Sex peptide causes mating costs in female Drosophila melanogaster

Conflicts between females and males over reproductive decisions are common . In Drosophila, as in many other organisms, there is often a conflict over how often to mate. The mating frequency that maximizes male reproductive success is higher than that which maximizes female reproductive success . In addition, frequent mating reduces female lifespan and reproductive success , a cost that is mediated by male ejaculate accessory gland proteins (Acps) . We demonstrate here that a single Acp, the sex peptide (SP or Acp70A), which decreases female receptivity and stimulates egg production in the first matings of virgin females , is a major contributor to Acp-mediated mating costs in females. Females continuously exposed to SP-deficient males (which produce no detectable SP ) had significantly higher fitness and higher lifetime reproductive success than control females. Hence, rather than benefiting both sexes, receipt of SP decreases female fitness, making SP the first identified gene that is likely to play a central role in sexual conflict.     

Why Do Female Callosobruchus maculatus Kick Their Mates?

Sexual conflict is now recognised as an important driver of sexual trait evolution. However, due to their variable outcomes and effects on other fitness components, the detection of sexual conflicts on individual traits can be complicated. This difficulty is exemplified in the beetle Callosobruchus maculatus, where longer matings increase the size of nutritious ejaculates but simultaneously reduce female future receptivity. While previous studies show that females gain direct benefits from extended mating duration, females show conspicuous copulatory kicking behaviour, apparently to dislodge mating males prematurely. We explore the potential for sexual conflict by comparing several fitness components and remating propensity in pairs of full sibling females where each female mated with a male from an unrelated pair of full sibling males. For one female, matings were terminated at the onset of kicking, whereas the other’s matings remained uninterrupted. While fecundity (number of eggs) was similar between treatments, uninterrupted matings enhanced adult offspring numbers and fractionally also longevity. However, females whose matings were interrupted at the onset of kicking exhibited an increased propensity to remate. Since polyandry can benefit female fitness in this species, we argue that kicking, rather than being maladaptive, may indicate that females prefer remating over increased ejaculate size. It may thus be difficult to assess the presence of sexual conflict over contested traits such as mating duration when females face a trade off between direct benefits gained from one mating and indirect benefits from additional matings.     

Age-dependent female responses to a male ejaculate signal alter demographic opportunities for selection

A central tenet of evolutionary explanations for ageing is that the strength of selection wanes with age. However, data on age-specific expression and benefits of sexually selected traits are lacking—particularly for traits subject to sexual conflict. We addressed this by using as a model the responses of Drosophila melanogaster females of different ages to receipt of sex peptide (SP), a seminal fluid protein transferred with sperm during mating. SP can mediate sexual conflict, benefitting males while causing fitness costs in females. Virgin and mated females of all ages showed significantly reduced receptivity in response to SP. However, only young virgin females also showed increased egg laying; hence, there was a narrow demographic window of maximal responses to SP. Males gained significant ‘per mating’ fitness benefits only when mating with young females. The pattern completely reversed in matings with older females, where SP transfer was costly. The overall benefits of SP transfer (hence opportunity for selection) therefore reversed with female age. The data reveal a new example of demographic variation in the strength of selection, with convergence and conflicts of interest between males and ageing females occurring over different facets of responses to a sexually antagonistic trait.     

No Effect of Male Courtship Intensity on Female Remating in the Butterfly <Emphasis Type="Italic">Pieris napi</Emphasis>

In Pieris napi, female fitness increases with number of matings, but wild females mate at an unexpectedly low rate. From a sexual conflict perspective this could be because males manipulate female remating, or alternatively, because wild females experience costs associated with remating which are not applicable under laboratory conditions. To get an indication which sex controls remating and/or the different sexes’ relative costs and benefits of remating, we here test whether female mating frequency is affected by male courtship intensity. We found no effect on female mating frequency or lifespan. This indicates that (i) females control remating and their optimal mating frequency is lower compared to males, or (ii) males can manipulate female remating. We argue that both these alternatives may apply simultaneously to P. napiand that they are inseparable.     

Nuptial gift consumption influences female remating in a scorpionfly: male or female control of mating rate?

