Mating performance of sterile Mexican fruit fly Anastrepha ludens (Dipt., Tephritidae) males used as vectors of Beauveria bassiana (Bals.

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1 J. Appl. Entomol. Mating performance of sterile Mexican fruit fly Anastrepha ludens (Dipt., Tephritidae) males used as vectors of Beauveria bassiana (Bals.) Vuill L. F. Novelo-Rincón 1, P. Montoya 2, V. Hernández-Ortíz 3, P. Liedo 1 & J. Toledo 1 1 Departamento de Entomología Tropical, El Colegio de la Frontera Sur (ECOSUR), Apartado Postal 36, Tapachula, Chiapas, México 2 Subdirección de Desarrollo de Métodos, Programa Moscamed, SAGARPA, Central Poniente 14, Tapachula, Chiapas, México 3 Departamento de Entomología, Instituto de Ecología, A.C. Apartado Postal 63 km, 2.5 Carretera Antigua a Coatepec # 351, Cong. El Haya, Xalapa, Veracruz, México Keywords Anastrepha ludens, Beauveria bassiana, entomopathogenic fungi, microbial control, reproductive behaviour, sterile insect technique Correspondence Luisa F. Novelo-Rincón (corresponding author), Departamento de Entomología Tropical, El Colegio de la Frontera Sur. Apartado Postal 36, Tapachula, Chiapas, 37 México. lnovelo@ecosur.mx Received: August 22, 28; accepted: May 13, 29. doi: /j x Abstract Field cage tests were carried out to determine the sexual competitiveness, copulation duration and sperm transfer of sterile and wild Mexican fruit fly, Anastrepha ludens (Loew), males that were treated or not treated with Beauveria bassiana (Balsamo) Vuillemin conidia immediately before mating tests. No significant differences in sexual competitiveness were found between untreated sterile males and sterile males treated with conidia, indicating that the presence of conidia did not significantly reduce mating performance. However, both types of sterile males were less competitive than wild untreated males. There were no significant differences in copulation duration and the quantity of sperm transferred between the two groups of sterile males. A positive correlation was found between copulation duration and the quantity of sperm transferred. The four treatments exhibited significant differences regarding the quantity of transferred spermatozoids stored in the spermathecae but there were no differences in the percentages of total sperm stored in each spermatheca. Our results support the idea of using sterile flies as vectors of B. bassiana conidia in controlling fruit fly populations, while, at the same time, reducing the exposure of and harmful effects on nontarget organisms. Introduction The Mexican fruit fly, Anastrepha ludens Loew (Dip., Tephritidae), is one of the most important pests affecting citrus and mango fruits in Mexico [Norma Oficial Mexicana (NOM) 1998] as it causes severe losses in these crops (Hernández-Ortíz 1996; Norrbom et al. 2). Its presence represents a constant threat for the United States citrus industry, where the detection of just one specimen results in the application of extensive and costly measures to eradicate incipient populations (Robacker et al. 23). The most common method to control this pest has been aerial or ground applications of insecticide-bait sprays. Although this method has been effective, it is frequently regarded as a serious threat to the environment (Moreno and Mangan 2). The search for alternative, more environmentally friendly control methods continues to be a priority. Several strains of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin are considered viable options in the development of alternative biocontrol methods. In addition, some strains have been successfully used to control pests ranging from the house fly Musca domestica L. (Barson et al. 1994; Watson et al. 1995, 1996), the tsetse fly Glossina spp. (Kaaya and Munyinyi 1995; Maniania and Odulaja 1998), and the coffee berry borer Hypothenemus hampei Ferrari (De la Rosa et al. 2). It has also been shown that this fungus can cause high 72 J. Appl. Entomol. 133 (29) ª 29 Blackwell Verlag, GmbH

