Exposure to plant compounds and analogues of juvenile hormone (JH) increase male mating success in several species of tephritid fruit flies. Most of these species exhibit a lek mating system, characterized by active female choice. Although the pattern of enhanced male mating success is evident, few studies have investigated what benefits, if any, females gain via choice of exposed males in the lek mating system. In the South American fruit fly, Anastrepha fraterculus, females mate preferentially with males that were exposed to volatiles released by guava fruit or treated with methoprene (a JH analogue). Here, we tested the hypothesis that female choice confers direct fitness benefits in terms of fecundity and fertility. We first carried out mate choice experiments presenting females with males treated and non-treated with guava volatiles or, alternatively, treated and non-treated with methoprene. After we confirmed female preference for treated males, we compared the fecundity and fertility between females mated with treated males and non-treated ones. We found that A. fraterculus females that mated with males exposed to guava volatiles showed higher fecundity than females mated to non-exposed males. On the other hand, females that mated methoprene-treated males showed no evidence of direct benefits. Our findings represent the first evidence of a direct benefit associated to female preference for males that were exposed to host fruit odors in tephritid fruit flies. Differences between the two treatments are discussed in evolutionary and pest management terms.
Citation: Bachmann GE, Devescovi F, Nussenbaum AL, Milla FH, Shelly TE, Cladera JL, et al. (2019) Mate choice confers direct benefits to females of Anastrepha fraterculus (Diptera: Tephritidae). PLoS ONE 14(6): e0214698. https://doi.org/10.1371/journal.pone.0214698
Editor: Nikos T. Papadopoulos, University of Thessaly School of Agricultural Sciences, GREECE
Received: March 16, 2019; Accepted: May 30, 2019; Published: June 14, 2019
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: Data uploaded as Supporting Information.
Funding: This research was funded by the Research Contract (16483 to JLC) and by FONCYT (PICT 2013 – 054 to DFS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Tephritid fruit flies (Diptera: Tephritidae) infest hundreds of different plant species, including many economically important fruits . One environmentally friendly control strategy used against fruit fly pests is the sterile insect technique (SIT) , which requires solid knowledge of the sexual behaviour of the target pest . SIT is based on the ability of mass-released, sterile males to mate with fertile, wild females and hence induce sterility on the target population . Many species of tephritid fruit flies exhibit lek mating systems characterized by female choice, and this has prompted considerable attention on the factors that influence male mating success [5,6]. The collective outcome of this research has been the identification of several pre-release treatments that boost the sexual competitiveness of male tephritids [7–10] including: pre-release diet containing a proteinaceous source [11–20]; exposure of males to semiochemicals [9,10,21,22]; and the use of methoprene, a juvenile hormone (JH) analogue, to both boost male mating success and accelerate their sexual maturation [7,17,18,23].
Although the drive to improve SIT has, in many cases, identified male traits associated with mating success, little is known about the ecological and evolutionary forces that shape female mate preferences, particularly the potential fitness benefits associated with mate choice . Kirkpatrick and Ryan  and Wyatt  proposed that female choice is generally associated with direct benefits, such as increased fecundity and longevity, reduced risk of predation, or access to material resources under male control [27–29]. Simultaneously, females may gain indirect benefits if mate choice positively affects the fitness of their offspring, such as those explained under the “sexy son” and good genes hypotheses [30–33]. For tephritids, female choice has been associated with direct and indirect benefits [34,35]. In particular, the association between female benefits and mate selection mediated via feeding or exposure of males to plant-borne compounds has been investigated in four tephritid species: Bactrocera dorsalis (Hendel) [36,37]; Bactrocera tryoni (Froggatt) [38,39]; Ceratitis capitata (Wiedemann) [13,40]; and Zeugodacus cucurbitae (Coquillett) . Although females of this species showed a preference for males either exposed or fed with plant-derived compounds, only Kumaran et al. [38,39] found evidence of direct and indirect benefits, respectively, for B. tryoni females.
The South American fruit fly, Anastrepha fraterculus (Wiedemann), is a polyphagous species that attacks more than 100 species of fruit plants , many of which with a high commercial value. Vera et al.  found an increase in the mating success of A. fraterculus males following exposure to the volatiles of guava fruits (Psidium guajava L.), a native plant and one of their main hosts in the wild. This phenomenon was later confirmed and extended by Bachmann et al. , who also found that males exposed to guava fruit volatiles released larger amounts of sex pheromone and performed courtship behaviours more frequently than non-exposed males. Similarly, topical applications of methoprene conferred a mating advantage to the males which also seems to be associated to larger amounts of pheromone being released by methoprene-treated males when competing with non-treated males .
