Cuernavaca); Myartseva & Ruíz: Mexican Marietta 297

Domínguez & Carrillo, 1976:195 (Chapingo, ex REFERENCES CITED Saissetia oleae Olivier); De Santis, 1979:317 (México); Hayat, 1986: 13 (México); Alvarado-Me- ALVARADO-MEJÍA, F. E., AND A. H. GONZÁLEZ-HERNÁN- jía & González-Hernández, 1990:43 (México, ex DEZ. 1990. Taxonomía de las especies de Aphytis Howard (Hymenoptera: Aphelinidae) y otros géneros Lecanodiaspis rufescens Cockerell, Quadraspid- en el área citrícola de Nuevo León. Biotam 2(3): 42-57. iotus perniciosus Comstock, Aphytis holoxanthus BEARDSLEY, J. W., AND D. M. TSUDA. 1990. Marietta De Bach). pulchella Howard (Hymenoptera: Aphelinidae), a primary parasite of Conchaspis angraeci Cockerell (Homoptera: Conchaspididae). Proc. Hawaiian Ento- Distribution: Nearctic, Neotropical, Japan. mol. Soc. 30: 151-153. CONTRERAS-CORONADO, A. 1972. Clave práctica para al- 3. M. picta (André, 1878). gunas familias de Hymenoptera relacionadas con el Literature: Rivera-Guillot, 1972:15 (México); combate biológico en la República Mexicana. Fitófilo De Santis, 1979:317-318 (México); Hayat, 1986: 25(67): 27-30. 13-15, 1998: 56-57 (México). CORONADO-PADILLA R., AND E. SOSA-ESQUILIANO. 1966. M. picta was has only been reported once in Campaña contra la mosca pinta y la escama alg- Mexican publications, more than 20 years ago. odonosa de los pastos. Fitófilo 19(50): 5-52. This species is widely distributed in the Palaearc- DE SANTIS, L. 1979. Catálogo de los himenópteros calci- tic region. doideos de América al sur de los Estados Unidos. La Plata (Argentina). 488 pp. DE SANTIS, L., AND P. FIDALGO. 1994. Catálogo de hi- Distribution: Nearctic, Palaearctic, India. menópteros calcidoideos. Serie de la Acad. Nac. de Agron. y Veterin. (Buenos Aires) 13: 1-154. 4. M. pulchella (Howard), 1881 DOMÍNGUEZ, J. R., AND J. L. CARRILLO S. 1976. Lista de Material: México, Tamaulipas, Ciudad Victo- los insectos en la colección entomológica del Instituto ria: 12-X-1998, 1 female; 30-XII-1998, 1 male; 8-I- Nacional de Investigaciones Agrícolas. Segundo suple- 1999, 1 female (S. Myartseva), all ex Saissetia mento. SAG. México. Folleto Miscélaneo No. 29: 1-245. GARCÍA-MARTELL, C. 1973. Primera lista de insectos en- oleae (Olivier) (Homoptera: Coccidae), new host tomófagos de interés agrícola en México. Fitófilo for M. pulchella, on Nerium oleander. 26(68): 1-47. Literature: Hayat, 1986: 15 (México); De San- HAYAT, M. 1986. Notes on some species of Marietta (Hy- tis & Fidalgo, 1994: 95 (México). menoptera: Aphelinidae), with a key to world spe- Distribution: Nearctic, Neotropical. cies. Colemania 2: 1-18. HAYAT, M. 1998. Aphelinidae of India (Hymenoptera: Chalcidoidea): A taxonomic revision. Memoirs of En- ACKNOWLEDGMENTS tomology, International. Vol. 13. Associated Publish- ers. Gainesville. 416 pp. We thank Dr. V. A. Trjapitzin (Universidad Au- HOWARD, L. O. 1895. Revision of the Aphelininae of tónoma de Tamaulipas and Zoological Institute of North America, a subfamily of hymenopterous para- the Russian Academy of Sciences), for collecting sites of the family Chalcididae. U.S. Dept. Agric. En- some specimens and for his suggestions for this tomol. Techn. Ser. 1: 4-44. study. To Dr. G. Evans for his advise, comments MILLER, D. R. 1996. Checklist of the scale insects (Coc- coidea: Homoptera) of México. Proc. Entomol. Soc. and review of this manuscript and Dr. A. B. Ha- Washington 98(1): 68-86. mon for the identification of Coccoidea hosts RIVERA-GUILLOT, R. 1972. La escama algodonosa de los (FSCA. Division of Plant Industry, Florida De- pastos (Antonina graminis Mask). Investigación, partment of Agriculture and Consumer Services). daños y control en el Suroeste de México. Fitófilo We also thank biologist Rafael Brito, who orga- 25(67): 1-26. nized the trip to the Sierra Madre Oriental, Re- TIMBERLAKE, P. M. 1925. Description of new chalcid- serve “El Cielo”. We also thank the Research flies from Panama and Hawaii (Hymenoptera). Proc. Center (UAMAC, UAT) for its continuous support Hawaiian Entomol. Soc. 6(1): 173-193. for the study of Mexican Hymenoptera. Funding WOOLLEY, J. B. 1997. Aphelinidae, pp. 134-150. In G. A. P. Gibson, J. T. Huber and J. B. Woolley (eds.). Anno- for the senior author was provided by CONA- tated keys to the genera of Nearctic Chalcidoidea CyT—México, through the Program “Cátedras (Hymenoptera). NRC Res. Press, Ottawa, Ontario, Patrimoniales de Excelencia”. Canada. 794 pp.

298 Florida Entomologist 84(2) June 2001

EFFECT OF TEMPERATURE ON EGG DEVELOPMENT OF DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONIDAE)

STEPHEN L. LAPOINTE USDA-ARS, U.S. Horticultural Research Laboratory, 2001 South Rock Road, Ft. Pierce, FL 34945

The Diaprepes root weevil, Diaprepes abbre- tion (Wolcott 1933). Eggs on wax paper strips viatus (L.), has become a key pest of citrus and or- were collected from cages twice daily and placed namental plants in Florida since its introduction in plastic vials with plastic caps in temperature- to the state in 1964. Adult D. abbreviatus feed and controlled growth chambers with a photoperiod of oviposit on leaves of many plant species in addi- 12:12 L:D and constant temperatures of 12, 15, tion to citrus including crops such as cassava, cot- 18, 19, 21, 22, 26, 30, and 32°C. Vials were misted ton, peppers, potatoes, sugar cane, and sweet periodically with sterile water and re-capped to potatoes. As neonate larvae emerge, they drop to avoid desiccation of the eggs. The wax paper the ground and crawl on the surface for a period of strips were gently separated 2 to 3 d after ovipo- up to three hours (Jones & Schroeder 1983) before sition to facilitate emergence of the neonate lar- burrowing into the soil to feed on roots. Eggs are vae. Vials were checked daily for neonate larvae subject to predation by ants and earwigs (Tryon and time to hatch was recorded. After egg hatch 1986) and parasitization by wasps such as the eu- commenced, the number of eggs that failed to lophid Quadrastichus haitiensis Gahan. Recent hatch after 7 consecutive days of no neonate efforts to establish exotic egg parasitoids in Flor- emergence in the vial was recorded. The mean ida have met with some initial success (Hall et al. and median number of days to egg eclosion and 2000), but a more complete understanding of host 95% conﬁdence intervals were calculated (Snede- biology may contribute to effective rearing and cor & Cochran 1967). The developmental rate (in- release. verse of median no. days to eclosion) was plotted Lapointe and Shapiro (1999) and Lapointe against temperature and a linear regression was (2000) described the effects of humidity and tem- calculated (Legg et al. 2000). perature on larval and pupal development of Wolcott (1936) reported that eggs of D. abbre- D. abbreviatus. To complete the description of the viatus hatched 7 d after oviposition and suggested thermal requirements for development of D. ab- that this was unaffected by temperature. In this breviatus, I report here the response of eggs of study, no eggs hatched when held at constant D. abbreviatus to a range of temperatures and de- temperatures of 12 or 32°C. The percentage of un- scribe the thermal upper and lower limits of egg hatched eggs ranged from 6% at 18°C to 43% at development. 30°C (Table 1). For the constant temperatures re- Male and female adult D. abbreviatus were ob- gimes tested here, there was a positive linear retained from a laboratory colony at the U.S. Horti- lationship (y = 0.009x - 0.108, r2 = 0.99) between cultural Research Laboratory, Orlando, FL, now developmental rate based on median values and located at Ft. Pierce, FL. Approximately 20 male/ temperature between 15 and 30°C (Fig. 1). Ex- female pairs were placed in a screened cage (30 by trapolation based on the regression equation 30 by 30 cm) and provided with bouquets of citrus yielded a median functional lower developmental foliage as food and strips of wax paper for oviposi- threshold of 12.0°C. The upper thermal limit for

TABLE 1. SURVIVAL OF EGGS AND NUMBER OF DAYS (MEDIAN AND MEAN ±95%CI) REQUIRED TO COMPLETE DEVELOP- MENT OF EGGS OF THE DIAPREPES ROOT WEEVIL AT 9 CONSTANT TEMPERATURES.

Days to hatch Temperature Survival (°C) n (%) Median Mean

12 1150 0.0 15 736 no data 27.0 ± 0.03 26.8 ± 1.04 15 818 78.0 28.1 ± 0.03 27.7 ± 1.07 18 2367 93.5 18.5 ± 0.01 18.0 ± 0.11 19 938 84.6 14.4 ± 0.05 13.9 ± 0.44 21 671 87.3 11.4 ± 0.07 10.9 ± 0.56 22 622 76.5 11.4 ± 0.15 11.3 ± 1.13 26 1045 80.8 7.1 ± 0.06 6.7 ± 0.67 30 566 57.4 5.8 ± 0.12 5.5 ± 0.69 32 750 0.0

Scientiﬁc Notes 299

time to hatch continued to decrease compared with cooler temperatures (Table 1). Of the temperatures tested, 26°C appears optimal based on survival and development rate. This information should be useful for those interested in rearing the Diaprepes root weevil or its egg parasitoids.

SUMMARY

No eggs of the Diaprepes root weevil survived constant temperatures of 12 or 32°C. There was a positive linear relationship (y = 0.009x - 0.108, r2 = 0.99) between developmental rate and temperature between 15 and 30°C. Extrapolation based on the regression equation yielded a median func- Fig. 1. Relationship of egg development rate versus tional lower developmental threshold of 12.0°C constant temperature for the Diaprepes root weevil in- and the upper thermal limit for constant temper- cluding extrapolation to the median functional lower atures occurred between 30 and 32°C. developmental threshold.

REFERENCES CITED constant temperatures occurred between 30 and 32°C because no eggs survived at 32°C. This is HALL, D. G., J. PEÑA, R. FRANQUI, R. NGUYEN, P. STANSLY, C. MCCOY, S. L. LAPOINTE, R. C. ADAIR, somewhat surprising since mean daily maximum AND B. BULLOCK. 2000. Status of biological control air temperatures in Florida citrus groves exceed by egg parasitoids of Diaprepes abbreviatus (Co- 32°C from May through September (Lapointe leoptera: Curculionidae) in citrus in Florida and Pu- 2000). On Puerto Rico, Wolcott and Martorell erto Rico. Biol. Control 46: 61-70. (1943) observed seasonality in occurrence of egg JONES, I. F., AND W. J. SCHROEDER. 1983. Study of first- clusters in sugarcane, with greatest abundance instar Diaprepes abbreviatus (Coleoptera: Curcu- occurring in June and a secondary peak in Sep- lionidae) activity for control purposes. J. Econ. Ento- tember and October. mol. 76: 567-569. It has been observed that female D. abbrevia- LAPOINTE, S. L. 2000. Thermal requirements for development of Diaprepes abbreviatus (Coleoptera: Cur- tus prefer to oviposit on mature, fully expanded culionidae). Environ. Entomol. 29: 150-156. citrus leaves and avoid flush or tender foliage. LAPOINTE, S. L., AND J. P. SHAPIRO. 1999. Effect of soil Various explanations can be put forth to explain moisture on development of Diaprepes abbreviatus this preference. First, the feeding preference of (Coleoptera: Curculionidae). Florida Entomol. 82: D. abbreviatus for tender foliage may have con- 291-299. tributed to a separate preference for oviposition LEGG, D. E., S. M. VAN VLEET, AND J. E. LLOYD. 2000. substrate (older leaves) to avoid egg consumption Simulated predictions of insect phenological events by feeding adults. Second, the shear forces ex- made by using mean and median functional lower erted by expanding leaves may dislodge egg developmental thresholds. J. Econ. Entomol. 93: 658-661. masses cemented between leaf surfaces, thereby SNEDECOR, G. W., AND W. G. COCHRAN. 1967. Statistical exposing the egg mass. Finally, temperature expe- methods. The Iowa State University Press, Ames, IA. rienced by eggs of D. abbreviatus sandwiched be- 593 pp. tween actively transpiring citrus leaves may vary TRYON, E. H., JR. 1986. The striped earwig, and ant from ambient air temperatures, particularly ma- predators of sugarcane rootstock borer, in Florida ture leaves within a shaded canopy, thereby pro- citrus. Florida Entomol. 69: 336-343. tecting eggs from lethal temperature extremes. WOLCOTT, G. N. 1933. Otiorhynchids oviposit between Lower thermal limits for neonate larvae and paper. J. Econ. Entomol. 26: 1172-3. pupae were estimated to be 15°C (Lapointe 2000). WOLCOTT, G. N. 1936. The life history of Diaprepes ab- ° breviatus L., at Rio Piedras, Puerto Rico. J. Agr. In this study, egg survival at 15 C was 78% and Univ. Puerto Rico 20: 883-914. ° development time was 4.8 times greater at 15 C WOLCOTT, G. N., AND L. F. MARTORELL. 1943. The sea- compared with that at 30°C. There appeared to be sonal cycle of insect abundance in Puerto Rican cane an increase in mortality at 30°C but the mean fields. J. Agr. Univ. of Puerto Rico 27: 85-104.