2 L. F. Novelo-Rincón et al. Mating performance of sterile Mexfly males as vectors of B. bassiana infection levels among adult fruit flies such as the Mediterranean fruit fly Ceratitis capitata (Wiedemann), other Ceratitis species, the olive fruit fly Bractocera oleae (Gmelin), and the Mexican fruit fly A. ludens (Castillo et al. 2; De la Rosa et al. 22; Ekesi et al. 22; Dimbi et al. 23; Konstantopoulou and Mazomenos 25). Recently, it was shown that adult Mexican fruit fly males treated with B. bassiana conidia were capable of transmitting them to receptor females during courtship and copulation (Toledo et al. 27). This suggests the possibility of combining the sterile insect technique (SIT) with the use of B. bassiana, using sterile A. ludens males as conidia vectors to introduce the fungus to wild populations as a new mortality factor. According to Klassen (25), the SIT is an inverse density-dependent strategy for pest management where the release of high quantities of sterile insects into the field is required to reach a favourable sterile insect:wild insect ratio. The goal is to induce high levels of sterility in wild populations, resulting in the eventual decrease in pest numbers. This strategy has been successfully applied against several insect pests, such as the new world screwworm [Cochliomyia hominivorax (Coquerel)], different species of fruit flies [Bractocea cucurbitae (Coquillett), Bractocea tryoni (Froggatt), and Ceratitis capitata (Wiedemann)] and tsetse flies (Glossina austenis, Glossina morsitans submorsitands Newstead) (Klassen and Curtis 25). The success of this control method is highly dependent on the sexual performance of the released sterile males, which compete for wild females during courtship. The number of copulations achieved, copulation duration, and sperm transfer are the main factors determining the effectiveness of the technique. Release recapture experiments have revealed that that the lifespan of sterile males is short (Baker et al. 1986; Hendrichs et al. 1993; Thomas and Loera- Gallardo 1998; Hernández et al. 27). There is also evidence that sterile males suffer high mortality due to predation (Hendrichs and Hendrichs 1998). Therefore, we hypothesized that the effectiveness of the technique could be increased if the sterile males, in addition to the induction of sterility into the wild population, could also induce mortality through the horizontal transmission of B. bassiana. A key point in this approach would be that the courtship behaviour and mating performance of the sterile male vectors should be unaffected by the presence of the fungi. Our aim in this study was to evaluate the mating performance of sterile A. ludens males treated with B. bassiana conidia in comparison with untreated and wild males. The number of copulations achieved with and without competition, and copulation duration and sperm transfer without competition were measured in order to determine the feasibility of using sterile males as vectors. Materials and Methods Biological material and experimental site Wild-strain (WS) flies were obtained as larvae from infested orange fruits (Citrus sinensis L.) which were collected on the outskirts of Tapachula, Chiapas. The fruits collected were carried to the laboratory where they were weighed, counted and placed in plastic trays ( cm) containing moist vermiculite and stored for 4 days at 28 1 C to allow larvae to mature. This incubation period allowed most of the larvae to reach physiological maturity optimal for pupation. Then, fruits were dissected in order to remove the third instar larvae, which were placed in.5 l plastic containers with moist vermiculite to encourage pupation and covered with no. 18 cloth mesh. Two days before adult emergence, the pupae were separated from the vermiculite with a fine sieve (18 mesh) and placed in glass cages (3 3 3 cm). The adults were fed with a mixture of enzymatically hydrolysed yeast (MP Biomedical, Irvine, CA, USA) plus sugar at a 1 : 3 ratio, and water was provided ad libitum in glass vials covered with cotton gauze. Sterile males [laboratory strain (LS)] were obtained as pupa from the MOSCAFRUT mass-rearing facility located in Metapa, Chiapas, Mexico, where they were bred following the procedures described by Domínguez et al. (2). The pupae were irradiated at 8 Gy with Cobalt 6, 36 h prior to adult emergence. Both WS and LS pupae were placed in cm glass cages and were provided with food and water as described above. Three days after emergence, flies were sorted by sex and males from both strains (WS and LS) were maintained in the laboratory at 27 2 C and 8 5% relative humidity (RH) until they were required for the tests. The males were used when they reached sexual maturity: 8-day-old in the case of LS and 14-day-old for WS. Females were used at 14-days. Beauveria bassiana entomopathogenic fungus, strain LCPP, were provided as dry conidia by the Entomopathogenic Organisms Production Laboratory of the Local Committee on Plant Protection, in Tapachula, Chiapas, Mexico. The concentration was conidia/g, with a >9% viability, and a LT 5 = 4.2 days (Toledo et al. 27). J. Appl. Entomol. 133 (29) ª 29 Blackwell Verlag, GmbH 73