Despite evidence showing that guava fruit volatiles and methoprene enhance male mating success in different Anastrepha species, no prior investigation has measured the potential fitness benefits gained by females that mate with those enhanced males. Even though methoprene is a synthetic compound, it still triggers behavioural and physiological effects in males similar to those triggered by exposure to guava volatiles (i.e., enhanced signalling and mating). Consequently, mating with a methoprene treated males could still provide a direct benefit to females. In the present work, we evaluated whether the preference of A. fraterculus females for males exposed to guava volatiles and those treated with methoprene derive from direct benefits in terms of increased fecundity and fertility. Because in A. fraterculus longer copulations result in longer refractory periods , which can decrease the need for future mates and consequently the costs in terms of energy and re-exposure to predators , we also evaluated copula duration as a potential direct benefit to females.
Materials and methods
Anastrepha fraterculus flies were obtained from a laboratory colony kept at Instituto de Genética E. A. Favret (IGEAF) which was originally established at Estación Experimental Agroindustrial Obispo Colombres (Tucumán, Argentina) in 1997 with pupae obtained from infested guavas collected in Tafí Viejo (26º43’25”S 65º16’43”W, Tucumán, Argentina) . Rearing followed standard procedures using an artificial diet based on yeast, wheat germ, sugar, and agar for larvae  and a mixture of sugar, hydrolysed yeast (MP Biomedicals, San Francisco, CA, USA), hydrolysed corn (ARCOR, Tucumán, Argentina) (4:1:1 ratio) and vitamin E (Parafarm, Buenos Aires, Argentina) for adults . Flies used in the tests were virgin and sexually mature (males were 10–14 days-old; females were 14–18 days-old)  and were kept under controlled environmental conditions (24 ± 2°C, 70 ± 10% RH, and a 12L: 12D photoperiod).
One day after adult emergence flies were sorted by sex, transferred to plastic containers, and provided with food and water. Females were placed in 1 L plastic cylindrical containers (15 cm tall, 12 cm in diameter) in groups of 25 individuals and fed with the standard diet. Males were placed in 21 L plastic containers (37 x 28 x 21 cm) in groups of 100 individuals and fed with sugar and brewer’s yeast (CALSA, Tucumán, Argentina) (3:1 ratio).
Experiment 1. Mating and reproductive output of females offered males exposed or not exposed to guava volatiles.
In order to determine whether the preference of A. fraterculus females for guava-exposed males  is associated with direct fitness benefits in terms of mating and reproductive output, females were first given the choice to mate with guava-exposed or non-exposed males. Then, we determined differences in mating and reproductive parameters between females that selected exposed or non-exposed males.
Treated males were exposed to guava volatiles without physical access to the fruit following procedures of Bachmann et al. . Non-exposed males were kept under the same environmental conditions but in a different room without access to guava odours. After exposure, males were kept in the 21 L plastic containers in separate rooms and under the same conditions described above. In the mating test, a total of 401 experimental arenas were established, each consisting of three virgin flies: one exposed male, one non-exposed male of the same age, and one female. Males were 12–14 d old; whereas females were 14–18 d old. These experimental arenas have been extensively used in A. fraterculus as a valid experimental approach to study female mate choice [42–44,51,52]. Males were marked on their thorax with a dot of non-toxic, water-based paint for identification . Randomly assigned colours identified different male treatments. Males and females were released in the experimental arenas early in the morning under semi-darkness. Males were released 15 min before females. Once all arenas were set up, fluorescent lights were turned on, and the occurrence of mating pairs was monitored continuously for 2 h during the natural period of mating activity for Argentinean populations of A. fraterculus (9–11 am) [50,53]. Whenever a couple was detected, male colour and time at which copulation started and ended were recorded. The recording of copula initiation and termination times was done continuously, and each mating couple was checked every 2–3 minutes. Due to inadvertent errors in recording times, 4 replicates were excluded from the analysis of mating duration. Experiments were conducted under laboratory conditions (25 ± 1 ºC and 70 ± 20% RH). Illumination was provided by fluorescent tubes and natural light coming from a window.
After the mating test, mated females were transferred to 3 L glass flasks (with water and food) in groups of three according to the type of male mated. We used groups of females instead of solitary individuals because several studies indicate that the presence of conspecific females stimulates the oviposition [54–56]. Each flask with three females was considered a replicate, with 25 and 22 replicates for females mated to exposed or non-exposed males, respectively. Every 3 days each replicate was provided an oviposition substrate (hereafter, oviposition unit) that consisted of a cylindrical plastic vial (2 cm tall, 2.5 cm in diameter) filled with water coloured with edible red dye (Fleibor, Tablada, Buenos Aires, Argentina) and covered with Parafilm M (Pechiney Plastic Packaging, Chicago, Illinois, USA). This oviposition unit is normally used in the laboratory rearing of A. fraterculus at IGEAF. After 24 h, the oviposition unit was removed, and the eggs were recovered using a plastic pipette and then transferred onto a black filter paper. This was, in turn, placed inside a plastic Petri dish (2 cm tall, 8 cm in diameter) on top of a wet cloth and then covered with its lid. The number of eggs from each oviposition unit was counted under a stereoscopic microscope (20x). Petri dishes were then placed inside an incubator at 25 ± 1°C and 70 ± 10% RH for 4 days to allow embryonic development. After this time, the numbers of hatched and unhatched eggs were counted under a stereoscopic microscope (20x). This entire process was repeated nine times with each replicate during 27 days, thus covering the peak period of egg-laying in this species . Whenever a dead female was detected, it was removed from the flask and recorded, thus allowing computation of ovipositions per capita for each day of egg collection. Due to logistic problems with the incubator, 7 and 1 replicates coming from females mated with exposed and non-exposed males, respectively, were discarded and not considered in the data analysis.