300 Florida Entomologist 84(2) June 2001

FIRST RECORD OF CHRYSOMYA MEGACEPHALA (DIPTERA: CALLIPHORIDAE) IN GEORIGA, U.S.A.

JEFFERY K. TOMBERLIN1, WILL K. REEVES2 AND D. CRAIG SHEPPARD1 1Department of Entomology, University of Georgia, P.O. Box 748 Tifton, GA 31793, USA

2Department of Entomology, Clemson University, 114 Long Hall, Clemson, SC 29634, USA

Four species of Old World Chrysomya Robin- Chrysomya megacephala has become estab- eau-Desvoidy (Diptera: Calliphoridae) have been lished in North America since being introduced introduced to the New World during the past 20 into Brazil (Baumgartner & Greenberg 1984). years (Wells 1991). Chrysomya megacephala (F.) The ﬁrst record of Chrysomya megacephala in the was ﬁrst collected from Brazil in 1975 and was United States was from a single specimen col- thought to have originated from southern Africa lected in Texas (Wells 1991). This species has since (Baumgartner & Greenberg 1984). been collected in California (Greenberg 1988),

TABLE 1. A SUMMARY OF CALLIPHORID SPECIES COLLECTED AT BAIT STATIONS IN GEORGIA AND SOUTH CAROLINA FROM 30 MARCH THROUGH 26 JULY 1999.

Species # Collected Date Location Bait

Calliphora vicina Robineau-Desvoidy 11 28 May Townes Co, GA Chicken liver 01 28 May Oconee Co, SC Chicken liver 01 01 June Dekalb Co, GA Chicken liver Chrysomya megacephala (Fabricius) 07 28 June Tift Co, GA Poultry facility Cochliomyia macellaria (Fabricius) 01 01 June Anderson Co, SC Chicken liver 03 14 June Pickens Co, SC Dung 05 28 June Tift Co, GA Poultry facility 13 23 July Pickens Co, SC Human dung 03 25 July Pickens Co, SC Human dung 01 26 July Pickens Co, SC Human dung Phaenicia cuprina (Weidemann) 43 28 May Jones Co, GA Chicken liver Phaenicia sericata (Meigen) 01 30 March Oconee Co, SC Chicken liver 23 28 May Jones Co, GA Chicken liver 02 28 May Pickens Co, SC Chicken liver 04 28 May Rabun Co, GA Chicken liver 07 01 June Fulton Co, GA Chicken liver 07 01 June Townes Co, GA Chicken liver 07 01 June Dekalb Co, GA Chicken liver 05 28 June Tift Co, GA Poultry facility Phormia regina (Meigen) 03 28 May Jones Co, GA Chicken liver 12 28 May Townes Co, GA Chicken liver 01 28 May Pickens Co, SC Chicken liver 01 28 May Oconee Co, SC Chicken liver 07 28 May Rabun Co, GA Chicken liver 05 01 June Anderson Co, SC Chicken liver 01 01 June Dekalb Co, GA Chicken liver 01 15 June Pickens Co, SC Sea urchin

Scientiﬁc Notes 301

Florida (Baumgartner 1993), New Mexico (DeJong did not contain any Chrysomya specimens col- 1995), and Alabama (Wells 2000). We present a lected in either state. new distribution record for Chrysomya megaceph- We thank Jeremy Greene and Gary Herzog for ala and associated calliphorid species in Georgia their helpful comments on an earlier draft of the and South Carolina. manuscript. We would also like to thank Peter We collected adult calliphorids by aerial net- Adler for providing transportation and the use of ting over various baits in Georgia and South his facilities while conducting this research. Carolina from 30 March through 26 July 1999. Baits were placed in open glass or metal contain- REFERENCES CITED ers at ground level. The University of Georgia Museum of Natural BAUMGARTNER, D. L. 1993. Review of Chrysomya rufifa- History and Clemson University Arthropod Col- cies (Diptera: Calliphoridae). J. Med. Entomol. 30: lection were examined for unreported records of 338-352. Chrysomya spp. from Georgia and South Caro- BAUMGARTNER, D. L., AND B. GREENBERG. 1984. The ge- lina. Voucher specimens of each species were de- nus Chrysomya (Diptera: Calliphoridae) in the New World. J. Med. Entomol. 21: 105-113. posited in the University of Georgia Museum of DE JONG, G. D. 1995. Report of Chrysomya megacephala Natural History and the Clemson University Ar- (Diptera: Calliphoridae) in northern New Mexico. thropod Collection. Adult calliphorid occurrences Entomol. News 106: 192. at bait stations are summarized in Table 1. GREENBERG, B. 1988. Chrysomya megacephala (F.) (Diptera: Calliphoridae) collected in North America and notes on Chrysomya species present in the New SUMMARY World. J. Med. Entomol. 25: 199-200. WELLS, J. D. 1991. Chrysomya megacephala (Diptera: Chrysomya megacephala was only collected in Calliphoridae) has reached the continental United States: Review of its biology, pest status, and spread Georgia, while five other calliphorid species were around the world. J. Med. Entomol. 28: 471-473. collected in both Georgia and South Carolina. The WELLS, J. D. 2000. Introduced Chrysomya (Diptera: Cal- University of Georgia Museum of Natural History liphoridae) flies in northcentral Alabama. J. Ento- and the Clemson University Arthropod Collection mol. Sci. 35: 91-92.

302 Florida Entomologist 84(2) June 2001

EASY-TO-HANDLE STICKY TRAP FOR FRUIT FLIES (DIPTERA: TEPHRITIDAE)

DAVID C. ROBACKER1 AND ROBERT R. HEATH2 1Crop Quality and Fruit Insects Research, Agricultural Research Service, U.S. Department of Agriculture, Kika de la Garza Suptropical Agricultural Research Center, 2413 E. Highway 83, Bldg. 200, Weslaco, TX 78596

2Subtropical Horticultural Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 1306 Old Cutler Rd., Miami, FL 33158

Heath et al. (1996, 1997) described a cylindrical sticky trap for tephritid fruit flies constructed with fruit fly adhesive paper (FFAP) (Atlantic Paste and Glue Co., Inc., Brooklyn, NY) which has an extremely tacky but non-messy adhesive. Capture rates with this trap were twice those of McPhail traps baited with the same lure. Unfortunately, the traps were difficult for users because they stick fast to almost everything they contact, including small birds in some field tests. In this work we describe a sticky trap made with the same paper but which avoids many of these problems. Experimental traps were constructed from fluorescent light green (E.I.C.MWG 17089) or yellow (fluorescent chartreuse, E.I.C.MWY 16823) FFAP. Green and yellow were chosen because they were the most attractive colors in previous experiments with many species of Tephritidae including the Mexican fruit fly (Katsoyannos 1989; Ro- backer et al. 1990). Except for the controls, all traps had black plastic mesh (Co-Polymer Gutter Guard, Amerimax Home Products, Lancaster, PA) stapled over the sticky surfaces. The FFAP was cut into 23 × 14 cm rectangles to equal the size of the Pherocon AM trap (Trece, Inc., Salinas, CA) that was used as the standard trap for this work. Plastic mesh was also cut into 23 × 14 cm rectangles. Mesh size (distance between plastic strands) initially was 0.7 × 0.7 cm. Mesh size was cut to 1.5 × 1.5 or 2.2 × 2.2 cm to test the effect of mesh size on trap efficacy. Thick- Fig. 1. Sticky trap (23 × 14 cm) for fruit flies con- ness of the plastic strands was approximately 1.1- structed with yellow or green fruit fly adhesive paper 1.4 mm. Two FFAP rectangles placed back to back covered with plastic mesh (2.2 × 2.2 cm mesh). with sticky surfaces outward, each with a mesh rectangle on its sticky surface, were stapled together as a unit (Fig. 1). Trap lures were AMPu one row of Dancy Tangerine (C. reticulata Blanco) vials (2 ml) containing an agar mixture of ammo- in a citrus orchard in Weslaco, TX. Two blocks of 8 nium carbonate, methylamine HCl, and putre- consecutive trees were used in each row. AMPu scine, described previously (Robacker 1995). vials were attached to the tops of traps. Traps Mexican fruit fly (Anastrepha ludens Loew) was were hung one to a tree, north of center, at 1-2 m used to evaluate the effectiveness of the trap. Flies height. Traps were placed in the orchard during were from a laboratory culture that originated in the morning and removed for fly counts on the fol- Nuevo Leon, Mexico, in 1987. Flies were irradiated lowing day. Approximately 2000 flies were distrib- with 70-92 Grays (Cobalt 60) before adult eclosion. uted equally among the test trees on the day Mixed-sex groups of 200 flies were kept in 473 ml before a test. cardboard cartons with sugar and water until re- The first experiment was a test of the 8 possi- leased in test plots 3 to 8 days after eclosion. ble trap types made from the 2 colors with the 3 Trap tests were conducted in one row of Ruby mesh sizes plus no mesh (Table 1). The 8 trap Red Grapefruit (Citrus paradisi MacFadyen) and types were randomized in each block. Four repli-

Scientiﬁc Notes 303

TABLE 1. NUMBERS OF MEXICAN FRUIT FLIES CAPTURED ON STICKY TRAPS WITH VARIOUS MESH SIZES COMPARED WITH TRAPS WITHOUT MESH.1,2

Test trap Males Females Total

Yellow, no mesh 12.0 c 17.9 d 29.9 d Yellow, 0.7 × 0.7 cm mesh 0.2 a 0.4 a 0.6 a Yellow, 1.5 × 1.5 cm mesh 1.2 ab 1.1 a 2.3 ab Yellow, 2.2 × 2.2 cm mesh 2.2 ab 2.9 ab 5.2 abc Green, no mesh 9.8 c 17.4 d 27.1 d Green, 0.7 × 0.7 cm mesh 2.2 ab 5.1 bc 7.3 bc Green, 1.5 × 1.5 cm mesh 2.8 b 5.8 bc 8.6 c Green, 2.2 × 2.2 cm mesh 3.5 b 6.6 c 10.1 c

1All traps were baited with an AMPu vial. 2Means in the same column followed by the same letter are not significantly different by Fishers protected LSD test (P < 0.05).

cations of the experiment over time were con- by using the plastic mesh to pull themselves from ducted for a total of 16 tests (4 blocks × 4 the sticky surface. replications) of each trap type. Replications over Results of experiments to evaluate FFAP traps time (test days) were treated like replications with different color/mesh combinations against over space (blocks of trees) for statistical analy- Pherocon AM traps are shown in Table 2. All 4 of ses. Data were subjected to analysis of variance the experimental traps were competitive with the using SuperANOVA (Abacus Concepts 1989). Pherocon AM traps. One experimental trap type was compared with Unlike Pherocon traps, FFAP traps with mesh the Pherocon AM (no bait) trap in each of 4 addi- are easy-to-handle. Unlike FFAP traps without tional experiments (Table 2). Experimental traps mesh, they do not trap birds because the mesh and Pherocon AM traps were alternated within prevents feathers from laying across the panel blocks. Replications of the 4 experiments were surface. Traps with mesh are also easy to stack conducted over time for a total of at least 24 tests into a box because they do not adhere to each of each trap type. Data were analyzed by t-tests. other. Also, FFAP is stickier than Tangletrap but In the first experiment, traps without plastic the adhesive does not run and leaves very little mesh were much more effective than any traps residue on the skin, much like contacting cello- with mesh (Table 1). This was especially true for phane tape. Disadvantages of these traps are yellow traps in which the traps without mesh cap- much reduced efficacy compared with FFAP traps tured 6× more flies than the most effective trap without mesh, and severe damage to FFAP by with mesh. Green traps without mesh captured rain. Further research is needed to optimize trap about 3× more flies than the most effective traps and mesh colors and sizes for different fruit flies, with mesh. Thus, mesh greatly reduced the effec- and to reduce rain damage. tiveness of the FFAP. Observations in flight cages We thank Maura Rodriguez and Jose Garcia showed equal attraction to traps with or without for technical assistance. Mention of a proprietary mesh. Thus, the black mesh was not repellent. product does not constitute an endorsement or However, flies often escaped from the mesh traps recommendation for its use by the USDA.

