Copulatory plugs inhibit the reproductive success of rival males

Ejaculated proteins play important roles in reproductive fitness. In many species, seminal fluid coagulates and forms what has been referred to as a copulatory plug in the female's reproductive tract. In mice, previous work demonstrated that knockout males missing a key seminal fluid protein were unable to form a plug and less successful at siring litters in noncompetitive matings (one female, one male), probably the result of reduced sperm transport or insufficient stimulation of the female. Here, we extend these previous studies to competitive matings (one female, two males) and make two key insights. First, when first males were unable to form a plug, they lost almost all paternity to second males to mate. Thus, the copulatory plugs of second males could not rescue the reduced fertility of first males. Second, we showed that the copulatory plug of first males effectively blocked fertilization by second males, even if first males were vasectomized. Taken together, our experiments demonstrated that first males lost almost all paternity if they never formed a plug. We discuss our results in the context of natural populations, where in spite of the strong effects seen here, pregnant female mice regularly carry litters fertilized by more than one male.     

Copulatory Plug Displacement Evidences Sperm Competition in Lemur catta

Male displacement of copulatory (sperm) plugs from female vaginas provides further evidence for sperm competition in ring-tailed lemurs (Lemur catta), a gregarious prosimian species with a multimale, multifemale mating system. During two mating seasons, I studied two groups of free-ranging ring-tailed lemurs on St. Catherines Island, GA, USA. I observed 22 mating pairs in which males achieved penile intromission. Copulatory plug displacement by males occurred in 9 cases. Plugs were displaced during copulation by male penes upon withdrawl following deep vaginal thrusting. In every case of copulatory plug displacement, the male displacing a plug mated to ejaculation with the estrous female. In a mating system in which females typically mate with more than one male during estrous, often in succession, copulatory plug displacement may function to disrupt or preclude other males' successful insemination of estrous females. The effects of sperm plug displacement on paternity in Lemur catta are unknown, as no study had heretofore documented copulatory plug displacement in this species. The first-male mating advantage suggested for Lemur catta should be re-evaluated where mating order is known, and copulatory plug displacement during mating, or lack thereof, is identified. Because there is a tendency for first-mating males to mate-guard for longer periods of time in Lemur catta, the latency period between the first mate's ejaculation and that of subsequent mates may be an important determinant of male fertilization success.     

Mutual adaptation between mouse transglutaminase 4 and its native substrates in the formation of copulatory plug

Formation of copulatory plugs by male animals is a common means of reducing competition with rival males. In mice, copulatory plugs are formed by the coagulation of seminal vesicle secretion (SVS), which is a very viscous and self-clotting fluid containing high concentration of proteins. In its native state, mouse SVS contains a variety of disulfide-linked high-molecular-weight complexes (HMWCs) composed of mouse SVS I-III, which are the major components of mouse SVS. Further, mouse SVS I-III are the substrates for transglutaminase 4 (TGM4), a cross-linking enzyme secreted from the anterior prostate. According to activity assays, mouse TGM4 prefers a mild reducing and alkaline environment. However, under these conditions, the activity of mouse TGM4 toward SVS I-III was much lower than that of a common tissue-type TGM, TGM2. On the other hand, mouse TGM4 exhibited much higher cross-linking activity than TGM2 when native HMWCs containing SVS I-III were used as substrates under non-reducing condition. By the action of TGM4, the clot of SVS became more resistant to proteolysis. This indicates that the activity of TGM4 can further rigidify the copulatory plug and extend its presence in the female reproductive tract. Together with the properties of TGM4 and the nature of its disulfide-linked SVS protein substrates, male mice can easily transform the semen into a rigid and durable copulatory plug, which is an important advantage in sperm competition.     

Female behaviour and the interaction of male and female genital traits mediate sperm transfer during mating

Natural selection and post‐copulatory sexual selection, including sexual conflict, contribute to genital diversification. Fundamental first steps in understanding how these processes shape the evolution of specific genital traits are to determine their function experimentally and to understand the interactions between female and male genitalia during copulation. Our experimental manipulations of male and female genitalia in red‐sided garter snakes (Thamnophis sirtalis parietalis) reveal that copulation duration and copulatory plug deposition, as well as total and oviductal/vaginal sperm counts, are influenced by the interaction between male and female genital traits and female behaviour during copulation. By mating females with anesthetized cloacae to males with spine‐ablated hemipenes using a fully factorial design, we identified significant female–male copulatory trait interactions and found that females prevent sperm from entering their oviducts by contracting their vaginal pouch. Furthermore, these muscular contractions limit copulatory plug size, whereas the basal spine of the male hemipene aids in sperm and plug transfer. Our results are consistent with a role of sexual conflict in mating interactions and highlight the evolutionary importance of female resistance to reproductive outcomes.     

