Tapachula, Chiapas, Mexico

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1 Proceedings of 6th International Fruit Fly Symposium 6 10 May 2002, Stellenbosch, South Africa pp Sexual compatibility, mating performance and sex pheromone release of mass-reared and wild Anastrepha obliqua (Diptera: Tephritidae) under field-cage conditions J.S. Meza-Hernández 1, E. Hernández 1, M. Salvador-Figueroa 3 & L. Cruz-López 2 * 1 Campaña Nacional contra Moscas de la Fruta Acuerdo SAGARPA-IICA, Tapachula, Chiapas, Mexico 2 El Colegio de la Frontera Sur. Carretera Antiguo Aeropuerto Km. 2.5, Tapachula, Chiapas, Mexico 3 Universidad Autónoma de Chiapas, Biotecnología. Carretera a Puerto Madero Km. 2 C.P , Tapachula, Chiapas, Mexico The isolation index (ISI),male relative performance index (MRPI),female relative performance index (FRPI) and the relative sterility index (RSI) were used to compare the mating compatibility and mating performance of mass-reared Anastrepha obliqua fruit flies with wild A. obliqua flies in field cages. Sexual maturity and amounts of the released male volatiles were also compared for both populations under field conditions. Wild A. obliqua adults started sexual activity at an age of 10 days, reaching peak sexual maturity at 16 days, while mass-reared flies started sexual activity at six days, reaching sexual maturity at eight days. The ISI value obtained (ISI 0.14 ) indicated random mating with a high degree of sexual compatibility. The FRPI value (FRPI 0.02 ) showed that laboratory reared and wild females mated equally well. The MRPI value (MRPI 0.25 ) showed a higher mating performance for wild flies than reared males. The RSI value (RSI 0.29 ) indicated that laboratory males performed well in competing for wild females, but that females tended to choose wild males. Gas chromatography mass spectrometry analysis of the male pheromone volatiles showed that they were first released by mass-reared males at an age of six days and by wild males at eight days. After eight days the amount of released volatiles from mass-reared flies remained constant,while that for the wild flies increased until an age of 16 days. From an age of 14 days, the amount of volatiles released by wild flies was three times larger than that from mass-reared flies. INTRODUCTION Sterile insect technique (SIT) programmes have been carried out against several Tephritidae species, including Ceratitis capitata (Wong et al. 1986), Bactrocera cucurbitae (Iwahashi et al. 1983) and several Anastrepha species (Holler et al. 1984; Holler & Harris 1993). SIT involves the colonization of insects in artificial habitats (Mason et al. 1987). In nature, tephritid males form leks to attract the females using acoustic signals (Sivinski et al. 1984) and sex pheromones (Nation 1989). Such behaviour is performed by males to choose the best partner (Liedo 1996), whereas in rearing cages no leks are formed (Cayol 2000), and the females have difficulty determining from which male a given pheromone plume is emitted because of the large amount of pheromone present (Calkins 1989). The above conditions create different selective pressures for laboratory-reared flies that can result in changes in sexual behaviour (Calkins 1984; Boake et al. 1996; Iwahashi 1996). Sexual compatibility between strains is one of the most important requirements for the success of SIT (Boller 1977). To assess sexual compatibility and mating performance, several mating indices have been used for *To whom correspondence should be addressed. lcruz@tap-ecosur.edu.mx C. capitata (Chambers et al. 1983; McInnis et al. 1996). Cayol et al. (1999) described three new indices, isolation index (ISI), male relative performance index (MRPI), and female relative performance index (FRPI), to measure sexual compatibility and mating performance between wild and laboratory-reared C. capitata. In this study we used the ISI, MRPI, FRPI and the relative sterility index (RSI) (McInnis et al. 1996) to assess the mating compatibility between massreared and wild Anastrepha obliqua fruit flies in field-cage conditions. In addition, we measured the sexual maturity and amount of the released sexual pheromone for both populations under field conditions. MATERIALS AND METHODS Biological material Wild A. obliqua fruit flies used in this study were obtained as larvae from jocote fruit (Spondias pupurea), while the mass-reared flies were obtained as pupae from the rearing facility of Moscafrut in Metapa de Domínguez, Chiapas, Mexico. Field cages Field cages (2.9 m diameter 2.0 m high) were made as described by Chambers et al. (1983). Each