TABLE 2. NUMBERS OF MEXICAN FRUIT FLIES CAPTURED ON STICKY TRAPS WITH MESH COMPARED WITH PHEROCON AM TRAPS IN CITRUS ORCHARD EXPERIMENTS.1,2

Test trap Pherocon trap

Experiment Test trap Males Females Total Males Females Total

1 Yellow, 1.5 cm 1.7 3.0 4.7 2.3 4.3 6.6 2 Yellow, 2.2 cm 4.3* 4.6 8.9 2.5 4.3 6.8 3 Green, 1.5 cm 2.7 4.4 7.2 2.2 4.5 6.7 4 Green, 2.2 cm 1.6 2.8 4.4 1.8 1.8 3.6

1All traps were baited with an AMPu vial. 1Within each experiment, means with an * are significantly different from Pherocon trap means at the 5% level by t tests.

304 Florida Entomologist 84(2) June 2001

SUMMARY 1996. Improved pheromone-based trapping systems to monitor Toxotrypana curvicauda (Diptera: Te- A sticky trap for fruit flies made from fruit fly phritidae). Florida Entomol. 79: 37-48. adhesive paper (FFAP) covered with a plastic HEATH, R. R., N. D. EPSKY, B. D. DUEBEN, J. RIZZO, AND mesh of either 1.5 × 1.5 or 2.2 × 2.2 cm mesh size F. JERONIMO. 1997. Adding methyl-substituted am- was as effective as Pherocon AM traps in captur- monia derivatives to a food-based synthetic attrac- ing Mexican fruit flies. FFAP traps without mesh tant on capture of the Mediterranean and Mexican captured 3× more flies than the best traps with fruit flies (Diptera: Tephritidae). J. Econ. Entomol. 90: 1584-1589. mesh. However, mesh eliminates many problems KATSOYANNOS, B. I. 1989. Response to shape, size and associated FFAP traps. The mesh-covered traps color, pp. 307-324 in A. S. Robinson and G. Hooper are simple, compact, easy to pack, and do not cap- (eds.), Fruit Flies: Their Biology, Natural Enemies ture birds or leave residue on users’ hands. and Control. Vol. 3A. Elsevier, Amsterdam. ROBACKER, D. C. 1995. Attractiveness of a mixture of REFERENCES CITED ammonia, methylamine and putrescine to Mexican fruit flies (Diptera: Tephritidae) in a citrus orchard. ABACUS CONCEPTS. 1989. SuperANOVA. Abacus Con- Florida Entomol. 78: 571-578. cepts, Inc., Berkeley, CA. ROBACKER, D. C., D. S. MORENO, AND D. A. WOLFEN- HEATH, R. R., N. D. EPSKY, A. JIMENEZ, B. D. DUEBEN, BARGER. 1990. Effects of trap color, height, and place- P. J. LANDOLT, W. L. MEYER, M. ALUJA, J. RIZZO, M. ment around trees on capture of Mexican fruit flies CAMINO, F. JERONIMO, AND R. M. BARANOWSKI. (Diptera: Tephritidae). J. Econ. Entomol. 83: 412-419.

Scientiﬁc Notes 305

EFFECT OF VARIABLE PHOTOPERIOD ON DEVELOPMENT AND SURVIVAL OF CIRROSPILUS SP. NR. LYNCUS (HYMENOPTERA: EULOPHIDAE), AN ECTOPARASITOID OF PHYLLOCNISTIS CITRELLA (LEPIDOPTERA: GRACILLARIIDAE)

ALBERTO URBANEJA1, ELENA LLÁCER, ANTONIO GARRIDO AND JOSEP-ANTON JACAS2 Departament de Protecció Vegetal i Biotecnologia; Institut Valencià d’Investigacions Agràries, IVIA Ctra. de Montcada a Nàquera km. 5; E-46113-Montcada, Spain

1Present address: R & D Department. Koppert Biological Systems. Finca Labradorcico del Medio s/n Apartado de Correos 286. 30880 Águilas, Spain

2Present address: Departament de Ciències Experimentals; Universitat Jaume I; Campus de Riu Sec E-12071-Castelló de la Plana, Spain

The eulophids Cirrospilus sp. near lyncus and Environmental chambers were used to check Pnigalio pectinicornis L. are the most abundant the effects of three different photoperiods: 16:8, parasitoids of the citrus leafminer, Phyllocnistis 12:12 and 8:16 (L:D). Temperature fluctuated citrella Stainton, in Spain (Urbaneja et al. 2000). from 10°C to 30°C in eight 3h-steps of 5°C (mean These were opportunistically recruited onto this temperature 20°C). Highest temperature always pest after its introduction in 1993. C. sp. near lyn- coincided with the mid-point of the photophase. cus is a late larval solitary idiobiont ectoparasi- This sequence mimics the regime of field temper- toid which can also behave as a hyperparasitoid. atures both in spring and autumn. Therefore re- Its impact on P. citrella relies on both parasitism sults obtained under both 16:8 and 8:16 (L:D) and host feeding (Urbaneja et al. 1998a). C. sp. photoperiods are presumed to reflect field condi- near lyncus populations in citrus orchards in- tions at those seasons. Insects were reared at the crease from mid-July until the end of October, but Institut Valencià d’Investigacions Agràries as de- remain almost undetectable during the rest of the scribed by Urbaneja et al. (1998b). Eggs of C. sp. year (Urbaneja et al. 1999). A previous study near lyncus were obtained by offering detached demonstrated that C. sp. near lyncus was very citrus leaves containing P. citrella third instar lar- well adapted to temperatures prevailing on the vae (LIII) to isolated mated females (12 LIII per western part of the Mediterranean Basin. That is female). Exposure took place in Petri dishes (140 the range between 7.1°C, mean of minimum tem- mm diameter) where leaves were placed on a layer peratures of the coldest month (January), and of agar (2% weight) under a temperature of 25 ± 29.0°C, mean of maximum temperatures of the 1°C during 4 hours. After exposure, leaves were hottest one (August) (Urbaneja et al. 1999). To checked under a stereoscopic binocular micro- further study the influence of environmental con- scope and those containing parasitized hosts (rec- ditions on the biology of this wasp, the effects of ognized by the presence of C. sp. near lyncus eggs photoperiod on development and survival were on them) were randomly transferred to the corre- investigated. sponding experimental photoperiod on a layer of

TABLE 1. MEAN DEVELOPMENT TIMES (DAYS) OF MALE AND FEMALE C. SP. NEAR LYNCUS UNDER THREE DIFFERENT PHOTOPERIODS (MEAN ± SE).

16:8 (L:D) 12:12 (L:D) 8:16 (L:D)

Egg 2.16 ± 0.18 2.28 ± 0.22 2.26 ± 0.15 Male Larva 5.39 ± 0.32 5.72 ± 0.28 5.74 ± 0.21 Pupa 8.84 ± 0.31 8.47 ± 0.41 8.50 ± 0.24 Total 16.39 ± 0.33 16.47 ± 0.24 16.50 ± 0.19 (n = 14) (n = 16) (n = 17) Egg 2.25 ± 0.22 2.11 ± 0.12 2.67 ± 0.25 Female Larva 5.71 ± 0.38 6.07 ± 0.22 5.88 ± 0.30 Pupa 9.14 ± 0.28 9.09 ± 0.25 9.29 ± 0.35 Total 17.11 ± 0.39 17.27 ± 0.36 17.83 ± 0.28 (n = 19) (n = 22) (n = 12)

306 Florida Entomologist 84(2) June 2001

TABLE 2. STAGE SPECIFIC SURVIVAL (%) OF C. SP. NEAR LYNCUS UNDER THREE DIFFERENT PHOTOPERIODS. INITIAL NUMBER OF REPLICATES WAS 60, BUT CALCULATIONS ARE BASED ON THE NUMBER OF NON-DECAYING LEAVES (=REPLICATES) AT THE END OF EACH PERIOD.

Photoperiod Egg-larva Larva-pupa Pupa-adult Total

16:8 (L:D) 93.3 85.7 91.7 73.3 12:12 (L:D) 93.0 97.5 97.4 88.4 8:16 (L:D) 92.5 89.2 87.9 72.5 agar (2% weight) in a Petri dish (55 mm diameter). this wasp in citrus orchards from autumn until Sixty replicates (= leaves) were used for each pho- early summer may be related to the extraordi- toperiod. Leaves decaying during the study period nary scarcity of P. citrella during winter months. were excluded from the analyses. Parasitoid de- We hypothesize that C. sp. near lyncus is forced to velopment was checked daily until adult emer- abandon citrus orchards and does not return until gence. Development times and stage-specific P. citrella populations have already peaked up. mortality were recorded and eventually emerging For this reason, conservation tactics aimed at fa- adults were sexed. These results were subjected to voring the permanence of C. sp. near lyncus could a two-way analysis of variance (STSC 1987). increase the impact of parasitism early in the sea- Development times for the three experimental son. C. sp. near lyncus was first noticed in Spain photoperiods are presented in Table 1. There were as a parasitoid of P. citrella, and its original host no differences among photoperiods (F = 2.951; df = range remains unknown. Therefore, as a first step 2, 98; P = 0.0572). Although there were differences to implement conservation strategies, its host between sexes (F = 48.059; df = 1, 98; P < 0.0001), range should be determined. no interaction between sex and photoperiod factors occurred (F = 1.820; df = 2, 98; P = 0.1677). SUMMARY Furthermore, there was good agreement between development times obtained in the present study No differences could be observed neither in de- and that reported previously (Urbaneja et al. velopment times nor on survival of C. sp. near lyn- 1999) under a constant temperature of 20°C and a cus exposed to three different photoperiods: 16:8; photoperiod of 16:8 (L:D): 17.20 ± 0.30 d. Survival 12:12 and 8:16 (L:D).Therefore, conservation tac- of the different stages are shown in Table 2. Lar- tics aimed at favoring the winter permanence of vae were the most sensitive stage, whereas both this species in citrus orchards could increase the eggs and pupae were more resistant. Similar re- impact of this opportunistic parasitoid on its host sults had already been found under different con- P. citrella. stant temperatures (Urbaneja et al. 1999). We thank J. E. Peña (TREC-University of Flor- However, no effect of photoperiod on survival was ida) and C. Rodríguez-Saona (USDA-ARS Phoe- found. Agreement between results obtained in nix, USA) for their comments on an early draft of this study and those obtained under a constant this note, and R. Hinarejos, A. Muñoz and J. temperature regime (Urbaneja et al. 1999) indi- Torres for technical assistance. A.U. and E.L. cates usefulness of the latter for prediction of field were recipients of a predoctoral grant from IVIA. performance based on a thermal constant. Photoperiod is one of the signals insects use to REFERENCES CITED perceive environmental changes and adjust their life cycle accordingly (Tauber et al. 1986). Abrams ABRAMS, P. A., O. LEIMAR, S. NYLIN, AND C. WIKLUND. et al. (1996) indicated shorter development times 1996. The effect of flexible growth rates on optimal should be expected in insects developing late in sizes and development times in a seasonal environment. American Nat. 147: 381-395. the season relative to either an upcoming winter SCHAUFF, M. E., J. LASALLE, AND G. A. WIJESEKARA. or an approaching optimal date for adult emer- 1998. The genera of Chalcid parasitoids (Hy- gence. Although C. sp. near lyncus is presumed to menoptera: Chalcidoidea) of citrus leafminer Phyl- be native to a temperate area, such as the Medi- locnistis citrella (Lepidoptera: Gracillariidae). J. terranean Basin (Schauff et al. 1998), where pho- Nat. Hist 32: 1001-1056. toperiod changes considerably along the year, no STSC. 1987. Statgraphics user’s guide, version 5.0. effects of this factor alone could be detected. How- Graphic software system STSC, Rockville, MD. ever, influence of the combined effect of photope- TAUBER, M. J., C. A. TAUBER, AND S. MASAKI. 1986. Sea- riod and with temperature cannot be excluded. sonal adaptations of insects. Oxford University Press. 441 pp. Based on these results and on those previously URBANEJA, A., E. LLÁCER, J. JACAS, AND A. GARRIDO. presented by Urbaneja et al. (1999), C. sp. near 1998a. Ciclo biológico de Cirrospilus sp. próximo a lyncus should be able to breed continuously under lyncus, parasitoide autóctono del minador de las ho- typical Mediterranean conditions. The absence of jas de los cítricos. Bol. San. Veg. Plagas 24: 704-714. Scientific Notes 307

URBANEJA, A., E. LLÁCER, R. HINAREJOS, J. JACAS, AND vival of Cirrospilus sp. near lyncus (Hymenoptera: A. GARRIDO. 1998b. Sistema de cría del minador de Eulophidae), a parasitoid of Phyllocnistis citrella las hojas de los cítricos, Phyllocnistis citrella Stain- (Lepidoptera: Gracillariidae). Environ. Entomol. 28: ton, y sus parasitoides Cirrospilus sp. próximo a lyn- 339-344. cus y Quadrastichus sp. Bol San. Veg. Plagas 24: URBANEJA, A., E. LLÁCER, Ó. TOMÁS, A. GARRIDO, AND 787-796. J. JACAS. 2000. Indigenous natural enemies associ- URBANEJA, A., E. LLÁCER, A. GARRIDO, AND J. JACAS. ated with Phyllocnistis citrella (Lepidoptera: Gracil- 1999. Effect of temperature on development and sur- lariidae) in Eastern Spain. Biol. Control 18: 199-207.