Paternity analyses in wild‐caught and laboratory‐reared Caribbean cricket females reveal the influence of mating environment on post‐copulatory sexual selection

Polyandry is ubiquitous in insects and provides the conditions necessary for male‐ and female‐driven forms of post‐copulatory sexual selection to arise. Populations of Amphiacusta sanctaecrucis exhibit significant divergence in portions of the male genitalia that are inserted directly into the female reproductive tract, suggesting that males may exercise some post‐copulatory control over fertilization success. We examine the potential for male–male and male–female post‐copulatory interactions to influence paternity in wild‐caught females of A. sanctaecrucis and contrast our findings with those obtained from females reared in a high‐density laboratory environment. We find that female A. sanctaecrucis exercise control by mating multiple times (females mount males), but that male–male post‐copulatory interactions may influence paternity success. Moreover, post‐copulatory interactions that affect reproductive success of males are not independent of mating environment: clutches of wild‐caught females exhibit higher sire diversity and lower paternity skew than clutches of laboratory‐reared females. There was no strong evidence for last male precedence in either case. Most attempts at disentangling the contributions of male–male and male–female interactions towards post‐copulatory sexual selection have been undertaken in a laboratory setting and may not capture the full context in which they take place – such as the relationship between premating and post‐mating interactions. Our results reinforce the importance of designing studies that can capture the multifaceted nature of sexual selection for elucidating the role of post‐copulatory sexual selection in driving the evolution of male and female reproductive traits, especially when different components (e.g. precopulatory and post‐copulatory interactions) do not exert independent effects on reproductive outcomes.     

The copulatory plug delays ejaculation by rival males and affects sperm competition outcome in house mice

Females of many species mate with multiple males (polyandry), resulting in male–male competition extending to post‐copulation (sperm competition). Males adapt to such post‐copulatory sexual selection by altering features of their ejaculate that increase its competitiveness and/or by decreasing the risk of sperm competition through female manipulation or interference with rival male behaviour. At ejaculation, males of many species deposit copulatory plugs, which are commonly interpreted as a male adaptation to post‐copulatory competition and are thought to reduce or delay female remating. Here, we used a vertebrate model species, the house mouse, to study the consequences of copulatory plugs for post‐copulatory competition. We experimentally manipulated plugs after a female's first mating and investigated the consequences for rival male behaviour and paternity outcome. We found that even intact copulatory plugs were ineffective at preventing female remating, but that plugs influenced the rival male copulatory behaviour. Rivals facing intact copulatory plugs performed more but shorter copulations and ejaculated later than when the plug had been fully or partially removed. This suggests that the copulatory plug represents a considerable physical barrier to rival males. The paternity share of first males increased with a longer delay between the first and second males' ejaculations, indicative of fitness consequences of copulatory plugs. However, when males provided little copulatory stimulation, the incidence of pregnancy failure increased, representing a potential benefit of intense and repeated copulation besides plug removal. We discuss the potential mechanisms of how plugs influence sperm competition outcome and consequences for male copulatory behaviour.     

Securing paternity: mating plugs in the dwarf spider Oedothorax retusus (Araneae: Erigoninae)

One of the various male strategies to prevent or impede female remating is the production of a mating plug that covers the female genital opening or remains inside of the female genital tract after mating. Such structures have been described for many species in many animal taxa; however, in most cases, we know little or nothing about their specific adaptive value. Our investigations demonstrate that females of the dwarf spider species Oedothorax retusus (Westring, 1851) (Linyphiidae, Erigoninae) exhibit a substance on one or both of her paired genital openings only after copulation. We performed double-mating trials and forced the second male to mate into the previously used or unused spermathecal duct of the female by amputating one of his paired male gonopods (pedipalps). Furthermore, to investigate whether the duration of the first mating has an effect on the size and efficiency of the mating plug, we interrupted first matings after either 1 or 3 min, categorized plug size and recorded mating behaviour of subsequent males. The amount of secretion transferred was larger in long compared to short copulations. A long first copulation successfully prevented subsequent males from mating into the used ducts, whereas mating success after short first matings was similar to matings into unused copulatory ducts of the females. The present study demonstrates that a male O. retusus can prevent a rival from transferring sperm into the same spermatheca by applying a mating plug, but only if he mates for long enough.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 574–583.     

