Male mating rate is constrained by seminal fluid availability in bedbugs, Cimex lectularius

Sexual selection, differences in reproductive success between individuals, continues beyond acquiring a mating partner and affects ejaculate size and composition (sperm competition). Sperm and seminal fluid have very different roles in sperm competition but both components encompass production costs for the male. Theoretical models predict that males should spend ejaculate components prudently and differently for sperm and seminal fluid but empirical evidence for independent variation of sperm number and seminal fluid volume is scarce. It is also largely unknown how sperm and seminal fluid variation affect future mating rate. In bedbugs we developed a protocol to examine the role of seminal fluids in ejaculate allocation and its effect on future male mating rate. Using age-related changes in sperm and seminal fluid volume we estimated the lowest capacity at which mating activity started. We then showed that sexually active males allocate 12% of their sperm and 19% of their seminal fluid volume per mating and predicted that males would be depleted of seminal fluid but not of sperm. We tested (and confirmed) this prediction empirically. Finally, the slightly faster replenishment of seminal fluid compared to sperm did not outweigh the faster decrease during mating. Our results suggest that male mating rate can be constrained by the availability of seminal fluids. Our protocol might be applicable to a range of other organisms. We discuss the idea that economic considerations in sexual conflict research might benefit from distinguishing between costs and benefits that are ejaculate dose-dependent and those that are frequency-dependent on the mating rate per se.     

Proteins within the seminal fluid are crucial to keep sperm viable in the honeybee Apis mellifera

Seminal fluid is a biochemically complex mixture of glandular secretions that is transferred to the females sexual tract as part of the ejaculate. Seminal fluid has received increasing scientific interest in the fields of evolutionary and reproductive biology, as it seems a major determinant of male fertility/infertility and reproductive success. Here we used the honeybee Apis mellifera, where seminal fluid can be collected as part of a male's ejaculate, and performed a series of experiments to investigate the effects of seminal fluid and its components on sperm viability. We show that honeybee seminal fluid is highly potent in keeping sperm alive and this positive effect is present over a 24 h time span, comparable to the timing of the sperm storage process in the queen. We furthermore show that the presence of proteins within the seminal fluid and their structural integrity are crucial for this effect. Finally, we activated sperm using fructose and provide evidence that the positive effect of seminal fluid proteins on sperm survival cannot be replicated using generic protein substitutes. Our data provide experimental insights into the complex molecular interplay between sperm and seminal fluid defining male fertility and reproductive success.     

Can paternal effects via seminal fluid contribute to the evolution of polyandry?

Genetic benefits from mating with multiple males are thought to favour the evolution of polyandry. However, recent evidence suggests that non-genetic paternal effects via seminal fluid might contribute to the observed effects of polyandry on offspring performance. Here, we test this hypothesis using the field cricket Teleogryllus oceanicus. Using interference RNA, we first show that at least one seminal fluid protein is essential for embryo survival. We then show that polyandrous females mated to three different males produced embryos with higher pre-hatching viability than did monandrous females mated with the same male three times. Pseudo-polyandrous females that obtained sperm and seminal fluid from a single male and seminal fluid from two additional males had embryos with viabilities intermediate between monandrous and polyandrous females. Our results suggest either that ejaculate mediated paternal effects on embryo viability have both genetic and non-genetic components, or that seminal fluids transferred by castrated males provide only a subset of proteins contained within the normal ejaculate, and are unable to exert their full effect on embryo viability.     

The evolution of sperm and non-sperm producing organs in male Drosophila

The male ejaculate is made up of two components: sperm and non-sperm. There has been little consideration of how these two basic compartments evolve. If they are subject to trade-offs, theory predicts that when the sperm competition raffle is unfair, when seminal fluid proteins stimulate fecundity and/or when ejaculate components alter fertilization success, there will be differential selection on sperm versus non-sperm ejaculate characteristics. However, the fundamental assumption that there are trade-offs between sperm and non-sperm ejaculate compartments in Drosophila has not yet been tested. To address this, we examined testis (sperm producing) and accessory gland (non-sperm producing) size across 22 species of Drosophila . We also examined how these characters varied with copulation duration, which may represent an additional target for sperm competition. The results showed no evidence of a trade-off between testis length and accessory gland length. Copulation duration correlated negatively with accessory gland length and there was a positive correlation with testis length, but only after correcting for body size. Overall, the results suggest no evidence for gross trade-offs in sperm versus non-sperm compartments across these Drosophila species, and motivate more detailed examination of ejaculate investment patterns.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 505–512.     