308 Florida Entomologist 84(2) June 2001

EFFECTS OF COOL TEMPERATURES ON OVIPOSITION AND DEVELOPMENT OF COTESIA MARGINIVENTRIS (HYMENOPTERA: BRACONIDAE)

A. SOURAKOV 1 AND E. R. MITCHELL2 1Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611

2Center for Medical, Agricultural and Veterinary Entomology, U. S. Department of Agriculture Agricultural Research Service, P. O. Box 14565, Gainesville, FL 32604, USA

C. marginiventris, an abundant parasitoid of 40.2 ± 7.4 (n = 9), and 39.6 ± 8.8 (n = 7) percent of noctuids in summer, practically disappears from host larvae were parasitised, respectively. The fe- fall to spring in northern Florida, when the cab- cundity of C. marginiventris varied greatly and no bage looper (Trichoplusia ni (Hübner)) can become significant (P > 0.05) differences in mean number a serious problem in crucifers. Temperature might of progeny/female were observed. be a limiting factor for the efficiency of C. margin- At 15°C, cabbage looper larvae develop faster iventris. In the present study we attempted to than beet armyworm larvae. However, the differ- evaluate the oviposition performance of C. mar- ence in development time of C. marginiventris giniventris and its development at different tem- larvae was non-significant (P > 0.05) when reared peratures. We used cabbage looper and beet in beet armyworm (47.3 ± 1.5 days SE) and in cab- armyworm (Spodoptera exigua (Hübner)) as hosts bage looper (45.4 ± 2.6), ranging from 37 to 62 to determine if the time of parasitoid development days (n = 23). Eighteen C. marginiventris’ cocoons differs in hosts with different growth rates. were transferred from 15 to 25°C: 6 took 6 days to C. marginiventris used in this study were ob- eclose; 6 emerged with a two-week delay; and the tained from the Mississippi State University rest never eclosed. Five cocoons that were left at rearing facility. Both host species were from 1.5 15°C never eclosed as well. year-old colonies maintained on pinto bean-based At 20°C, mean development time was 17.6 ± diet (Guy et al. 1985) at USDA-ARS, Gainesville, 0.3 days in beet armyworm larvae (n = 69, wasp Florida. First- and second-instar larvae of either progeny were from six different females). No sig- beet armyworm or cabbage looper were offered in nificant difference was found (P > 0.05) when first clusters of 100 to C. marginiventris females. The instar (17.4 ± 0.3 days (n = 19)) vs. second instar larvae on individual leaves of pigweed (Amaran- host larvae (17.7 ± 0.3 days (n = 50)) were parasi- thus sp.) were placed in transparent 400 ml plas- tised. In cabbage looper, mean development time tic containers, with a top of nylon screen. Females was more rapid (16.1 ± 0.3 days (n = 53, six differ- of C. marginiventris were chilled to 10°C and a ent females)) than in beet armyworm, and this single female was then placed into each container difference was significant (P < 0.0001). The devel- and supplied with a streak of honey and a moist opment time at this temperature ranged from 14 cotton ball (placed on the nylon top). The contain- to 21 days. Duration of the pupal stage was 6.6 ± ers were placed in temperature-controlled envi- 0.1 days (n = 28), ranging from five to eight days. ronmental chambers (Forma Scientific Diurnal At 25°C, development took 8.4 ± 0.1 days in Growth Chambers with a 16 h light : 8 h dark beet armyworm larvae (n = 152, progeny of six dif- photoperiod) at 10, 15, 20, and 25°C. After 24 h, ferent females). Unlike 20°C, at 25°C the parasi- the wasps were removed, and feeding cups with toids developed more slowly in cabbage looper artificial diet were added to each container. Some (9.7 ± 0.1 days (n = 48, progeny of five different fe- containers were held in their respective environ- males)) (P < 0.0001). Duration of the pupal stage mental chambers to determine time of parasitoid at this temperature was 4 days. development at different temperatures. Other Maximum life-time fecundity of C. margini- containers were transferred to the 25°C chamber ventris was previously found to be 111 ± 16 (Jalali to maximize the rearing of progeny. A total of 60 et al. 1987). Here, we report data on maximum C. marginiventris females were tested. Contain- 24-h fecundity and how it changes with time. At ers were checked daily for parasitoid cocoons. Co- 22°C, we supplied single C. marginiventris fe- coons and larvae with a parasitoid-produced exit males with ca. 150 one-day-old beet armyworm hole were removed and counted to assess parasit- larvae on young collard leaves in tightly sealed 15 ism. The statistical analyses (ANOVA and t-test) cm Petri dishes. A streak of honey was placed on were performed using “JMP” at α = 0.05 (SAS In- the inside of the lid. After 24 hours, the wasps stitute Inc., ©1989- 95). were removed and offered a new group of larvae. At 10°C, no parasitism occurred. Apparently, The old larvae were fed collard leaves for two the wasps remained completely inactive at this more days and then dissected for parasitoids. temperature. When oviposition occurred at 15°C, Mean fecundity was 85.8 ± 1.7 eggs on the first 20°C, and 25°C, a mean (±SE) of 28.4 ± 5.3 (n = 5), day, and 16.3 ± 6.8 eggs on the second day.

Scientiﬁc Notes 309

Twenty C. marginiventris were reared outdoors SUMMARY in November-December, using beet armyworm larvae as hosts. Larvae on cabbage leaves were placed Host species had no or small effect on the time in a 30 × 30 × 30 cm plexiglass cage with two net- of C. marginiventris development at different ting sides for ventilation. Thus, the light cycle and temperatures, while temperature at the time of temperature inside the cage were ambient. We oviposition had no effect on the numbers of result- monitored the cage daily for C. marginiventris co- ing progeny. In the laboratory, most eggs (85/fe- coons and later, for adults. During the test period, male) were laid by wasps during the first 24- temperatures fluctuated between 15-25°C during hours. In winter, C. marginiventris reproduced the day and –2-5°C at night. The average monthly more rapidly outdoors than at controlled constant temperature in the region in November and De- temperatures with the same average, and at- cember is 14°C and 16°C, respectively, equal to our tacked sentinel host larvae in cabbage fields. coldest rearing setting in the laboratory. Neverthe- less, the parasitoids pupated in only 14.2 ± 0.3 days; the duration of the pupal stage was 11.4 ± 0.2 REFERENCES CITED days. During November-January, we also placed ca. 200 beet armyworm larvae on cabbage plants JALALI, S. K., S. P. SINGH, AND C. R. BALLAL. 1987. in several cabbage fields in northeastern Florida to Studies on host age preference and biology of exotic determine the presence of naturally-occurring parasite, Cotesia marginiventris (Cresson) (Hy- C. marginiventris. A mixture of two-to-five day-old menoptera: Braconidae). Entomon 12: 59-62. GUY, R. H., N. C. LEPPLA, J. R. RYE, C. W. GREEN, S. L. larvae were used, which fed on collard leaves prior BARRETTE, AND K. A. HOLLIEN. 1985. Trichoplusia to release. Larvae were collected after three-four ni, pp. 487-494. In Pritam Singh and R. F. Moore days and dissected for parasitoids. They yielded (eds.). Handbook of Insect Rearing, Vol. 2, Elsevier eggs and larvae of C. marginiventris of all instars, Science Publishers B. V., Amsterdam. which indicated that C. marginiventris was successfully developing through.

310 Florida Entomologist 84(2) June 2001

A NEW HOST RECORD FOR PSEUDACTEON CRAWFORDI (DIPTERA: PHORIDAE)

JAMES P. PITTS1 AND THERESA L. PITTS-SINGER2 1Department of Entomology, University of Georgia, Athens, GA 30602

2USDA Forest Service, Forestry Sciences Laboratory, 320 Green Street, Athens, GA 30602

The biology of the phorid fly Pseudacteon was mediate-sized workers. A series of the phorid flies reviewed by Porter (1998). The female fly ovipos- was collected with the ants. Solenopsis aurea its into the thorax of its ant host. After hatching, workers were also collected the next day from the larva works its way into the host’s head cap- 0800-1200 h, but no phorid flies were seen. The sule where it continues to grow and ultimately de- phorid flies were identified as Pseudacteon craw- capitates the host. The fully grown larva then fordi by Sanford Porter (Medical and Veterinary uses the head capsule of the host as a pupal case. Entomology Research Laboratory, USDA-ARS, Because of their parasitic lifestyle, flies of this ge- Gainesville, Florida) and the fire ants were iden- nus are of great interest as potential biocontrol tified as S. aurea by senior author (JPP). This rep- agents against the imported fire ants, Solenopsis resents a new host record for Pseudacteon invicta Buren and Solenopsis richteri Forel, in crawfordi. North America. Voucher specimens of both the ants and flies Four species of Solenopsis fire ants are native are deposited in the University of Georgia, Collec- to North America: S. amblychila Wheeler, S. au- tion of Arthropods, Athens, Georgia, USA. rea Wheeler, S. geminata (Fab.) and S. xyloni (MacCook). All four of these species overlap in SUMMARY their ranges in the southwestern United States, mainly in Arizona and Texas (Trager 1991). Pseu- Solenopsis aurea in Arizona is reported as a dacteon crawfordi was described by Coquillett in new host species for Pseudacteon crawfordi. 1907 and is known to be parasitic on S. geminata (Disney 1994) and S. xyloni (Feener 1987). Here we report that P. crawfordi attacks S. aurea. Sole- REFERENCES CITED nopsis aurea is a species of fire ant that occurs in COQUILLETT, D.W. 1907. A new phorid fly genus with xeric conditions in the southwestern United horny ovipositor. Canadian Entomol. 39: 207-208. States and is found mainly in Texas and Arizona DISNEY, R. H. L. 1994. Scuttle flies: the Phoridae. Chap- (Trager 1991). man & Hall, London. 467 pp. Collection: Near dusk, on August 3, 1999, a FEENER, D. H., JR. 1987. Size-selected oviposition in nest of S. aurea was excavated. The nest was lo- Pseudacteon crawfordi (Diptera: Phoridae), a para- cated close to mile marker 13 north of Portal, Ar- site of fire ants. Ann. Entomol. Soc. America 80: 148- izona on Speed Road. Foragers from the colony 151. were initially found under a pile of semi-moist PORTER, S. D. 1998. Biology and behavior of Pseudacteon cow manure. The nest was located close to a pool decapitating flies (Diptera: Phoridae) that parasitize Solenopsis fire ants (Hymenoptera: Formicidae). of water created by a leaking irrigation line. Florida Entomol. 81: 292-309. Shortly after the cow manure was disturbed and TRAGER, J. C. 1991. A revision of the fire ants, Solenopsis the ants were uncovered, six female phorid flies geminata group (Hymenoptera: Formicidae: Myr- were seen hovering around and attacking inter- micinae). J. New York Entomol. Soc. 99: 141-98.

