SIT, MASS-REARING AND RELATED SUBJECTS First Meeting of TEAM. Palma of Mallorca. 7-8 th April 2008.

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1 SIT, MASS-REARING AND RELATED SUBJECTS Rearing of the European cherry fruit fly Rhagoletis cerasi optimized egg laying devices and larval diets KIRSTEN KÖPPLER, THOMAS KAFFER and HEIDRUN VOGT Julius Kuehn Institute (JKI), Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and Viticulture, Schwabenheimer Str. 101, 69221, Dossenheim, Germany. The European cherry fruit fly (Rhagoletis cerasi) is the most serious cherry pest in cherry orchards in Germany. Currently, no sufficient management strategy is available, neither for conventional nor for organic farming, nor for house gardens as infestation sources. Hence, there is an urgent need to establish environment-friendly control strategies. To do so, rearing methods must be developed to ensure effective research work in laboratory and semi field. P-generations of fruit flies were obtained by collecting pupae from infested cherries, which were stored for diapause at 4 to 6 C. Adults were kept under controlled conditions (25/18 C, photoperiod 16:8hrs, RH 65%), and supplied with brewer s yeast, sugar and water. According to the literature, black dyed wax domes were used for egg laying. But, in contrast to the proposed wax type, ceresin type 1577, a new wax combination was developed, which resulted in a much higher rate of eggs deposited into the inner of the domes. The main components of an agar-based diet were sucrose, brewer s yeast, vitamins, and cherry juice. Adult flies emerged in the P- generation with 73% success after a diapause of 5 to 9 month. F1 eggs hatched at a rate of 50 to 60%. Average pupation rate was 40% after 3 to 4 weeks. Subsequent lab generations were reared up to F5. F2 to F5 generations had decreasing numbers of flies. Despite of the still existing dependence on the field population, reliable egg laying behaviour and development of R. cerasi larvae in the laboratory is an urgent requirement for research on R. cerasi. This could be realized in our work with the improved egg laying device and the new larval diet. Key: 1735

2 Mass-rearing and field cage performance of a transgenic sexing strain of Mediterranean fruit fly NEIL I. MORRISON 1,2, DIEGO F. SEGURA 3, VIWAT WORNOAYPORN 3, ISLAM SM AMIRUL 3, KIRSTY C STAINTON 1, CARLOS CACERES 3, GERALD FRANZ 3, LUKE ALPHEY 1,2 and ALAN S. ROBINSON 3 1 Department of Zoology, Oxford University, South Parks Road, Oxford OX1 3PS, UK. 2 Oxitec Ltd, 71 Milton Park, Oxford OX14 4RX, UK. 3 FAO/IAEA Agriculture and Biotechnology Laboratory, A-2444 Seibersdorf, Austria. neil.morrison@oxitec.com A strain of Mediterranean fruit fly (Ceratitis capitata), OX3376B, carrying a female-lethal transgene insertion has been reared for four generations under simulated mass-rearing conditions and its mating competitiveness assessed in field cages with a wild-type strain. The lethal effect of the transgene insertion is repressed in the presence of tetracycline or related compounds in the larval and adult diet. The strain is homozygous for the insertion and marked with a green fluorescent protein. Standard QC parameters for the strain were assessed but expanded to include the phenotypic stability of the transgene. With chlortetracycline hydrochloride (CTC) (>70 mg/kg) in both the larval and adult diet and drinking water, fecundity of the OX3376B strain was comparable with that of the conventional genetic sexing strain, VIENNA-8. The egg hatch of OX3376B was also comparable with that of VIENNA-8, although this drops after 5-7 days, possibly as a result of a lack of CTC available to the adult flies. Rearing of OX3376B larvae on CTC supplemented diet results in eclosion rates comparable with those of the VIENNA-8 strain, although the percentage of emerging females is lower than that of males. The presence of the green fluorescent marker in pupae was monitored each generation and no non-fluorescent pupae were detected. The intensity of the fluorescence, however, is generally low and not clearly visible in dead flies. From larvae reared in the absence of CTC, no female adults were found from multiple large collections of pupae, as expected for a female-lethal strain. Comparison of the mating competitiveness of un-irradiated OX3376B males with those from a wild-type laboratory strain and VIENNA-8 was carried out following the standard protocol in field cage conditions. OX3376 performance was shown to be good in comparison with these other strains. In summary, these preliminary data indicate that, despite limitations associated with this specific transgene insertion, the use of a transgenic sexing strain may provide a viable alternative to conventional Mediterranean fruit fly genetic sexing strains. Key: 1752