3 Mating performance of sterile Mexfly males as vectors of B. bassiana L. F. Novelo-Rincón et al. The experiments were performed in semi-natural conditions using clear cylindrical nylon screen field cages (3 m diameter 2 m high). Two potted mango (Mangifera indica L.) trees (1.2 m high) and one coffee (Coffea arabica L.) tree were placed in the centre of each field cage, as suggested by Calkins and Webb (1983). The field cages were set up in the gardens of El Colegio de la Frontera Sur in Tapachula, Chiapas, México. In this locality, the average temperature is 26 C, and the rainy season extends from May to October, with an annual rainfall range of mm (García 24). During the experiment, the photoperiod (light : dark) was 12 : 12 h. Sexual competitiveness of sterile males treated with B. bassiana This study was carried out in field cages using a 4 : 1 ratio of males to females. To identify males of each treatment, they were marked through the addition of 5 ll of vegetable food colouring (McCormick and Company Inc., Naucalpan, México, DF ) to 3 g of the food mixture of enzymatically hydrolysed yeast and sugar. The colours used for each treatment were: (a) untreated wild males = green; (b) treated wild males = blue; (c) untreated sterile males = pink; and (d) treated sterile males = yellow. Adding the natural colouring to fruit fly food is an easy way to distinguish males because the colouring is visible through the abdomen, in the insect digestive system and it has been shown that it has no effect on behaviour (P. Liedo, pers. obs.). For treatment with B. bassiana conidia, 3 min prior to the start of experiments, 2 wild males and 2 sterile males were placed separately in test tubes and, to facilitate their management during the inoculation process, insects were placed in a refrigerator at C for 3 min. Subsequently, the lethargic males were treated with B. bassiana conidia at a proportion of 1 g per 1 males. The tubes were gently shaken until the insects bodies were completely covered by conidia. For controls, we used 2 untreated wild males and 2 untreated sterile males that were subject to the same cooling and handling process. Twenty males from each treatment and 2 wild virgin females (for a total of 1 flies per cage) were released into each field cage. Copulating pairs were collected, removed from the field cage and placed in glass vials covered with cotton gauze. The type of male and treatment was recorded. Considering that the mating behaviour of A. ludens is crepuscular (Aluja et al. 1983; Robacker et al. 23), all experiments lasted 4 h, starting at 16: hours and observations were undertaken until 2: hours, where darkness was reached at approximately 19:3 hours. Recordings were made in two field cages per day over six consecutive days, for a total of 12 replicates. During the experiment, the average temperature was C and average RH was %. Copulation duration and sperm storage patterns To carry out this test, four field cages were set up as described above, one for each treatment. The treatments were: (a) untreated wild males; (b) treated wild males; (c) untreated sterile males; and (d) treated sterile males. Males were marked and exposed to B. bassiana conidia as described above. Then, 4 males from each treatment were transferred along with 4 wild females into each field cage. After the initiation of mating, each copulating pair was collected in a test tube, covered with cotton gauze, and the duration of copulation was recorded. The experiments lasted 4 h, starting at 16: hours, and observations were carried out until 2: hours. Six replicates were performed, for a total of 24 observed males per treatment. Twelve hours after copulation, the amount of sperm and how it was stored in the female spermatheca was recorded. Using a binocular dissecting microscope, the female reproductive apparatus was separated from the abdomen and the fatty tissue was removed in order to locate the three