Effect of mating on sperm distribution in the reproductive tract of the male rat

Extragonadal sperm reserves in male rats were measured in different regions of the genital tract before and subsequent to normal ejaculation. In sexually rested rats, the sperm count (million spermatozoa for the paired organs) in different regions was: distal vas, 18; proximal vas, 9.8; cauda epididymidis, 229; caput + corpus epididymidis, 154. Following mating, the sperm count was reduced in the proximal and distal vas deferens and in the cauda epididymidis. The reproductive tract of mated females was found to contain 29% (no copulatory plug) or 59% (with copulatory plug) of the estimated mean ejaculate, which was estimated from the difference between the sperm counts in the sexually rested rat and following ejaculation. It is concluded that in the rat the immediate source of spermatozoa for ejaculation is the cauda epididymidis, with a smaller contribution arising from the vas deferens.     

Genetic and phenotypic influences on copulatory plug survival in mice

Across a diversity of animals, male seminal fluid coagulates upon ejaculation to form a hardened structure known as a copulatory plug. Previous studies suggest that copulatory plugs evolved as a mechanism for males to impede remating by females, but detailed investigations into the time course over which plugs survive in the female''s reproductive tract are lacking. Here, we cross males from eight inbred strains to females from two inbred strains of house mice (Mus musculus domesticus). Plug survival was significantly affected by male genotype. Against intuition, plug survival time was negatively correlated with plug size: long-lasting plugs were small and relatively more susceptible to proteolysis. Plug size was associated with divergence in major protein composition of seminal vesicle fluid, suggesting that changes in gene expression may play an important role in plug dynamics. In contrast, we found no correlation to genetic variation in the protein-coding regions of five genes thought to be important in copulatory plug formation (Tgm4, Svs1, Svs2, Svs4 and Svs5). Our study demonstrates a complex relationship between copulatory plug characteristics and survival. We discuss several models to explain unexpected variation in plug phenotypes.     

Male Performance and Body Size Affect Female Re-Mating Occurrence in the Orb-Web Spider Leucauge mariana (Araneae, Tetragnathidae)

Females can affect male probabilities of paternity success through behavioural, morphological and/or physiological processes occurring during or after copulation. These processes under female-control include the acceptance or rejection of mating attempts by subsequent males. Leucauge mariana is an orb weaving spider that shows male mate guarding of penultimate females, male–male competition on female webs and copulatory plugs, suggesting a polyandric mating system. The aim of the present study was to ascertain whether male behaviour during courtship and copulation in L. mariana relate with female re-mating decisions. Forty-three virgin females were exposed to up to three males until they mated. In 24 cases, the copulatory plug was absent after mating and females were exposed the next day to up to three other males. Eighteen females accepted a second mating. Relatively larger females were more receptive to second matings and were more likely to copulate if the second male was smaller. Longer duration of female tapping and abdominal bobbing during courtship, and first copulations with less short insertions and more flubs, were associated with increased female acceptance to second matings. The results indicate cryptic female choice on male courtship and copulatory performance and suggest female-control over the determination of male mating success in this spider species.     

Immunofluorescent localization of a seminal vesicle proteinase inhibitor in the female reproductive tract of naturally inseminated mice

The indirect immunofluorescent technique was used to localize a low molecular weight, acid-stable proteinase inhibitor of seminal vesicle origin in the female reproductive tract of mice. In recently inseminated animals (0, 2, and 4 hr postcoitus) the inhibitor was localized in the copulatory plug, on the epithelia of the vaginal fornix and cervix, in the uterine lumen, and on the apical surface of the uterine epithelium. Ten hours postcoitus the inhibitor was found in localized areas on the uterine epithelium, in a sperm-leucocyte mass in the uterine lumen, and in the copulatory plug. The inhibitor was not found in females 24 hr postcoitus. The inhibitor could not be localized in the oviducts of any of the animals tested. The data are interpreted to mean that the inhibitor, transported to the female at ejaculation, coats the surface of the female reproductive tract protecting it from acrosomal enzymes or from invasion by spermatozoa or pathogens.     