3 The use of different concentrations of ginger root oil to improve the mating propensity of the Medfly sterile males S. FADHL 1, M. M SAAD GUERFALI 1, A. KAHLANI 1, H. HAITHEM 1 and C. CHEVRIER 2 1 Centre National des Sciences et technologies Nucléaires, Technopole de Sidi Thabet, Tunis Tunisie. 2 Laboratoire d étude des mécanismes de la reproduction, Institut de Recherche sur la Biologie de l Insecte, Tours, France. Selma_cnstn@hotmail.com The Sterile Insect Technique is an efficient technique used to control fruit flies such as Medfly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Unfortunately, this technique involves mass breeding of huge quantities of target insects in the factory and sterilizing the males by exposing them to low doses of radiation. This procedure reduces the mating propensity of the sterile males which are the active agent of the technique. It was reported that the exposition of the sterile males to ginger root oil [Zingiber official Roscoe (Zingiberaceae)] improves the mating propensity of the sterile males in comparison with the non exposed ones. In this study, we exposed irradiated males at two different doses (100 and 110 Gy) to different concentrations of the ginger oil (20 µl; 50 µl; 80 µl; 0.25 ml; 0.5 ml; 1 ml) the third day after emergence. This study shows that the irradiated males at 100 Gy are more competitive in comparison with those irradiated at 110 Gy with a mating propensity percentage respectively of 60 and 35%. Regarding the exposition dose of the ginger oil, 1 ml gave the best mating propensity (42%) in comparison with the other concentrations. Key: 1767

4 Mating compatibility and competitiveness of a lab reared, sterilized, hybrid strain of olive fruit fly Bactrocera oleae (Rossi) (Diptera:Tephritidae), compared with wild flies originated from Israel POLYCHRONIS REMPOULAKIS 1, ESTHER NEMNY-LAVY 2, IOANNIS DIMOU 1, ARISTIDES ECONOMOPOULOS 1 and DAVID NESTEL 2 1 Laboratory of Applied Entomology, Biology Department, University of Crete, P.O Box 2208, Herakleion, Crete, Greece. 2 Department of Entomology, Agricultural Research Organization, The Volcani Center, P.O.Box 6, Bet Dagan 50250, Israel chronis@edu.biology.uoc.gr The Sterile Insect technique (SIT) is a promising, environmentally friendly, methodology for control or eradication of insect pests. Following successful application against insects of major economic importance, such as screwworm and Mediterranean fruit fly, new target species are under examination for potential control with the release of sterile insects. In the case of olive fruit fly, a big amount of knowledge on basic biology, ecology, artificial rearing and sterilization has been collected; nevertheless, to date, pilot field application of SIT has short record and controversial results. For the successful application of SIT, adequate mating compatibility and competitiveness of mass reared insects are factors of key importance. In the case of the olive fly, it has been demonstrated that mating timing differences between wild and lab insects may lead to the failure of the technique, due to mating isolation. The aim of the present study was to evaluate the performance of a new hybrid strain of the olive fly in comparison with wild insects from Israeli olive plantations. The hybrid strain originated by backcrossing for 4 generations wild male olive flies from Israel with a laboratory colony from Vienna, originated from a mass reared strain from Greece. Hybrid insects were sterilized with 100 Gray of γ-rays from a Co 60 source. The experimental design included the use of field cages for outdoors observations, and large tunnel cages for indoor observations under natural light conditions. In order to discriminate between the two strains at copula, insects were painted on the notum surface with a non toxic enamel paint. For mating compatibility, a 1:1:1:1 (wild male: wild female: lab sterile male: lab sterile female) ratio was used, while for mating competitiveness a 1:1:1 (wild male: lab sterile male: wild female) ratio was used. Results showed that, compared to the Israeli wild flies, the hybrid strain has similar timing of mating initiation and peak activity. Also, the various isolation indices used to determine mating compatibility and competitiveness in field cage and laboratory experiments, proved that the hybrid strain is considerably compatible and competitive. The duration of copula with sterile hybrid males was in general significantly higher in all the couples including those formed with wild females. Nevertheless, effective sperm

5 transfer occurred in all crossings, showing that the hybrid strain is capable of successfully mating. The above results are encouraging for a wider, pilot field application of the sterile insect technique against the olive fruit fly, using a sterilized, mass reared hybrid strain. Key: 1770