2 100 Proceedings of the 6th International Fruit Fly Symposium cage was supported by a metallic frame (Calkins & Webb 1983),and contained small mango and citrus trees (2 m high) alternately distributed in the centre and the inside periphery. Field-cage testing protocol Bioassays were carried out in the Irene orchard located in the highland of Tapachula, Chiapas, Mexico. To determine sexual maturity, five previously marked wild flies and five mass-reared flies of both sexes, at ages 4, 6, 8, 10, 12, 14, 16, 18 and 20 days, were released in separate cages. We recorded the number of matings per fly of each age. Later, 20 flies of each sex and each population were released into the field cages at 06:00 (dawn), and the test lasted until 12:30. Male flies were released 30 min before the females (Prokopy & Hendrichs 1979). The numbers of calling males were recorded every 30 minutes in each cage. The type of males participating in the leks which formed (wild, mass-reared and mixed) were also recorded. To estimate compatibility and mating performance, the ISI, MRPI, FRPI and RSI were calculated using each possible mating combination between strains [8Wild 9Wild (WW), 8Wild 9Laboratory (WL), 8Laboratory 9Laboratory (LL) and 8Laboratory 9Wild (LW)]. Three replicates of the tests were run on the same day for three days. Sex pheromone collection Volatiles emitted by males of A. obliqua were collected using an air-entrapment technique. Ten males were confined in a 100 ml glass entrapment container (4.8 cm internal diameter 12.5 cm high). Volatiles were drawn from the container using purified air that had previously passed through an activated charcoal trap, onto a glass volatile collection trap (4 mm ID 40 mm long) containing 50 mg Super Q adsorbent (Alltech Associates, Deerfield, II., U.S.A.) (Heath & Manukian 1992). Air was drawn through the trap at a rate of 1 l/minute for two hours by a vacuum pump. At the conclusion of each air entrapment the volatiles were eluted from the adsorbent with 200 µl of methylene chloride (Baker, HPLC grade), and 100 ng of tridecane was added as an internal standard for subsequent quantification. Chemical analysis Gas chromatography mass spectrometry (GC MS) was conducted using a Varian Star 3400 CX gas chromatograph linked to a Varian Saturn 4D mass spectrometer. The samples were analysed using a fused silica column (30 m 0.25 mm) coated with poly (5%-diphenyl-95%-dimethylsiloxane) programmed from 50 to 250 C at 15 C/minute. The carrier gas was helium. The injector port temperature was held at 200 C. Statistical analysis To determine significant differences in the collected data we used an analysis of variance (ANOVA), using the software StatView for Windows (SAS Institute Inc., , ver. 5). RESULTS Sexual maturity Maximum sexual activity of wild A. obliqua was reached at an age of 16 days, while that for reared flies was reached when eight days old. There was no significant difference between the numbers of matings of wild flies between 12 and 20 days old. By contrast, the mass-reared flies showed a decrease in number of matings after eight days of age, when the highest number of matings occurred (Fig. 1). Sexual compatibility The mating proportion was higher that 0.8, indicating that 80% of the total number of possible matings took place; these data can be used for compatibility studies (International Atomic Energy Agency 1977). The ISI 0.14 value obtained indicated random mating and sexual compatibility with a slight tendency towards isolation.the FRPI 0.02 value showed that mass-reared and wild females performed equally well at mating. The MRPI 0.25 value showed a higher mating performance for wild flies than for mass-reared males. The RSI 0.29 value indicated that laboratory males performed well in competing for wild females, but there was a tendency for wild females to choose wild males (Fig. 2). On the other hand, the ANOVA test of independence for each individual combination of mass-reared and wild strains did not show significant differences in the selection of partners by mass-reared females (with wild male = 28.30%; lab male = 22.65%;F = ;d.f. = 3,32;P = ), while wild females showed significant differences in the choice of mating partners (with wild male = 34.55%; lab male = 14.48%; F = ; d.f. = 3,32; P = <0.0001) (Table 1). Male calling activity By counting the number of calling males throughout the day it is possible to identify any

3 Meza-Hernández et al.: Mating and sex pheromone release of mass-reared and wild Anastrepha obliqua 101 Fig. 1. Effect of age on the mating of Anastrepha obliqua. Thin line = mass-reared males; bold line = wild males difference in activity rhythms between the massreared and wild males (Cayol et al. 1999). No qualitative differences were found in the male calling activity pattern of the two types of males (Fig. 3). Leks formed Of 157 leks formed, ± 2.365% (mean ± S.E.) were composed of reared males, ± 6.814% of wild males, and ± 8.012% were composed of both male populations (Fig. 4). Volatile emission GC MS analysis of the volatiles collected from male flies showed that they comprised mainly three components: (Z,E)- -farnesene, (E,E)- -farnesene and (Z)-3-nonanol. Pheromone was first released by mass-reared males at an age of six days, and by wild males after eight days. After eight days the amounts of pheromone released by mass-reared flies remained constant,and for the wild flies, there was no significant difference in Fig. 2. Means of isolation index (ISI), female relative performance index (FRPI), male relative performance index (MRPI) and relative sterility index (RSI) of Anastrepha obliqua.