Mate plugging via genital mutilation in nephilid spiders: an evolutionary hypothesis

Nephilid spiders are known for gigantic females and tiny males. Such extreme sexual dimorphism and male-biased sex ratios result in fierce male–male competition for mates. Intense sperm competition may be responsible for behaviors such as mate guarding, mate binding, opportunistic mating, genital mutilation, mating plugs and male castration (eunuchs). We studied the mating biology of two phylogenetically, behaviorally and morphologically distinct south-east Asian nephilid spider species ( Herennia multipuncta, Nephila pilipes ) in nature and in the laboratory. Specifically, we established the frequencies and effectiveness of plugging (a plug is part of the male copulatory organ), and tested for male and female copulatory organ reuse. Both in nature and in the laboratory, plug frequencies were higher in H. multipuncta (75–80% females plugged) compared with N. pilipes (45–47.4%), but the differences were not significant. Plugs were single and effective (no remating) in H. multipuncta but multiple and ineffective (remating possible) in N. pilipes . In Herennia , the males plugged when the female was aggressive and in Nephila plugging was more likely when mating with previously mated and larger females. Further differences in sexual biology are complete palpal removal and higher sexual aggressiveness in Herennia (sexual cannibalism recorded for the first time), and mate binding in Nephila . Thus, we propose the following evolutionary hypothesis: nephilid plugging was ancestrally successful and enabled males to monopolize females, but plugging became ineffective in the phylogenetically derived Nephila . If the evolution of nephilid sexual mechanisms is driven by sexual conflict, then the male mechanism to monopolize females prevailed in a part of the phylogeny, but the female resistance to evade monopolization ultimately won the arms race.     

Sexual selection,seminal coagulation and copulatory plug formation in primates

This study examines the question of whether multipartner matings by female primates, with resulting sperm competition among males, may have favored the evolution of biochemical mechanisms to enhance seminal coagulationand copulatory plug formation. Comparative ratings of seminal coagulation (using a four-point scale where 1 = no coagulation and 4 = copulatory plug formation) were obtained for 40 species representing 26 primate genera. Coagulation ratings were highest (mean = 3.64) in those genera where females commonly mate with multiple partners, and lowest (mean = 2.09) in genera where females are primarily monogamous or belong to polygynous (one male) units(p < 0.0001). This result remained significant (p < 0.001) after the use of comparative analysis of independent contrasts (CAIC) to control for possible phylogenetic biases in the data set. Results indicate that sexual selection has played an important role in the evolution of seminal coagulation, and copulatory plug function, in primates.     

Double insurance of paternity by a novel type of mating plug in a monandrous solitary mason bee Osmia bicornis (Hymenoptera: Megachilidae)

Males of some hymenopteran species are able to behaviourally induce unreceptivity to mating in females by a post‐copulatory display (post‐copulatory courtship). This method of preventing further inseminations requires a high degree of female cooperation by the female and is especially susceptible to manipulations. If mating chances are low and sexual competition between males high, males can be expected to evolve additional mechanisms to protect their fertilization opportunities. This hypothesis was checked in the red mason bee Osmia bicornis (Linnaeus, 1758; syn. Osmia rufa). Males of this species were found to use a mating plug as a reinsurance to protect their paternity, although they induce females' monandry by a post‐copulatory display. Male accessory gland secretions transferred during ejaculation form a plastic, spongy plug in the females' vagina that also contains the spermatozoa. This mating plug does not hinder rivals to mate with the female but prevents intermixing of the sperm. Thus, the sperm precedence of the first male is secured. The plug is ejected by the female after approximately 1 day when the spermatheca has been completely filled with sperm. The male accessory gland supply is sufficient for the formation of two to three plugs only and is not replenished. This pattern fits well with the low mating opportunities of O. bicornis males. The evolution of a mating plug in addition to the behavioural induction of unreceptivity to mate in females is discussed. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 115 , 28–37.     

