Pablo Liedo, ECOSUR, Carretera Antiguo Aeropuerto Km 2.5, Tapachula, Chiapas, México 30700. E-mail: email@example.com
The effect of post-teneral diets on the mating performance, pheromone production and longevity of sterile Anastrepha ludens and Anastrepha obliqua fruit flies (Diptera: Tephritidae) was investigated. Enriched pre-release diets improved male mating performance. Wild and sterile males fed on mango and orange fruits achieved the greatest copulatory success, followed by those fed on a 3 : 1 sugar : yeast (SY) diet. Males fed only on a sugar diet achieved the lowest number of matings. The mean numbers of copulas achieved by wild males were significantly higher than those by sterile males fed on a sugar-only diet, while there were no significant differences between wild males and sterile males fed on yeast diets. There was a trend of reduced mean number of copulas as the proportion of yeast was reduced in the diet, but differences were not significant. Pre-release diets had a significant effect on pheromone production. Males fed on a 3 : 1 SY diet produced the greatest amount of the three main pheromone components in A. ludens males and two major components in A. obliqua males, followed by males fed on fruits or pasteurized fruit juice. Males fed on a sugar only diet produced the lowest amounts. The longevity response to post-teneral diets was complex. The most yeast-rich diet and the poorest diet (sugar only) resulted in the lowest life expectancies. Flies fed on 24 : 1 SY diets showed the highest life expectancies for both males and females of the two species. Considering the tradeoffs between mating performance and longevity, the 24 : 1 SY diet would be recommended for programmes integrating the sterile insect technique, but the effects of these diets on field survival and dispersal still need to be investigated.
Anastrepha fruit flies are important pests of fruits in the Americas (Aluja 1994). In Mexico, the technology has been developed to apply the sterile insect technique (SIT) for the control of the Mexican fruit fly, Anastrepha ludens (Loew) and the West Indies fruit fly or mango fly, Anastrepha obliqua (Macquart). Over 100 million sterile flies of each species are produced weekly at the MOSCAFRUT facility, in Metapa, Chiapas, to be released in different places of Mexico and in the southern USA (Orozco et al. 2004).
Wild A. ludens flies were obtained as larvae from infested sour oranges (Citrus aurantium L.) and wild A. obliqua were obtained as larvae from infested yellow mombin fruits or jobos (Spondias mombin L.) collected in the Soconusco region of Chiapas, Mexico. The mass-reared non-irradiated and irradiated flies were obtained as pupae from the MOSCAFRUT mass-rearing facility at Metapa, Chiapas, Mexico. Sterile flies were γ-irradiated as pupae at 80 Gy 2 days before adult emergence. The mass-reared flies were from strains with approximately 130 and 110 generations under mass rearing conditions, for A. ludens and A. obliqua, respectively.
Initially, a series of five field cage mating tests were carried out with A. ludens and one with A. obliqua. The tests were done following the quality control protocol commonly used at the MOSCAFRUT facility (FAO/IAEA/USDA 2003).
After emergence, adult flies were sorted by sex and placed in groups of 40 individuals in 30 cm × 30 cm × 30 cm Plexiglas cages. Before the tests, these cages were maintained at the laboratory at 24 ± 2°C, 70 ± 10% RH, 550 ± 50 lux light intensity, with a 12 : 12 L : D photoperiod. Water and food were provided ad libitum. The types of foods were: (i) orange, Citrus sinensis (L.) Osbeck cv Valencia or mango, Mangifera indica L. cv Ataulfo (≈200 g pieces); (ii) full diet, which was the standard 3 : 1 sugar : hydrolysed yeast laboratory diet; and (iii) only sugar (standard cane sugar, dry sucrose). Wild females always were fed a full diet.
Five field cages, 2.9 m diameter × 2 m height (Calkins and Webb 1983), were set up at a mango orchard near Tapachula, Chiapas, Mexico (14° 55′ 08.9′′ N, 92° 16′34.2′′W and 137 msl). Six small orange and mango potted trees (≈1.5 m tall) were placed at the periphery and central part of each field cage. Each field cage was considered as a replicate. Thirteen males per treatment and 39 wild females were released per field cage in the five A. ludens tests (table 1). Twelve males per treatment and 48 wild females in the A. obliqua test (table 2). Forty-eight hours before the test, flies were individually marked by gluing a small piece of paper printed with a number on their thorax for treatment identification. This type of mark does not interfere with the sexual activity of the flies (Meza et al. 2005). Mating pairs were vial collected and the number and type of matings was recorded. The five tests for A. ludens were made between 15:00 and 19:00 hours, and the test for A. obliqua was between 06:00 and 12:00 hours. In the tests for A. ludens we first compared wild males fed on orange with sterile males fed on sugar only and full diet. In the second, third and fourth tests we compared the effect of orange, sugar only and full diet on the performance of sterile, fertile and wild males, respectively. In the fifth test the effect of two fruits, mango and orange and full diet was compared on sterile males. For A. obliqua only one test was done where wild males were fed on orange and sterile males were fed on orange, full diet and only sugar.
