Tropical tephritids are ideally suited for studies on population divergence and speciation because they include species groups undergoing rapid radiation, in which morphologically cryptic species and sister species are abundant. The fraterculus species group in the Neotropical genus Anastrepha is a case in point, as it is composed of a complex of up to seven A. fraterculus morphotypes proposed to be cryptic species. Here, we document pre‐ and post‐zygotic barriers to gene flow among adults of the Mexican A. fraterculus morphotype and three populations (Argentina, Brazil, and Peru) belonging to two separate morphotypes (Brazilian 1 and Peruvian). We unveiled three forms of pre‐zygotic reproductive isolation resulting in strong assortative mating. In field cages, free‐ranging male and female A. fraterculus displayed a strong tendency to form couples with members of the opposite sex belonging to their own morphotype, suggesting that male pheromone emission, courtship displays, or both intervene in shaping female choice before actual contact and coupling. In addition, males and females of the Peruvian morphotype became receptive and mated significantly later than adults of the Mexican and Brazilian 1 morphotypes. After contact, Mexican females exhibited greater mating discrimination than males when facing adults of the opposite sex belonging to either the Peruvian or the Brazilian 1 morphotype as evidenced by vigorous resistance to penetration once they had been forcefully mounted by heterotypic males. Forced copulations resulted in production of F1 hybrids that were either less viable (and partially fertile) than parental crosses or even sterile. Our results suggest that the Mexican morphotype is a distinct biological entity and that pre‐zygotic reproductive isolation through divergence in courtship or male‐produced pheromone and other mechanisms appear to evolve faster than post‐zygotic isolation in the fraterculus species group.

Anastrepha fraterculus is a major fruit pest in South America. Ongoing studies support the implementation of the sterile insect technique (SIT) against this pest. Sexual readiness of sterile males is a key point for SIT application. The pre‐copulatory period of A. fraterculus males has not been reported before, but it is expected to last several days. An acceleration of sexual maturation was achieved in other Anastrepha species after topical applications of juvenile hormone analogues, like methoprene. Here, we studied the effect of methoprene on male sexual maturation, mating duration and sperm transfer in A. fraterculus as well as the impact of acetone (methoprene solvent) on survival. We also explored a method to deliver methoprene massively. Pheromone‐calling and mating ability were evaluated daily from adult emergence, and used as indicators of sexual maturity. Anastrepha fraterculus males showed a long pre‐copulatory period (7 days approximately), as other Anastrepha species. This process was accelerated after methoprene treatment (2.5 μg/μl), both in non‐irradiated and irradiated males which matured 2–3 days earlier. Mating duration for methoprene‐treated males was longer than for mature untreated males, however, no differences in sperm transfer were detected. Survival was not affected by acetone. Dipping pupae in methoprene allowed emerging males to mature as fast as those receiving topical application as adults. Dipping of pupae is a promising method to deliver massively methoprene and should be further investigated.

Sexual maturation of Anastrepha fraterculus is a long process. Methoprene (a mimic of juvenile hormone) considerably reduces the time for sexual maturation in males. However, in other Anastrepha species, this effect depends on protein intake at the adult stage. Here, we evaluated the mating competitiveness of sterile laboratory males and females that were treated with methoprene (either the pupal or adult stage) and were kept under different regimes of adult food, which varied in the protein source and the sugar:protein ratio. Experiments were carried out under semi-natural conditions, where laboratory flies competed over copulations with sexually mature wild flies. Sterile, methoprene-treated males that reached sexual maturity earlier (six days old), displayed the same lekking behaviour, attractiveness to females and mating competitiveness as mature wild males. This effect depended on protein intake. Diets containing sugar and hydrolyzed yeast allowed sterile males to compete with wild males (even at a low concentration of protein), while brewer´s yeast failed to do so even at a higher concentration. Sugar only fed males were unable to achieve significant numbers of copulations. Methoprene did not increase the readiness to mate of six-day-old sterile females. Long pre-copulatory periods create an additional cost to the management of fruit fly pests through the sterile insect technique (SIT). Our findings suggest that methoprene treatment will increase SIT effectiveness against A. fraterculus when coupled with a diet fortified with protein. Additionally, methoprene acts as a physiological sexing method, allowing the release of mature males and immature females and hence increasing SIT efficiency.

