Reactive oxygen species (ROS) in large amounts have been shown to cause peroxidative damage to tissues, thereby influencing life‐history trade‐offs such as between reproduction and survival. Reactive oxygen species production is heightened in stressful environments, such as after exposure to toxins. Our study analyzes the effects of antioxidants in reducing the damaging effects of ROS to rescue pre‐ and post‐copulatory reproductive efforts in Drosophila melanogaster Meigen (Diptera: Drosophilidae). We hypothesized that if male fruit flies were fed antioxidant‐enriched diets prior to environmental stress, then the antioxidants would quench the ROS. This would then reduce the lipid peroxidative damage to male sperm, resulting in increased pre‐ and post‐copula reproductive efforts. Two groups of fruit flies were fed antioxidant‐rich diets of lipoic acid (2.15 mM) or melatonin (0.43 mM). Males from all treatments were then fed an herbicide, paraquat (10 mM), which functions as an environmental stressor to shock their immune systems and increase ROS production. All males were then mated to virgin females and copula behavior, sperm viability, male fertility, and total antioxidant capacity were assessed. Our results showed significantly longer mating latencies, higher levels of viable sperm, and more offspring sired in antioxidant‐fed treatments compared to controls. Antioxidant assays reveal that post‐paraquat challenge, males up‐regulate the amount of antioxidants produced. These results draw light on the basis of life‐history trade‐offs such as between reproduction and survival under the context of the molecular mechanisms that govern them, such as pre‐ and post‐copulatory behavior, ROS production, fertility, and sperm viability.

All animals are under the constant threat of pathogenic infection. However, little is known regarding the influence of acute infection on sperm viability, particularly in female insects. This information is crucial for our understanding of mating and immune system coevolution, considering that females store sperm and serve as the site of sperm competition. Using the fruitfly, Drosophila melanogaster, we examined the influence of infection on sperm viability and storage. Twenty-four hours after haemocoel inoculation with a pathogen mimic (peptidoglycan, PGN) both sexes exhibited reduced sperm viability, indicating that systemic immune activation played a significant role in gamete survival. Surprisingly, sperm death did not appear to result from a reproductive-immune system trade-off, considering that sperm survived 24 h in vitro once removed from their somatic resources. Instead, our results are most consistent with death owing to immune effector collateral damage. We also examined the potential for sexually transmitted pathogens to influence sperm storage. Females mated with ‘infected’ males (created by dipping genitalia into a PGN solution) exhibited a higher proportion of empty sperm stores 48 h after mating compared to their controls. Remarkably, these data indicate that females may increase their fitness by removing ‘infected’ ejaculates from storage over time.

Sperm viability has been associated with the degree of promiscuity across species, as well as the degree of reproductive success within species. Thus, sperm survival within the female reproductive tract likely plays a key role in how mating systems evolve. In the fruit fly, Drosophila melanogaster, however, the extent and cause of sperm death has been the subject of recent debate. Here, we assess sperm death within the female reproductive tract of D. melanogaster following single and multiple matings in order to elucidate the extent of death and its potential mechanisms, including an acute female response to mating, female age and/or sperm senescence. We found no evidence that sperm viability was influenced by an acute female response or female age. We also found that rival ejaculates did not influence viability, supporting recent work in the system. Instead, the majority of death appears to be due to the aging of male gametes within the female, and that at least some dead resident sperm remain in the female after multiple mating. In contrast to earlier in vivo work, we found that overall sperm death was minimal (8.7%), indicating viability should have a negligible influence on female remating rates.

Copula duration and sperm storage patterns can directly or indirectly affect fitness of male and female insects. Although both sexes have an interest in the outcome, research has tended to focus on males. To investigate female influences, we compared copula duration and sperm storage of Queensland fruit fly females that were intact, or had been incapacitated through decapitation or abdomen isolation. We found that copulations were far longer when females had been incapacitated, indicating that constraints imposed on copula duration by intact females had been relaxed. Repeatability of copula duration for males was very low regardless of female treatment, and this is also consistent with strong female influence. Number of sperm in the spermathecae was not influenced by female treatment, suggesting that female abdominal ganglia control the transport of sperm to these long-term storage organs. However, more sperm were found in the ventral receptacles of incapacitated females compared to intact females. Overall, results implicate cephalic ganglia in regulation of copula duration and short-term sperm storage in the ventral receptacle and abdominal ganglia in regulation of long-term sperm storage in the spermathecae.

Ultrastructure of male reproductive accessory glands and ejaculatory duct in the Queensland fruit fly (Q-fly), Bactrocera tryoni, were investigated and compared with those of other tephritid flies. Male accessory glands were found to comprise one pair of mesodermic glands and three pairs of ectodermic glands. The mesodermic accessory glands consist of muscle-lined, binucleate epithelial cells, which are highly microvillated and extrude electron-dense secretions by means of macroapocrine transport into a central lumen. The ectodermic accessory glands consist of muscle-lined epithelial cells which have wide subcuticular cavities, lined with microvilli. The electron-transparent secretions from these glands are first extruded into the cavities and then forced out through small pores of the cuticle into the gland lumen. Secretions from the two types of accessory glands then flow into the ejaculatory duct, which is highly muscular, with epithelial cells rich in rough endoplasmic reticulum and lined with a thick, deeply invaginated cuticle. While there are some notable differences, reproductive accessory glands of male Q-flies generally resemble those of the olive fruitfly, Bactrocera oleae, and to a lesser extent the Mediterranean fruit fly, Ceratitis capitata.