spermathecae. Each spermatheca was placed on a microscope slide and then 12.5 ll of phosphate-buffered saline solution (PBS), which consisted of 1 ml of distilled deionized water,.5 m NaHPO 4 and.85 m NaCl (Moritz 1984), was applied. The spermathecae were perforated with a # entomological needle to extract sperm, which was extended over the microscope slide and then covered with a 2 2 mm cover slip. Finally, the preparation was sealed with clear nail varnish. The spermatozoids from each spermatheca were quantified using a 1X phase contrast microscope, observing the whole preparation to obtain the total number of spermatozoids per spermatheca and for each female. Statistical analysis The number of copulations per treatment was adjusted with a logistical regression using the Splus v7 program. Differences between treated and untreated sterile males and between treated and untreated wild males were determined using a 74 J. Appl. Entomol. 133 (29) ª 29 Blackwell Verlag, GmbH

4 L. F. Novelo-Rincón et al. Mating performance of sterile Mexfly males as vectors of B. bassiana chi-square test. Copulation duration, and transfer and distribution of sperm data were subjected to a chi-square test. Differences between copulation duration and the total amount of transferred spermatozoids were analysed using anova. A Spearman correlation test was applied to data regarding copulation duration and the total amount of spermatozoids per treatment. The amount of spermatozoids from treated and untreated males transferred to the spermathecae was analysed using a G-test contingency table, as well as a discriminant analysis to identify the relationships between individuals of each treatment and between treatments. The data were analyzed with Statistica version 6. statistical software (Statsoft 23). Results Sexual competitiveness of sterile males treated with B. bassiana The greatest number of copulations was exhibited by untreated wild males, whereas the treated wild males displayed the lowest number (table 1). There were significant differences between treated and untreated wild males (v 2 = 22.5, d.f. = 1, P <.1), but no significant differences were observed between treated and untreated sterile males (v 2 = 2.25, d.f. = 1, P =.133). Data on the number of copulations in competitive conditions were adjusted to a logistic regression model (v 2 = 3.26, d.f. = 3, P <.1), revealing the influence of treatment on the probability of attaining copulation (v 2 = 26.7, d.f. = 3, P <.1). A significant difference in the probability of achieving copulation was observed between untreated wild males and the three other treatments (table 2). Table 1 Average and percentage of copulations obtained by Anastrepha ludens males corresponding to the four treatments in competition (N = 24 males/treatment) Treatments Copulations Average SE Untreated wild males a B. bassiana-treated wild males b 5.42 Untreated sterile males ns B. bassiana-treated sterile males ns 1.83 % achieved of total pairs released *Values in the same column with the same letter are not significantly different (P >.5). Table 2 Confidence intervals for probability of achieving copulation by wild and laboratory male A. ludens treated and non-treated with Beauveria bassiana Treatments Probability 95% confidence intervals Lower Copulation duration and sperm storage pattern Higher Untreated wild males.212 a B. bassiana-treated wild males.54 b Untreated sterile males.158 b B. bassiana-treated sterile males.18 b *Values in the same column with the same letter are not significantly different (P >.5). A total of 88 females mated with untreated wild males, 25 mated with treated wild males, 86 with treated sterile males, and 36 with untreated sterile males. The treatments groups with the highest numbers of copulations and successful copulations (resulted in sperm transfer) were by untreated wild and sterile males, respectively. Significant differences between the four treatments were recorded vis-à-vis the total number of copulations (v 2 = 55.4, P <.1). There were differences between treatments regarding the percentage of copulations with sperm transfer (v 2 = 58.47, d.f. = 3, P <.1) (table 3). Untreated sterile males displayed the shortest copulation duration (2 min) but also the longest copulation (28 min); however, no significant differences in copulation duration between treatments were found (F =.26, d.f. = 3, 231, P =.855) (table 3). In relation to the total number of spermatozoids transferred to the spermatheca for each treatment, untreated wild males transferred the largest amount of sperm and the difference with the other three treatments was significant (F = 1.2, d.f. = 3, 231, P <.1) (table 3). Copulation duration and the total number of spermatozoids exhibited a significant positive correlation for all four treatments (fig. 1). Discriminant analysis revealed significant differences between treatments in the total number of spermatozoids for each spermatheca (F = 5.6, d.f. = 9, 557, P <.1). The major distance occurred between untreated wild males and treated sterile males (F = 1.147, d.f. = 3, 229, P <.1). These differences were determined by the quantity of spermatozoids stored by the females in the spermathecae located as 1 and 2 (Wilks k =.88 and.87, respectively; P =.1 and.4). However, J. Appl. Entomol. 133 (29) ª 29 Blackwell Verlag, GmbH 75

5 Mating performance of sterile Mexfly males as vectors of B. bassiana L. F. Novelo-Rincón et al. Table 3 Successful copulations, copula duration and average number and percentages of spermatozoids by spermatheca transferred by male A. ludens for the four treatments without competition Treatment Successful copulas (with spermatozoids) Copula duration (min) Number and percentage of spermatozoids by spermatheca* 1 % 2 % 3 % Total number of spermatozoids Untreated wild males a a B. bassiana-treated wild males a b Untreated sterile males a b B. bassiana-treated sterile males a b Averages SE in the same column with the same letter are not significantly different (P >.5) according to ANOVA. *No significant differences in percentage of spermatozoids by spermatheca according by G-test (G = 1.865, d.f. = 6, P =.932). 3 (a) y =.688x R 2 = (b) y =.879x R 2 = Copula duration (min) (c) y =.332x R 2 = (d) y =.925x R 2 = Total number of spermatozoids Fig. 1 Relationship between copula duration (min) and total number of spermatozoids for the four treatments: (a) non-treated wild males; (b) treated wild males; (c) non-treated sterile males; (d) treated sterile males. no significant differences were found in the percentage of total sperm stored in each spermatheca (G = 1.865, d.f. = 6, P =.932). Discussion The results obtained in this study suggest that the use of sterile insects as vectors of B. bassiana is feasible only if the conditions for their use are carefully considered. In a previous study, Toledo et al. (27) demonstrated that horizontal transmission of B. bassiana conidia was feasible through copulation and copulation attempts between treated males and healthy females of A. ludens, and that infected females suffered a significant reduction in survival, fecundity and fertility after the fungus attack. Quesada-Moraga 76 J. Appl. Entomol. 133 (29) ª 29 Blackwell Verlag, GmbH

6 L. F. Novelo-Rincón et al. Mating performance of sterile Mexfly males as vectors of B. bassiana et al. (28), who inoculated C. capitata adults with Metarhizium anisopliae conidia, reported similar results. The data regarding sexual competition indicate that the wild males not treated with the fungal conidia performed better sexually when compared with the other treatments. This wild male superiority over sterile males has been consistently reported for several fruit fly species (e.g. Wong et al. 1983; Cayol 2; Lux et al. 22; Allinghi et al. 27; Orozco- Davila et al. 27). Although sterile males join leks, release pheromones, court approaching females, compete for mating, mate, transfer sperm and induce refractory periods in females (the period between matings), they have traditionally shown less competitiveness than wild males (Whittier et al. 1992; Hendrichs et al. 22). The presence of the fungus conidia demonstrated differential effects on sexual competitiveness. In the case of wild males, the fungi significantly reduced their sexual performance. However, no significant difference was detected in sterile males. This suggests that sterile males were less susceptible to the presence of conidia, thus supporting the alternative of using inoculated sterile insects to facilitate the introduction of fungus conidia in wild populations of this pest. Campos et al. (28) reported that, in natural conditions (preliminary data), 65% of C. capitata wild females were infected with B. bassiana conidia