Effects of male nutrition on sperm storage and remating behavior in wild and laboratory Anastrepha fraterculus (Diptera: Tephritidae) females

Male physiological condition can affect his ability to modulate female sexual receptivity. Thus, studying this aspect can have biological and practical implications. Here, we examine how male nutritional status affected the amount of sperm stored, remating rate and refractory period of the tephritid fruit fly Anastrepha fraterculus (Wiedemann) females. Both wild and laboratory flies were evaluated. We also examine female sperm storage patterns. Experiments were carried out by manipulating male adult diet and exposing these males to virgin females. Females mated with differently treated males were either dissected to count the amount of sperm stored or exposed to virgin males to determine remating rate and the length of the refractory period. We found that male nutritional status affected the amount of the sperm stored and the renewal of sexual receptivity in wild flies. For laboratory flies, male nutritional status affected the length of the refractory period but not the amount of sperm stored by females. In addition, we report that the ventral receptacle is not an important organ of sperm storage in this species. We conclude that male nutritional condition influences the ability to modulate female sexual receptivity, possibly through a combination of the quantity and quality of the ejaculate. From an applied perspective, providing males with an enriched diet will likely result in increased efficacy of the sterile insect technique.     

Regulation of sexual receptivity of female Mediterranean fruit flies: old hypotheses revisited and a new synthesis proposed

The objective of this study was to examine the relative contributions of copula duration and sperm transfer to the inhibition of sexual receptivity of female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae). Females choosing to remate had significantly fewer sperm in their spermathecae than females who chose not to remate. Duration of a female's first copulation did not affect her subsequent receptivity. Furthermore, on the first day following copulation significantly more females whose first mate was sterile and from a laboratory strain (sterile males transfer fewer sperm than wild males) chose to copulate than did females whose mate was fertile and recently derived from wild stock. Finally, we offer a synthesis of the available information on remating in this species, and suggest that while females are facultatively polyandrous, copula duration, sperm transfer and male accessory gland secretions act in succession to inhibit female receptivity.     

Remating by female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae): Temporal patterns and modulation by male condition

We determined the temporal pattern of female remating in the Mediterranean fruit fly, Ceratitis capitata, and how mating with sterile males affects remating. In addition, we examined the hypotheses that sterile male nutrition and age affect the subsequent receptivity of their mates. Temporally, female receptivity varied significantly throughout the experimental period. Relatively high levels of remating (14%) on the days following the first copulation were followed by a decline, with a significantly low point (4.1%) 2 weeks after mating. Subsequently, receptivity is gradually restored (18%) 3 and 4 weeks after the initial copulation. When females were first mated to sterile males, significantly higher remating percentages were recorded. The ability of sterile males to inhibit receptivity of both wild and laboratory reared females on the day of first mating was significantly improved when they were fed a nutrient rich diet. Male age at first mating also affected female receptivity: sterile males of intermediate age (11 days old) inhibited female remating significantly more than younger or older flies. Although further studies are needed to determine the relative roles of natural and sexual selection in modulating patterns of female sexual receptivity, the Sterile Insect Technique may be improved by releasing well nourished, older sterile males.     

Remating inhibition in female Queensland fruit flies: effects and correlates of sperm storage

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.     

Male irradiation affects female remating behavior in Anastrepha serpentina (Diptera: Tephritidae)

Female remating in target pest species can affect the efficacy of control methods such as the Sterile Insect Technique (SIT) but very little is known about the postcopulatory mating behavior of these pests. In this study, we investigated the remating behavior of female Anastrepha serpentina (Diptera: Tephritidae), an oligophagous pest of Sapotaceae. First, we tested how long the sexual refractory period of females lasted after an initial mating. Second, we tested the effect of male and female sterility, female ovipositing opportunities and male density on female propensity to remate. Lastly, we tested if the amount of sperm stored by females was correlated to the likelihood of females to remate. We found that receptivity of mass-reared A. serpentina females had a bimodal response, with up to 16% of mass-reared A. serpentina females remating five days after the initial copulation, decreasing to 2% at 10 and 15 days and increasing to 13% after 20 days. Compared to fertile males, sterile males were less likely to mate and less likely to inhibit females from remating. Copula duration of sterile males was shorter compared to fertile males. Remating females were less likely to mate with a sterile male as a second mate. Sterile females were less likely to mate or remate compared to fertile females. Opportunity to oviposit and male density had no effect on female remating probability. Sperm numbers were not correlated with female likelihood to remate. Information on the post-copulatory behavior of mass-reared A. serpentina will aid fruit fly managers in improving the quality of sterile males. We discuss our results in terms of the differences this species presents in female remating behavior compared to other tephritids.     