Table 1. Mean number of matings (SE) achieved by Anastrepha ludens males from different strains in five separate field cage tests (five replicates per test) after exposure to different food sources1
Sterile and wild males
Sterile males/two fruits
No. of matings
No. of matings
No. of matings
No. of matings
No. of matings
1Notation for treatments: the first letter corresponds to the strain and the second to the type of food. Strains: W: wild, S: mass-reared sterile, F: mass-reared fertile. Food type: O: orange, M: mango, F: full diet (3 : 1 SY hydrolysate) and S: sugar only. Means follow by the same letter within each column were not statistically different at P > 0.05.
6.4 (1.077) b
Table 2. Mean number of matings (SE) achieved by A. obliqua sterile and wild males exposed to different adult food sources, with wild females in field cages
No. of matings (SE)
Wild – Orange
Sterile – Orange
Sterile – Full
Sterile – Sugar
The age of the flies, at the time of the test was, 18–19 days for the wild flies and 12 days for the mass-reared fertile and sterile flies in the case of A. ludens, and 15 days for wild and 8 days for mass-reared sterile A. obliqua flies.
A second set of tests were carried out to look at the effect of different sugar : hydrolysed yeast (SY) ratios. Laboratory and field cage conditions were as described above. Groups of 200 males were provided one of the following four diets: Only sugar (1 : 0), and 24 : 1, 9 : 1 and 3 : 1 SY ratios. In the case of A. ludens, eight sterile males from each diet treatment, eight wild males and 40 wild females fed with the standard 3 : 1 SY diet were released per field cage. In the case of A. obliqua, 10 sterile males of each diet treatment, 10 wild males and 50 wild females fed on full diet were released in each field cage. Six replicates were carried out per species.
Pheromone volatiles emitted by males were collected using an air-entrainment technique. Ten males were confined in a 100 ml glass entrainment container [4.8 cm inner diameter (ID) × 12.5 cm long]. Volatiles were drawn from the container, using purifier 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 of Super Q adsorbent (Alltech Associates, Deerfield, IL) (Heath and Manukian 1992). Air was drawn through the trap at a rate of 1 l/min by a vacuum pump. At the conclusion of each air entrainment, which lasted 4 h, the volatiles were eluted from the adsorbent with 200 μl of methylene chloride (Baker, HPLC grade) and 100 ηg of tridecane was added as an internal standard for subsequent quantification. The samples were kept at −20°C before analysis.
Volatiles were analysed by means of gas chromatography-mass spectrometry using a Varian Star 3400 CX gas chromatograph linked to a Varian Saturn 4D mass spectrometer (GC-MS). The samples were analysed using a fused silica column (30 m × 0.25 mm) coated with poly (5%-diphenyl-95%-dimethylsiloxane) programmed from 50°C to 250°C at 15 °C/min. The carrier gas was helium. The injector port temperature was held at 200°C. Compounds were identified using their retention times, Kovat index (KI), and mass spectra and comparing these data with those of synthetic standards. Synthetic standards of farnesene (mixture of isomers that includes (E,E)-α-farnesene) and (Z)-3-nonenol were supplied by Aldrich (Toluca, Mexico).
Adult A. ludens sterile males were exposed to five types of food: (i) standard laboratory full diet (3 : 1 SY dry mixture); (ii) pulp of orange fruits cv. Valencia; (iii) pulp of mango fruits cv Ataulfo; (iv) commercial pasteurized peach juice (Herdez ®); and (v) sugar only. These males were placed in 30 cm × 30 cm × 30 cm Plexiglas cages after emergence. Every day a sample of 10 males per treatment was taken and this was repeated during six consecutive days, from 6- to 12-day-old flies. Four known main pheromone components were quantified, (E,E)-α-farnesene, suspensolide, anastrephin and epianastrephin.
Adult A. obliqua males were exposed to three food sources: (i) standard laboratory 3 : 1 SY full diet: (ii) pulp of mango fruits cv Ataulfo; and (iii) sugar only. Pheromone components were collected from males that were 8 to 10 days old. Three known compounds, z-3-nonenol, (Z, E)-α-farnesene, and (E, E)-α-farnesene, and one unknown compound, were quantified.