The sterile insect technique (SIT) has been proposed as an area-wide method to control the South American fruit fly, Anastrepha fraterculus (Wiedemann). This technique requires sterilization, a procedure that affects, along with other factors, the ability of males to modulate female sexual receptivity after copulation. Numerous pre-release treatments have been proposed to counteract the detrimental effects of irradiation, rearing and handling and increase SIT effectiveness. These include treating newly emerged males with a juvenile hormone mimic (methoprene) or supplying protein to the male's diet to accelerate sexual maturation prior to release. Here, we examine how male irradiation, methoprene treatment and protein intake affect remating behavior and the amount of sperm stored in inseminated females. In field cage experiments, we found that irradiated laboratory males were equally able to modulate female remating behavior as fertile wild males. However, females mated with 6-day-old, methoprene-treated males remated more and sooner than females mated with naturally matured males, either sterile or wild. Protein intake by males was not sufficient to overcome reduced ability of methoprene-treated males to induce refractory periods in females as lengthy as those induced by wild and naturally matured males. The amount of sperm stored by females was not affected by male irradiation, methoprene treatment or protein intake. This finding revealed that factors in addition to sperm volume intervene in regulating female receptivity after copulation. Implications for SIT are discussed.

1 The avoidance of parasitized or infested hosts, which is a common phenomenon in parasitic wasps and phytophagous insects, may act both intra-and interspecifically. Most studies on chemically-mediated avoidance of interspecific competition in insects have been conducted at the individual level. The role of this behaviour on the spatial distribution of offspring of sympatric species with overlapping host ranges has been overlooked. 2 In the present study, two analytical approaches were used to investigate the co-infestation patterns of the fruit flies Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) and Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), aiming to unravel the importance of cross-species infestation recognition in nature. 3 Guava fruit were sampled in an area of coexistence of these two fruit flies and individually categorized as non-infested, infested by one of the species or infested by both species. The frequency of each type of fruit was compared with the frequency distributions expected under two models: an independent oviposition model and a competition avoidance model. As an alternative approach, co-occurrence patterns were evaluated using null models. 4 The results showed that avoidance of competition could be occurring in nature, although only in a few cases in which infestation levels are moderate. The two approaches revealed that the spatial scale has significant impact on the resulting co-occurrence patterns, such that opposite behaviours towards infested fruit are inferred at the largest (mainly aggregated oviposition pattern) versus the smallest scale (mainly independent oviposition pattern). 5 For the system under investigation, our findings suggest that the avoidance of infested fruit does not contribute, or at least not strongly, to the coexistence of the two species.

Anastrepha fraterculus (Diptera: Tephritidae) and Ceratitis capitata (Diptera: Tephritidae) are fruit fly pests whose larvae are frequently found sharing the same fruit with conspecifics. Because larvae are incapable of leaving the fruit in search of non‐infested fruit, they are forced to share nutritional resources and eventually compete with other larvae for these. In the present study, we investigated the effect of intraspecific competition on the development of A. fraterculus and C. capitata larvae, and compared the strategies adopted by these species. To this end, newly‐hatched larvae were transferred into a container with a fixed amount of larval diet at increasing larval densities and several developmental parameters were measured. The two species showed similarities and differences in the way in which they responded to an increasing density. In A. fraterculus, pupal weight and the duration of the larval stage decreased as the competition levels increased. Larvae of C. capitata showed a consistent reduction in pupal weight and larval survival as the larval density increased. In A. fraterculus, the reduction in pupal weight was heterogeneous, with most pupae showing a marked decrease in weight and only few pupae showing slight negative effects or no effect at all, whereas, in C. capitata, the reduction was similar for all pupae. The differences in the way in which these species responded to competition suggest that there are different patterns of resource distribution among conspecific larvae. The implications of these findings for oviposition behaviour and the life history of the two species are discussed.

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1. Unravelling the strength and modes of interspecific interactions between resident and introduced species is necessary in order to understand the basis of their coexistence or the displacement of the former by the latter. In Argentina, the indigenous Tephritidae fly Anastrepha fraterculus overlaps its distribution and host fruit with the introduced species Ceratitis capitata.2. This study focused on the relative strength of intra-and interspecific competition during the larval stage as a potential factor supporting coexistence. Classical competition experiments (addition and substitution) were conducted between larvae of the two species reared in artificial larval diet. The study evaluated whether a temporal separation between oviposition events affects the outcome of the competition.3. When both species started to consume the resource at the same time, A. fraterculus experienced a negative effect in larval survival, pupal weight and duration of larval stage, while for C. capitata, pupal weight decreased. When A. fraterculus started feeding 1 day earlier than C. capitata, the negative effects became milder, and when the temporal separation increased, these effects were reversed. Substitution experiments showed an increase in pupal weight when larvae had to share the resource with heterospecific larvae, and showed negative effects suffered for both species when they shared the resource with conspecific individuals.4. These results suggest that intraspecific competition is stronger than interspecific competition, and a differential oviposition preference could generate an asynchrony of these species in nature. Such mechanisms could favour coexistence between A. fraterculus and C. capitata in an environment previously occupied only by the former.