Remating inhibition in the melon fly,Bactrocera (=Dacus) cucurbitae (Diptera: Tephritidae): Copulation with spermless males inhibits female remating

Eight hour copulation of the melon fly,Bactrocera cucurbitae, which usually mates at dusk and finishes copulation at dawn, inhibited female remating, while 3 h copulation did not. Copulation of females with either normal or virgin sterile males inhibited female remating. Sperm-depleted sterile males inhibited female remating at the same rate as normal males when the copulation duration was 8 h, indicating that existence or amount of sperm in females' spermathecae is not important in remating inhibition. Females of a wild strain remated later than females of a mass-reared strain, irrespective of strains of 1st and 2nd males. This suggests that the females may control their own remating, or that there is a difference between wild and mass-reared strains in their sensitivity to a male substance that inhibits females' receptivity.     

Female Age Determines Remating Behavior in Wild Mexican Fruit Flies

Male and female age are important factors that can influence mating and remating behavior. Females can discriminate against or prefer older males, but there have been relatively fewer studies on how female and male age influence female remating. Here we showed in wild flies of the Mexican fruit fly Anastrepha ludens (Loew), that when females were given a choice between males of different ages, younger females preferred to mate with younger males over older males, while older females were less selective. Also, when given a choice between males of different ages, older females had longer copulation durations than younger females. On the other hand, older males and females had lower mating success, compared with young and middle-aged flies under no choice conditions. However, middle-aged females mated faster compared to young females and young males mated faster compared to middle-aged males. Male age did not influence female remating, while female age strongly determined female remating, with no females remating when they were old. It is unclear if female receptivity mechanisms are switched off at older ages, or if females are reluctant to remate due to possible costs of mating. We discuss our results in terms of how male and female age can influence mating decisions.     

Strategic ejaculation and level of polyandry in <Emphasis Type="Italic">Callosobruchus chinensis</Emphasis> (Coleoptera: Bruchidae)

Variation in the level of polyandry of females produces a difference in the risk of sperm competition among males. As a consequence, investment in ejaculate expenditure by males should vary. We compared the number of sperm ejaculated by males into the female reproductive organ of six strains of the adzuki bean beetle, Callosobruchus chinensis (Coleoptera: Bruchidae), when males were reared at different larval densities in a bean. A significant positive correlation was found between the remating frequency of females and the ratio of the ejaculate sizes of high-density and low-density males as a measure of the response to the risk of sperm competition among males. The measure was estimated by dividing the number of sperm ejaculated by males reared at high larval density in a bean with the number of sperm ejaculated by males reared alone. The number of sperm transferred by a male to a female was not correlated with the duration of copulation. The results suggest an evolutionary relationship between ejaculatory expenditure and the level of polyandry in C. chinensis.     

Sperm competition promotes the exploitation of rival ejaculates

In many polyandrous species, the second male to mate with a female has fertilization priority. Strategic mechanisms generating this pattern tend to involve facultative increases in ejaculate size by informed males. Here we suggest a novel mechanism to generate second male precedence in internal fertilizers, based on the established fact that the female site of insemination is hostile to sperm, and that non-sperm components of the ejaculate frequently act to ameliorate these hostile conditions. We suggest that in species where female remating is frequent and rapid, second male sperm precedence may be due to the prior buffering of the female tract by previous males' ejaculates. In this scenario, second male costs are lower due to reduced sperm mortality and/or the redundancy of seminal fluid. Contrary to many classical predictions, second males may gain a paternity advantage despite investing less in an ejaculate.     

A mating plug protein reduces early female remating in Drosophila melanogaster

Mating plugs are formed within the female reproductive tract during mating from male ejaculate constituents or even from male genitalia themselves. Across species, mating plugs have roles in sperm storage and the prevention of female remating. In the fruitfly Drosophila melanogaster, accessory gland proteins such as the sex peptide are known to reduce female remating, however this effect can take some time to establish, hence other ejaculate components must also be involved. We hypothesised a role for the PEBII mating plug protein in the prevention of early female remating. Using RNA interference we produced PEBII knockdown males. We found that these males were significantly less able to prevent female remating in the 4 h following mating. The mating plugs produced by PEBII knockdown males also showed lower levels of autofluorescence in the first 10 min after the start of mating, suggesting they differed in composition to those of control males. Reduced levels of PEBII had no effect, however, on fecundity, progeny production or egg-adult viability in the first 24 after mating, suggesting there were no short-term effects of PEB II on sperm transfer, storage or use. Our results show that PEBII has a subtle but significant role in the prevention of early female remating.     

Mating-induced inhibition of remating in female Mediterranean fruit flies Ceratitis capitata

Mechanisms producing inhibition of remating in mated female Mediterranean fruit flies Ceratitis capitata, were investigated by matings with surgically altered males. Comparison of remating by females mated with either intact control males or males with a shortened penis, showed that ejaculate or a physical stimulus of penis insertion caused remating inhibition for at least 10 days after first mating. Remating frequency at two days after mating was significantly higher in females mated to castrated (spermless) males than in females mated to sham-operated control males. This difference disappeared by day four after mating, indicating that sperm cause a shorter-term inhibition of remating than does a normal first mating. Other factors in addition to sperm must therefore play a role in inhibition of remating.     