The effect of sugar : hydrolysed yeast ratios on the longevity of mass-reared non-irradiated A. ludens and A. obliqua fruit flies was evaluated under laboratory conditions by comparing four diets (only sugar 1 : 0, and 24 : 1, 9 : 1, and 3 : 1 S: : Y). Aluminium frame, mesh covered, 80 cm × 50 cm × 15 cm cages, were used, three cages per treatment. In each cage about 2000 adult flies were released. Food, according to each treatment, and water, were provided ad libitum. Dead flies were removed daily from the cages. The number and sex of the dead flies were recorded.
The data from the mating tests were analysed by Fisher’s PLSD statistical test and P values were calculated for pair-wise comparisons in each test. For the pheromone analysis, the relative amount of each compound was estimated from the GC graphs. These figures were square root transformed for analysis of variance (anova). In the case of A. obliqua, a Kruskal–Wallis test was used. Life tables were constructed for demographic analysis of longevity (Carey 1993). Survival data were analysed using the Cox proportional hazard model (Everitt and Pickles 2004). Independent analyses were done for males and females. Survival of flies exposed to 3 : 1, 9 : 1 and 24 : 1 SY diets was compared with survival of flies fed with only sugar (control). Treatments were compared with 95% CI. Statistical analyses were performed using R software (R v.2.9.2. 2009, the R Foundation for Statistical Computing, http://www.r-project.org/).
A total of 713 copulas were observed in the five initial tests with A. ludens, 56.1% were achieved by males fed on fruit (even though only 40% of males had been exposed to fruit), 28.7% by males fed on full diet (33% of males on this sugar-yeast hydrolysed diet) and 15.1% by males on a sugar only diet (27% of males present). The mean number (SE) of matings recorded for A. ludens males for different combinations of strain (wild, mass-reared fertile and mass-reared sterile) and food type (orange and mango fruit pieces, sugar only and full diet) in the five initial tests is shown in table 1.
In the first of the five tests, with wild males fed on orange and sterile males fed on sugar only or full diet, the difference between wild males and sterile males was significant (P < 0.0001), with the wild ones achieving significantly more matings. The difference between sterile males fed on a full diet or sugar only was not significant (P > 0.05). In order to separate the effect of ‘wild’, from the effect of ‘fruit’, the second test was designed using only mass-reared sterile males (table 1). In this second test, the differences in matings between orange fruit and full diet and orange fruit and sugar fed males were significant in favour of fruit feeding (P = 0.0328 and P = 0.0181, respectively). The difference between full diet and sugar only fed males was not significant (P > 0.05). In the third test, mass-reared not sterilized males showed a similar pattern (table 1). The difference in mating success between males fed on fruit and males fed on full diet and sugar only was significant (P = 0.0023 and P = 0.0009, respectively), whereas the difference between males fed on full diet and males fed on sugar only was not significant (P > 0.05). Wild males showed a slightly different mating pattern in the fourth test. There was no significant difference between orange fruit and the full diet (P > 0.05) and full diet was significantly different from sugar only fed males (P = 0.0341) (table 1). In the last test, while sterile males fed on mango showed a significantly better mating performance than those fed on the full diet (P = 0.0213), there was no significant difference between males fed on mango and those fed on orange, nor between those fed on orange and those fed on full diet (P > 0.05) (table 1).
In the field cage test with A. obliqua, a total of 104 matings were observed, 42.3% were by wild males fed on orange, 25.0% by sterile males fed on orange, 18.3% by sterile males fed on full diet and 14.4% by males fed on sugar only. The mean numbers of matings (SE) are shown in table 2. There was no significant difference between sterile and wild males fed with orange, while orange fed wild males performed significantly better than sterile males fed on the full diet or sugar. No significant differences were found among sterile insect treatments.
Results from the second series of field cage mating tests with males exposed to different SY ratios are shown in table 3. A total of 123 and 86 matings were observed for A. ludens and A. obliqua, respectively. In both species, the mean numbers of matings by wild males were significantly different from those by sterile males fed on a sugar-only diet. There were no significant differences between wild males and sterile males fed on yeast diets, although the mean number of matings tended to decline as the proportion of yeast was reduced in the diet.
Table 3. Mean number (SE) of matings achieved by wild and sterile males of A. ludens and A. obliqua fed on diets with different SY hydrolysate ratios in tests conducted in field cages
Strain – SY ratio
Means followed by the same letter were not statistically different at P > 0.05 according to Fisher’s PLSD.