Scientiﬁc Notes 311

EFFECT OF SPINOSAD ON ORIUS INSIDIOSUS (HEMIPTERA: ANTHOCORIDAE) WHEN USED FOR FRANKLINIELLA OCCIDENTALIS (THYSANOPTERA: THRIPIDAE) CONTROL ON GREENHOUSE POT CHRYSANTHEMUMS

SCOTT LUDWIG1 AND RONALD OETTING2 1Department of Entomology, 501 Agricultural Sciences and Industries Building, Pennsylvania State University, University Park, PA 16802

2Department of Entomology, College of Agricultural and Environmental Sciences, Griffin Campus University of Georgia, Griffin, GA 30223

Western flower thrips, Frankliniella occidenta- tered daily with 200 ppm N [20-10-20 Peter’s Peat- lis (Pergande), is a major pest of greenhouse veg- lite Special® (Scotts, Marysville, OH)] using a etables and ornamentals. This is a result of both spaghetti drip watering system that entered each feeding damage and the thrips ability to transmit cage through a hole in the fabric under the pots. tospoviruses (Tommasini & Maini 1995; van de The cages were arranged in a randomized com- Wetering et al. 1996). Control of this thrips spe- plete block design with four treatments and six cies has been difficult because it has developed re- replicates in the first trial and five replicates in sistance to the major classes of insecticides used the second trial. The four treatments investigated for its control (Brødsgaard 1994; Zhao et al. 1995). were O. insidiosus (four females and two males Orius insidiosus (Say) is capable of reducing per cage), spinosad (0.5 ml / liter), O. insidiosus + thrips populations on greenhouse vegetables (van spinosad, and a control. Spinosad was applied to den Meiracker & Ramakers 1991) and ornamen- runoff using a hand sprayer at 241 kPa. Sampling tals (Fransen et al. 1993). for thrips and O. insidiosus was conducted weekly Spinosad (Dow AgroSciences, Indianapolis, by collecting ten flowers per cage and placing IN) is a new insecticide derived from the fermen- them into a jar containing 200 ml of 50% ethyl al- tation of Saccharpolyspora spinosa. Spinosad ef- cohol. The flowers were then rinsed, removed fectively controls western flower thrips under from the jars and discarded. The number of adult laboratory conditions (Eger et al. 1998). Funder- and immature thrips and O. insidiosus were de- burk et al. (2000) demonstrated that spinosad termined by microscopic evaluation. was more effective than broad-spectrum insecti- In the first trial, O. insidiosus adults were re- cides in suppressing F. occidentalis populations in leased at bud break and again 14 days later. Spi- field peppers. This suppression was partially due nosad was applied approximately 24 h after each to the spinosad applications not reducing O. in- O. insidiosus release. In the second trial, an at- sidiosus populations compared to the standard tempt was made to establish O. insidiosus before insecticide treatment. Our objective was to evalu- the plants set bud, but poor establishment oc- ate the impact of spinosad on O. insidiosus when curred. A second O. insidiosus release was made used in combination to control F. occidentalis on one week after bud formation and spinosad was greenhouse grown pot chrysanthemums. applied 14 and 28 days after the second O. insid- Three rooted chrysanthemum ‘Charm’, Den- iosus release. Sampling in both trials was con- dranthema × grandiflora (Ramat.) Kitamura, (Yo- ducted every seven days. der Brothers Inc., Barberton, OH) cuttings were A logarithmic transformation [log10 (x + 1)] of planted per 15 cm plastic pot containing Pro-Gro the data was used to make the variance indepen- Professional Growing Medium® 300 (Pro-Gro dent of the means (Sokal & Rohlf 1995). Data Products Inc., McCormick, SC). Plant terminals were subjected to Analysis of Variance (GLM pro- were removed to promote branching two weeks af- cedure). The means were separated using the ter the plants were potted. A foliar application of least significant difference test (LSD) at the P < 5,000 ppm daminozide (B-Nine®, Uniroyal, Mid- 0.05 level (SAS Institute 1985). The effects of the dlebury, CT) was applied four weeks after potting. treatments were determined by comparing thrips The western flower thrips population was a natu- populations in each of the four treatments. The ral infestation which occurred prior to the plants effects of spinosad on O. insidiosus was deter- being placed into the cages. Screen cages measur- mined by comparing the O. insidiosus present in ing 105 × 57 × 57 cm (L × W × H) were placed over the O. insidiosus treatment and the O. insidiosus groups of eight plants on cloth covered greenhouse + spinosad treatment. benches. The cages were constructed using PVC In the first trial O. insidiosus, spinosad, and pipe frames covered with mesh screen (25 threads the combination treatments resulted in lower per cm). Cages were lifted off of the plants to apply thrips populations (adults + larvae) than the con- treatments and collect samples. Plants were wa- trol (Table 1). Both treatments containing spi-

312 Florida Entomologist 84(2) June 2001

TABLE 1. MEAN DENSITIES ±SD OF ORIUS INSIDIOSUS (A) AND FRANKLINIELLA OCCIDENTALIS (B) PER 10 CHRYSAN- THEMUM FLOWERS IN THE FIRST O. INSIDIOSUS AND SPINOSAD COMPATIBILITY TRIAL.

Days after ﬁrst spinosad application1,2

Treatment 7 14212835

A (Orius insidiosus) Control 0.2 ± 0.4 a 0.8 ± 0.8 b 1.2 ± 1.2 b 1.0 ± 1.0 b 1.8 ± 1.8 b Orius 0.7 ± 1.0 a 3.2 ± 3.2 a 4.5 ± 4.5 a 3.2 ± 3.2 a 4.2 ± 4.2 a Spinosad 0.0 a 0.0 b 0.0 b 0.0 b 0.0 b Spinosad + Orius 0.2 ± 0.4 a 1.7 ± 1.7 b 0.0 b 0.2 ± 0.2 b 0.0 b

F(8,15) = 2.1 F(8,15) = 2.3 F(8,15) = 6.3 F(8,15) = 3.1 F(8,15) = 5.1 P = >0.05 P > 0.05 P = 0.001 P < 0.05 P < 0.01 B (Frankliniella occidentalis) Control 124.5 ± 61.5 a 96.0 ± 25.8 a 184.2 ± 59.8 a 389.3 ± 130.9 a 614.0 ± 286.9 a Orius 60.0 ± 24.4 b 59.8 ± 17.1 a 73.5 ± 48.4 b 159.0 ± 56.8 b 148.8 ± 51.2 b Spinosad 20.3 ± 21.5 c 22.0 ± 25.3 b 7.0 ± 3.4 c 7.7 ± 7.4 d 68.2 ± 44.9 c Spinosad + Orius 19.0 ± 17.5 c 19.8 ± 19.4 b 6.5 ± 5.4 c 24.2 ± 17.5 c 80.5 ± 57.4 c

F(8,15) = 8.6 F(8,15) = 7.1 F(8,15) = 15.4 F(8,15) = 26.7 F(8,15) = 7.5 P < 0.001 P < 0.001 P < 0.0001 P < 0.0001 P < 0.001

1Means in each column for each species followed by the same letters are not significantly different (P > 0.05; LSD). 2A second treatment spinosad was made on day 14 and Orius release on day 13. nosad resulted in lower populations than O. While the O. insidiosus treatment had successful insidiosus alone on all five dates. A low popula- predator establishment, they failed to establish tion of Orius was observed in the control although in the O. insidiosus + spinosad treatment. none were released. This was most likely due to Lower thrips populations (adults + larvae) Orius eggs or nymphs on a few plants before cag- were observed in the second trial than in the first. ing. The spinosad + O. insidiosus treatment never Orius insidiosus suppressed thrips population on resulted in significantly lower thrips populations the first sample date. The thrips populations were than the spinosad treatment. This can be ex- statistically lower in the spinosad and spinosad + plained by the O. insidiosus population data. O. insidiosus treatments on the final two sample

TABLE 2. MEAN DENSITIES ±SD OF ORIUS INSIDIOSUS (A) AND FRANKLINIELLA OCCIDENTALIS (B) PER 10 CHRYSAN- THEMUM FLOWERS IN THE SECOND O. INSIDIOSUS AND SPINOSAD COMPATIBILITY TRIAL (GRIFFIN, GA, SPRING 1999).

Days after ﬁrst spinosad application1,2

Treatment 0 101724

A (Orius insidiosus) Control 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a 0.0 ± 0.0 a Orius 0.2 ± 0.4 a 0.2 ± 0.4 a 1.0 ± 1.4 a 2.0 ± 2.6 a Spinosad 0.3 ± 0.5 a 1.2 ± 1.6 a 0.7 ± 1.2 a 0.7 ± 1.2 a Spinosad + Orius 0.2 ± 0.4 a 0.3 ± 0.8 a 0.0 ± 0.0 a 0.2 ± 0.4 a

F(8,15) = 1.0 F(8,15) = 1.3 F(8,15) = 1.5 F(8,15) = 1.6 P > 0.5 P > 0.5 P > 0.5 P > 0.5 B (Frankliniella occidentalis) Control 4.3 ± 2.4 a 8.3 ± 10.8 ab 18.0 ± 2.6 a 30.0 ± 20.9 a Orius 0.8 ± 1.6 c 2.5 ± 2.8 b 26.5 ± 26.2 a 52.7 ± 50.2 a Spinosad 4.8 ± 5.7 ab 9.6 ± 10.3 a 5.0 ± 6.0 b 6.0 ± 8.7 b Spinosad + Orius 1.5 ± 2.3 bc 4.3 ± 5.0 ab 3.0 ± 1.4 b 4.8 ± 4.7 b

F(8,15) = 5.7 F(8,15)) = 6.4 F(8,15) = 6.7 F(8,15) = 5.3 P < 0.01 P < 0.01 P < 0.05 P < 0.05

1Means in each column for each species followed by the same letters are not significantly different (P > 0.05; LSD). 2A second spinosad application was made on day 17. Scientiﬁc Notes 313

dates (Table 2). The O. insidiosus populations REFERENCES CITED were not significantly different. Funderburk et al. (2000) demonstrated Orius BRØDSGAARD, H. F. 1994. Insecticide resistance in Euro- pean and African strains of western flower thrips was capable of establishing and controlling thrips (Thysanoptera: Thripidae) tested in a new residual- in field peppers. Our data obtained in the green- on-glass test. J. Econ. Entomol. 87: 1141-1146. house differ from those of Funderburk et al. There EGER, J. E., JR., J. STAVISKY, AND J. E. FUNDERBURK. may be at least two explanations for this differ- 1998. Comparative toxicity of spinosad to Franklin- ence. First, O. insidiosus was capable of moving iella spp. (Thysanoptera: Thripidae), with notes on a between treated and untreated areas in the field bioassay technique. Florida Entomol. 81: 547-551. pepper trials, while under the greenhouse cage FRANSEN, J. J., M. BOOGAARD, AND J. TOLSMA. conditions its movement was restricted. Second, 1993. The minute pirate bug, Orius insidiosus (Say) although in both studies spinosad was applied at (Hemiptera: Anthocoridae), as a predator of western flower thrips, Frankliniella occidentalis (Pergande), the same rate, there was a difference in the vol- in chrysanthemum, rose and saintpauli. Bull. IOBC/ ume of insecticide applied between the two stud- WPRS. 16(8): 73-77. ies. Spinosad was applied at 174 liters per acre in FUNDERBURK, J., J. STAVISKY, AND S. OLSON. 2000. Pre- the field studies while in the greenhouse the ap- dation of Frankliniella occidentalis (Thysanoptera: plication rate was approximately 750 liters per Thripidae) in field peppers by Orius insidiosus acre. Additional studies need to be conducted to (Hemiptera: Anthocoridae). Environ. Entomol. 29: evaluate the timing of insecticide applications 376-382. and predator releases. SAS INSTITUTE. 1985. SAS User’s Guide: Statistics, ver- We thank Stan Malloy and Sherrie Stevens for sion 5 ed. SAS Institute, Cary NC. SOKAL, R. R., AND F. J. ROHLF. 1995. Biometry: the prin- assistance with plant maintenance and insect ciples and practice of statistics in biological research. sampling and Jerry Davis for help with data anal- W. H. Freeman and Company, New York. 887 pp. ysis. We also thank Yoder Brothers Inc., Barber- TOMMASINI, M. G., AND S. MAINI. 1995. Frankliniella oc- ton OH, for supplying chrysanthemums and Dow cidentalis and other thrips harmful to vegetable and AgroSciences, Indianapolis, IN, for the spinosad. ornamental crops in Europe, p. 1- 42. In: A. J. M. Loomans, J. C. van Lenteren, M. G. Thomasini, S. Maini, and J. Riudavets (eds.). Biological Control of Thrips Pests. Wageningen Agric. Univ. Papers 95-1, SUMMARY Wageningen, The Netherlands. VAN DEN MEIRACKER, R. A. F., AND P. M. J. RAMAKERS. Orius insidiosus and spinosad were used in 1991. Biological control of the western flower thrips combination against F. occidentalis on green- Frankliniella occidentalis, in sweet pepper, with the house pot chrysanthemums to test for compatibil- anthocorid predator Orius indisiosus. Med. Land- ity. Orius insidiosus failed to establish in the first bouww. Rijksuniv. Gent 56: 241-249. trial when exposed to spinosad, but was more VAN DE WETERING, F., R. GOLDBACH, AND D. PETERS. 1996. Tomato spotted wilt tospovirus ingestion by compatible in the second trial. This indicates that first instar larvae of Frankliniella occidentalis is a spinosad may have an effect on Orius populations prerequisite for transmission. Phytopathology 86: when there was little free movement of the thrips 900-905. and Orius between plants. ZHAO, G, W. LIU, J. M. BROWN, AND C. O. KNOWLES. 1995. Insecticide resistance in field and laboratory strains of western flower thrips (Thysanoptera: Thripidae). J. Econ. Entomol. 88: 1164-1170.