Methyl eugenol effects on Bactrocera dorsalis male total body protein,reproductive organs and ejaculate

Methyl eugenol (ME) and inclusion of protein into the adult diet increase the mating competitiveness of the Oriental fruit fly, Bactrocera dorsalis (Hendel). Exposing males to ME or protein is a promising post‐teneral treatment for males being released in the sterile insect technique (SIT). However, the effect of this post‐teneral treatment on male reproductive organs or the male ejaculate is unknown. During mating, males transfer sperm and accessory gland products (AGPs) to females and these compounds are reported to modulate female sexual inhibition. We studied the impact of male exposure to ME and a yeast hydrolysate (YH) diet on the protein reserves of males, male reproductive organ size, and the male ejaculate through sperm and AGPs. We show that males exposed to ME regardless of access to YH accumulated a greater amount of whole body protein. Males fed on YH also had increased total body protein and had bigger reproductive organs than YH‐deprived males, but no apparent effect of ME exposure was observed on reproductive organ size. Females stored less sperm when mated with males fed on YH and ME compared to males not fed on ME. YH and ME had no effect on male AGPs. Females injected with AGPs of males fed on YH and exposed to ME were just as likely to mate as females injected with AGPs of non‐treated males. However, females injected with AGPs of males exposed to ME mated faster than females injected with AGPs of non‐exposed males. We conclude that while exposure to ME increases male copulatory success and protein reserves in the male body, there seem to be some potential trade‐offs such as lower sperm stored by females. We discuss our results in terms of pre‐release protocols that may be used for B. dorsalis in SIT application.     

Sperm storage and copulation duration in a sexually cannibalistic spider

Female St Andrew’s Cross spiders control copulation duration by timing sexual cannibalism and may thereby control paternity if cannibalism affects sperm transfer. We have investigated the effect of copulation duration on sperm transfer and documented sperm storage patterns when we experimentally reduced the ability of females to attack and cannibalise the male. Virgin males and females were paired and randomly allocated either to a control treatment, where females were allowed to attack and cannibalise the male during copulation, or to an experimental treatment, where females were unable to cannibalise the male. The latter was achieved by placing a paintbrush against her chelicerae during copulation. Our experimental manipulation did not affect copulation duration or sperm storage. However, the number of sperm stored by the female increased with copulation duration only if the male was cannibalised, suggesting that cannibalism increases relative paternity not only through prolonged copulation duration following a fair raffle model but also through the cannibalism act itself. Future studies should explore whether cannibalised males ejaculate more sperm or whether females selectively store the sperm of cannibalised males.     

Copulation Song in Drosophila: Do Females Sing to Change Male Ejaculate Allocation and Incite Postcopulatory Mate Choice?

Drosophila males sing a courtship song to achieve copulations with females. Females were recently found to sing a distinct song during copulation, which depends on male seminal fluid transfer and delays female remating. Here, it is hypothesized that female copulation song is a signal directed at the copulating male and changes ejaculate allocation. This may alter female remating and sperm usage, and thereby affect postcopulatory mate choice. Mechanisms of how female copulation song is elicited, how males respond to copulation song, and how remating is modulated, are considered. The potential adaptive value of female signaling during copulation is discussed with reference to vertebrate copulation calls and their proposed function in eliciting mate guarding. Female copulation song may be widespread within the Drosophila genus. This newly discovered behavior opens many interesting avenues for future research, including investigation of how sexually dimorphic neuronal circuits mediate communication between nervous system and reproductive organs.     

Copula duration and sperm storage in Mediterranean fruit flies from a wild population

In the Mediterranean fruit fly (Ceratitis capitata Weidemann, ‘medfly’), a lekking tephritid, evidence from laboratory studies of flies from laboratory strains suggests that copulation is shorter, and sperm storage more abundant, if males are large or protein‐fed, and that copulation is longer when females are large. In addition, sperm tend to be stored asymmetrically between the female’s two spermathecae and this asymmetry declines with abundance of stored sperm. The primary objective of this study was to investigate whether these trends persist in other experimental contexts that bear closer resemblance to nature. Accordingly, we carried out experiments in a field‐cage using males derived as adults from a wild population and virgin females reared from naturally infested fruit. The results of this study were consistent with laboratory studies in that copula duration increased with female size, that sperm were stored asymmetrically between the females’ spermathecae, and that this asymmetry declined with number of sperm stored. However, we also found some previously unreported effects of female size; large females stored more sperm and stored sperm more asymmetrically between their two spermathecae than did small females. Unlike the laboratory studies, copula duration and sperm storage patterns were unaffected by male size and diet. This may be due to overwhelming variation from other sources in the wild‐collected males used, as well as environmental variability in the semi‐natural setting.     