6 Genetic strategies for the construction of genetic sexing strains in the insect Bactrocera oleae D. LAGOS 1, S. SOURMELI 1, C. SAVAKIS 2, R. LECANIDOU 1 and K. KOMITOPOULOU 1 1 Division of Genetics and Biotechnology, Dept. of Biology, University of Athens, Greece. 2 Institute of Molecular Biology and Biotechnology, FORTH, Heraklion, Crete, Greece. akomitop@biol.uoa.gr Genes involved in sexual differentiation of insect pests have been proposed as the potential targets for developing new genetic control strategies for improvement of the SIT method. We isolated the sex determining genes tra, tra2, dsx and fruitless genes, in Bactrocera oleae. We also characterized the direct target of dsx gene, yolk protein gene (yp1). All these genes are suitable candidates for the modification of the sex-ratio in insect pests. RNA interference (RNAi) technologies are very promising for their application in pest insect control. RNAi occurs in a wide variety of organisms, by which introduction of exogenous double-stranded RNA (dsrna) results in post-transcriptional depletion of the homologue endogenous mrna and thus the knock down of the corresponding gene. We utilized the RNAi technique against two of the above sex-determining genes, tra and dsx as a mean of producing only males in an improved genetic sexing system. In the case of tra gene, the introduction of Botra dsrna in embryos resulted in almost complete transformation of XX flies into fertile males, whereas Bodsxf dsrna reduced specifically its own transcript and inhibited the expression of the yolk protein gene Boyp1. In addition the ovaries were underdeveloped and the number of the mature eggs was reduced. However, in this case, the sex-ratio of offspring was not really affected. Our results lead to the hypothesis that the Bactrocera oleae tra gene seems to initiate an auto regulatory mechanism that provides the continued production of Tra protein and thus the memory of the female identity. Tra/Tra2 protein complexes, participate in the female-specific splicing of its own primary transcript. Thus, XX embryos follow the female developmental route. However, XY embryos are able to follow the male route. Botra gene is the most promising candidate for the construction of a stable transgenic sexing strain for the biological control of Bactrocera oleae. Key: 1772

7 The Sterile Insect Technique for the Control of Ceratitis Capitata (Weideman) in Oranges Orchards in Beni Khaled, Tunisia M. M SAAD GUERFALI 1 and F. LOUSSAIEF 2 1 Unité de Production des mâles Stériles de la Cératite, Centre National des Sciences et Technologies Nucléaires, Technopole Sidi Thabet, 2020, Tunis, Tunisie. 2 Laboratoire d entomologie, Direction Générale de la Protection et du Contrôle de la Qualité des Produits Agricoles. msaad_tn@yahoo.fr The Mediterranean fruit fly (Ceratitis capitata) is the key pest of citrus in the Mediterranean Basin including Tunisia. Tunisia grows 13,000 hectares of citrus with an annual production of around 200,000 tons valued at ca. US $59 million. Despite repeated malathion-bait applications the residual damage is still in the order of 10 to 15%. In addition, malathion use will be phased out in Tunisia in the near future and the European Union is pressing trading partners to comply with insecticide residue levels. Given this problems and the plan of the government of Tunisia to significantly expand its citrus production areas, mass-rearing and sterilization facility was built and has been in operations since The facility has a maximum rearing capacity of 12 million pupae per week. Quality control tests were routinely conducted. In parallel, the field operation component is being developed by installation of a trapping network which was serviced weekly to monitor the distribution and abundance of the sterile flies. At the beginning the selected area was Hammamet, and then due to some technical constraints was changed to Beni Kalled where citrus production is more intensive and where 4 to 5 aerial bait sprays applications are routinely made from September to November immediately followed by sterile males release. One thousand sterile males per hectare are released weekly in the commercial area and 2000 sterile male per hectare in the buffer area to protect form reinfestations. A control area was also included in the programme with 2500 ha of citrus. Key: 1756