Male insects that are unable to replenish sperm supplies between matings can suffer fitness costs either because their mates are more likely to accept subsequent suitors, or because their sperm are outnumbered when females do remate. We assessed the ability of fertile and sterile (irradiated) male Queensland fruit flies, Bactrocera tryoni ('Q-flies'), to have sperm stored by five sequential mates as well as the association between sperm depletion and female remating tendency. Sequential mates of fertile males stored similar numbers of sperm, indicating ample ability to replenish and maintain constant supplies between their once-daily mating opportunities. In contrast, sequential mates of sterile males stored progressively fewer sperm, with only trivial numbers of sperm stored by females mated by sterile males that had mated with two or more females previously. Despite the massive reduction in sperm storage by sequential mates of sterile males, fertile and sterile males were similar in their ability to induce sexual inhibition in their mates (to at least 30 days) and neither showed any decline in this ability across sequential matings. The ability of multiple-mated sterile males to induce sexual inhibition in their mates despite near or complete absence of sperm provides compelling evidence that sperm abundance plays no role in the induction of sexual inhibition in this species. (C) 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Insect seminal fluid commonly comprises a complex cocktail of proteins and other biochemical components that migrate away from the female reproductive tract to sites elsewhere in the female body and elicit changes in female reproductive behaviour. The transfer of male seminal fluid molecules to reproductive and somatic tissues of the female Queensland fruit fly ('Q-fly') Bactrocera tryoni is examined and some putative target sites identified. Male Q-flies are fed a diet containing radiolabelled (S-35) amino acids, which are incorporated into male accessory gland products. Radioactivity diminishes within the accessory glands and increases in all assessed parts of the female body during copulation, indicating the transfer of these products into the female soma via the reproductive tract. There are significant changes in the absolute and proportional radioactivity profiles among female tissues over the next 22 h, with substantial reductions in the thorax and increases in the head. This is consistent with accumulation of behaviour-modifying male products at binding sites in the female head. Parallels can be drawn between the data in the present study and seminal fluid distribution profiles and receptor binding documented in other insects.

Mating success of male insects is commonly determined by their ability to find and copulate with multiple females, but is also determined by their ability to transfer an effective ejaculate. In order to succeed in these tasks, males must first succeed in replenishing the necessary reproductive reserves between mating opportunities. We here investigate the ability of male Queensland fruit flies (‘Q-fly’) to recover from their first matings in time to both mate again the following day and to induce sexual inhibition in successive mates. We have previously found that accessory gland fluids (AGFs) transferred in the ejaculate of male Q-flies are directly responsible for induction of sexual inhibition in their mates. We here investigate changes in male accessory gland, testis and ejaculatory apodeme dimensions that are likely to reflect depletion and recovery of contents. We found no differences between virgin and previously mated males in their ability to obtain matings or to induce sexual inhibition in their mates, indicating a full recovery of the necessary reproductive reserves between mating opportunities. Whereas no changes were detected in testis or ejaculatory apodeme size following mating, the recovery of male ability to inhibit female remating was closely reflected in the mesodermal accessory gland dimensions; these accessory glands greatly diminished in size (length and area) immediately after mating, with recovery commencing between 5.5 and 11h after mating. The accessory glands then expanded to reach their original size in time to mate the following day and induce sexual inhibition in the next mate.

Molecules in male seminal fluid transferred to female insects during mating can have potent effects on their subsequent sexual and reproductive behaviour. Like many other tephritids, female Queensland fruit flies (Bactrocera tryoni) typically have diminished sexual receptivity after their first mating. Also, copulations of females that do remate tend to be shorter than those of virgins. We here find that virgin females injected with small doses (0.1, 0.2 or 0.5 male equivalents) of extracts from the male reproductive tract accessory tissues, which consist of male accessory glands, ejaculatory apodeme and ejaculatory duct (AG/EA/ED), have diminished receptivity and short copula duration very similar to naturally mated females. In contrast, virgin females injected with saline or with high doses of AG/EA/ED (1 or 2 male equivalents) that likely exceed the range of natural variation retain the higher levels of sexual receptivity and longer copulations of un-injected virgins. We conclude that reduced sexual receptivity and shorter copulations of mated female Q-flies are mediated by products in the male seminal fluid derived from the male reproductive tract accessory tissues.

Reproductive success of male insects commonly hinges both on their ability to secure copulations with many mates and also on their ability to inseminate and inhibit subsequent sexual receptivity of their mates to rival males. We here present the first investigation of sperm storage in Queensland fruit flies (Tephritidae: Bactrocera tryoni; a.k.a. 'Q-flies') and address the question of whether remating inhibition in females is directly influenced by or correlated with number of sperm stored from their first mates. We used irradiation to disrupt spermatogenesis and thereby experimentally reduce the number of sperm stored by some male's mates while leaving other aspects of male sexual performance (mating probability, latency until copulating, copula duration) unaffected. Females that mated with irradiated rather than normal males were less likely to store any sperm at all (50% vs. 89%) and, if some sperm were stored, the number was greatly reduced (median 11 vs. 120). Despite the considerable differences in sperm storage, females mated by normal males and irradiated males were similarly likely to remate at the next opportunity, indicating (1) number of sperm stored does not directly drive female remating inhibition and (2) factors actually responsible for remating inhibition are similarly expressed in normal and irradiated males. While overall levels of remating were similar for mates of normal and irradiated males, factors responsible for female remating inhibition were positively associated with presence and number of sperm stored by mates of normal but not irradiated males. We suggest seminal fluids as the most likely factor responsible for remating inhibition in female Q-flies, as these are likely to be transported in proportion to number of sperm in normal males, be uninfluenced by irradiation, and be transported without systematic relation to sperm number in irradiated males.