Strategic ejaculation in the common dung fly Sepsis cynipsea

Sperm competition has been a major selective force acting on male and female behaviour. Theory predicts that when sperm compete numerically, selection will favour males that vary the number of sperm they transfer with sperm competition risk. This often leads to increased copula duration when sperm competition risk is high, the selective advantage of which is increased paternity. We investigated the copulatory behaviour of the common dung fly Sepsis cynipsea in relation to male and female size, female mating status, age, and presence or absence of dung. This fly is unusual in that males mate-guard before copula while females use the sperm of previous males for their current clutch. Body size had no effect on copula duration, but duration of first copulations depended on female age, with older females having longer copulations. For females that copulated twice, there was an interaction between female age and mating status influencing copula duration: old females had longer copulations than young females, but second copulas were longer for young females. Residual testis size of nonvirgin males was smaller than for virgins, and testis shrinkage was significantly associated with copula duration, which indicates that males transfer more ejaculate with longer copulations. We therefore conclude that copulation duration and ejaculate transfer vary in accordance with sperm competition theory.     

Evolutionary Consequences of Desiccation Resistance in the Male Ejaculate

Avoiding water loss for insects is critical for survival. Selection for reduced water loss will depend on trade-offs between resources allocated for reproduction and those allocated for resisting desiccation. However, we lack knowledge on how selection for desiccation resistance can affect the male ejaculate. Furthermore, as male ejaculate composition is complex, desiccation resistant females could evolve traits that enable them to derive longevity benefits from mating. Here, we assessed how selection for desiccation resistance impacts male testes and accessory gland size, protein content of these organs, female sperm storage and male ability to inhibit female remating behavior, in the Mexican fruit fly Anastrepha ludens. Additionally, we tested if mating increased longevity and fecundity in desiccation resistant females. Males selected for resistance to desiccation stress had smaller accessory glands and seminal vesicles and females mating with these males stored less sperm compared to control males. Females mating with resistant males had lower fecundity compared to females mating with control males. Desiccation resistant females lived longer than control females, yet this was irrespective of mating. Rapid evolutionary responses to hydric stress can have correlated effects in reproductive capabilities, which are not restricted to pre-copulatory traits. Trade-offs between resistance to desiccation stress are reflected in decreased allocation of resources to reproductive organs. Thus, production of the ejaculate may be costly for A. ludens males. Knowledge on the evolution of ejaculate traits and reproductive organ size in response to directional selection for desiccation resistance, will aid our understanding of differential sex-specific responses to environmental stress.     

The role of sperm numbers in sperm competition and female remating in Drosophila melanogaster

Single mating productivities (used as estimates of the relative number of sperm transferred) are highly correlated with several parameters used to quantify sperm competition in D. melanogster. Matings that result in the transferal of large numbers of sperm are associated with longer delay of female remating than are matings that transfer fewer sperm. Males that transfer larger numbers of sperm also suffer a smaller proportional reduction in reproductive success (smaller COST) than males transferring fewer sperm. The number of sperm transferred by a female's second mate is not related to the COST to the first male. However, there is a high positive correlation between the number of sperm transferred by the second male and P₂ (the proportion of second male progeny following female remating). Thus, large sperm numbers apparently increase the reproductive success of males whether they mate with virgin or non-virgin females. Because female receptivity mediates these events, there is no need to invoke sperm displacement to explain the reproductive outcome of female remating.The timing of female remating is evaluated in terms of a receptivity-threshold model. This model suggests that female receptivity returns when some small, relatively constant, number of sperm remain in storage.     

Variation in the number of sperm transferred during mating among males of the Colorado potato beetle (Coleoptera: Chrysomelidae)

We suggest an explanation for the bimodal distribution in the number of sperm transferred into the female during mating by males of the Colorado potato beetle (Leptinotarsa decemlineata). While in most matings females receive 20,000 sperm, in others they receive less than 1000. A variety of potential proximate causes for this bimodal distribution including experimental procedures and several physiological and behavioral causes were examined: male body size, female body size, male mating history, socio-sexual environments, duration and number of mounts, and duration and number of copulations. Surprisingly, none of these explained the observed distribution. However, we did find that the total time spent in copulation was significantly less variable among males that transferred more than 20,000 sperm than among males that passed less than 1000 sperm. In addition, males transferring large amounts of sperm resulted in proportionally more sperm inside the female’s spermatheca within a short period of time than males passing less than 1000 sperm. The lack of pattern in male sperm ejaculate suggests definite differences in sperm delivery tactics and may be related to the female condition rather than the male.