Experiment 2. Mating and reproductive output of females offered males treated or not treated with methoprene.
To determine whether the preference of A. fraterculus females for methoprene-treated males  is associated with a direct benefit, we followed a protocol similar to that described for experiment 1.
The procedure for methoprene application followed Teal et al. . Briefly, on the day of emergence males were topically treated by applying 1 μL of a solution of methoprene dissolved in acetone (5 μg/μl) into their thorax. Females, treated males, and non-treated males were kept in separate rooms under the same environmental and feeding conditions as described above. In the mating tests a total of 166 experimental arenas were set up (4 replicates were excluded from the analysis of mating duration due to mistakes during the recording of duration times). At the day of the mating test, males were 12 d old and females were 14–18 d old. Afterwards, mated females were transferred in groups of three to cylindrical, 1 L plastic containers. Fecundity and fertility were assessed following the same procedures as described for experiment 1, except that oviposition units were offered seven times to each replicate, covering a total of 21 days. The number of replicates was 24 for females mated to methoprene-treated males and 16 for females mated to non-treated males.
The numbers of copulations achieved by guava-exposed and non-exposed males (experiment 1) or methoprene-treated and non-treated males (experiment 2) were compared by means of a G test of goodness of fit to an equal proportion hypothesis. The latency to mate (i.e., time elapsed between female release and mating), the mating duration (i.e., time elapsed between the start and end of mating), the overall fecundity (i.e., number of eggs laid across all the egg collections), and the fertility (i.e., average of hatch rate across all egg collections) were compared between females mated to treated or non-treated males by means of a t-test for independent samples. Assumptions of normality of the residuals and homoscedasticity were checked prior to each test. In order to meet the homoscedasticity assumption, mating duration from experiment 1 and fertility in experiment 2 were ln- and logit-transformed, respectively. Also, fertility was logit-transformed in experiment 2. Finally, we carried out a series of survival analyses (Kaplan-Meier estimation, Log-rank test) to evaluate the temporal pattern of female fecundity after mating with exposed or non-exposed males (experiment 1) or treated or non-treated males (experiment 2). The accumulated total number of eggs laid on each egg collection date was used in the survival analysis. STATISTICA 7  and GraphPad Prism 6  were used for statistical analyses and preparation of figures.
Experiment 1. Mating and reproductive output of females offered males exposed or not exposed to guava volatiles
A total of 401 mating arenas were established of which 387 resulted in mating. Females mated significantly more often with males exposed to guava than with non-exposed males (G = 19.73, N = 387, d.f. = 1, p < 0.001) (Fig 1). The latency to mate was not affected by the type of male chosen for mating (t = 0.284, d.f. = 385, p = 0.776) (Fig 2A). Mating duration, on the other hand, was longer in matings that involved males exposed to guava volatiles (t = 2.626, d.f. = 381, p < 0.01) (Fig 2B). Female fecundity was significantly different between females mated with exposed and non-exposed males (t = 2.145, d.f. = 45, p = 0.037), as females mated with guava exposed males laid significantly more eggs (Fig 2C). Fertility was not statistically different between the two types of females (t = 0.372, d.f. = 37, p = 0.712) (Fig 2D).
Numbers above bars represent numbers of matings. * statistically significant difference (α = 0.05).
Latency to mate (A), mating duration (B), fecundity (per capita) (C) and fertility (D) for females mated to males exposed or non-exposed to guava volatiles. Bars represent mean values and SE for each variable. Results from the independent t-tests are indicated as follows: * statistically significant differences (α = 0.05); n.s. non statistically significant differences.
The temporal pattern in which the total number of eggs laid by females mated with exposed males was deployed was similar to that of females mated with non-exposed males(Survival analysis: χ2 = 1.02, d.f. = 1, p = 0.312). Fig 3 shows that the cumulative percentage of eggs laid across the nine egg collection dates extensively overlaped for the two types of females.