The influence of male ejaculate quantity on female fitness: a meta‐analysis

Although the primary function of mating is gamete transfer, male ejaculates contain numerous other substances that are produced by accessory glands and transferred to females during mating. Studies with several model organisms have shown that these substances can exert diverse behavioural and physiological effects on females, including altered longevity and reproductive output, yet a comprehensive synthesis across taxa is lacking. Here we use a meta‐analytic approach to synthesize quantitatively extensive experimental work examining how male ejaculate quantity affects different components of female fitness. We summarize effect sizes for female fecundity (partial and lifetime) and longevity from 84 studies conducted on 70 arthropod species that yielded a total of 130 comparisons of female fecundity and 61 comparisons of female longevity. In response to greater amounts of ejaculate, arthropod females demonstrate enhanced fecundity (both partial and lifetime) but reduced longevity, particularly for Diptera and Lepidoptera. Across taxa, multiply mated females show particularly large fecundity increases compared to singly mated females, indicating that single matings do not maximize female fitness. This fecundity increase is balanced by a slight negative effect on lifespan, with females that received more ejaculate through polyandrous matings showing greater reductions in lifespan compared with females that have mated repeatedly with the same male. We found no significant effect size differences for either female fecundity or longevity between taxa that transfer sperm packaged into spermatophores compared to taxa that transfer ejaculates containing free sperm. Furthermore, females that received relatively larger or more spermatophores demonstrated greater lifetime fecundity, indicating that these seminal nuptial gifts provide females with a net fitness benefit. These results contribute to our understanding of the evolutionary origin and maintenance of non‐sperm ejaculate components, and provide insight into female mate choice and optimal mating patterns.     

Rat dorsal prostate is necessary for vaginal adhesion of the seminal plug and sperm motility in the uterine horns

The rat prostate comprises dorsal, ventral and lateral lobes that are morphologically and biochemically distinct. Lesions to these structures are expected to affect the quality of the ejaculate and male fertility. In experiment 1, we analyzed ejaculate parameters of males that had chemical lesions of the dorsal or ventral lobes. At pre-lesion and at 5 and 20 days post-lesion males were mated, and after ejaculation, seminal fluid and seminal plug were obtained from the mated females. In experiment 2, the ventral lobes were ablated, and the ejaculate was analyzed. In experiment 3, the fertility of males with chemically-lesioned dorsal lobes or ablation of the ventral lobes was evaluated. Chemical lesion of the dorsal lobe prevented the adhesion of the seminal plug to vaginal walls. When these males were tested at 5-days postlesion, no sperm were found in uterus, and at 20-days post-lesion, the few sperm encountered showed slow progressive motility. None of the females that mated with dorsal lobe-lesioned males became pregnant. However, chemical lesion or ablation of the ventral lobes did not affect ejaculate or fertility. Our results indicate that the dorsal prostatic lobes are indispensable for reproductive success in males, and define parameters of ejaculate with which fertility can be estimated.     

Condition-dependent ejaculate size and composition in a ladybird beetle

Sexually selected male ejaculate traits are expected to depend on the resource state of males. Theory predicts that males in good condition will produce larger ejaculates, but that ejaculate composition will depend on the relative production costs of ejaculate components and the risk of sperm competition experienced by low- and high-condition males. Under some conditions, when low condition leads to poorer performance in sperm competition, males in low condition may produce ejaculates with higher sperm content relative to their total ejaculate and may even transfer more sperm than high-condition males in an absolute sense. Previous studies in insects have shown that males in good condition transfer larger ejaculates or more sperm, but it has not been clear whether increased sperm content represents a shift in allocation or simply a larger ejaculate, and thus the condition dependence of ejaculate composition has been largely untested. We examined condition dependence in ejaculate by manipulating adult male condition in a ladybird beetle (Adalia bipunctata) in which males transfer three distinct ejaculate components during mating: sperm, non-sperm ejaculate retained within the female reproductive tract, and a spermatophore capsule that females eject and ingest following mating. We found that high condition males indeed transferred larger ejaculates, potentially achieved by an increased rate of ejaculate transfer, and allocated less to sperm compared with low-condition males. Low-condition males transferred ejaculates with absolutely and proportionally more sperm. This study provides the first experimental evidence for a condition-dependent shift in ejaculate composition.     

