M.T. Vera

Methoprene treatment reduces the pre-copulatory period in Anastrepha fraterculus (Diptera: Tephritidae) sterile males

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.

Precocious sexual signalling and mating in Anastrepha fraterculus (Diptera: Tephritidae) sterile males achieved through juvenile hormone treatment and protein supplements

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.

Remating behavior in Anastrepha fraterculus (Diptera: Tephritidae) females is affected by male juvenile hormone analog treatment but not by male sterilization

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 males 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.

Fruit compounds affect male sexual success in the South American fruit fly, Anastrepha fraterculus (Diptera: Tephritidae)

Although exposure to plants has been shown to influence sexual behaviour in a number of phytophagous insect species, a relatively small number of fruit flies have been investigated in that respect. Here, we evaluated the effect of exposure to the pulp of guava and mango and to essential oils emanating from glands in the flavedo area of lemons on the mating success of Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) males. We also evaluated different durations of exposure and the need for physical contact with the fruit. Results showed that exposure to guava increased the mating success of both wild and laboratory males relative to non‐exposed males. In addition, exposed wild flies copulated earlier than non‐exposed males. Physical contact with the fruit or ingestion of compounds was not a prerequisite for this phenomenon to occur, since just the exposure to volatiles resulted in a significant enhancement in mating success. Exposure to mango did not affect male sexual performance. In contrast, exposure to lemon for 1 day decreased mating success of males relative to unexposed males, whereas exposure for longer periods seemed to eliminate this effect resulting in comparable mating success rates between treated and untreated males. These results provide a better understanding of the sexual behaviour of this species and may also find application for its control.

Management of protein intake in the fruit fly Anastrepha fraterculus

This work tested if carbohydrates and proteins ingestion is regulated in the South American fruit fly, Anastrepha fraterculus, to optimize survival and reproduction. Adult food treatments were established by providing sugar and hydrolyzed yeast in various combinations either alone or mixed at a standard 3:1 ratio (sugar:hydrolyzed yeast). Individual food consumption was assessed and related to survival patterns. The effects of adult feeding on fecundity and fertility patterns were investigated in groups of flies. Sugar consumption was the lowest in the treatment where it was provided with hydrolyzed yeast at a fixed 3:1 ratio. Consumption of hydrolyzed yeast did not differ between this treatment and the one in which this solution was complemented with one solution of sugar. It seems that a mixture of sugar and hydrolyzed yeast at a fixed ratio of 3:1, respectively, restricts extra ingestion of sugar; most probably because of negative response of the fly to overconsumption of protein. Survival was affected by the treatments, being lower in those cases where protein was at the fixed ratio. Group experiments revealed that protein restriction expanded longevity and decreased egg production. In contrast, egg production was enhanced when flies were kept continuously with a mixture of yeast and sugar plus an extra source of sugar, and this was not in detriment of survival. Our results suggest that fixed sugar-protein ratios in which protein is in excess affects fitness components such as longevity and reproduction. These findings are discussed from a theoretical and applied perspective in the context of pest control by means of the sterile insect technique.

Dynamics of genetic variability in Anastrepha fraterculus (Diptera: Tephritidae) during adaptation to laboratory rearing conditions

BackgroundAnastrepha fraterculus is one of the most important fruit fly plagues in the American continent and only chemical control is applied in the field to diminish its population densities. A better understanding of the genetic variability during the introduction and adaptation of wild A. fraterculus populations to laboratory conditions is required for the development of stable and vigorous experimental colonies and mass-reared strains in support of successful Sterile Insect Technique (SIT) efforts.MethodsThe present study aims to analyze the dynamics of changes in genetic variability during the first six generations under artificial rearing conditions in two populations: a) a wild population recently introduced to laboratory culture, named TW and, b) a long-established control line, named CL.ResultsResults showed a declining tendency of genetic variability in TW. In CL, the relatively high values of genetic variability appear to be maintained across generations and could denote an intrinsic capacity to avoid the loss of genetic diversity in time.DiscussionThe impact of evolutionary forces on this species during the adaptation process as well as the best approach to choose strategies to introduce experimental and mass-reared A. fraterculus strains for SIT programs are discussed.

Differences in sperm storage and remating propensity between adult females of two morphotypes of the Anastrepha fraterculus (Diptera: Tephritidae) cryptic species complex.