in coffee areas subject to ground release of treated sterile males. This experiment was carried out under conditions of strong competition. Each type of male had to compete simultaneously with three other males for each wild female (at a ratio of 4 males : 1 female). The standard competition test, which is carried out under quality control protocols at SIT facilities, uses a sterile:wild ratio of 3 : 1 (FAO/IAEA/USDA 23). This test is a fundamental parameter in the application of SIT (McInnis et al. 1994). The success of the SIT depends on the relative density of both sterile and wild populations (sterile : wild ratio) which should clearly be inclined to the former, so that mating between sterile males and wild females is favoured (Hendrichs et al. 25; Knipling 1955). Therefore, it can be expected that, when inoculating and releasing large quantities of sterile males treated with conidia, these insects will be present in favourable proportions compared with wild males in the field, thus incorporating a new mortality factor into the system. Treated sterile males could have three possible effects on the wild population: (1) induction of sterility (i.e. lower egg hatching as result of sterile sperm transfer to wild females); (2) reduction in the probability of a second mating, due to female infection and death because of B. bassiana; and (3) in the case of no copulation or sperm transfer, B. bassiana horizontal transmission could result in wild female mortality. The number of attained copulations can be used as an approximation of reproductive success in males. However, the fact that a male achieves copulation does not determine whether it is successful (which requires the transfer of sperm) (Arita and Kaneshiro 1985). Successful fertilization can vary, depending on the number or distribution of the sperm stored in the females (Eady 1995; Ward 1998) and could also be positively correlated with copulation duration (Field and Yuval 1999; Field et al. 1999; Taylor and Yuval 1999; Taylor et al. 2). In short, reproductive success is the result of the interaction between copulation and sperm competition. In our study, sterile males with or without the presence of conidia displayed similar performance regarding copulation duration and the total number of spermatozoids transferred to wild females. Therefore, inoculation had no effect on sterile male mating performance, and copulation duration makes conidia transmission feasible. In contrast to established knowledge, fewer spermatozoids transferred to the female spermathecae could indirectly increase the efficiency of this method because females treated with conidia would experience a shorter refractory period (resulting in a higher chance of copulating again) and could infect other wild males. This effect would be consistent with previous work, which showed that A. ludens females have a shorter period between copulations following mating with sterile males (Bloem et al. 1993; Rull et al. 25). The proposal to use sterile males as vectors of entomopathogenic fungi has some disadvantages, but we still believe that its application has great potential in fruit fly action programmes. Since its conception, this proposal has focused on areas where other control methods are not compatible with existing production systems or conditions, such as organic farms, areas of difficult access for pest control personnel, areas with social resistance to traditional control methods such insecticide-bait spraying, urban areas and home gardens (Toledo et al. 27). Additional knowledge is required on the survival, movement, dispersal and spore transmission of treated sterile flies under natural field conditions, as well as the development of methods for inoculation just before aerial release. These are new challenges that need to J. Appl. Entomol. 133 (29) ª 29 Blackwell Verlag, GmbH 77