Copulation, the dynamics of sperm transfer and female refractoriness in the leafhopper Balclutha incisa (Hemiptera: Cicadellidae: Deltocephalinae)

Abstract.  Mature sperm of the leafhopper Balclutha incisa (Matsumara) (Cicadellidae: Auchenorrhyncha: Hemiptera) are stored as a series of sperm bundles within seminal vesicles prior to ejaculation. During transfer, sperm are pumped from the vesicles into the ejaculatory duct to the complex aedeagus. Sperm transfer is marked by a c . 30-fold expansion of the spermatheca to accommodate both sperm and seminal fluid. Sperm number increases exponentially with male age, reaching a maximum of 700 000 after 14 days, while the number of sperm available on days 2–5 is between 70 000 and 100 000. During mating, maximum sperm transfer occurs after 7 min and mating is complete after about 10 min. Ejaculate size, defined by both sperm and associated accessory gland fluid, is influenced by male mating status and the interval since the previous mating. There is a positive correlation between duration of copulation and both ejaculate and the time to subsequent mating. Sperm are more likely to be retained in the testes during mating by males of 2–5 days post-emergence than older males. The number of sperm received by the female can be manipulated experimentally by mating males once (medium ejaculate) or twice (small ejaculate) immediately after their first mating. Females that receive small ejaculates from sperm-depleted males have a far shorter refractory period than females receiving medium to large ejaculates. Both ejaculate size and the time after males have mated influence the female post-mating refractory period as measured by the female's responsiveness to male sexual signalling.     

Black goby territorial males adjust their ejaculate's characteristics in response to the presence of sneakers

In many species, males can rapidly adjust their ejaculate performance in response to changing levels of sperm competition, an ability that is probably mediated by seminal fluid adaptive plasticity. In the black goby, Gobius niger, territorial males attach viscous ejaculate trails to the nest roof, from which sperm are slowly released into the water during the long-lasting spawning events. Sneaker males release their sperm in the vicinity of the nest, and territorial males try to keep them at a distance by patrolling their territory. We show here that territorial males'' ejaculate trails released a higher proportion of their sperm in the presence of a single sneaker, but this proportion decreased when there were three sneakers, an effect that is most likely mediated by a change in the seminal fluid composition. Field observations showed that when multiple sneaking attempts occurred, territorial males spent more time outside the nest, suggesting that ejaculation rate and territory defence are traded-off. Altogether, these results suggest that the adjustment of sperm release from the ejaculate may be strategic, guaranteeing a more continuous concentration of the territorial male''s sperm in the nest, although at a lower level, when he is engaged in prolonged territory defence outside the nest.     

Heterogenous turnover of sperm and seminal vesicle proteins in the mouse revealed by dynamic metabolic labeling

Plasticity in ejaculate composition is predicted as an adaptive response to the evolutionary selective pressure of sperm competition. However, to respond rapidly to local competitive conditions requires dynamic modulation in the production of functionally relevant ejaculate proteins. Here we combine metabolic labeling of proteins with proteomics to explore the opportunity for such modulation within mammalian ejaculates. We assessed the rate at which proteins are synthesized and incorporated in the seminal vesicles of male house mice (Mus musculus domesticus), where major seminal fluid proteins with potential roles in sperm competition are produced. We compared rates of protein turnover in the seminal vesicle with those during spermatogenesis, the timing of which is well known in mice. The subjects were fed a diet containing deuterated valine ([(2)H(8)]valine) for up to 35 days, and the incorporation of dietary-labeled amino acid into seminal vesicle- or sperm-specific proteins was assessed by liquid chromatography-mass spectrometry of samples recovered from the seminal vesicle lumen and cauda epididymis, respectively. Analyses of epididymal contents were consistent with the known duration of spermatogenesis and sperm maturation in this species and in addition revealed evidence for a subset of epididymal proteins subject to rapid turnover. For seminal vesicle proteins, incorporation of the stable isotope was evident from day 2 of labeling, reaching a plateau of labeling by day 24. Hence, even in the absence of copulation, the seminal vesicle proteins and certain epididymal proteins demonstrate considerable turnover, a response that is consonant with the capacity to rapidly modulate protein production. These techniques can now be used to assess the extent of phenotypic plasticity in mammalian ejaculate production and allocation according to social and environmental cues of sperm competition.     

