Methoprene treatment and its effect on male reproductive organ size and female remating in a fruit fly.

Methoprene, a juvenile hormone analog, is used to accelerate sexual maturation in males of species of economic importance in support to the sterile insect technique (SIT). In the SIT, mass-reared sterile males are released into the field and need to survive until they reach sexual maturation, find a wild female, mate with her and then induce female sexual refractoriness, so she will not remate with a wild counterpart. The use of methoprene shortens the time between release and copulation. However, in South American fruit flies, Anastrepha fraterculus, the ability of methoprene-treated males to inhibit female remating has been shown to be lower than wild males, when methoprene was applied by pupal immersion or topical application. Here we evaluated the possibility of incorporating methoprene into the male diet at different doses and the ability of those males to inhibit female remating, as well as the effect of methoprene on male reproductive organ size, due to the possible correlation between male accessory gland size and their content, and the role of male accessory gland proteins in female inhibition. We found that A. fraterculus males fed with methoprene in the adult protein diet at doses as high as 1% were less likely to inhibit female remating, however, at all other lower doses males had the same ability as untreated males to inhibit female remating. Males fed with methoprene had bigger male accessory glands and testes compared to methoprene-deprived males. We demonstrate that the incorporation of methoprene in adult male diets is possible in this species and potentially useful as a post-teneral, pre-release supplement at doses as low as 0.01%. Even at higher doses, the percentage of females remating after 48 h from the first copulation is sufficiently low in this species so as not compromise the efficiency of the SIT.

Post-mating gene expression of Mexican fruit fly females: disentangling the effects of the male accessory glands.

Mating has profound physiological and behavioural consequences for female insects. During copulation, female insects typically receive not only sperm, but a complex ejaculate containing hundreds of proteins and other molecules from male reproductive tissues, primarily the reproductive accessory glands. The post-mating phenotypes affected by male accessory gland (MAG) proteins include egg development, attraction to oviposition hosts, mating, attractiveness, sperm storage, feeding and lifespan. In the Mexican fruit fly, Anastrepha ludens, mating increases egg production and the latency to remating. However, previous studies have not found a clear relationship between injection of MAG products and oviposition or remating inhibition in this species. We used RNA-seq to study gene expression in mated, unmated and MAG-injected females to understand the potential mating- and MAG-regulated genes and pathways in A. ludens. Both mating and MAG-injection regulated transcripts and pathways related to egg development. Other transcripts regulated by mating included those with orthologs predicted to be involved in immune response, musculature and chemosensory perception, whereas those regulated by MAG-injection were predicted to be involved in translational control, sugar regulation, diet detoxification and lifespan determination. These results suggest new phenotypes that may be influenced by seminal fluid molecules in A. ludens. Understanding these influences is critical for developing novel tools to manage A. ludens.

Antipredator behavior of the new mass-reared unisexual strain of the Mexican Fruit Fly.

Tephritid pests controlled through the sterile insect technique (SIT) are mass-reared and subsequently released in affected areas. Several quality parameters are currently used to test adults, but none take into account interactions with a predator. When sterile males are released in the field, they will need to avoid predators until they reach sexual maturity and survive long enough to mate with wild females. Spiders are one of the most common predators that flies may encounter in release sites. In this study, we evaluated the antipredator behavior of a mass-reared sterile unisexual strain ('Tapachula-7') of the Mexican fruit fly Anastrepha ludens (Diptera: Tephritidae) against their spider predators. We sampled spiders in citrus trees to determine which families could be more common. We established the baseline activity rates of sterile Tapachula-7 (Tap-7) flies in comparison with wild flies. We also tested the behavior of the fertile and sterile bisexual strain and wild flies against hunting spiders (Family Salticidae) and orb building spiders (Family Tetragnathidae). We recorded 18 spider families, with Salticidae being the most dominant. Tap-7 flies diminished their activity in comparison with wild males at 1800 h but showed similar activity levels earlier in the day. When exposed to orb-web spiders (Leucauge venusta), Tap-7, fertile and sterile males from the bisexual strain had similar rates of survival, but Tap-7 males showed lower survival than wild males. Against hunting spiders (Phidippus audax), wild males had higher probability of defensive wing displays, but there was no difference in spider attack rates. In general, sterile Tap -7 males performed as well as males from the bisexual strain, although they had lower survival than wild males. This could be due to either mass-rearing and/or irradiation effects. We recommend the use of the defensive wing display behavior as a quality parameter and propose a rapid and effective method to evaluate fly activity. The efficiency of SIT will be improved if released sterile males have the same antipredator repertoire as their wild counterparts.