7 Mating performance of sterile Mexfly males as vectors of B. bassiana L. F. Novelo-Rincón et al. be met in order to fortify this innovative approach for pest control. Acknowledgements We thank two anonymous reviewers for their comments that improved this work, and also thank Jorge Hendrichs for critical review and suggestions. Our thanks are due to Gustavo Rodas, Ezequiel de León and Salvador Flores for their help with the fieldwork, and to Javier Valle for his assistance with the statistical analysis. We also thank the MOSCAMED- MOSCAFRUT Program (SAGARPA) for providing sterile flies, and the Consejo Nacional de Ciencia and Tecnología (CONACYT) for the scholarship granted to Luisa F. Novelo Rincón. This work was carried out as part of Luisa F. Novelo Rincón Ph.D. thesis at ECOSUR. References Allinghi A, Calcagno G, Petit-Marty N, Gómez Cendra P, Segura D, Vera T, Cladera J, Gramajo C, Willink E, Vilardi JC, 27. Compatibility and competitiveness of a laboratory strain of Anastrepha fraterculus (Diptera: Tephritidae) after irradiation treatment. Fla. Entomol. 9, Aluja M, Cabrera M, Hendrichs J, General behavior and interactions between Anastrepha ludens and A. obliqua under seminatural conditions. I. Lekking behavior and male territoriality. In: Fruit flies of economic importance Ed. by Cavalloro R, Balkema, Rotterdam, Arita LH, Kaneshiro KY, The dynamics of the lek system and success in males of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Proc. Hawaiian Entomol. Soc. 25, Baker PS, Chan AST, Jimeno-Zavala MA, Dispersal and orientation of sterile Ceratitis capitata and Anastrepha ludens (Tephfitidae) Chiapas, Mexico. J. Appl. Ecol. 23, Barson G, Renn N, Bywater F, Laboratory evaluation of six species of entomopathogenic fungi for the control of the house fly (Musca domestica L.) a pest of intensive animal units. J. Invertebr. Pathol. 64, Bloem K, Bloem S, Rizzo N, Chambers D, Female medfly refractory period: effect of male reproductive status. In: Fruit flies: biology, and management. Ed. by Aluja M, Liedo P, Springer, New York, Calkins CO, Webb JC, A cage and support framework for behavioral tests of fruit flies in the field. Fla. Entomol. 66, Campos SE, Flores S, Espinoza E, Montoya P, Villaseñor A, Toledo J, 28. Control de Ceratitis capitata en zonas cafetaleras mediante liberaciones de adultos estériles transmisores de conidios de Beauveria bassiana (Bals.) Vuill. In: Proceedings of the 7th Meeting of the Working Group on Fruit Flies of the Western Hemisphere. Ed. by Montoya P, Diaz F, Flores S, Mazatlán, Sinaloa, Mexico, 156. Castillo MA, Moya P, Hernández E, Primo-Yúfera E, 2. Susceptibility of Ceratitis capitata Wiedemann (Diptera: Tephritidae) to entomopathogenic fungi and their extracts. Biol. Control, 19, Cayol JP, 2. Changes in sexual behavior and life history traits of tephritid species caused by mass-rearing processes. In: Fruit flies (Tephritidae): phylogeny and evolution of the behaviour Ed. by Aluja M, Norrbom AL, CRC Press, Boca Ratón, FL, De la Rosa W, Alatorre R, Barrera JF, Toriello C, 2. Effect of Beauveria bassiana and Metarhizium anisopliae (Deuteromycetes) upon the coffee berry borer (Coleoptera: Scolytidae) under field conditions. J. Econ. Entomol. 93, De la Rosa W, López FL, Liedo P, 22. Beauveria bassiana as a pathogen of the Mexican fruit fly (Diptera: Tephritidae) under laboratory conditions. J. Econ. Entomol. 95, Dimbi S, Maniania NK, Lux SA, Ekesi S, Mueke M, 23. Pathogenicity of Metarhizium anisopliae (Metsch.) Sorokin and Beauveria bassiana (Balsamo) Vuillemin, to three adult fruit fly species: Ceratitis capitata (Weidemann), C. rosa var. fasciventris Karsch and C. cosyra (Walker) (Diptera: Tephritidae). Mycopathologia, 156, Domínguez J, Castellanos D, Hernández-Ortiz E, Martínez A, 2. Métodos de cría masiva de moscas de la fruta. In: Proceedings XIII Curso Internacional sobre Moscas de la Fruta. Centro Internacional de Capacitación en Moscas de la Fruta. Metapa de Domínguez, Chiapas, Mexico, Eady PE, Why do males Callosobruchus maculatus beetles inseminate so many sperm? Behav. Ecol. Sociobiol., 36, Ekesi S, Maniania NK, Lux SA, 22. Mortality in three African tephritid fruit fly puparia and adults caused by the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana. Biocontrol Sci. Technol. 12, FAO/IAEA/USDA, 23. Manual for product quality control and shipping procedures for sterile mass-reared Tephritid fruit flies, version 5.. International Atomic Energy Agency, Vienna, 85. Field SA, Yuval B, Nutritional status affects copula duration in the Mediterranean fruit fly, Ceratitis capitata (Insecta: Tephritidae). Ethology, Ecology and Evolution, 11, Field SA, Taylor PW, Yuval B, Sources of variability in copula duration of Mediterranean fruit flies. Entomol. Exp. Appl. 92, J. Appl. Entomol. 133 (29) ª 29 Blackwell Verlag, GmbH

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