4 102 Proceedings of the 6th International Fruit Fly Symposium Table 1. Mating proportion between combinations of wild (W) and mass-reared (L) strains of Anastrepha obliqua (mean ± S.E., n = 9). Mating proportion (%) (male female) W W W L L L L W ± a ± b ± b ± c Means followed by the same letters do not differ significantly (P = 0.05). Fig. 3. Percentage of calling males of Anastrepha obliqua at different times during the evaluation. Open bar = laboratory males; solid bar = wild males. the amounts of pheromone release after 14 days (Fig. 5). The amount of pheromone released by wild flies was greater than that released by mass-reared flies, with maximum pheromone release by mass-reared flies occurring on the sixteenth and eighteenth day (reared males = ± ng/m/h; wild males = ± ng/m/h). DISCUSSION An examination of the mating patterns by age between wild and mass-reared A. obliqua males showed that mass-reared flies reached sexual maturity earlier than wild flies, as has been observed in other fruit fly species (Rössler 1975; Suzuki & Koyawa 1980; Liedo et al. 1996). This indicates that the mass-rearing process may provoke sexually-linked behavioural changes. The ISI, MRPI, FRPI and RSI data showed that the mass-reared A. obliqua in Metapa, Chiapas, Mexico, are compatible in mating with the wild population in the region of the Soconusco, Chiapas, although there was a slight tendency toward isolation. This isolation was due to the high proportion of wild fly matings (WW) recorded during the test, possibly due to the less aggressive sexual behaviour of the Fig. 4. Types of male Anastrepha obliqua participating in lek formation (ANOVA; P = 0.05, mean ± S.E., n = 9). laboratory-reared males versus wild males, since there was 25% less mating by laboratory-reared males than by wild flies. We also found that mass-reared males were accepted by only 29% of the wild population females. Although this figure is low in relation to the acceptability of wild males, it is normally not severe enough to preclude the effective use of SIT (Cayol et al. 1999). However, low male acceptability, together with altered male mating behaviour and lower mating compatibility due to the effects of irradiation, will probably result in the need to

5 Meza-Hernández et al.: Mating and sex pheromone release of mass-reared and wild Anastrepha obliqua 103 Fig. 5. Changes in the concentration of the major compound (Z,E)- -farnesene found in the volatiles of male pheromones with increasing age. Open bar = mass-reared males; solid bar = wild males (ANOVA; P = 0.05, mean ± S.E., n = 5). release more sterile males per unit area in an SIT programme (Lux & Gaggl 1996). This will reduce the cost-effectiveness of a SIT programme. In our experiments calling behaviour and formation of leks appeared similar for both strains of flies. It was therefore not possible to determine possible causes of poor selection by wild females of the mass-reared males. Possibly, wild females are able to detect not only qualitative but quantitative differences in male behaviour. For example, video recordings of the mating of C. capitata have shown differences between mass-reared males and wild males in the frequency and duration of certain components of the courtship sequence (Briceño & Eberhard 1998). Also, quantification of the acoustic signals emitted during the calling of A. suspensa indicated significant differences in sexual behaviour between mass-reared and wild males (Sivinski et al. 1984). We found a direct correlation between pheromone release and mating performance in both male strains. We also observed that mass-reared males released less sex pheromone which likely puts them at a disadvantage, since the female response depends on the amount of the male pheromone released (Epsky & Heath 1993). These findings could explain why selection against mass-reared males was observed. So, the periodical determination of the levels of sex pheromone released by mass-reared flies could provide objective information about when the insects may have performance problems in the field. On the other hand, it is possible that the high concentration of pheromone in the laboratory colony was the cause of this effect (Calkins 1989) due to it acting as a mating inhibitor. Therefore, to counteract this undesirable characteristic it is recommended that colonies be maintained at low population densities with good ventilation, and using air extraction systems to remove excess pheromone. ACKNOWLEDGEMENTS We thank the field evaluation work team in the Moscafrut facility, Marco P. Pérez Gómez, José L. Zamora Palomeque and Jesús A. Escobar Trujillo for their technical support; Antonio Santiesteban for the support in the GC MS analysis; D. Orozco Davila for her comments on sexual compatibility; and J.C. Domínguez Gordillo, Moscafrut Manager. The study received financial support from SIBEJ and through a SIBEJ studentship for J.S. M-H. REFERENCES BOAKE, C.R., SHELLY, T.E. & KANESHIRO, K.Y Sexual selection in relation to pest-management strategies Annual Review of Entomology 41: BOLLER, E.F Sexual activity. In: Boller, E.F. & Chambers, D.L. (Eds) Quality Control: An Idea Book for Fruit Fly Workers. International Organization of Biological Control of Noxious Animals and Plants, WPRS Bulletin 1977/5. BRICEÑO, R.D. & EBERHARD, W.G Medfly courtship duration: a sexually selected reaction norm changed by crowding. Ethology, Ecology and Evolution 10: CALKINS, C.O The importance of understanding fruit fly mating behavior in sterile male release programs (Diptera: Tephritidae). Folia Entomologica Mexicana 61: CALKINS, C.O Lekking behavior in fruit flies and its implications for quality assessments. In: Cavalloro, R. (Ed.) Fruit Flies of Economic Importance A.A. Balkema, Rotterdam. CALKINS, C.O. & WEBB, J.C A cage and support framework for behavioral tests of fruit flies in the field. Florida Entomologist 66: CAYOL, J.P Changes in sexual behavior and life history traits of tephritid species caused by mass-

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