8 Optimisation of the Medfly irradiation dose for sterile insect release programs M. M SAAD GUERFALI 1, S. FADHL 1, H. HAITHEM 1, A. KAHLANI 1 and C. CHEVRIER 2 1 Centre National des Sciences et technologies Nucléaires, Technopole de Sidi Thabet, Tunis Tunisie. 2 Laboratoire d étude des mécanismes de la reproduction, Institut de Recherche sur la Biologie de l Insecte, Tours, France. msaad_tn@yahoo.fr A pilot Sterile Insect Technique programme was established in Tunisia in order to control medfly. Mass breeding of huge quantities of the target insect in the factory and sterilization of the males by exposing them to low doses of radiation, may reduce their fitness. In order to optimise the irradiation sterility dose for the pilot SIT program and to obtain the best balance between sterility and quality, sterility tests were set up using males pupae of the Genetic Sexing Strain Vienna 8. Irradiation was carried out in a cobalt 60 big facility irradiator of a current activity Ci. Special cartography was done in a polystyrene boxes allowing to irradiate 10 bags of 2 l pupae. The dose of 110 Gy gave the best sterility percent in relation to the routine quality control parameters tests of the sterile males (Emergence and flight ability). Other complementary tests were developed in order to optimise more the quality of the males and to measure the impact of irradiation on their fitness. Sperm counts were done for irradiated males at different doses and the sperm storage in female spermatheca was evaluated as well as competitivity of the irradiated males. Both 100 and 110 Gy gave the lower sperm count in testis and the best mating behaviour (latency time and mating duration). Key: 1757

9 Enhanced mating competitiveness following exposure to different citrus oils in males of Ceratitis capitata NIKOS A. KOULOUSSIS 1, BYRON I. KATSOYANNOS 1, NIKOS T. PAPADOPOULOS 2, CHARALAMBOS S. IOANNOU 1 and JOHN V. ILIADIS 1 ¹Laboratory of Applied Zoology and Parasitology, School of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece. 2 Department of Agriculture, Crop Production and Rural Environment, University of Thessaly, Phytokou St N. Ionia (Volos) Greece. nikoul@agro.auth.gr Previous work has demonstrated that exposure of males of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), to the scent of sweet orange oil increases their mating success relative to non-exposed males. Here, we describe laboratory experiments that investigate the effect on mating of the scents of five citrus species and four different sweet orange varieties. Results showed that exposure to sweet oranges, mandarin oranges and lemons, which were superficially wounded in the flavedo region of the peel, conferred to males a significant mating advantage over control non-exposed males, whereas exposure to bitter orange and grapefruit did not confer a significant advantage. When instead of peel-wounded fruits we used commercial essential oils from the above species, a significant mating advantage was conferred upon exposed males in all cases. In a final test males were exposed to peelwounded sweet orange fruits of the varieties Merlin, Newhole, Navel and Artas (a local Greek variety). Although exposure to all four varieties conferred to males a mating advantage over control males, the differences observed were significant only in the case of Merlin, Newhole and Navel oranges. These results suggest that the chemicals responsible for mating enhancement are present in several citrus fruit species and varieties. Key: 1763

10 Evaluation of mating competitiveness for Bactrocera dorsalis adults reared as larvae in liquid vs standard wheat-based diets CHIOU LING CHANG and DONALD O. MCINNIS U. S. Pacific Basin Agricultural Research Center, USDA-ARS, 2727 Woodlawn Drive, Honolulu, Hawaii, 96822, USA. Three strains (standard lab, DTWP pupal color sexing strain, and wild strain) of Bactrocera dorsalis adults reared as larvae in a liquid diet, mill feed diet (Tanaka s diet), or natural host fruit diet were evaluated for mating competitiveness in both outdoor field cages and indoor Boller s mating cages. The Relative Sterility Index (RSI) was used as an indicator of mating capability. The RSI between standard strain adults that grew as larvae in liquid diet (LD) and those in Tanaka s mill feed diet (MF) indicated no statistically significant difference in Boller s mating cage results after irradiation and dye marking, although the LD strain mated significantly more than the nonirradiated standard lab strain. These results show that irradiation and dye marking had no effect on laboratory mating ability. Adults of the DTWP sexing strain that were reared as larvae in a liquid diet (DTWP-LD) or mill feed diet (DTWP-MF) had a lower mating ability than the standard lab strain in Boller s cage, and against the wild strain in both field and Boller s cages. However, there was no statistically significant difference between DTWP-LD or DTWP-MF strains in competition with the standard lab or wild strain in Boller s or field cages. These findings indicate that: (1) the quality of DTWP may be not as capable in mating as the standard lab strain; (2) liquid diet reared larvae can produce adults of equal quality compared to adults reared from conventional diet. Liquid diet reared fruit flies should be ready for implementation into mass scale rearing for SIT programs following the completion of other field quality control tests including those measuring dispersal abilities. Key: 1804