The South American fruit fly, Anastrepha fraterculus, is a complex of cryptic species composed of at least seven morphotypes. Some of them, such as the Peruvian and Brazilian 1 morphotypes (which include Argentinean populations), exhibit strong pre-copulatory isolation, yet it is possible to obtain heterotypic crosses when forcing copulation of adults under laboratory conditions. The cross involving Peruvian males and Argentinean females produces F1 offspring with reduced viability in terms of egg hatch. This low hatchability could be caused by a reduced amount of sperm transferred to and stored by females mated with heterotypic males, which in turn could affect their post-copulatory behaviour. To test these hypotheses, we investigated sperm transfer and female mating and remating behaviour for homotypic and heterotypic crosses between adults of two morphotypes (Brazilian 1 [Argentina] and Peruvian [Peru]) of the A. fraterculus cryptic species complex. As reported before, Argentinean males and females mated earlier in the day than the other three mating combinations. Peruvian females engaged in shorter copulation times than Argentinean females. Peruvian females tended to store smaller quantities of sperm than Argentinean females, and almost a half of the crosses involving Argentinean males and Peruvian females were unsuccessful (no sperm transfer). However, there was no evidence that the cross between Peruvian males and Argentinean females resulted in storage of a critically small amount of sperm (posing risk of sperm shortage). Argentinean females were more willing to remate than Peruvian females, irrespective of male morphotype, but latency to remating was not affected by male or female morphotype. This study shows that mating behaviour differs between some of the A. fraterculus complex morphotypes, with female but not male morphotype determining female likelihood to remate.

Patterns of resource distribution among conspecific larvae in two fruit fly species: Anastrepha fraterculus and Ceratitis capitata (Diptera: Tephritidae)

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.

Male sexual enhancement after methoprene treatment in Anastrepha fraterculus (Diptera: Tephritidae): A sustained response that does not fade away after sexual maturation

The juvenile hormone (JH) of insects triggers physiological changes related to reproduction in adults of both sexes. Methoprene is a sesquiterpene with some effects that are analogous to those of JH. Treatments with methoprene accelerate sexual maturation in males of the South American fruit fly Anastrepha fraterculus, giving young males a mating advantage over non-treated males of the same age. Here, we evaluated the effects of methoprene treatment on A. fraterculus males after the sexual maturation phase and tested whether this compound provides a long-term mating advantage. Moreover, we took the first step to unravel the mechanisms that underlie male sexual enhancement. We treated males 1day or 8days after adult emergence and compared mate choice between recently matured (young) females and females that had been mature for ca. 10days (aged females). We also addressed methoprene treatment effects on male sexual signalling. We found that methoprene treatment enhanced male sexual competitiveness even after the sexual maturation phase, and the effect did not decrease until males were older than 20days. However, when methoprene treatment was carried out close to sexual maturity, the mating enhancement was no longer observed, suggesting a non-immediate effect and excluding the possibility that methoprene acts as a pheromonal compound. Young and aged females tended to mate more frequently with treated-males. This might indicate that in a context of sexual selection, the potential benefits associated with reproductive success would be similar for females of both ages. Treated males released larger amounts of pheromonal compounds than non-treated males, but their courtship behaviour was not altered to the same extent, suggesting that methoprene treatment may accelerate differently the components of male courtship. We discuss potential benefits of using methoprene to increase the efficiency of the sterile insect technique, which is an environmentally safe method to control this important South American fruit pest.

Gut bacterial diversity and physiological traits of Anastrepha fraterculus Brazilian-1 morphotype males are affected by antibiotic treatment

Background The interaction between gut bacterial symbionts and Tephritidae became the focus of several studies that showed that bacteria contributed to the nutritional status and the reproductive potential of its fruit fly hosts. Anastrepha fraterculus is an economically important fruit pest in South America. This pest is currently controlled by insecticides, which prompt the development of environmentally friendly methods such as the sterile insect technique (SIT). For SIT to be effective, a deep understanding of the biology and sexual behavior of the target species is needed. Although many studies have contributed in this direction, little is known about the composition and role of A. fraterculus symbiotic bacteria. In this study we tested the hypothesis that gut bacteria contribute to nutritional status and reproductive success of A. fraterculus males. Methods Wild and laboratory-reared males were treated with antibiotics (AB) and provided sugar (S) or sugar plus protein (S+P) as food sources. The effect of AB on the gut bacteria diversity was assessed through DGGE and sequencing of the V6-V9 variable region of the bacterial 16S rRNA gene. Results AB affected the bacterial community of the digestive tract of A. fraterculus, in particular bacteria belonging to the Enterobacteriaceae family, which was the dominant bacterial group in the control flies (i.e., non-treated with AB). AB negatively affected parameters directly related to the mating success of laboratory males and their nutritional status. AB also affected males’ survival under starvation conditions. The effect of AB on the behaviour and nutritional status of the males depended on two additional factors: the origin of the males and the presence of a proteinaceous source in the diet. Conclusions Our results suggest that A. fraterculus males gut contain symbiotic organisms that are able to exert a positive contribution on A. fraterculus males’ fitness, although the physiological mechanisms still need further studies.