Wild –3 : 1
Sterile –3 : 1
Sterile –9 : 1
Sterile –24 : 1
Sterile –1 : 0
In A. ludens, diet induced differences in the amount of the main pheromone compounds produced by males were significant for (E,E)-α-farnesene (F = 4.27, d.f. = 4, 12, P = 0.0223), anastrephin (F = 4.20, d.f. = 4, 12, P = 0.0234) and epianastrephin (F = 5.57, d.f. = 4, 12, P = 0.0089). Differences in the amount produced of suspensolide were not significant (F = 2.42, d.f. = 4, 12, P = 0.1057). In A. obliqua, differences were significant for Z-3-nonenol (H = 3.86, d.f = 1, P = 0.05) and the unknown compound (H = 6.49, d.f. = 2, P = 0.039), but non-significant in the other two compounds (H = 4.36, d.f. = 2, P = 0.113 and H = 4.27, d.f. = 2, P = 0.118 for Z,E-α-farnesene, and E,E-α-farnesene, respectively).
In most cases, males fed with the standard full diet produced the greatest amount of each compound and males fed with only sugar produced the lowest amount (figs 1 and 2). A. ludens males fed with mango, orange or peach juice produced an intermediate amount of each compound. The only exception was with (E, E)-α-farnesene in A. obliqua, where males fed on mango produced greater amounts than those fed on the full diet.
Anastrepha ludens longevity response to pre-release diets was complex. In males, the lowest expectation of life was observed on the most yeast rich diet (3 : 1 SY), followed by the non-yeast diet (sugar-only). In females, the lowest life expectancy was observed on the 3 : 1 diet, followed by the 9 : 1 diet and the sugar only diet. The effect of the four SY ratios on the survival of A. ludens can be seen in fig. 3. According to the Cox proportional hazard model and the Likehood Ratio test, differences in survival were highly significant (table 4). The SY ratio that resulted in the highest life expectancy was the 24 : 1 ratio for both males and females.
Table 4. Z-values obtained from survival analysis using the Cox proportional hazard model, comparing each SY hydrolysate ratio with the sugar only diet (control) for both sexes of Anastrepha species
Likelihood ratio test
***Highly significant differences, P < 0.0001; *significant difference, P < 0.05; NS: non-significant difference, P > 0.05.
The daily mortality rates (qx) during the first 30 days of A. ludens adult life are shown in fig. 4. In males, the sugar only diet showed the lowest mortality rates during the first 20–24 days, then mortality increased and was the highest after 30 days. In females, the mortality rates under full diet (3 : 1 SY) were the highest during the initial 30 days. The 9 : 1 and 24 : 1 SY diets showed the lowest mortality rates through most of the lifespan of males and females.
The survival response of A. obliqua to the four SY diets are shown in fig. 5 and the daily mortality rates (qx) in fig. 6. Differences between flies fed on sugar only and all other treatments were significant, except with A. obliqua males fed on the 24 : 1 SY diet, where the difference was non-significant (table 4). The most protein rich diet (SY 3 : 1) showed the lowest life expectancies for both, males and females. The highest life expectancy was observed with the SY 24 : 1 diet for both males and females.
Correspondingly, the highest mortality rates for both A. obliqua males and females were found in the 3 : 1 SY diet and the lowest mortality rates were observed in males and females fed on sugar only and the 24 : 1 SY diet. The cross-over of the mortality schedules observed in A. ludens (fig. 4) was not seen in A. obliqua females during the first 40 days of adult life and was observed in males around 37 to 40 days old (fig. 6).
The best performance for sterile males was observed when they were provided with fruit. In contrast, differences between the mating performance of males fed on a full diet or on sugar only were not significant in most cases. The better performance of males fed on fruits could be explained as a nutritional and/or as a microbiological effect. Also, it could be attributed to the effect of possible semiochemicals as has been reported for Ceratitis capitata (Wiedemann) and several Bactrocera species (Shelly et al. 1996, 2005; Shelly and McInnis 2001; Papadopulous et al. 2006; Shelly and Edu 2007).
There was a non-significant trend for the mean number of matings to increase as the yeast concentration in the diets increased. The best mating performance was achieved by males exposed to the diet with the highest ratio (3 : 1, or 25% yeast). Prabhu et al. (2008) found that 25% yeast was the optimal concentration for mating performance of Bactrocera tryoni (Froggatt). Above this concentration, the percent of matings showed a decreasing tendency. We did not test greater yeast concentrations because our own unpublished observations and results by Cresoni-Pereira and Zucoloto (2001) on A. obliqua have indicated that these adversely affect survival.