Sperm competition and the significance of female multiple mating in the predatory miteParasitus fimetorum

Females of the predatory miteParasitus fimetorum (Gamasida; Parasitina) inhabiting animal manure indiscriminately copulate with many mates. The sperm competition between the males was estimated by electrophoresis of allozymes and the effects of multiple mating on female reproduction were investigated. When females were forced to mate only once, their fecundity decreased drastically compared to the case of multiple mating (but longevity was unaffected). When one female mated with two males, the outcome of sperm competition depended greatly upon the mating interval. When the second mating occurred immediately after the first, the female fecundity decreased as in the case of single mating and the second male fertilized only a few eggs. However, when there was an interval of 1 day between the two matings, the females achieved normal fecundity and the second male fertilized approximately half the eggs. This suggests that the spermatophore deposited by the first male may act as a short-term copulatory ‘plug’ in the female's genital opening. When one female mated with several males with 1 day intervals, three or more males shared fertilization of the eggs. This study suggests that the multiple mating of females is a necessary stimulus to continue oogenesis and some physiological factors for this stimulation may exist in spermatophores.     

Heterosexual interactions in laboratory-housed stumptail macaques (Macaca arctoides): observations during the menstrual cycle and after ovariectomy.

Heterosexual interactions of pairs of stumptail macaques (Macaca arctoides) were studied in relation to the female menstrual cycle and after ovariectomy. Five intact male and 10 tubal-ligated female macaques were observed in laboratory pair tests of 20-mins duration, and data were obtained on various male and female behaviors. Each male was tested with the same two females during four 40-day observation periods. Males were tested daily and females were tested every other day. After two 40-day testing periods, one female partner of each male was ovariectomized and the other was sham-operated. Blood was collected regularly from the females during the course of observation and serum levels of estradiol and progesterone were determined by radioimmunoassay. The midcycle peak of estradiol was observed to occur approximately 18 days prior to menses. A distinct secondary peak in estradiol was observed to occur during the luteal phase of all cycles examined. Of 28 different male and female behaviors studied only female presentation to male sexual contact showed a significant midcycle peak related to the endogenous estradiol surge. After ovariectomy a significant decrease in the frequency of several male copulatory behaviors was observed, but most males continued to copulate regularly with their spayed partners throughout the period of this study. Thus, the pattern of copulatory behavior observed in stumptail macaques over the cycle of the female partner and subsequent to ovariectomy differs from that observed in other macaque species studied in the laboratory. It is concluded that cyclical fluctuations in the level of ovarian hormones are not significantly related to measures of sexual interactions in laboratory tests of this species, although the maintenance of copulation and associated behaviors at high levels depends to some degree upon the ovary.     

EVIDENCE FOR WIDESPREAD COURTSHIP DURING COPULATION IN 131 SPECIES OF INSECTS AND SPIDERS,AND IMPLICATIONS FOR CRYPTIC FEMALE CHOICE

Male courtship behavior is generally thought to function prior to copulation, as an inducement to the female to allow the male to copulate with her; this study indicates however, that male courtship during and following copulation (“copulatory courtship”) is common in insects and spiders (81% of 131 species in 102 genera and 49 families, mostly Coleoptera, Hemiptera, Diptera, and Araneioidea). Copulatory courtship is apparently evolutionarily labile, as expected if it is under sexual selection; intrageneric variation occurred in all 17 genera in which more than one species was observed. In 81% of 94 species with copulatory courtship, the male abandoned the female soon after copulation ended; thus, copulatory courtship appears not to function generally to induce acceptance of further copulatory attempts. The most likely explanation for copulatory courtship is that it represents attempts by males to influence cryptic female choice. This suggests that an aspect of sexual selection by female choice not considered by Darwin may be more important than previously appreciated and that the common practice in evolutionary studies of measuring male reproductive success by counting numbers of copulations may sometimes be misleading because of cryptic female choice during and after copulation.     