11 A liquid larval diet and its effect on the reproductive behaviour of the melon fly, Bactrocera cucurbitae (Diptera: Tephritidae) PREEADUTH SOOKAR 1, CHIOU LING CHANG 2, MALINI ALLECK 1, FAZILLA KHAYRATTEE 1 and SHRADANAND PERMALLOO 1 1 Entomology Division, Ministry of Agro Industry &Fisheries, Reduit, Mauricia. 2 U.S. Pacific Basin Agricultural Research Center, USDA-ARS, 2727 Woodlawn Dr., Honolulu, HI 96822, USA. psookar@mail.gov.mu A liquid larval diet and its effect on the reproductive behaviour of the melon fly, Bactrocera cucurbitae Coquillett was assessed. The liquid diet was composed of brewer s yeast, sugar, antifungal agents (sodium benzoate and nipagen), citric acid, and distilled water. Sponge cloth placed in rearing trays was used as a support substrate for larvae. The control diet was made up of sugarcane bagasse, maize powder, wheat bran, brewer s yeast, antifungal agents, sugar and tap water. There was no significant difference in egg hatch, adult emergence, adult fliers and the pupal weight between the liquid and the control diets. However, pupal recovery in the liquid diet was 64% less as compared to the control diet. The flies reared from the liquid diet showed a reduction in calling by 20% while the mating behaviour was not affected. Key: 1805

12 Evaluation of the chemosterilization experiment against the Mediterranean Fruit Fly Ceratitis capitata (Wied.) (Diptera: Tephritidae) in the Sóller Valley (Mallorca, Balearic Islands, Spain) A. ALEMANY 1, A. GONZÁLEZ 1, A. JUAN 2 and C. TUR 1 1 Department of Biology (Zoology), University of the Balearic Islands, Ctra. Valldemossa km 7.5, Palma de Mallorca, Spain. 2 Dirección Gral. de Recursos Hídricos, Govern Balear, Palma de Mallorca, Spain. analem@uib.es A three year experimentation ( ) programme using the chemosterilant lufenuron against the C. capitata (Wied.)(Mediterranean Fruit Fly) in the Sóller Valley (Mallorca, Spain) is reported. The insect growth regulator lufenuron (a phenylbenzoylurea) was placed into Sevep devices with proteinaceous gel mixed with the synthetic based female attractants (ammonium acetate, putrescine and amines), as well as the male attractant Trimedlure (TML). A total of 7200 devices were set up on 300 ha at the municipalities of Fornalutx and Soller, at a density of 24 devices/ha. The C. capitata population was evaluated by using Probodelt traps, baited either with the female attractants or TML, placed in three citrus orchards located at different altitudes. Fruit damage was also evaluated. Results showed and average of 20 % of reduction in the medfly population, but sex-wise very different: 37.2% for males vs. 2.7% for females. Orchard location was important too. In the Fornalutx plot, the female captures decreased 63,28%, whereas at Biniaraix one increased until 19,9%. The fruit damage followed the same pattern. Farm management allows to correctly interpreting the results. Thus, in Fornalutx, fallen oranges were removed and treatments were applied, resulting in a notably diminishing of the medfly population. On the contrary, the pest increased in Biniaraix, where fallen fruits were neglected and no treatment was applied. This was also favoured by the neighbour plots improperly managed. It may be inferred that the method based of lufenuron applied in the Soller Valley was successful in decreasing medfly population. The presence of nearby improperly managed citrus orchards, as well as other hosts for C. capitata, decreases the efficacy of the method. This should be applied to selected orchards which are well managed. This project was financed by the Balearic Government (Conselleria d Agricultura i Pesca, Crop Protection Service). Key: 1872