Experiment 2. Mating and reproductive output of females offered males treated or not treated with methoprene
Successful matings were recorded in 152 out of 166 mating arenas. Females mated significantly more often with males treated with methoprene than with non-treated males (G = 11.76, N = 152, d.f. = 1, p < 0.001) (Fig 4). However, male type had no effect on any of the measured variables (latency to mate: t = 0.357, d.f. = 150, p = 0.722; mating duration: t = 0.257, d.f. = 146, p = 0.797; fecundity: t = 0.715, d.f. = 38, p = 0.476; fertility: t = 1.100, d.f. = 38, p = 0.278) (Fig 5A–5D). Likewise, survival analysis showed that the temporal pattern in which females deployed the total number of eggs acroos the 7 egg collection dates did not differ between females mated to methoprene treated males and females mated to non-treated males (Survival analysis: χ2 = 1.61, d.f. = 1, p = 0.205) (Fig 6).
Numbers above bars represent numbers of matings. * statistically significant difference (α = 0.05).
Latency to mate (A), mating duration (B), fecundity (per capita) (C) and fertility (D) for females mated to males treated or non-treated with methoprene. Bars represent mean values and SE for each variable. n.s. indicates non statistically significant differences between mean values after independent t-tests.
Temporal pattern of egg deposition by females mated to males treated or non-treated with methoprene. The cumulative percentage of eggs laid (y axis) across 7 egg collection samplings (x axis) is presented for the two types of females. Each sampling was carried out every three days.
Documenting female preference based on particular male traits is more easily, and thus more frequently, accomplished than demonstrating fitness benefits to females accruing from such preferences. Vera et al.  and Bachmann et al. [43,44] observed that A. fraterculus females prefer males that had been exposed to volatiles of guava fruit (one of its main hosts) or treated with methoprene (an artificial analogue of JH). Here, we verified these patterns of sexual selection and investigated whether female preferences can be explained on the basis of a direct fitness benefit. We found that females that selected males exposed to guava volatiles had higher fecundity than those selecting non-exposed males. Therefore, such enhanced egg output is consistent with the existence of a direct fitness benefit. In other words, preference for males exposed to guava volatiles represents an adaptive, fitness-based decision. Additionally, matings that involved guava exposed males lasted longer, which, as discussed below, may also be considered beneficial for the females. On the other hand, for methoprene treatment, we did not detect any association between preference and fitness benefits for females. Female latency to mate did not differ with male status for both guava volatile and methoprene treatments. Thus, although exposure to guava volatiles and application of methoprene boosted male mating success, neither treatment stimulated more rapid mating decisions by females.
The difference in fecundity between females mated to guava-exposed and non-exposed males was constant over time, which indicates that the benefit derived from mating with exposed males was long-lasting and not restricted to the beginning of the oviposition period. The higher fecundity observed here is consistent with Kumaran et al. , who recorded increased fecundity in B. tryoni females mated to males that were previously exposed to zingerone, a floral compound produced by orchids, that enhaces male mating success. Particularly, our findings constitute the first evidence of such mechanism involving volatiles from the host fruit. For tephritids, that study and the present one provide the only clear evidence of a direct benefit associated with female mate choice, where female preference is mediated by male exposure to plant-derived compounds. Other related studies on tephritids found no support for this phenomenon . For example, females of B. dorsalis mate preferentially with methyl eugenol-fed males, but they did not show an increase in their fecundity, fertility, or survival [37,61]. Similarly, C. capitata females did not exhibit any reproductive or survival benefits after mating with preferred, ginger root oil treated males .
One mechanism by which fecundity might have increased is the acquisition of higher quality male accessory gland products (AGPs) during copulation, which occurs in many insects including tephritids [62–64]. There is a large variety of physiologically active substances, such as proteins and juvenile hormone in the ejaculate, that may inhibit female remating propensity and induce egg maturation [62,65,66]. The perception of guava volatiles by males could act as an indicator of host availability and alter male reproductive physiology, stimulating the production of AGPs that would be transferred in higher amounts to the females with their concomitant impact on fecundity. The possibility that males spend more energetic sources in reproduction (e.g. signalling and AGPs production) when a host is present can be a reasonable explanation, but it is, of course, conjectural, and studies on the effects of guava exposure on male reproductive physiology are required.