Sperm competition roles and ejaculate investment in a promiscuous mammal

Theoretical models of sperm competition predict how males should allocate sperm and seminal fluid components to ejaculates according to their mating role (dominant vs. subordinate). Here, we present a detailed analysis of ejaculate expenditure according to male roles in the bank vole (Myodes glareolus). Sperm competition occurs regularly in this species, and dominant males typically achieve higher fertilization success than subordinates. Contrary to theoretical predictions, we found that dominant male bank voles invest more sperm per ejaculate than subordinates, both absolutely and relative to body and testes mass. The testes of dominant males were also absolutely (although not relatively) larger than those of subordinates. However, we found no evidence that subordinate males compensate for lower sperm numbers per ejaculate by increasing ejaculation frequency or sperm velocity. Similarly, we found no evidence for differential investment in copulatory plug size according to male roles in sperm competition, although dominant males had significantly larger seminal vesicles (both absolutely and relative to body mass) compared with subordinates. We conclude that sperm competition roles can have significant but unexpected influences on ejaculate investment in mammals with clearly defined differences in male social status.     

Effects of nutrient limitation on sperm and seminal fluid: a systematic review and meta‐analysis

Theory predicts that costly sexual traits should be reduced when individuals are in poor condition (i.e. traits should exhibit condition‐dependent expression). It is therefore widely expected that male ejaculate traits, such as sperm and seminal fluid, will exhibit reduced quantity and quality when dietary nutrients are limited. However, reported patterns of ejaculate condition dependence are highly variable, and there has been no comprehensive synthesis of underlying sources of such variation in condition‐dependent responses. In particular, it remains unclear whether all ejaculate traits are equally sensitive to nutrient intake, and whether such traits are particularly sensitive to certain dietary nutrients, respond more strongly to nutrients during specific life stages, or respond more strongly in some taxonomic groups. We systematically reviewed these potential sources of variation through a meta‐analysis across 50 species of arthropods and vertebrates (from 71 papers and 348 effect sizes). We found that overall, ejaculate traits are moderately reduced when dietary nutrients are limited, but we also detected substantial variation in responses. Seminal fluid quantity was strongly and consistently condition dependent, while sperm quantity was moderately condition dependent. By contrast, aspects of sperm quality (particularly sperm viability and morphology) were less consistently reduced under nutrient limitation. Ejaculate traits tended to respond in a condition‐dependent manner to a wide range of dietary manipulations, especially to caloric and protein restriction. Finally, while all major taxa for which sufficient data exist (i.e. arthropods, mammals, fish) showed condition dependence of ejaculate traits, we detected some taxonomic differences in the life stage that is most sensitive to nutrient limitation, and in the degree of condition dependence of specific ejaculate traits. Together, these biologically relevant factors accounted for nearly 20% of the total variance in ejaculate responses to nutrient limitation. Interestingly, body size showed considerably stronger condition‐dependent responses compared to ejaculate traits, suggesting that ejaculate trait expression may be strongly canalised to protect important reproductive functions, or that the cost of producing an ejaculate is relatively low. Taken together, our findings show that condition‐dependence of ejaculate traits is taxonomically widespread, but there are also many interesting, biologically relevant sources of variation that require further investigation. In particular, further research is needed to understand the differences in selective pressures that result in differential patterns of ejaculate condition dependence across taxa and ejaculate traits.     