The Sterile Insect Technique: Success and Perspectives in the Neotropics.

The sterile insect technique (SIT), an environmentally friendly means of control, is currently used against plant, animal, and human pests under the area-wide integrated pest management. It consists in the mass production, sterilization, and release of insects in an affected area where sterile males mate with wild females leading to no reproduction. Here, we review SIT in the Neotropics and focus on particular recent successful cases of eradication of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), as well as effective programs used against the Mexican fruit fly Anastrepha ludens (Loew), the New World screwworm fly Cochliomyia hominivorax (Coquerel)), and the Cactus moth Cactoblastis cactorum (Berg). We examine when SIT does not work and innovations that have made SIT more efficient and also highlight complimentary techniques that can be used in conjunction. We address potential candidate species that could be controlled through SIT, for example Philornis downsi Dodge & Aitken. Finally, we consider the impact of climate change in the context of the use of the SIT against these pests. Given the recent dramatic decline in insect biodiversity, investing in environmentally friendly means of pest control should be a priority. We conclude that SIT should be promoted in the region, and leadership and political will is needed for continued success of SIT in the Neotropics.

Random mating among Anastrepha ludens (Diptera: Tephritidae) adults of geographically distant and ecologically distinct populations in Mexico.

The Mexican fruit fly Anastrepha ludens (Loew) (Diptera: Tephritidae) is a polyphagous pestiferous insect with a geographical range encompassing highly variable environmental conditions. Considering that cryptic species have been recently found among South American representatives of the same taxonomic group as A. ludens, we tested whether or not some populations of A. ludens have evolved assortative mating as an isolating mechanism that maintains intrapopulation genetic differences and behavioral adaptations to local conditions. Males and females stemming from widely separated locations with similar environmental conditions and males and females stemming from populations within individual-flight range, but collected in different hosts (a native and an exotic one), mated randomly amongst themselves when placed in a field cage. Despite the fact that sibling males and females from two distinct populations also mated randomly amongst themselves, siblings engaged in significantly longer copulations than non-siblings, indicating that perhaps adults discriminated mates with similar genetic compositions. Our results have important practical implications as A. ludens is the most devastating pest of citrus in Mexico and Central America, and large-scale releases of sterile flies are used to control it.

When Less Is More: Sex Ratios for the Mass-Rearing of Anastrepha ludens (Diptera: Tephritidae).

Anastrepha ludens (Loew) is one of the most important pests of citrus and mango crops in Mexico. A method used to control this pest is the sterile insect technique, which consists in the mass production, irradiation, and release of insects in affected areas. The production of insects begins with the establishment of colonies to produce eggs, which must be highly fertile to ensure an adequate production of larvae. However, female fecundity and fertility can be affected by adult density and sex ratio, thus an optimal sex ratio in mass-rearing cages must be used. The genetic sexing strain of A. ludens (Tapachula-7) allows the identification of the sex at the pupal stage, making it possible to establish rearing cages with different sex ratios. We determined if different sex ratios have an effect on egg production. Two sex ratios (4♀: 1♂ and 1♀: 1♂) were compared. Fecundity, fertility and survival at different ages were also determined. Higher fertility and fecundity per female were observed at a ratio of 4:1. However, females with higher fecundity had reduced survival probabilities. In conclusion, maintaining colonies with a lower proportion of males in cages ensures a greater fecundity and fertility. Further research is necessary to understand whether results can be attributed to lower male harassment in cages.