314 Florida Entomologist 84(2) June 2001

A MORPHOLOGICAL MEANS OF DISTINGUISHING FEMALES OF THE CRYPTIC FIELD CRICKET SPECIES, GRYLLUS RUBENS AND G. TEXENSIS (ORTHOPTERA: GRYLLIDAE)

DAVID A. GRAY1,4, THOMAS J. WALKER2, BRENDA E. CONLEY1 AND WILLIAM H. CADE1,3 1Department of Biological Sciences, Brock University, St. Catharines, Ontario, L2S 3A1, Canada

2Department of Entomology and Nematology, University of Florida, Gainesville, FL, 32611-0620, USA

3Present Address: The President’s Office, The University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada

4Present Address: Department of Biology, The University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada

The field crickets Gryllus rubens Scudder and lection locality alone (G. rubens: Gainesville, FL G. texensis Cade and Otte (previously G. integer (n =15); G. texensis: Austin, TX (n = 14), Dallas, from Texas, see Cade & Otte (2000)) are the most TX (n = 6)) or based on the songs of their brothers commonly collected crickets throughout the (G. texensis: Uvalde, TX (n = 2), Austin, TX (n = southeastern United States from North Carolina 1)). Females from sympatric sites (G. rubens: Mil- to Texas. The two species are sympatric from ton, FL (n = 8), Marianna, FL (n = 12), Pensacola, western Florida to eastern Texas (Walker 1998). FL (n = 5), Mobile, AL (n = 1), Decatur, AL (n = 1); These two species are the only known trilling field G. texensis: Milton, FL (n = 32), Starkville, MS (n crickets in the southeastern US, and are cur- = 9), Tuscaloosa, AL (n = 4), Pensacola, FL (n = 4), rently separated by song differences alone. Male Mobile, AL (n = 2), Carrollton, GA (n = 6)) were G. rubens produce trilled calling song with pulse identified to species based on the songs of their rates averaging ca. 55 pulses per second (p/s) at brothers. 25°C, whereas G. texensis produce trills with The crickets were measured as two replicates; pulse rates averaging about 80 p/s at 25°C (Sour- replicates 1 and 2 represent crickets in the collec- oukis et al. 1992, Walker 1998, 2000 Martin et al. tions of DAG and the Florida State Collection of 2000). The pulse rates of the two species become Arthropods, Gainesville, respectively. The data more similar at lower temperatures, but provided are presented together in Figure 1 and both sepa- that the temperature is above approximately rately and pooled in Table 1. We found that female 20°C, males can unambiguously be identified to G. rubens have ovipositors ca. 2.5 to 3 mm longer species by song (Walker 1998; Gray & Cade 2000). relative to their body size than do female G. texen- No morphological means of distinguishing either sis. For the pooled data, we tested the effect of males or females has previously been reported species with pronotal width as a covariate using (see Nickle & Walker (1975) for other species of an ANCOVA. The model r2 was 0.88 (i.e., 88% of the southern United States). Here we report that the variation in ovipositor length was accounted

most females of the two species can be separated for). Both species (F(1,119) = 170.96, P < 0.0001) and

on the basis of body-size relative ovipositor pronotal width (F(1,119) = 440.93, P < 0.0001) were length: G. rubens have longer ovipositors relative signiﬁcant predictors. A comparison of the slopes to body size than do G. texensis. of ovipositor length on pronotal width was made We compared the ovipositor lengths and the by testing for a species x pronotal width interac- pronotal widths of 122 females from several local- tion in a separate ANCOVA. The slopes did not ities across the species’ geographic ranges. Fe- differ (G. rubens, slope = 2.285, G. texensis, slope

males were either laboratory reared from field- = 2.374; F(1,118) = 0.10, P < 0.7507). The intercepts caught nymphs, laboratory reared offspring of of the lines were 1.34 mm for G. rubens and -1.31 field-caught field-inseminated females, or were mm for G. texensis. Thus it should be possible to field caught females. Females from allopatric separate accurately most females of these two sites were identified to species on the basis of col- species.

TABLE 1. MEAN ±SD OVIPOSITOR LENGTHS (MM) OF G. RUBENS AND G. TEXENSIS.

Replicate G. rubens G. texensis t (df) P -value ± ± 1 14.42 0.79 (n = 24) 11.46 0.77 (n = 31) t (53) = 13.91 <0.0001 ± ± 2 14.7 2.0 (n = 18) 11.2 2.4 (n = 49) t (65) = 5.53 <0.0001 ± ± Combined data 14.53 1.44 (n = 42) 11.30 1.91 (n = 80) t (120) = 9.60 <0.0001

Scientiﬁc Notes 315

Fig. 1. Ovipositor length as a function of body size (pronotal width) in Gryllus rubens (filled symbols) and G. texensis (open symbols). Triangles represent individuals identified to species based on the songs of their brothers, whereas circles represent individuals identified to species based on locality alone. G. rubens have longer ovipositors than G. texensis.

SUMMARY MARTIN, S. D., D. A. GRAY, AND W. H. CADE. 2000. Fine- scale temperature effects on cricket calling song. Females of the field crickets Gryllus rubens Can. J. Zool. 78: 706-712. and G. texensis can generally be distinguished on NICKLE, D. A., AND T. J. WALKER. 1975. A morphological the basis of their ovipositor length relative to key to field crickets of Southeastern United States body size. G. rubens females have longer oviposi- (Orthoptera: Gryllidae: Gryllus). Florida Entomol. tors than do G. texensis. 57: 8-12. SOUROUKIS, K., W. H. CADE, AND G. ROWELL. 1992. Fac- tors that possibly influence variation in the calling REFERENCES CITED song of field crickets: temperature, time, and male size, age, and wing morphology. Can. J. Zool. 70: 950- CADE, W. H., AND D. OTTE. 2000. Gryllus texensis n. sp.: 955. A widely studied field cricket (Orthoptera; Gryllidae) WALKER, T. J. 1998. Trilling field crickets in a zone of from the southern United States. Trans. Am. Ento- overlap (Orthoptera: Gryllidae: Gryllus). Ann. Ento- mol. Soc. 126: 117-123. mol. Soc. Am. 91: 175-184. GRAY, D. A., AND W. H. CADE. 2000. Sexual selection WALKER, T. J. 2000. Pulse rates in the songs of trilling and speciation in field crickets. Proc. Natl. Acad. Sci. field crickets (Orthoptera: Gryllidae: Gryllus). Ann. USA. 97: 14449-14454. Entomol. Soc. Am. 93: 565-572.

316 Florida Entomologist 84(2) June 2001

BEMISIA AFER SENS. LAT. (HOMOPTERA: ALEYRODIDAE) OUTBREAK IN THE AMERICAS

PAMELA K. ANDERSON1, JON H. MARTIN2, PILAR HERNANDEZ1 AND AZIZ LAGNAOUI3 1International Center for Tropical Agriculture, A.A. 6713, Cali, Colombia

2Entomology Department, The Natural History Museum, London, United Kingdom

3International Potato Center, Apartado 1558, Lima, Peru

Bemisia tabaci (Gennadius) was reported on (housed at USDA, Beltsville, MD), Martin noted a sweetpotato (Ipomoea batatas Lam.) from the cen- small number of Bemisia afer-group samples that tral coast of Peru in the late 1980s, noting that it are likely to be conspecific with the samples from was not a significant pest (Redolfi 1989, cited in B. frutescens in Belize. These samples were either Nuñez 1995). However, in the 1997-1998 agricul- field-collected in, or intercepted by US quarantine tural season, unusually large populations of Be- authorities from Honduras, Mexico and El Salva- misia tabaci were reported to be significantly dor. Quoted host plants include Pouteria sp (Sapo- affecting sweetpotato yields in the coastal valleys taceae), Hibiscus sp (Malvaceae), Origanum sp of Peru (Valencia et al. 2000). In August of 2000, P. (Labiateae), Ficus sp or spp (Moraceae), Serjania Anderson (CIAT) made a field visit to the Cañete sp (Sapindaceae) and Psidium guajava (Myrta- Valley, approximately 100 km south of Lima, with ceae). There are also two additional slides from Cristina Fonseca of the International Potato Cen- Belize in BMNH, one from an unidentified woody ter (CIP) and Ing. Jose M. Valencia of the Cañete vine and matching the Bocconia puparia, and the Experimental Station, to explore the problem. other (possibly a smooth-leaf form of the same The nymphs that were actively reproducing on species) from a wild cassava plant growing on a sweetpotato were Bemisia. However, the adult forest track remote from agriculture. From this whiteflies, which were abundant on sweetpotato material, it appears that this taxon is widespread and pepino (Solanum muricatum Ait.) were larger and oligophagous in Central America. and whiter (more Trialeurodes-like) than typical Bink-Moenen (1983) proposed the synonymy of for Bemisia tabaci. Thus, nymphs were collected Bemisia hancocki Corbett (1936) with B. afer from sweetpotato for taxonomic verification. (Priesner & Hosny 1934). This synonymy was Whitefly nymphs were slide-mounted and ten- based on examination of one badly damaged syn- tatively identified as Bemisia afer, by P. Hernan- type of B. afer deposited in the BMNH. Based on dez at the International Center for Tropical Martin’s subsequent examination of a complete Agriculture (CIAT) in Cali, Colombia. The identi- syntype puparium of B. hancocki deposited in fication was verified as Bemisia afer sens. lat., by USNM, this synonymy may have been prema- J. Martin at the Natural History Museum in Lon- ture. However, with the considerable degree of don, UK (BMNH). Voucher specimens were de- puparial morphological plasticity now becoming posited in the BMNH. evident within the B. afer group, formally resur- This is the first outbreak we have observed of recting B. hancocki could cause further nomencla- Bemisia afer sens. lat. in an agricultural situation tural confusion at this point. in the Americas. B. afer has been recorded from B. hancocki was first described from cotton Egypt, Greece, Sicily, the Middle East, the Ethio- (Gossypium hirsutum L.) in Uganda by Corbett pian region, Comoro Islands, India, Pakistan, New (1936). Mound (1965) examined B. hancocki spec- Guinea, Fiji, Tonga (Martin 1987), Sudan, Sierra imens from cotton, peanut (Arachis hypogaea L.), Leone, Cote d’Ivoire, Nigeria, Niger, Chad, Came- and Vigna (catjang) unguiculata (L.) Walp., and roon, Congo, Zaire, Uganda, Rhodesia, Malawi, noted B. hancocki collections from cassava (Mani- South Africa (Bink-Moenen 1983), and Australia hot esculenta Cranz) in Sierra Leone, Nigeria, (Martin 1999). B. afer has hitherto been consid- Cameroon, and Sudan. He further described the ered as a common and widespread pest species, variation in the puparial morphology of B. han- feeding on a wide variety of plants (Martin 1987). cocki as being almost as great as that of B. tabaci. In Belize in 1994 and 1996, plants of a papav- Personal observations made by Martin, Estrella eraceous host, Bocconia frutescens L., were found Hernández-Suarez (ICIA, Canary Islands, Spain) to be colonized by very large populations of a spe- and by Raymond Gill (CDFA, Sacramento, USA) cies of Bemisia with highly characteristic puparia. indicate that the B. afer group actually displays This belongs to B. afer sens. lat., but the puparial considerably greater puparial morphological vari- characteristics fall outside those normally ob- ation than does B. tabaci and its forms/biotypes. served in areas of the world where B. afer is wide- Although specimens from the B. afer group spread. While studying the whitefly collection of have been previously discovered in non-agricul- the US National Museum of Natural History tural situations in the Americas, this is the first

Scientiﬁc Notes 317

report of a Bemisia afer sens. lat. outbreak on an CORBETT, G. H. 1936. New Aleurodidae (Hem.). Proc. important crop host in the New World. The extent Royal Entomol. Soc. London (B) 5: 18-22. of B. afer dissemination and its host-associations MARTIN, J. H. 1987. An identification to common white- in Peru need to be investigated. Furthermore, the fly pest species of the world (Homoptera, Aley- taxonomy of Bemisia afer and Bemisia hancocki rodidae). Tropical Pest Management 33: 298-322. MARTIN, J. H. 1999. The whitefly fauna of Australia should be re-visited and, the possible role of B. afer (Sternorrhyncha: Aleyrodidae): a taxonomic account in virus transmission needs to be clarified. and identification guide. CSIRO, Canberra, Austra- lia (CSIRO Entomology Technical Paper No. 38). SUMMARY MOUND, L. A. 1965. An introduction to the Aleyrodidae of Western Africa (Homoptera). Bulletin of the Brit- The first outbreak of Bemisia afer sens. lat. in ish Museum (Natural History) 17: 115-160. an agricultural situation in the Americas is re- NUÑEZ, E. Y. 1995. Reporte de Perú. CEIBA (Honduras) ported. B. afer was discovered on sweetpotato 36: 157-162. (Ipomoea batatas Lam.) in the Cañete Valley in PRIESNER, H., AND M. HOSNY. 1934. Contributions to a knowledge of the white flies (Aleurodidae) of Egypt the central coast of Peru. (III). Bulletin Ministry of Agriculture of Egypt, Tech- nical and Scientific Service 145: 1-11. REFERENCES CITED VALENCIA, L., N. MUJICA, AND F. CISNEROS. 2000. In- formé de Perú. pp. 194-198. In IX Taller Latinoamer- BINK-MOENEN, R. M. 1983. Revision of the African icano y del Caribe sobre Mosca Blanca y whiteflies. Monografieen van de Nederlandse Ento- Geminivirus. MIDA-IDIAP, Panama. mologisch Vereniging No. 10. 211 pp.