Female life span and fertility are increased by the ejaculates of preferred males

In animals with internal fertilization, sperm competition among males can favor the evolution of male ejaculate traits that are detrimental to females. Female mating preferences, in contrast, often favor traits in males that are beneficial to females, yet little is known about the effect of these preferences on the evolution of male ejaculates. A necessary condition for female preferences to affect the evolution of male ejaculate characteristics is that females select mates based on a trait correlated with ejaculate quality. Previous work has shown that females of the variable field cricket, Gryllus lineaticeps, prefer males that produce calling songs containing faster and longer chirps. In this study, we tested the hypothesis that females receive more beneficial ejaculates from preferred males. Females were placed on either a high- or a reduced-nutrition diet then mated twice to a male of known song phenotype. Females received only sperm and seminal fluid from males during these matings. There was no effect of male song phenotype on any fitness component for females on the high-nutrition diet. Reduced-nutrition females mated to males that produced preferred song types, however, lived longer, produced more eggs, produced more fertile eggs, and had a higher proportion of their eggs fertilized than those mated to other males. The life-span benefit was positively associated with male chirp duration, and the reproductive benefits were positively associated with male chirp rate. We explored two possible mechanisms for the life span and reproductive benefits. First, a path analysis suggested that part of the effect of male chirp duration on female life span may have been indirect; females mated to males that produced longer chirps showed delayed oviposition, and females that delayed oviposition lived longer. Males that produce longer chirps may thus transfer fewer or less potent oviposition stimulants to females in their seminal fluid. Second, there was a positive correlation between male chirp rate and the number of sperm transferred to females. The fertility benefit may thus have resulted from females receiving more sperm from males that produce faster chirps. Finally, there was a negative phenotypic correlation between male chirp rate and chirp duration, suggesting that females may have to trade off the life span and reproduction benefits when selecting a mate.     

Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions

Background

No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating.

Methodology and Principal Findings

Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera.

Conclusion and Significance

This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post-mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti.     

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.     

Song and Sperm in Crickets: A Trade‐off between Pre‐ and Post‐copulatory Traits or Phenotype‐Linked Fertility?

When females mate multiply (polyandry) both pre‐ and post‐copulatory sexual selection can occur. Sperm competition theory predicts there should be a trade‐off between investment in attracting mates and investment in ejaculate quality. In contrast, the phenotype‐linked fertility hypothesis predicts a positive relationship should exist between investment in attracting mates and investment in ejaculate quality. Given the need to understand how pre‐ and post‐copulatory sexual selection interacts, we investigated the relationship between secondary sexual traits and ejaculate quality using the European house cricket, Acheta domesticus. Although we found no direct relationship between cricket secondary sexual signals and ejaculate quality, variation in ejaculate quality was dependent on male body weight and mating latency: the lightest males produced twice as many sperm as the heaviest males but took longer to mate with females. Our findings are consistent with current theoretical models of sperm competition. Given light males may have lower mating success than heavy males because females take longer to mate with them in no‐choice tests, light males may be exhibiting an alternative reproductive tactic by providing females with more living sperm. Together, our findings suggest that the fitness of heavy males may depend on pre‐copulatory sexual selection, while the fitness of light males may depend on post‐copulatory fertilization success.     

The Drosophila melanogaster seminal fluid protease "seminase" regulates proteolytic and post-mating reproductive processes

Proteases and protease inhibitors have been identified in the ejaculates of animal taxa ranging from invertebrates to mammals and form a major protein class among Drosophila melanogaster seminal fluid proteins (SFPs). Other than a single protease cascade in mammals that regulates seminal clot liquefaction, no proteolytic cascades (i.e. pathways with at least two proteases acting in sequence) have been identified in seminal fluids. In Drosophila, SFPs are transferred to females during mating and, together with sperm, are necessary for the many post-mating responses elicited in females. Though several SFPs are proteolytically cleaved either during or after mating, virtually nothing is known about the proteases involved in these cleavage events or the physiological consequences of proteolytic activity in the seminal fluid on the female. Here, we present evidence that a protease cascade acts in the seminal fluid of Drosophila during and after mating. Using RNAi to knock down expression of the SFP CG10586, a predicted serine protease, we show that it acts upstream of the SFP CG11864, a predicted astacin protease, to process SFPs involved in ovulation and sperm entry into storage. We also show that knockdown of CG10586 leads to lower levels of egg laying, higher rates of sexual receptivity to subsequent males, and abnormal sperm usage patterns, processes that are independent of CG11864. The long-term phenotypes of females mated to CG10586 knockdown males are similar to those of females that fail to store sex peptide, an important elicitor of long-term post-mating responses, and indicate a role for CG10586 in regulating sex peptide. These results point to an important role for proteolysis among insect SFPs and suggest that protease cascades may be a mechanism for precise temporal regulation of multiple post-mating responses in females.     