In contrast to the guava treatment, mating with methoprene-treated males did not result in increased fecundity, fertility, or copulation duration. The lack of evidence of direct benefits can be interpreted in different ways. First, because methoprene is a synthetic compound, it may be possible that looking at the behavior of females from an evolutionary perspective (i.e. relating their preference with direct fitness benefits) is misleading. In this scenario, methoprene would induce a higher rate of pheromone release in males, which triggers female acceptance over non-treated males  by exploiting females’ sensory channel with no reward in terms of their own potential fecundity. Second, because males treated with methoprene release more pheromone than no-treated males, females could still be obtaining a direct benefit if treated males are more easily located, consequently reducing predation risks and mate location costs . Finally, even though we did not find evidence of direct benefits associated to females preference for methoprene treated males, indirect (genetic) benefits may be involved this preference. In B. tryoni, Kumaran et al.  found that the sons of males that were treated with cuelure and zingerone detected and located these chemicals more effectively than sons of non-exposed males, thus showing evidence of a potential “sexy son” mechanism. Potential indirect benefits, like those reported for B. tryoni [38,39], should also be studied for A. fraterculus females mated not only with methoprene-treated males but also in females mated with guava-exposed males, as direct and indirect effects can both be influencing female choice.
Copula lasted longer for guava exposed males than for non-exposed ones. Anastrepha fraterculus females appear to remate primarily to replenish sperm , and this tendency is negatively associated to copula duration . Thus, long copulations may be adaptive, because the transfer of large amounts of sperm would eliminate or delay the expenditure of energy and time to find males. Because predation risk increases during courtship and mating  delaying remating would also be beneficial in terms of survival. These benefits may, of course, be offset to some degree by any increase in predation risk during a single but lengthy copulation and the fact that overall genetic variability of the progeny is expected to be lower. In the case of methoprene, copula duration did not differ between treated and non-treated males. This agrees with Haq et al.  for Z. cucurbitae and shows a general lack of other types of benefits associated to methoprene.
The preference of females for males treated with sexual enhancers, together with a resulting increased fecundity could have practical implications in the context of SIT. In a mass rearing facility, an increase in fecundity would mean a higher yield which directly translates into lower costs of maintenance (because fewer reproductive adults would be needed). Also, if enhanced, but sterile, males were able to induce sterility in wild females, then a higher proportion of the reproductive output of the wild population would be unviable. Nonetheless, in order to extend our results to the context of SIT, further research is needed. For instance, the impact of irradiation on both the enhancement of males mating success and females fecundity were not assessed.
To conclude, previous work  showed that the preference of females for males exposed to guava volatiles could be explained at a proximal level by a higher rate of sexual displays and sex pheromone release by exposed males. Here, at least for guava volatiles, we found evidence that female preference could also be explained at an evolutionary level, because females that mate with guava-exposed males obtained a direct benefit in terms of increased fecundity. Our results contribute to a better understanding of the mechanisms related to mate choice and their evolutionary implications. However, the proximate (physiological) causes of an increased fecundity after mating with a sexually enhanced male still remain unknown and represent an interesting field of study. It is worth mentioning that, in all the experiments of the present study, females were fed with diet of high nutritional quality and presumably met, to a large extent, their nutritional and physiological needs (e.g., oocytes development). Aluja et al.  found that Anastrepha ludens and Anastrepha obliqua females fed on protein diets developed more oocytes than those fed without protein. It is thus possible that a rich diet actually reduced the positive effects of mating with guava-exposed males and that females feeding on a lower quality diet, as expected in the wild, would show an even greater increase in fecundity than found here. The interaction between female nutritional status and potential benefits gained through mate choice should be considered in future studies.
The authors are grateful to our colleagues at IGEAF, INTA, for their collaboration during the tests.
- 1. White IM, Elson-Harris MM. Fruit Flies of Economic Significance: Their Identification and Bionomics (1992). CAB International.
- 2. Knipling EF. Possibilities of insect control or eradication through the use of sexually sterile males. J Econ Entomol. 1955; 48(4): 459–462.
- 3. Robinson AS, Hendrichs J. Prospects for the future development and application of the sterile insect technique. In: Dyck VA, Hendrichs J, Robinson AS, editors. Sterile Insect Technique. Principles and Practice in Area-Wide Integrated Pest Management. Springer; 2005. pp. 727–760.
- 4. Lance DR, McInnis DO. Biological basis of the sterile insect technique. In: Dyck VA, Hendrichs J, Robinson AS, editors. Sterile Insect Technique. Principles and Practice in Area-Wide Integrated Pest Management. Springer; 2005. pp. 69–94.
- 5. Hendrichs J, Robinson AS, Cayol JP, Enkerlin W. Medfly areawide sterile insect technique programmes for prevention, suppression or eradication: the importance of mating behavior studies. Fla Entomol. 2002; 85(1): 1–13.
- 6. Robinson AS, Cayol JP, Hendrichs J. Recent findings on medfly sexual behavior: implications for SIT. Fla Entomol. 2002; 85(1): 171–181.
- 7. Teal PEA, Gomez-Simuta Y, Dueben BD, Holler TC, Olson S. Improving the efficacy of the sterile insect technique for fruit flies by incorporation of hormone and dietary supplements into adult holding protocols. In: Vreysen MJB, Robinson AS, Hendrichs J, editors. Area-Wide Control of Insect Pests; 2007. pp. 163–173.