In the scorpionfly Panorpa cognata, males provide females with saliva secretions as nuptial food gifts. Consequently, females derive material benefits and possibly also genetic benefits from multiple matings. Females therefore generally should have a high motivation to remate. Males, on the other hand, do not share this interest, which will generate a sexual conflict over remating interval, possibly leading to male adaptations that prevent females from remating with other males. In this study, I found that mated females were less prone to copulate than virgin females, despite female benefits of multiple matings. Further, I found that the remating interval was significantly longer if the first copulation was long compared to shorter matings. This effect does not entirely depend on copulation duration per se, but on the amount of saliva, that a female is consuming during copulation. These results suggest a mating-induced refractory period and can be interpreted as male manipulation of female remating behaviour mediated through substances in the nuptial gift. Alternatively, receiving large nuptial gifts may decrease the prospective direct fitness benefits from further copulations, and thus change optimal female remating rate. Furthermore, gift size has been shown to correlate with male nutritional condition, which may be an indicator of male genetic quality. Females may therefore benefit indirectly by not remating following copulations involving large saliva gifts. In this scenario, female remating interval would be an effect of cryptic female choice.     

Impaired sperm quality,delayed mating but no costs for offspring fitness in crickets winning a fight

The outcome of male–male contest competition is known to affect male mating success and is believed to confer fitness benefits to females through preference for dominant males. However, by mating with contest winners, females can incur significant costs spanning from decreased fecundity to negative effects on offspring. Hence, identifying costs and benefits of male dominance on female fitness is crucial to unravel the potential for a conflict of interests between the sexes. Here, we investigated males' pre‐ and post‐copulatory reproductive investment and its effect on female fitness after a single contest a using the field cricket Gryllus bimaculatus. We allowed males to fight and immediately measured their mating behaviour, sperm quality and offspring viability. We found that males experiencing a fight, independently of the outcome, delayed matings, but their courtship effort was not affected. However, winners produced sperm of lower quality (viability) compared to losers and to males that did not experience fighting. Results suggest a trade‐off in resource allocation between pre‐ and post‐mating episodes of sexual selection. Despite lower ejaculate quality, we found no fitness costs (fecundity and viability of offspring) for females mated to winners. Overall, our findings highlight the importance of considering fighting ability when assessing male reproductive success, as winners may be impaired in their competitiveness at a post‐mating level.     

Receptive females mitigate costs of sexual conflict

Males typically gain fitness from multiple mating, whereas females often lose fitness from numerous mating, potentially leading to sexual conflict over mating. This conflict is expected to favour the evolution of female resistance to mating. However, females may incur male harassment if they refuse to copulate; thus, greater female resistance may increase costs imposed by males. Here, I show that the evolution of resistance to mating raises fitness disadvantages of interacting with males when mating is harmful in female adzuki bean beetles, Callosobruchus chinensis. Females that were artificially selected for higher and lower remating propensity evolved to accept and resist remating, respectively. Compared with females that evolved to accept remating, females that evolved to resist it suffered higher fitness costs from continuous exposure to males. The costs of a single mating measured by the effect on longevity did not differ among selection line females. This study indicates that receptive rather than resistant females mitigate the fitness loss resulting from sexual conflict, suggesting that even though mating is harmful, females can evolve to accept additional mating.     

Male‐induced costs of mating for females compensated by offspring viability benefits in an insect

Sexual conflict facilitates the evolution of traits that increase the reproductive success of males at the expense of components of female fitness. Theory suggests that indirect benefits are unlikely to offset the direct costs to females from antagonistic male adaptations, but empirical studies examining the net fitness pay‐offs of the interaction between the sexes are scarce. Here, we investigate whether matings with males that invest intrinsically more into accessory gland tissue undermine female lifetime reproductive success (LRS) in the cricket Teleogryllus oceanicus. We found that females incur a longevity cost of mating that is proportional to the partner’s absolute investment into the production of accessory gland products. However, male accessory gland weight positively influences embryo survival, and harmful ejaculate‐induced effects are cancelled out when these are put in the context of female LRS. The direct costs of mating with males that sire offspring with higher viability are thus compensated by direct and possibly indirect genetic benefits in this species.     