13 Commercialization of fertile male only eggs or male only sterile pupae of the Mediterranean fruit fly produced at El Pino Guatemala modular mass rearing facility CARLOS CACERES 1, OSCAR ZELAYA 1, CRISTOBAL PEZZAROSSI 1, PEDRO RENDON 2 and TERRY MCGOVERN 1 1 USDA-APHIS-PPQ-IS, Guatemala. 2 Methods USDA-APHIS-PPQ-CPHST, Guatemala. terry.e.mcgovern@aphis.usda.gov The "El Pino" facility for rearing Mediterranean fruit fly located 48 Km Southeast from Guatemala City has a modular design. The facility comprises four modules with single floor plus 2 modules with two floors (10,000 m 2 of rearing area). The whole complex facility is capable to produce 3,500 million of male only sterile pupae per week of the temperature sensitive lethal strain (tsl). Recently the facility has been certified and is currently operating under International Standard ISO 9001:2000. During 2007, it has produced 300 million per week to support Florida's Eradication campaign and California's prophylactic fly program, and 1,500 million/wk used in Mexico- Guatemala's Eradication Project. In addition El Pino has been shipping ca. 15 liters of male only viable eggs/day to Metapa facility in Chiapas, Mexico. These shipments have made possible to produce 500 million of sterile male only pupae per week. Shipping low cost eggs to Mexico has allowed to centralize in Guatemala the Filter Rearing System and respective amplification colonies. This strategy has optimized the use of the Metapa facility that is now fully dedicated to the male only production without risk of keeping a fertile colony in a Medfly free Area. Currently El Pino has in place appropriate protocols for long distance shipments either for eggs or pupae. So, El Pino currently is in the position to supply high quality standards sterile male only pupae or fertile male only eggs at very completive production cost to any Medfly eradication project around the world. Key: no number yet

14 Influence of methoprene and protein on survival, maturation and sexual performance of male Ceratitis capitata (Diptera: Tephritidae) MARIA JOÃO FARIA 1, RUI PEREIRA 2, THOMAS DELLINGER 1 and PETER E. A. TEAL 3 1 Departamento de Biologia, Universidade da Madeira, Funchal, Portugal. 2 Insect Pest Control Section. Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, Vienna, Austria. 3 Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, Florida. ldantas.sra@gov-madeira.pt The Mediterranean fruit fly (medfly), Ceratitis capitata (Wied.), like many other polyphagous tephritids (Diptera: Tephritidae), adopts a lek as mating system. The sterile insect technique (SIT) requires released males to survive in the field, and to compete with wild males in attracting, courting and mating with wild females. The effects of the juvenile hormone analogue, methoprene, and the incorporation of hydrolysed yeast protein into the adult diet, on survival, sexual maturation and sexual performance of male C. capitata were evaluated for the four possible combinations of methoprene-treated or untreated, and protein-fed or deprived flies. The incorporation of protein had a positive impact on laboratory survival and accelerates the sexual maturation of mass reared males. In addition, in the laboratory, male sexual performance was significantly higher in treatments with methoprene. However, field cage tests with mature and protein-fed wild males and females failed to show any effects of either factor on male sexual performance. The increased survival and the earlier sexual maturation of sterile males due to the addition of protein to the adult diet can positively influence the efficacy of SIT application against C. capitata. Key: 1745

15 Effect of juvenile hormone analog and dietary protein on lekking behavior of Melon fly Bactrocera cucurbitae Coquillett (Diptera: Tephritidae) IHSAN UL HAQ 1, C. CACERES 1, A. S. ROBINSON 1, J. HENDRICHS 2, P. E. A. TEAL 3 and C. STAUFFER 4 1 Entomology Unit, Joint FAO/IAEA Division, A-2444, Seibersdorf, Austria. 2 Insect Pest Control Section, Joint FAO/IAEA Division,Wagramerstrasse 5, P.O.Box.100, A-1400, VIC, Vienna, Austria. 3 Chemistry Research Unit, USDA, ARS, SW 23 RD DRIVE, Gainesville, FL 32608, Florida, USA. 4 Institute of Forest Entomology, Forest Pathology and Forest Protection, University of Natural Resources and applied Life Sciences, Vienna, Austria. imihsan@yahoo.com The effect of Juvenile hormone analog (Methopre) applied topically and dietary protein on mating behavior of male Melon fly Bactrocera cuccurbitae white pupae strain was studied in semi natural conditions. Males congregated and engaged in wing vibration for sexual calling in late after noon on non host tree Citrus sinensis in field cages. The young males treated with methoprene fed on protein (M+P+) initiate the wing vibration earlier than any of the males fed on protein (M-P+),males treated with methoprene fed on only sugar (M+P-) having the same age and sexually mature males without methoprene fed on sugar only (M-P-) when all males have a competition for mating with females. lek participation and sexual success was significantly higher in males (M+P+) than that of any of the males (M-P+,M+P-and M-P- ).The males M+P+ were better defender of the lekking site than any other type of males. Methoprene influenced the mating behavior of males when fed on protein that synergized the efficacy of methoprene. Without protein diet, methoprene has not effect on the mating behavior of males. Key: 1807