- 8. Yuval B, Maor M, Levy K, Kaspi R, Taylor P, Shelly T. Breakfast of champions or kiss of death? Survival and sexual performance of protein-fed, sterile Mediterranean fruit flies (Diptera: Tephritidae). Fla Entomol. 2007; 90(1): 115–122.
- 9. Papadopoulos NT, Kouloussis NA, Katsoyanos BI. Effect of plant chemicals on the behavior of the Mediterranean fruit fly. In: Sugayama RL, Zucchi RA, Ovruski SM, Sivinski J, editors. Proceedings of the 7th International Symposium on fruit Flies of Economic Importance. Press Color Graficos Especializados, Brotas, Bahia, Brazil. 2008
- 10. Shelly TE, Cowan AN, Edu J, Pahio E. Mating success of male Mediterranean fruit flies following exposure to two sources of α-copaene, manuka oil and mango. Fla Entomol. 2008a; 91(1): 9–15.
- 11. Aluja M, Díaz-Fleischer F, Papaj DR, Lagunes G, Sivinski J. Effects of age, diet, female density, and the host resource on egg load in Anastrepha ludens and Anastrepha obliqua (Diptera: Tephritidae). J Insect Physiol. 2001; 47(9): 975–988. pmid:11472760
- 12. Yuval B, Kaspi R, Field SA, Blay S, Taylor P. Effects of post-teneral nutrition on reproductive success of male Mediterranean fruit flies (Diptera: Tephritidae). Fla Entomol. 2002; 85(1): 165–170.
- 13. Shelly TE. Does mating with ginger root oil-exposed males confer fitness benefits to female Mediterranean fruit flies, Ceratitis capitata (Diptera: Tephritidae)? Proc Hawaii Entomol Soc. 2005; 37: 65–71.
- 14. Pérez-Staples D, Prabhu V, Taylor PW. Post‐teneral protein feeding enhances sexual performance of Queensland fruit flies. Physiol Entomol. 2007; 32(3): 225–232.
- 15. Pérez‐Staples D, Weldon CW, Smallridge C, Taylor PW. Pre‐release feeding on yeast hydrolysate enhances sexual competitiveness of sterile male Queensland fruit flies in field cages. Entomol Exp Appl. 2009; 131(2): 159–166.
- 16. Pereira R. Sivinski J, Teal PE. Influence of methoprene and dietary protein on male Anastrepha suspensa (Diptera: Tephritidae) mating aggregations. J Insect Physiol. 2009; 55(4): 328–335. pmid:19185584
- 17. Pereira R, Sivinski J, Teal PE. Influence of a juvenile hormone analog and dietary protein on male Anastrepha suspensa (Diptera: Tephritidae) sexual success. J Econ Entomol. 2010; 103(1): 40–46. pmid:20214366
- 18. Haq I, Cáceres C, Hendrichs J, Teal P, Wornoayporn V, Stauffer C, et al. Effects of the juvenile hormone analogue methoprene and dietary protein on male melon fly Bactrocera cucurbitae (Diptera: Tephritidae) mating success. J Insect Physiol. 2010; 56(11): 1503–1509. pmid:20438735
- 19. Haq I, Cáceres C, Liedo P, Soriano D, Jessup A, Hendrichs J, et al. Effect of methoprene application, adult food and feeding duration on male melon fly starvation survival. J Appl Entomol. 2013; 137(s1): 61–68.
- 20. Gavriel S, Jurkevitch E, Gazit Y, Yuval B. Bacterially enriched diet improves sexual performance of sterile male Mediterranean fruit flies. J Appl Entomol. 2011; 135(7): 564–573.
- 21. Tan KH, Nishida R, Jang EB, Shelly TE. Pheromones, male lures, and trapping of tephritid fruit flies. In: Shelly T, Epsky N, Jang E, Reyes-Flores J, Vargas R, editors. Trapping and the Detection, Control, and Regulation of Tephritid Fruit Flies. 2014. pp. 15–74. Springer Netherlands.
- 22. Segura DF, Belliard SA, Vera MT, Bachmann GE, Ruiz MJ, Jofre-Barud F, Fernández PC, López ML, Shelly TE. Plant Chemicals and the Sexual Behavior of Male Tephritid Fruit Flies. Ann Entomol Soc Am. 2018; 111: 239–264.
- 23. Collins SR, Reynolds OL, Taylor PW. Combined effects of dietary yeast supplementation and methoprene treatment on sexual maturation of Queensland fruit fly. J Insect Physiol. 2014; 61: 51–57. pmid:24424344
- 24. Shelly TE, Nishimoto J. Does female mate choice confer direct fitness benefits? Results from a tephritid fruit fly. Ann Entomol Soc Am. 2016; 110(2): 204–211.