Remating, sperm transfer, and sperm displacement in the cactophilic species Drosophila buzzatii Patterson & Wheeler (Diptera: Drosophilidae)

The mating system of Drosophila buzzatii is characterized by short copulation duration, frequent remating in both males and females, and male ejaculate partitioning. Additional features of the system are strong sperm displacement and a high frequency of sterile matings. Remating frequencies and the effects of remating on various mating parameters were studied. In order to characterize variation, five isofemale lines from geographically distant localities in Australia (three localities), Brazil and the Canary Islands were used. Mating parameters studied were: premating time, copulation duration, interval between successive matings, and progeny number as a measure of sperm transfer. Variation for sperm displacement was studied in crosses between laboratory stocks and a number of isofemale lines from Australia. There were significant between‐line differences in female remating frequencies, premating time, copulation duration, interval between successive matings, and progeny numbers, indicating genetic variation for these traits. Females from the five lines mated on average 1.6 to 3.1 times in 4 h, with a maximum of eight matings for one female. The males were given a maximum of ten virgin females in sequence and more than one‐third of the males mated all ten females in the 2 h observation period. Copulation duration decreased and interval between matings increased with copulation number in multiply mated males. Mean copulation duration was c. 2 min. Sperm transfer, measured as the average number of progeny from a single mating, was low (c. 25) and multiply mated females gave more progeny than single mated females, although with much lower progeny numbers than observed in wild‐caught non‐virgin females. A surprisingly high proportion of observed matings gave no progeny, i.e. they were sterile matings. Sperm displacement was strong in most crosses and remained strong in multiply mated females. The results are discussed in relation to the evolution of mating patterns in Drosophila.     

Female wolf spiders exert cryptic control drastically reducing ejaculate size

Sexes' roles in post‐copulatory processes have important effects on individual fitness and are promising to study in species showing complex mating behaviours. In the spider Schizocosa malitiosa, males perform two different copulatory patterns, pattern 1 includes 80% of total pedipalp insertions and pattern 2 includes 20%. Both patterns produce similar number of offspring, but pattern 1 induces higher female reluctance to remating than pattern 2. We hypothesised that the complex copulatory patterns are linked to post‐copulatory sexual selection, affecting males' sperm transfer and the resulting sperm storage by females. First, we examined amounts of sperm in males and live females from uninterrupted (pattern 1 + 2) and interrupted matings (pattern 1, pattern 2). Second, in order to disentangle male and female actions, we induced males to mate with dead females and examined amounts of sperm. Males transfer in total 71% of the sperm available in their pedipalps, being higher but not significant in pattern 1 than in pattern 2. Females drastically reduced the amount of sperm stored in their spermathecae and such control is stronger in pattern 1 compared to pattern 2 matings. We propose that cryptic female control is a main factor driving males to strengthen sperm transfer. Active female reduction in ejaculate most probably diminished her reluctance to remate.     

Fitness effects of female mate choice: preferred males are detrimental for Drosophila melanogaster females

The evolution of female mate choice, broadly defined to include any female behaviour or morphology which biases matings towards certain male phenotypes, is traditionally thought to result from direct or indirect benefits which females acquire when mating with preferred males. In contrast, new models have shown that female mate choice can be generated by sexual conflict, where preferred males may cause a fitness depression in females. Several studies have shown that female Drosophila melanogaster bias matings towards large males. Here, we use male size as a proxy for male attractiveness and test how female fitness is affected by reproducing with large or small males, under two different male densities. Females housed with large males had reduced lifespan and aged at an accelerated rate compared with females housed with small males, and increased male density depressed female fitness further. These fitness differences were due to effects on several different fitness components. Female fitness covaried negatively with male courtship rate, which suggests a cost of courtship. Mating rate increased with male size, whereas female fitness peaked at an intermediate mating rate. Our results suggest that female mate choice in D. melanogaster is, at least in part, a by-product of sexual conflict over the mating rate.     