Post-teneral diets increased the amount produced of three out of four main pheromone compounds in A. ludens and two out of four in A. obliqua.Epsky and Heath (1993) found similar effects of food on pheromone production in A. suspensa (Loew). These results are consistent with our field cage mating tests, confirming that adult nutrition has an effect on mating performance and suggest that this effect could be mediated, at least in part, by the effect of food on pheromone production. However, the amount produced by males fed on the full diet, was greater than the amount produced by males fed on fruits or fruit juice, whereas mating performance of males fed on fruits was better than those fed on full diet. This suggests that although there is an effect of adult diet on pheromone production, there is not a linear association between pheromone production and mating success. Landolt and Sivinski (1992) also found that overripe fruit increased pheromone calling behaviour in A. suspensa. Thus, these results suggest that the contribution of diet to male fitness may be realized through several pathways – contribution to quantity and quality of pheromones, increased levels of courtship activity in the pre-copulatory phase and possibly other post copulatory effects as well (e.g. Aluja et al. 2008; Gavriel et al. 2009).
As in the case of mating performance, the results obtained here on pheromone production suggest the there might be nutritional, microbial or semiochemical factors that could have an effect on pheromone synthesis. Because of possible basic and applied implications, we believe the effect of fruits on mating behaviour and the relationship between pheromone production and mating performance, justify further research.
The diets with the highest and the lowest protein content (3 : 1 SY and sugar only) adversely affected male and female longevity in both species. Sugar fed flies showed low mortality rates in early adult life, but in old flies (>40 days old), mortality rates of sugar fed flies were higher than those fed on yeast enriched diets. Mortality rates of flies fed on the richest diet (3 : 1 SY) were among the highest throughout the lifespan of males and females in both species. This longevity paradox was reported for A. ludens (Carey et al. 2008) and studies on other fruit flies have shown the adverse effect of yeast enriched diets on male longevity (Carey et al. 1999; Jácome et al. 1999; Kaspi and Yuval 2000; Prabhu et al. 2008). The reduced longevity of flies fed on the sugar only diet could be interpreted as malnutrition or depletion of reserves (Warburg and Yuval 1996; Nestel et al. 2005). The adverse effect of yeast enriched diets could be explained as the trade-offs between longevity and reproduction. Protein enriched diets stimulate reproduction, and reproductive effort (egg production for females, calling, intrasexual interactions, courtship and sperm and accessory gland substances production for males, among others) could result in higher mortality (Carey et al. 1999, 2001, 2002a,b, 2008; Müller et al. 2001). The adverse effect of pre-release diets on longevity could be overcome by the ability of flies to forage for natural food sources (Maor et al. 2004). Utges et al. (unpublished data) found that A. ludens and A. obliqua flies fed on fruit and sugar before release showed the greater recapture and dispersal distance, compared with flies fed on the full or sugar only diets in release–recapture field tests.
In terms of SIT application, since sterile males have only limited sperm available for very few matings, improved mating competitiveness should have priority over extended lifespan.
After careful analysis of pre-release feeding trade-offs, Yuval et al. (2007) concluded that yeast enriched diets will be highly recommended for C. capitata SIT programmes. Following the same approach and considering mating performance, laboratory longevity and field survival and dispersal, the 24 : 1 SY diet could be recommend for A. ludens and A. obliqua SIT programmes. The mating competitiveness of males fed on this 24 : 1 SY diet was similar to those males fed on the most protein rich diet, without the detrimental effect on longevity.
Further research to understand the mechanisms underlying the enhanced mating performance of fruit fed males, similar to those by Papadopolous et al. (2006) and development of methods for pre-release feeding using fruits or fruit products, are highly recommended.
We thank R. Bustamante, E. de León, J. A. Escobar-Trujillo, M.P. Pérez-Gómez, R. Rincón, G. Rodas, S. Rodríguez, S. Salgado, and J. L. Zamora-Palomeque for technical assistance. We also thank J. Valle-Mora for advice on statistical analysis and B. Yuval for review of earlier versions. This research was supported by the Food and Agriculture Organization/International Atomic Energy Agency (FAO/IAEA) through contract 12860/RBF, the MOSCAFRUT facility (SENASICA – SAGARPA-IICA) and ECOSUR. The Consejo Nacional de Ciencia y Tecnología (CONACYT) and ECOSUR provided support through sabbatical leave to PL.