- 25. Kirkpatrick M, Ryan MJ. The evolution of mating preferences and the paradox of the lek. Nature. 1991; 350(6313): 33–38.
- 26. Wyatt TD. Pheromones and Animal Behaviour: Communication by Smell and Taste. Cambridge university press (2003).
- 27. Gwynne DT. Courtship feeding increases female reproductive success in bushcrickets. Nature. 1984; 307(5949): 361–363.
- 28. Reinhold K. Paternal investment in Poecilimon veluchianus bushcrickets: beneficial effects of nuptial feeding on offspring viability. Behav Ecol Sociobiol. 1999; 45(3): 293–299.
- 29. Arnqvist G, Nilsson T. The evolution of polyandry: multiple mating and female fitness in insects. Anim Behav. 2000; 60(2): 145–164. pmid:10973716
- 30. Andersson MB. Sexual Selection. Princeton University Press. 1994.
- 31. Johnstone RA. Sexual selection, honest advertisement and the handicap principle: reviewing the evidence. Biol Rev. 1995; 70(1): 1–65. pmid:7718697
- 32. Ryan MJ. Sexual selection and mate choice. In: Krebs JR, Davies NB, editors. Behavioural ecology: an evolutionary approach. 4th edition. Oxford: Blackwell Science; 1997. pp. 179–202.
- 33. Fedorka KM, Mousseau TA. Material and genetic benefits of female multiple mating and polyandry. Anim Behav. 2002; 64(3): 361–367.
- 34. Kasuya E. Female mate preference and offspring fitness in the melon fly. Ecol Res. 1992; 7(3): 277–281.
- 35. Briceño RD, Eberhard WG. Possible fisherian changes in female mate-choice criteria in a mass-reared strain of Ceratitis capitata (Diptera: Tephritidae). Ann Entomol Soc Am. 2000; 93(2): 343–345.
- 36. Shelly TE. Fecundity of female oriental fruit flies (Diptera: Tephritidae): effects of methyl eugenol-fed and multiple mates. Ann Entomol Soc Am. 2000b; 93(3): 559–564.
- 37. Shelly TE, Nishida R. Larval and adult feeding on methyl eugenol and the mating success of male oriental fruit flies, Bactrocera dorsalis. Entomol Exp Appl. 2004; 112(2): 155–158.
- 38. Kumaran N, Balagawi S, Schutze MK, Clarke AR. Evolution of lure response in tephritid fruit flies: phytochemicals as drivers of sexual selection. Anim Behav. 2013; 85(4): 781–789.
- 39. Kumaran N, Clarke AR. Indirect effects of phytochemicals on offspring performance of Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae). J Appl Entomol. 2014; 138(5): 361–367.
- 40. Morelli R, Paranhos BJ, Coelho AM, Castro R, Garziera L, Lopes F, et al. Exposure of sterile Mediterranean fruit fly (Diptera: Tephritidae) males to ginger root oil reduces female remating. J Appl Entomol. 2013; 137(s1): 75–82.
- 41. Norrbom AL. Host plant database for Anastrepha and Toxotrypana (Diptera: Tephritidae: Toxotrypanini). Diptera Data Dissemination Disk, 2. 2004.
- 42. Vera MT, Ruiz MJ, Oviedo A, Abraham S, Mendoza M, Segura DF, et al. Fruit compounds affect male sexual success in the South American fruit fly, Anastrepha fraterculus (Diptera: Tephritidae). J Appl Entomol. 2013; 137(s1): 2–10.
- 43. Bachmann GE, Segura DF, Devescovi F, Juárez ML, Ruiz MJ, Vera MT, et al. Male sexual behavior and pheromone emission is enhanced by exposure to guava fruit volatiles in Anastrepha fraterculus. PLOS ONE. 2015; 10(4): e0124250. pmid:25923584
- 44. Bachmann GE, Devescovi F, Nussenbaum AL, Cladera JL, Fernández PC, Vera MT, et al. Male sexual enhancement after methoprene treatment in Anastrepha fraterculus (Diptera: Tephritidae): A sustained response that does not fade away after sexual maturation. J. Insect. Physiol. 2017; 101: 7–14. pmid:28623148
- 45. Abraham S, Goane L, Cladera J, Vera MT. Effects of male nutrition on sperm storage and remating behavior in wild and laboratory Anastrepha fraterculus (Diptera: Tephritidae) females. J Insect Physiol. 2011a; 57(11): 1501–1509.
- 46. Hendrichs J, Katsoyannos BI, Wornoayporn V, Hendrichs MA. Odour-mediated foraging by yellowjacket wasps (Hymenoptera: Vespidae): predation on leks of pheromone-calling Mediterranean fruit fly males (Diptera: Tephritidae). Oecol. 1994; 99(1–2): 88–94.