Ejaculate size, second male size, and moderate polyandry increase female fecundity in a seed beetle

Several hypotheses have been proposed to explain the evolutionof polyandry in species that provide nuptial gifts. When nuptialgifts are in the form of nutritional elements in the ejaculateand ejaculate size is correlated with male body size, femalescan accrue both direct (nutritional) and indirect (genetic)benefits from multiple mating. We examined remating decisionsin females of the seed beetle Stator limbatus and, using pathanalysis, examined the effects of male body size on the sizeof his ejaculate, the amount of ejaculate that was successfullytransferred to females, and the overall effect of these variableson female fecundity. Larger males produced larger ejaculatesand consequently transferred a larger ejaculate to females,but the effects on female fecundity differed between the females'first and second mates. Both larger first and second males wereable to transfer more of their ejaculate to females than weresmaller males. Both the total amount of ejaculate transferredby these males and polyandry (number of matings) were positivelycorrelated to female fecundity independently of each other.However, larger second males were more successful at stimulatingfemale fecundity independently of how much ejaculate they transferred.We also provide evidence that females are choosy during theirsecond mating opportunity. Both female choosiness and higherfemale investment after mating with larger second males suggestthat females may benefit from both direct and indirect effectsfrom multiple mating. We also conclude that male body size isunder both directional fecundity selection and directional sexualselection.     

The consequences of genetic variation in sex peptide expression levels for egg laying and retention in females

The accessory gland proteins (Acps) that male Drosophila melanogaster produce and transfer to females during copulation are key to male and female fitness. One Acp, the sex peptide (SP), is largely responsible for a dramatic increase in female egg laying and decrease in female receptivity after copulation. While genetic variation in male SP expression levels correlate with refractory period duration in females, it is unknown whether male SP expression influences female egg laying or if any effect of SP is mediated by SP retention in the female reproductive tract. Here we measured the amount of SP retained in the female reproductive tract after mating and female egg laying after copulating with virgin males. We found no correlation between male SP expression levels and egg laying, or the amount of SP in the female reproductive tract after mating. Additionally, the amount of SP retained in the female did not influence egg laying. These finding suggests that additional factors, such as variation in other Acps, are important for the retention of SP in females and its quantitative effects on egg laying. It also shows that egg laying and refractory period response to SP is at least partially uncoupled.     

Cross-generational fitness benefits of mating and male seminal fluid

In many species, the physical act of mating and exposure to accessory gland proteins (Acps) in male seminal fluid reduces female survival and offspring production. It is not clear what males gain from harming their sexual partners or why females mate frequently despite being harmed. Using sterile strains of Drosophila melanogaster that differ in their production of Acps, we found that both the physical act of mating and exposure to male seminal fluid in mothers increase the fitness of daughters. We show that the changes in daughter fitness are mediated by parental effects, not by sexual selection involving good genes or owing to variation in maternal egg production. These results support the idea that male harm of females might partly evolve through cross-generational fitness benefits.     

The Effects of Experimentally Induced Polyandry on Female Reproduction in a Monandrous Mating System

Females of most insect species maximize their fitness by mating more than once. Yet, some taxa are monandrous and there are two distinct scenarios for the maintenance of monandry. While males should always benefit from inducing permanent non‐receptivity to further mating in their mate, this is not necessarily true for females. Since females benefit from remating in many species, cases of monandry may reflect successful male manipulation of female remating (i.e. sexual conflict). Alternatively, monandry may favor both mates, if females maximize their fitness by mating only once in their life. These two hypotheses for the maintenance of monandry make contrasting predictions with regards to the effects of remating on female fitness. Here, we present an experimental test of the above hypotheses, using the monandrous housefly (Musca domestica) as a model system. Our results showed that accessory seminal fluid substances that males transfer to females during copulation have a dual effect: they trigger female non‐receptivity but also seem to have a nutritional effect that could potentially enhance female fitness. These results suggest that monandry is maintained in house flies despite potential benefits that females would gain by mating multiply.     

Ability of male Queensland fruit flies to inhibit receptivity in multiple mates, and the associated recovery of accessory glands

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 11 h 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.     

Remating responses are consistent with male postcopulatory manipulation but not reinforcement in D. pseudoobscura

Reinforcement occurs when hybridization between closely related lineages produces low‐fitness offspring, prompting selection for elevated reproductive isolation specifically in areas of sympatry. Both premating and postmating prezygotic behaviors have been shown to be the target of reinforcing selection, but it remains unclear whether remating behaviors experience reinforcement, although they can also influence offspring identity and limit formation of hybrids. Here, we evaluated evidence for reinforcing selection on remating behaviors in Drosophila pseudoobscura, by comparing remating traits in females from populations historically allopatric and sympatric with Drosophila persimilis. We found that the propensity to remate was not higher in sympatric females, compared to allopatric females, regardless of whether the first mated male was heterospecific or conspecific. Moreover, remating behavior did not contribute to interspecific reproductive isolation among any population; that is, females showed no higher propensity to remate following a heterospecific first mating than following a conspecific first mating. Instead, we found that females are less likely to remate after initial matings with unfamiliar males, regardless of species identity. This is consistent with one scenario of postmating sexual conflict in which females are poorly defended against postcopulatory manipulation by males with whom they have not coevolved. Our results are generally inconsistent with reinforcement on remating traits and suggest that this behavior might be more strongly shaped by the consequences of local antagonistic male–female interactions than interactions with heterospecifics.     