318 Florida Entomologist 84(2) June 2001

RESISTANCE OF IMMATURE MANGO FRUITS TO CARIBBEAN FRUIT FLY (DIPTERA: TEPHRITIDAE)

MICHAEL K. HENNESSEY1 AND R. J. SCHNELL2 1U. S. Environmental Protection Agency, Ofﬁce of Pesticide Programs 12000 Pennsylvania Avenue NW, Mailbox 7503C, Washington, DC 20460

2USDA-ARS, Subtropical Horticulture Research Station, 13601 Old Cutler Road, Miami, FL 33158

Both mature and immature mangoes, Mangifera tices included fertilization, pruning, and chemical indica L., grown in subtropical Florida are mar- weed control. No insecticides were applied during keted. Food use of immature mangoes includes testing and for at least four months prior to test- being cooked for sauce, powdered for a flavoring ing. The methods for rearing flies, determining agent, pickled, and processed into chutney. Imma- mango maturity, and bioassaying of fruits were ture mangoes are distinguished from mature previously described in detail (Hennessey & mangoes in that they do not ripen after harvest Schnell 1995). Flies were reared on an agar-based (Reid 1992). Immature mangoes have a whitish diet (Hennessey 1994) which was used as the con- flesh, lack juiciness, and have a sour flavor, while trol in the present study. Stage of maturity of the mature mangoes have yellow flesh, are juicy, and mangoes was determined for each accession and have a sweet flavor. harvest date by holding 5-10 fruits in the labora- Mango has been listed as a host of Caribbean tory and observing flesh color, juiciness, and fla- fruit fly, Anastrepha suspensa (Loew) (Norrbom & vor after 5-8 days. Each accession was bioassayed Kim 1988). California, where the largest imma- on three dates: early (March-April), middle (May), ture mango market is, maintains a Caribbean and late (June) immature period (Table 1). Slices, fruit fly quarantine on both mature and imma- including peel, from five fruits per accession were ture imported mangoes. Mangoes can only be bioassayed for each date. Ten mature Caribbean shipped from Florida to California if they have fruit fly eggs were inoculated on each fruit slice undergone an acceptable postharvest treatment and corresponding agar control portions. The cor- that disinfests them of fruit flies. rected mean percentage adult emergence was the To our knowledge, there have been no studies criterion used to evaluate resistance. This value to determine to what extent immature mangoes was obtained by dividing the percentage of eggs may represent a lower risk for fruit fly infestation reaching adulthood from each slice by the mean than mature mangoes. Peña & Moyhuddin (1997) percentage reaching adulthood from five agar reviewed mature mango fruit resistance to Anas- controls of the same date. trepha obliqua (Macquart) and infestability dif- The experiment was completely randomized. ferences among cultivars were suggested to be Corrected mean percentage adult emergence data caused by differences in toxic chemicals, nutri- from the three harvest dates combined (n = 15) were ents, or resin ducts. Hennessey & Schnell (1995) analyzed with PROC ANOVA and compared among determined that for mature fruit, some genotypes cultivars with the LSD test (SAS Institute 1992). are more resistant to Caribbean fruit fly than oth- The corrected mean percentage of emergence of ers. They also suggested highly resistant germ- adult flies from mangoes varied from 0.0% for plasm accessions could be employed in breeding ‘Saigon Seedling’ M13269 to 35.7% for ‘Keitt’ (Table efforts and integrated pest management systems 1). The most resistant group included 15 cultivars for control of Caribbean fruit flies. The present in- that supported less than 28.9% emergence. Hen- vestigations were conducted to determine if some nessey & Schnell (1995) observed that the cor- germplasm accessions in the immature state were rected mean percentage emergence varied from more resistant to Caribbean fruit fly than others. 26.6% for mature ‘Tobago Small Red’ to 119.0% (a Fruits used in the experiments were from the higher emergence rate than the control) for mature USDA National Clonal Germplasm Repository at ‘Sabre’. In that study, the group that contained the the Subtropical Horticulture Research Station in most resistant cultivar (P = 0.10) consisted of Miami, FL, where over 150 Mangifera accessions ‘Peach’, ‘Irwin’, ‘Tommy Atkins’, ‘Rumani’, ‘Turpen- are maintained. The accessions comprise a valu- tine’, ‘Keitt’, ‘13-1’, ‘Sandersha’, ‘Zilate’, and ‘Tobago able source of genetic diversity utilized by breed- Small Red’ (Table 1). Mature mangoes served as a ers and researchers from all over the world. better substrate for development than did imma- Fruits from 18 M. indica accessions (individual ture mangoes of the same cultivar. Over all culti- trees) were selected based on availability and in- vars, immature mangoes supported from 2-59% of clusion in a previous study of mature mangoes the emergence compared with mature fruits of the (Table 1). Immature mangoes were available from same cultivars did. This could be interpreted to March 21 to June 23, 1996. Tree cultivation prac- mean that, within cultivars, most immature man-

Scientiﬁc Notes 319

TABLE 1. MANGO CULTIVARS, SAMPLING DATES, AND CORRECTED MEAN PERCENTAGE ADULT CARIBBEAN FRUIT FLY EMERGENCE FROM IMMATURE AND MATURE MANGO FRUITS, MIAMI, FL.

Sampling dates for immature mangoes Mean % emergence (SEM)1

Cultivar Accession # Early Middle Late Immature mangoes Mature mangoes2

‘Keitt’ M17451 April 18 May 30 June 20 35.7a (15.9) 59.0b (6.7) ‘S-10’ M22097 March 21 May 16 June 13 35.2a (20.9) 88.4a (9.7) ‘Peach’ M14278 March 28 May 16 June 20 28.9ab (9.6) 76.8b (22.0) ‘Winters’ M20222 April 4 May 9 June 6 23.9abc (13.0) 85.6a (16.2) ‘Golek’ M12329 March 21 May 16 June 20 20.5abc (9.5) 92.4a (24.7) ‘Sabre’ M14277 April 25 May 9 June 20 17.3abc (10.2) 119.0a3 (27.1) ‘Irwin’ M17452 April 18 May 23 June 20 14.3abc (7.1) 76.2b (24.3) ‘Jo-Z’ M27187 April 25 May 30 June 20 13.8abc (7.7) 100.0a (17.5) ‘Zilate’ M24476 April 11 May 23 June 13 13.7abc (5.8) — ‘Carabao’ M04336 April 11 May 2 June 6 7.0bc (7.0) 88.4a (9.7) ‘13-1’ — April 4 May 23 June 13 6.9bc (5.2) — ‘Eldon’ M19833 March 21 May 16 June 13 6.1bc (6.1) 92.2a (11.1) ‘Aeromanis’ M12327 March 28 May 9 June 13 5.9bc (4.4) 80.8a (16.6) ‘Rumani’ M17699 April 25 May 30 June 20 2.8c (2.0) 73.8b (9.3) ‘Tommy Atkins’ M23457 April 4 May 9 June 6 2.7c (1.9) 76.0b (20.5) ‘Turpentine’ — March 28 May 2 June 13 1.5c (1.5) 65.2b (21.5) ‘Becky’ M26401 April 16 May 23 June 23 1.1c (1.1) 38.4b (9.6) ‘Saigon M13269 April 11 May 2 June 6 0.0c (0.0) 42.2b (16.6) Seedling’

1Column means (n = 15) followed by the same letter do not differ (P = 0.05; LSD). 2Hennessey & Schnell (1995). 3Percentage emergence exceeded that for controls. goes would present less than half the risk of intro- from 0.0% for ‘Saigon Seedling’ (M13269) to ducing Caribbean fruit flies to an importing area 35.7% for ‘Keitt’. Immature mangoes supported than mature mangoes would. only 2-59% of the emergence compared with ma- Resistant cultivars may be coupled with other ture fruits of the same cultivars. Resistant culti- technology including monitoring traps and pre- vars may possibly be coupled with preharvest harvest insecticide treatments in a systems ap- insecticide treatments to reduce fruit fly infesta- proach to reduce fruit fly infestations to negligible tions to below the level of quarantine concern. levels, thus achieving quarantine security. More information on the accessions used in the REFERENCES present studies may be obtained from the Germ- plasm Resources Information Network website HENNESSEY, M. K. 1994. Depth of pupation of Caribbean http://www.ars-grin.gov. Propagative materials fruit fly in soils in the laboratory. Environ. Entomol. from the Miami collection are available free to re- 23: 1119-1123. searchers and breeders. HENNESSEY, M. K., AND R. J. SCHNELL. 1995. Screening Florida mango fruits from germplasm for resistance The authors thank P. Mendez, E. Schnell, and to immature stages of Caribbean fruit fly. Insect Sci. W. Wasik, USDA-ARS, Miami, FL, for their assis- Applic. 16: 119-121. tance in conducting the experiments, and R. J. NORRBOM, A. L., AND K. C. KIM. 1988. A list of the re- Knight, Jr. (University of Florida, Homestead) ported host plants of Anastrepha (Diptera: Tephriti- and L. Chang (USDA-APHIS, Riverdale, MD) for dae). USDA, APHIS, PPQ Report 81-52. viii, 114 pp. reviewing the article. PEÑA, J. E., AND A. I. MOYHUDDIN. 1997. Insect Pests, p. 327-362. In R. E. Litz (ed.). The mango. CAB Inter- national, Wallingford, UK. UMMARY S REID, M. S. 1992. Maturation and maturity indices, p. 21-28. In A. A. Kader (ed.). Postharvest technology Immature mango slices were artificially in- for horticultural crops. Univ. California Div. Agric. fested with Caribbean fruit fly eggs in the labora- and Natural Resources Publ. 3311. tory. The mean percentage of emergence of adults, SAS INSTITUTE, INC. 1992. SAS, version 6.04. SAS Insti- relative to emergence from control media, varied tute, Cary, NC.