- 47. Jaldo HE, Gramajo MC, Willink E. Mass rearing of Anastrepha fraterculus (Diptera: Tephritidae): a preliminary strategy. Fla Entomol. 2001; 84(4): 716–718.
- 48. Salles LAB. Bioecologia e Controle das Moscas das Frutas Sul-Americanas. Embrapa Clima Temperado-Livros técnicos (INFOTECA-E). Pelotas, Brazil. 1995
- 49. Jaldo HE, Willink E, Liedo P. Demographic analysis of mass-reared Anastrepha fraterculus (Diptera: Tephritidae) in Tucumán, Argentina. Revista Industrial y Agrícola de Tucumán. 2007; 84: 15–20.
- 50. Petit-Marty N, Vera MT, Calcagno G, Cladera JL, Segura DF, Allinghi A, et al. Sexual behavior and mating compatibility among four populations of Anastrepha fraterculus (Diptera: Tephritidae) from Argentina. Ann Entomol Soc Am. 2004; 97(6), 1320–1327.
- 51. Segura DF, Utgés ME, Liendo MC, Rodríguez MF, Devescovi F, Vera MT, et al. Methoprene treatment reduces the pre‐copulatory period in Anastrepha fraterculus (Diptera: Tephritidae) sterile males. J Appl Entomol. 2013; 137: 19–29.
- 52. Juárez ML, Pimper LE, Bachmann GE, Conte CA, Ruiz MJ, Goane L, et al. Gut bacterial diversity and physiological traits of Anastrepha fraterculus Brazilian-1 morphotype males are affected by antibiotic treatment. BMC Microbiol, in press.
- 53. Vera MT, Cáceres C, Wornoayporn V, Islam A, Robinson AS, De la Vega MH, et al. Mating incompatibility among populations of the South American fruit fly Anastrepha fraterculus (Diptera: Tephritidae). Ann Entomol Soc Am. 2006; 99(2): 387–397.
- 54. Clayton DA. Socially facilitated behavior. Q Rev Biol. 1978; 53(4): 373–392.
- 55. Prokopy RJ, Duan JJ. Socially facilitated egg-laying behavior in Mediterranean fruit flies. Behav Ecol Sociobiol. 1998; 42: 117–122.
- 56. Rull J, Prokopy RJ, Vargas RI. Effects of conspecific presence on arrival and use of host in Ceratitis capitata flies. J Insect Behav. 2003; 16: 329–346.
- 57. Oviedo A, Nestel D, Papadoupulos NT, Ruiz MJ, Prieto SC, Willink E, et al. Management of protein intake in the fruit fly Anastrepha fraterculus. J Insect Physiol. 2011; 57(12): 1622–1630. pmid:21896276
- 58. Teal PEA, Gomez-Simuta Y, Proveaux AT. Mating experience and juvenile hormone enhance sexual signaling and mating in male Caribbean fruit flies. Proc Natl Acad Sci. 2000; 97(7): 3708–3712. pmid:10706642
- 59. StatSoft, Inc. STATISTICA (data analysis software system), version 7. 2004. www.statsoft.com.
- 60. GraphPad Software, Inc. 1992–2012. Prism 6 for Windows, Version 6.1.
- 61. Shelly TE. Effects of raspberry ketone on the mating success of male melon flies (Diptera: Tephritidae). Proc Hawaii Entomol Soc. 2000a; 34: 143–147.
- 62. Gillott C. Male accessory gland secretions: modulators of female reproductive physiology and behavior. Annu Rev Entomol. 2003; 48(1): 163–184.
- 63. Avila FW, Sirot LK, LaFlamme BA., Rubinstein CD, Wolfner MF. Insect seminal fluid proteins: identification and function. Annu Rev Entomol. 2011; 56: 21–40. pmid:20868282
- 64. Abraham S, Cladera J, Goane L, Vera MT. Factors affecting Anastrepha fraterculus female receptivity modulation by accessory gland products. J Insect Physiol. 2012; 58(1): 1–6. pmid:21907717
- 65. Pszczolkowski MA, Tucker A, Srinivasan A, Ramaswamy SB. On the functional significance of juvenile hormone in the accessory sex glands of male Heliothis virescens. J Insect Physiol. 2006; 52(8): 786–794. pmid:16806257
- 66. Clifton ME, Correa S, Rivera-Perez C, Nouzova M, Noriega FG. Male Aedes aegypti mosquitoes use JH III transferred during copulation to influence previtellogenic ovary physiology and affect the reproductive output of female mosquitoes. J Insect Physiol. 2014; 64: 40–47. pmid:24657670
- 67. Abraham S, Goane L, Rull J, Cladera J, Willink E, Vera MT. Multiple mating in Anastrepha fraterculus females and its relationship with fecundity and fertility. Entomol Exp Appl. 2011b; 141(1): 15–24.