Sperm: seminal fluid interactions and the adjustment of sperm quality in relation to female attractiveness

An important predictor of male fitness is the fertilizing efficiency of their ejaculates. Ejaculates are costly to produce and males are predicted to devote greater resources to copulations with reproductively superior females. It is well established that males allocate different numbers of sperm to ejaculates. However, less is known about how males adjust their sperm quality, which has important implications for our understanding of fertilization and the evolution of sexual strategies. Here we test in the fowl, Gallus gallus, whether males adjust their sperm velocity by differentially allocating seminal fluid to copulations with attractive and unattractive females. To disentangle the contributions of sperm and seminal fluid to sperm velocity, we separated and remixed sperm and seminal fluid from ejaculates allocated to females of different attractiveness. We show that dominant males increase the velocity of the sperm they invest in more attractive females by allocating larger ejaculates that contain seminal fluid that increases sperm velocity. Furthermore, we find weak evidence that males also allocate sperm with higher velocity, irrespective of seminal fluid, to more attractive females.     

Functions and analysis of the seminal fluid proteins of male Drosophila melanogaster fruit flies

The study of insect seminal fluid proteins provides a unique window upon adaptive evolution in action. The seminal fluid of Drosophila melanogaster contains over 80 proteins and peptides, which are transferred together with sperm by mating males. The functions of many of these substances are not yet known. However, those that have been characterized have marked effects on the reproductive success of males and females. For example, seminal fluid proteins and peptides can decrease female receptivity, can increase egg production and can increase sperm storage, and are necessary for sperm transfer and success in sperm competition. In this review we focus on the currently known functions of seminal fluid molecules and on new technologies and approaches that are enabling novel questions about their form and function to be addressed. We discuss how techniques for disrupting the production of seminal fluid proteins, such as homologous recombination and RNA interference, along with the use of microarrays and yeast two hybrid systems, should allow us to address ever more sophisticated questions about seminal fluid protein function. These and similar techniques promise to reveal the function of naturally-occurring variants of these proteins and hence the evolutionary significance of genetic variation for them.     

A nonsemen copulatory fluid influences the outcome of sperm competition in Japanese quail

Sperm competition is a powerful and widespread evolutionary force that drives the divergence of behavioural, physiological and morphological traits. Elucidating the mechanisms governing differential fertilization success is a fundamental question of sperm competition. Both sperm and nonsperm ejaculate components can influence sperm competition outcomes. Here, we investigate the role of a nonsemen copulatory fluid in sperm competition. Male Japanese quail possess a gland that makes meringue‐like foam. Males produce and store foam independent of sperm and seminal fluid, yet transfer foam to females during copulation. We tested whether foam influenced the outcome of sperm competition by varying foam state and mating order in competitive matings. We found that the presence of foam from one male decreased the relative fertilization success of a rival, and that foam from a given male increased the probability he obtained any fertilizations. Mating order also affected competitive success. Males mated first fertilized proportionally more eggs in a clutch and had more matings with any fertilizations than subsequent males. We conclude that the function of foam in sperm competition is mediated through the positive interaction of foam with a male's sperm, and we speculate whether the benefit is achieved through improving sperm storage, fertilizing efficiency or retention. Our results suggest males can evolve complex strategies to gain fertilizations at the expense of rivals as foam, a copulatory fluid not required for fertilization, nevertheless, has important effects on reproductive performance under competition.