320 Florida Entomologist 84(2) June 2001

CONCERNING THE OCCURRENCE OF ANASTREPHA OBLIQUA (DIPTERA: TEPHRITIDAE,)IN FLORIDA

GARY J. STECK Florida Department of Agriculture & Consumer Services, Division of Plant Industry, Gainesville FL 32614-7100

Regulatory agencies worldwide take careful West, which “either have dates from 1931-33 or note of published literature that describes the dis- (the majority) don’t have a date other than ‘Nov.’, tribution of various pests, which they take into but the latter were reared by L. C. McAlister, so account when establishing their quarantine rules they should have originated from the same time for importing agricultural commodities from period” (A. L. Norrbom, USDA-Systematic Ento- other countries. Thus, timely and accurate report- mology Laboratory, pers. comm.). ing of pest distributions is of great commercial The 1957 record noted above by Weems (1970) importance. is doubtful. DPI identification records show that The West Indian fruit fly, Anastrepha obliqua R. H. Foote did identify three specimens of A. obli- (Macquart) (Diptera: Tephritidae), a serious and qua in 1957, but these were specimens that had highly polyphagous fruit pest, is documented as been collected in Key West in 1935. There are no present in Florida in numerous publications, in- voucher specimens in the FSCA or NMNH to con- cluding some prominent and recent ones. The firm presence of A. obliqua in Florida in 1957. widely used CABI Distribution Maps of Pests Other DPI identification records indicate detection (1988, citing Foote 1965) show A. obliqua present of fruit fly larvae in fruit which were identified as in Florida. Likewise, Foote et al. (1993) include “Anastrepha mombinpraeoptans”: (1) “Tampa, the Florida Keys in their distribution map for A. 1946, fruit?, det[ermination] A. Stone?”; (2) obliqua. Weems (1970) wrote that Anastrepha “Miami, hog plum, 1947, det Merrill and G.W. obliqua (as A. mombinpraeoptans) “apparently Dekle”; (3) “Miami, 1946, guava, det Dr. A. Stone?”; . . . exists at a threshold level in Florida. Most re- (4) “Miami, 1947, mango, det Merrill?”; (5) cent Florida records are for several adult females “Ft. Lauderdale, 1963, Anastrepha sp. (possibly in 1957 from Key West and one larva in mango mombinpraeoptans Sein), mango, det R. H. Foote”. from Ft. Lauderdale, June 25, 1963, which was Fortunately, slide-mounted voucher specimens identified by Dr. R. H. Foote as ‘Anastrepha spe- were retained for instances (1) through (4). Label cies, possibly mombinpraeoptans’.” A similar com- data for each of these show that larvae were taken ment, “the West Indian fruit fly . . . occurs in from intercepted fruit originating in Puerto Rico. southern Florida” was uncritically repeated by The Ft. Lauderdale larva of instance (5) came from Heppner (1991). a residential address. Foote correctly left its iden- I believe that these publications do not cor- tification as uncertain, as it is not possible to sepa- rectly reflect the present situation in Florida, and rate larvae of West Indian fruit fly from those of they likely have been in error for several decades. the Caribbean fruit fly, Anastrepha suspensa This is based on an examination of the entire col- (Loew), and several other species of Anastrepha lection of this species from the Florida State Col- (Steck et al. 1991). In fact this larva may have been lection of Arthropods (FSCA, Gainesville), the a harbinger of the large colonization by Caribbean official repository for insect specimens of regula- fruit fly in south Florida, where adults were first tory importance in Florida, and the National Mu- detected in 1965. Unfortunately, the 1963 larva seum of Natural History (NMNH, Washington was not retained as a voucher in the FSCA. The DC), plus all of the official identification records of final DPI identification record of A. obliqua was the Division of Plant Industry (DPI) regarding de- from two adult specimens trapped in Key West, tections of this species from 1915 to the present. 1971, determined by Weems. These specimens are Briefly, the history of A. obliqua in Florida is as in the FSCA. I have re-examined them, and found follows (see Clark et al. 1996 and references them to be mis-identified A. suspensa. therein). It was first discovered in Florida in In summary, there is no confirmed evidence of 1930. As a result of that discovery, a large fruit fly the presence of A. obliqua in Florida since 1935. survey and eradication campaign was conducted Apparently, the control actions of 1931-1936 in- from 1930 until 1936. Eradication actions began deed eradicated this pest from Florida as no adult in 1934 and included widespread fruit removal A. obliqua has ever again been detected in the and destruction, and biweekly insecticidal sprays. field, despite the presence of many thousands of During this time, numerous A. obliqua specimens fruit fly detection traps that have been run were collected, all from Key West, excepting a sin- throughout the Keys and peninsular Florida con- gle specimen from mainland Florida. The FSCA tinuously and year-round since 1956. I think it is has 63 specimens taken in Key West from 1931 to safe to say that Florida is completely free of Anas- 1935 and one specimen from Redlands [Dade Co.] trepha obliqua and probably has been so for the in 1935. The NMNH has 48 specimens from Key past 65 years.

Scientiﬁc Notes 321

Contribution No. 904, Bureau of Entomology, tropics: Integrated Pest Management—A Florida Nematology & Plant Pathology—Entomology Perspective. Intercept Ltd., Andover, UK. Section. FOOTE, R. H. 1965. Family Tephritidae, pp. 658-678. In A. Stone, C. W. Sabrosky, W. W. Wirth, R. H. Foote, and J. R. Coulson (eds.). A catalog of the Diptera of SUMMARY America north of Mexico. U.S. Dept. of Agric., Agric. A comparison of published and other docu- Handbook No. 276. Washington, DC. mented identification records of West Indian fruit FOOTE, R. H., F. L. BLANC, AND A. L. NORRBOM. 1993. Handbook of the fruit flies (Diptera: Tephritidae) of fly, Anastrepha obliqua, in Florida and their asso- America north of Mexico. Cornell Univ. Press, Ithaca ciated museum specimens shows that this pest NY. 571 pp. has not re-established in Florida after its eradica- HEPPNER, J. B. 1991. Larvae of fruit flies, 7. Anastrepha tion in about 1935. obliqua (West Indian fruit fly) (Diptera: Tephriti- dae). Entomology Circular 339, Florida Dept. of REFERENCES CITED Agriculture & Consumer Services, 2 pp. STECK, G. J., L. E. CARROLL, H. CELEDONIO-H., AND J. C.A.B. International Institute of Entomology. 1988. Dis- GUILLEN-A. 1990. Methods for identification of tribution maps of pests. Anastrepha obliqua (Mac- Anastrepha larvae (Diptera: Tephritidae), and key to quart). Series A (Agricultural), Map No. 90 (revised). 13 species. Proc. Entomol. Soc. Washington 92: 356- 2 pp. 369. CLARK, R. A., G.J. STECK, AND H. V. WEEMS, JR. 1996. WEEMS, H. V., JR. 1970. West Indian fruit fly Anas- Detection, quarantine, and eradication of fruit flies trepha mombinpraeoptans Sein. Entomology Circu- in Florida, pp. 29-54. In D. Rosen, F. D. Bennett and lar 101, Florida Dept of Agriculture & Consumer J. L. Capinera (eds.). Pest Management in the Sub- Services, 2 pp.

322 Florida Entomologist 84(2) June 2001

ERRATUM

Caymanis, a New Genus of Antillocorini from the Cayman Islands (Hemiptera: Lygaeidae). By R. M. Baranowski and Julieta Brambila 84(1): 117-118.

The name Caymanis should be substituted for Abroxis in the description of the genus Caymanis on page 117, right column.

Holotype deposited in the Florida State Collection of Arthropods.

Book Reviews 323

BOOK REVIEWS

KRITSKY, G., AND R. CHERRY. 2000. Insect mythology. Writers Club Press; San Jose, California. xiii + 140 pp. ISBN 0-595-15017-9. Paperback. $11.95.

The role of insects in human culture is a subject dered about the origin of some insect family of growing interest, and this book treats one of the names, so it was interesting to read about some most interesting elements, insects in mythology. minor Roman and Greek deities, and how their Myths seemingly convey history, but actually rep- roles in mythology relate to the behavior and mor- resent natural history, and religious, philosophi- phology of insects. Anyone teaching a course in in- cal, psychological aspects of culture. That insects troductory entomology or insect classification will should be important elements of myths is an idea find numerous tidbits of information to spice up that is quite foreign to modern western cultures, lectures, and make those horrible, unpronounce- but nevertheless widespread when viewed from able order and family names more appealing. The an historical and world-wide perspective. Kritsky chapter on re-interpretation of the references to and Cherry introduce the reader to several cul- insects in the bible was also quite revealing, both tures where insects occur in myths, and provide because it reinforces the importance of locusts to insight that allows an appreciation of insects in residents of the Middle East, and because it high- ancient and diverse civilizations. lights the tractability of the “gospel.” There are three major sections to this book: gen- The major strengths of this little book are that eral mythology, old world mythology, and new world it brings together a diverse set of very interesting mythology. The first section points out that insect literature, and provides an introduction to litera- symbols are common. For example, ants and bees ture that is scattered and often overlooked. The are often symbols of industriousness and organiza- major weakness is that it truly is a little book, tion, whereas grasshoppers represent destruction. and in most cases the reader hungers for more de- Quite interesting is the observation that there are tail and explanation than is provided. Although many parallels in mythology; cultures distant in some chapters have been published previously, time or geography often use the same insect sym- this book brings them together in a concise and bols. The section on old world mythology consists of useful format. It is a reference that many will chapters on insect names derived from Greek and want for a quick, enjoyable read, and for a valu- Roman mythology, insects in Egyptian mythology, able instructional aid. Even if you do not engage insects in the bible, and cicadas in Chinese mythol- in formal teaching, think of the conversational ogy. The section on new world mythology consists of value of being able to explain why mosquito im- chapters on insects in Mesoamerican astronomy, ages appear on totem poles! insects in the mythology of Native Americans, and John L. Capinera mosquito origin myths. Department of Entomology and Nematology Almost everyone will find something of inter- University of Florida est in this little book. Personally, I’ve often won- Gainesville, FL 32611-0620

324 Florida Entomologist 84(2) June 2001

FOELIX, R. F. 1996. Biology of Spiders. Oxford Univ. Press; New York. Second edition. 330 pp. ISBN 0-19-509593-6. Hardback. $60.00 (also ISBN 0-19-509594-4. Paperback. $29.95).

Ten well-written chapters with outstanding il- idea that jumping spider courtship is always pri- lustrations quickly give the reader the sense that marily visual is maintained, with no mention of this author knows his subject and knows how to the vibratory courtships which can be performed convey this information. An introductory chapter in complete darkness. One jumping spider is mis- explains some gross morphological features, dis- identified as a lynx spider (Oxyopidae) (Fig. 13b, cusses numbers and types of spiders, and intro- p. 16); another is misspelled: Phiddipus is cor- duces some of the more common families. The rectly Phidippus (p. 197). Another misspelling (p. remaining chapters cover anatomy, metabolism, 5) is Scytotidae (= Scytodidae), the family of spit- neurobiology, webs, locomotion and prey capture, ting spiders. The bolas spider Mastophora is not reproduction, development, ecology, and phylog- credited with using pheromones to attract prey eny and systematics. The coverage is sufficiently (p. 147), which is well-known, but a relative is so thorough for an introductory text. This book credited. Ctenidae (wandering spiders) are sur- would have to be the primary textbook for an prisingly still sometimes classified as a subfamily arachnology course, and it would be an excellent of Lycosidae (wolf spiders) (p. 9). The character- supplement for an entomology or invertebrate zo- ization of wandering tarantulas (Theraphosidae) ology course. versus climbing tarantulas (Avicularidae) on p. This second edition has been updated and ex- 165 is simply wrong. Aviculariidae (proper spell- panded from the original edition published in ing) is a synonym of Theraphosidae. Genera and 1982. Particularly Chapter 4 on neurobiology has species names are not italicized on several occa- had considerable new material added, such as sions, especially in figure legends. A glossary sections on temperature perception, peripheral would have been useful. nerves, and the sub- and supraesophageal gan- The above criticisms are relatively minor and glia. Chapter 10 on phylogeny and systematics likely only to be noticed by another arachnologist. briefly discusses the most current hypothesis They do not detract from the flow of the book, and about phylogenetic relationships available at the can be corrected by an instructor or through fol- time. However, it is clear from the coverage that low-up with the bibliography provided. Unfortu- the author’s strengths are in morphology and nately, the bibliography does not directly cite physiology. Other chapters vary in the amount of some of the more useful volumes (e.g., Ecophysi- updating that has been done, which is reflected in ology of Spiders, 1987, ed. Nentwig; Spider Com- the changes, or lack thereof, made in the nomen- munication, 1982, eds. Witt and Rovner), so that clature of suborder, family, genus, and species one has to search for articles cited from these names. On p. 4, the author explains why the sub- books in order to find the book itself. order names Orthognatha and Labidognatha are No book can be all things to all people, and cer- no longer in use, then proceeds to use them on p. tainly individual subjects are covered in more 16. Other names no longer in use, such as Eu- depth elsewhere. Nevertheless, this is the most rypelma and Dugesiella among the tarantulas, complete summary of what is known of spider bi- are still cited on several occasions. Elsewhere, ology presently available. I would highly recom- both the old and new names are given (e.g., Ly- mend it. cosa pullata = Pardosa pullata, p. 236). More con- G. B. Edwards sistency in making these changes would have Florida State Collection of Arthropods been helpful. Distances given for jumping spider Division of Plant Industry (Salticidae) visual and stalking distances (pp. 11, P. O. Box 147100 90) are low and do not reflect recent research. The Gainesville, FL 32614-7100