Female Remating Behavior in a Lekking Moth

In polyandrous species, male reproductive success will at least partly be determined by males' success in sperm competition. To understand the potential for post‐mating sexual selection, it is therefore important to assess the extent of female remating. In the lekking moth Achroia grisella, male mating success is strongly determined by female choice based on the attractiveness of male ultrasonic songs. Although observations have indicated that some females will remate, only little is known about the level of sperm competition. In many species, females are more likely to remate if their first mating involved an already mated male than if the first male was virgin. Potentially, this is because mated males are less well able to provide an adequate sperm supply, nutrients, or substances inhibiting female remating. This phenomenon will effectively reduce the strength of pre‐copulatory sexual selection because attractive males with high mating success will be more susceptible to sperm competition. We therefore performed an experiment designed both to provide a more precise estimate of female remating probability and simultaneously to test the hypothesis that female remating is influenced by male mating history. Overall, approximately one of five females remated with a second male. Yet, although females mated to non‐virgin males were somewhat more prone to remate, the effect of male mating history was not significant. The results revealed, however, that heavier females were more likely to remate. Furthermore, we found that females' second copulations were longer, suggesting that, in accordance with theory, males may invest more sperm in situations with an elevated risk of sperm competition.     

Selection on female remating interval is influenced by male sperm competition strategies and ejaculate characteristics

Female remating rate dictates the level of sperm competition in a population, and extensive research has focused on how sperm competition generates selection on male ejaculate allocation. Yet the way ejaculate allocation strategies in turn generate selection on female remating rates, which ultimately influence levels of sperm competition, has received much less consideration despite increasing evidence that both mating itself and ejaculate traits affect multiple components of female fitness. Here, we develop theory to examine how the effects of mating on female fertility, fecundity and mortality interact to generate selection on female remating rate. When males produce more fertile ejaculates, females are selected to mate less frequently, thus decreasing levels of sperm competition. This could in turn favour decreased male ejaculate allocation, which could subsequently lead to higher female remating. When remating simultaneously increases female fecundity and mortality, females are selected to mate more frequently, thus exacerbating sperm competition and favouring male traits that convey a competitive advantage even when harmful to female survival. While intuitive when considered separately, these predictions demonstrate the potential for complex coevolutionary dynamics between male ejaculate expenditure and female remating rate, and the correlated evolution of multiple male and female reproductive traits affecting mating, fertility and fecundity.     

Comparative evidence for a cost to males of manipulating females in bushcrickets

Recent theoretical and empirical research on sexual conflicthas tended to focus on the costs to females of being manipulatedby males. The costs to males associated with the productionof manipulative traits have received relatively little attention.In numerous insects, including bushcrickets (Orthoptera: Tettigoniidae),males are known to transfer substances in the ejaculate thatinhibit the receptivity of females to further matings in a dose-dependentmanner. The aim of this study was to test the prediction that,across bushcricket taxa, larger ejaculates and nuptial giftswill be associated with, on the one hand, longer sexual refractoryperiods in females and, on the other hand, longer sexual refractoryperiods in males. Data on the duration of the sexual refractoryperiod in both males and females, together with ejaculate mass,spermatophylax mass, and male body mass, were obtained for 23species of bushcricket. Both comparative analysis by independentcontrasts and species regression revealed a positive relationship,across taxa, between the duration of the female's sexual refractoryperiod and both relative ejaculate mass and relative nuptialgift mass. Positive relationships were also found between theduration of the male's sexual refractory period and both relativeejaculate mass and relative nuptial gift mass, indicating thatthere is a trade-off between resources spent on spermatophoresize and the male's potential mating rate. This appears to bethe first comparative evidence that there is a cost to malesassociated with manipulating the remating behavior of theirmates.