High-resolution X-ray computed tomography scanning of primate copulatory plugs

In this study, high-resolution computed tomography X-ray scanning was used to scan ring-tailed lemur (Lemur catta) copulatory plugs. This method produced accurate measures of plug volume and surface area, but was not useful for visualizing plug internal structure. Copulatory plug size was of interest because it may relate to male fertilization success. Copulatory plugs form from coagulated ejaculate, and are routinely displaced in this species by the penis of a subsequent mate during copulation (Parga [2003] Int. J. Primatol. 24:889-899). Because one potential function of these plugs may be to preclude or delay other males' successful insemination of females, we tested the hypothesis that larger plugs are more difficult for subsequent males to displace. Plugs were collected opportunistically upon displacement during data collection on L. catta mating behavior on St. Catherines Island, Georgia (USA) during two subsequent breeding seasons. Copulatory plugs exhibited a wide range of volumes: 1,758-5,013.6 mm3 (n = 9). Intraindividual differences in plug volume were sometimes greater than interindividual differences. Contrary to predictions, larger plugs were not more time-consuming for males to displace via penile intromission during copulation. Nor were plugs with longer vaginal residence times notably smaller than plugs with shorter residence times, as might be expected if plugs disintegrate while releasing sperm (Asdell [1946] Patterns of Mammalian Reproduction; Ithaca: Comstock). We found a significant inverse correlation between number of copulatory mounts leading to ejaculation and copulatory plug volume. This may indicate that if males are sufficiently sexually aroused to reach ejaculation in fewer mounts, they tend to produce ejaculates of greater volume.     

Male Post‐Ejaculatory Mounting in the Ring‐Tailed Lemur (Lemur catta): A Behavior Solicited by Females?

Male mate guarding can take many forms but often involves aggression toward male conspecifics and continued proximity with a female. This study describes a previously undocumented behavior in a promiscuous primate, the ring‐tailed lemur: post‐ejaculatory (PE) mounting. PE mounting was documented across eight mating seasons in a ring‐tailed lemur colony on St. Catherines Island (SCI), USA. During PE mounting, a male remounts a female following ejaculation and clasps her midsection as if to mate again, but copulation does not occur; males showing this behavior typically lack erections, and their mounts show an absence of penile intromission and rhythmic thrusting. Male PE mounting was more common among males mating earlier in the queue, and when PE mounting occurred, it accompanied mate guarding. Four non‐mutually exclusive hypotheses to explain PE mounting were evaluated as follows: (1) gaining additional copulations, (2) prevention of re‐mating, (3) lengthening sperm residence time, and (4) re‐mounting as a function of female proceptivity. Male PE mounting did not aid males in gaining additional copulations nor did PE mounting prevent females from mating with new males. Equivocal support was found for Hypothesis 3: although there was much overlap in copulatory plug residence times for males who did and did not show PE mounting, one‐third of males who practiced PE mounting had plug residence times of 2 h or more, much longer than that of males who did not show PE mounting. PE mounting may therefore be related to increased plug residence time, which may provide an advantage to males in sperm competition. Strong evidence was found in support of Hypothesis 4: males overwhelmingly performed PE mounts in response to continued female sexual presentations, suggesting that females can solicit this male behavior. Females consequently exercise an even greater degree of control over males than was previously realized in this ‘female dominant’ primate.     

Copulation anatomy of Drosophila melanogaster (Diptera: Drosophilidae): wound-making organs and their possible roles

Males of several insect species inflict wounds on female genitalia during copulation. Such copulatory wounding also occurs in the fruit fly Drosophila melanogaster Meigen, 1830, one of the most important model organisms. Using a flash fixation technique with mating pairs of D. melanogaster, I examined the use and functions of the male phallic organ within the female reproductive tract. Paired components of the phallic organ (gonopods and two pairs of branches of the basal processes of the aedeagus) opened sequentially, from outer to inner components, during copulation. The dorsal branches of the aedeagal basal processes pierced the intima of the female reproductive tract at the lateral shallow folds. Consequently, mated females usually had a pair of melanized patches from repaired copulatory wounds. The sites that were stabbed by the dorsal branches were also clutched on the outside of the female oviscape (ovipositor) by the posterior process, which is a component of the periphallic organ. These structures likely function together as a mate-holding device. Male ejaculate labeled with rhodamine-B fluorescent dye entered the copulatory wounds in D. eugracilis Bock and Wheeler (Univ Texas Publ 7213:1–102, 1972), a related species, but not in D. melanogaster. Thus, copulatory wounds may function as an entrance for male seminal chemicals into the female circulatory system in D. eugracilis, but might not in D. melanogaster.