Identification of Ceratitis capitata (Diptera: Tephritidae) Sperm From Females in Traps: The Importance of the Ventral Receptacle.

The monitoring of a pest population often relies on the identification of individuals from traps. For area-wide programs utilizing the sterile insect technique, the further identification of the mated status of females found in traps is of utmost importance. For the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), this is usually verified through the assessment of sperm in the spermathecae. However, this can be misleading for species where there are other sperm storage organs such as the ventral receptacle. Here, we studied the relative importance of sperm storage in the ventral receptacle compared to the spermathecae for females from 5 to 18 d of age. Furthermore, we studied how sperm can be identified in the ventral receptacle or spermathecae after females were recovered from traps. We found no effect of female age on likelihood of sperm storage. Sperm could be identified in both sperm storage organs at 7 or 14 d after females had been placed in traps. We found that the ventral receptacle is a more reliable indicator of female mated status. Thus, we propose that if no sperm are found in the spermathecae, program managers should revise the ventral receptacle before assuming that females are not mated. This test may also be relevant to other pest tephritids that store even more sperm in the ventral receptacle than C. capitata.

Effects of size, sex and teneral resources on the resistance to hydric stress in the tephritid fruit fly Anastrepha ludens.

Water availability is recognized as one of the most important factors in the distribution and activity of terrestrial organisms. In the case of insects, hydric stress imposes a major challenge for survival because of the small surface-area-to-volume ratio they exhibit. In general, stress resistance is expected to co-vary positively with size; however, this pattern can become obscured in insects that exhibit sexual size dimorphism, as sexes differ in size and/or shape and have dissimilar resource allocations. In the present study, we use an allometric-based approach to (i) assess the desiccation and starvation stress resistance of teneral Anastrepha ludens flies, (ii) disentangle the relationships between resistance, size and sex and (iii) examine the adult fly body differences in water and lipid contents before and after exposure to stress. After controlling for sexual size dimorphism, an allometric increase of resistance with overall size was observed for all stress-based treatments. The scaling exponents that define the proportion of increase resistance varied with size traits and with type and degree of hydric stress. In this allometric relationship, and also in the relationships between mass and wing length and between size and teneral resources, the sexes maintained similar scaling exponents but differed in the intercepts. Males were more resistant to stress than females; this competitive advantage is probably linked to greater amounts of teneral lipids and more water use during stress.

Male and female condition influence mating performance and sexual receptivity in two tropical fruit flies (Diptera: Tephritidae) with contrasting life histories.

Recent recognition of widespread polyandry in insects has generated considerable interest in understanding why females mate multiple times and in identifying factors that affect mating rate and inhibit female remating. However, little attention has been paid to understanding the question from both a female and male perspective, particularly with respect to factors that may simultaneously influence female remating rates. Here, we report on a study aimed at ascertaining the possible interactive effects that male and female size and diet, and female access to a host could have on mating latency, probability, and duration and female refractory period using two tropical fruit fly species with contrasting life histories. Of all factors tested, adult diet played the most significant role. Both Anastrepha ludens and Anastrepha obliqua males which had constant access to protein and sucrose mated more often, had shorter copulations and induced longer refractory periods in females than males fed a low quality diet (sucrose offered every third day). Female size and the interaction with male diet determined how quickly female A. ludens mated for the first time. Smaller females mated sooner with low quality fed males than with high quality fed males while there was no difference for large females, suggesting that male choice may be at play if high quality fed males discriminate against smaller females. Copulation duration also depended on both male and female nutritional condition, and the interaction between male diet and female size and diet. Large and high quality fed females had shorter copulations regardless of male condition. Importantly, for A. ludens, female refractory period depended on male size and the nutritional condition of both males and females, which could indicate that for this species, female receptivity does not depend only on the condition of the male ejaculate. For A. obliqua refractory period was associated with the interaction between male size and diet and male diet and host presence. We discuss our results in terms of male ability to inhibit female remating and the relative contribution of female condition to this behavior. We also address the importance of studying effects simultaneously on species with contrasting life histories.