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.     

Evaluating the post‐copulatory sexual selection hypothesis for genital evolution reveals evidence for pleiotropic harm exerted by the male genital spines of Drosophila ananassae

The contemporary explanation for the rapid evolutionary diversification of animal genitalia is that such traits evolve by post‐copulatory sexual selection. Here, we test the hypothesis that the male genital spines of Drosophila ananassae play an adaptive role in post‐copulatory sexual selection. Whereas previous work on two Drosophila species shows that these spines function in precopulatory sexual selection to initiate genital coupling and promote male competitive copulation success, further research is needed to evaluate the potential for Drosophila genital spines to have a post‐copulatory function. Using a precision micron‐scale laser surgery technique, we test the effect of spine length reduction on copulation duration, male competitive fertilization success, female fecundity and female remating behaviour. We find no evidence that male genital spines in this species have a post‐copulatory adaptive function. Instead, females mated to males with surgically reduced/blunted genital spines exhibited comparatively greater short‐term fecundity relative to those mated by control males, indicating that the natural (i.e. unaltered) form of the trait may be harmful to females. In the absence of an effect of genital spine reduction on measured components of post‐copulatory fitness, the harm seems to be a pleiotropic side effect rather than adaptive. Results are discussed in the context of sexual conflict mediating the evolution of male genital spines in this species and likely other Drosophila.     

Challenging the Aggressive Spillover Hypothesis: Is Pre‐Copulatory Sexual Cannibalism a Part of a Behavioural Syndrome?

Pre‐copulatory cannibalism – females devouring males during courtship – may bring no benefit to either sex. The ‘aggressive spillover hypothesis’ (ASH) posits that pre‐copulatory cannibalism represents a spillover of female aggressiveness from the juvenile foraging context, when aggressiveness is advantageous, to the adult mating context, when aggressiveness may be non‐adaptive or maladaptive. The ASH suggests that individuals exhibit limited plasticity in aggressive behaviours because they are genetically canalised for indiscriminate aggressiveness towards prey and conspecifics, including males. Hence, a tendency to employ pre‐copulatory cannibalism is a part of the female aggression syndrome, an assertion generally accepted in the personality field. We here re‐evaluate the previous findings in the light of personality criteria, which we propose for ASH validation: between‐individual differences, repeatability and heritability in tendency for pre‐copulatory attacks (and pre‐copulatory cannibalism) and voracity towards prey, and their correlation. To re‐evaluate ASH and to allow for additional or alternative explanations, we ask whether pre‐copulatory cannibalism depends on female hunger, mating status, size and/or male quality. Finally, we ask whether cannibalistic females have a reduced reproductive success as predicted by the ASH. While repeatability and heritability in voracity towards prey and its correlation with the tendency to engage in pre‐copulatory cannibalism were found in certain systems, we lack any evidence for repeatability and heritability in pre‐copulatory cannibalistic attempts and for its maladaptiveness. Rather than only resorting to the ASH, foraging and mate choice hypotheses may also explain pre‐copulatory cannibalism. We suggest clarifying the use of the terms sexual cannibalism (effect) and female aggressiveness or tendency to attack and devour males (cause), and argue that male strategies to avoid cannibalism should be considered. We propose testing the ASH as the explanation for pre‐copulatory cannibalism in those cases where female tendency to devour males correlates with actual pre‐copulatory cannibalism and when all the above criteria are fulfilled. Finally, we propose future directions for studying the ASH.     

Genitalia Fitting,Mating Behaviour and Possible Hybridization in Millipedes of the Genus Craspedosoma (Diplopoda,Chordeumatida, Craspedosomatidae)

Abstract The functional anatomy of the extremely complex copulatory organs of the millipede Craspedosoma transsilvanicum Verhoeff, 1897, was investigated by scanning electron microscopy and serial semi-thin sectioning after fixation of the animals by freezing in copula. There is no male organ for sperm transfer which inserts into the receptacula seminis. Sperm secretion is only brushed over the openings of the receptacula by cuticular projections of the anterior gonopods (‘brush’). Other parts of the gonopods (projections of the cheirites, podosternite) bring the brush and the openings of the receptacula in the proper position. Examination of the gonopods from series of Craspedosoma from locations near Vienna showed that there are intermediary specimens between the nominal species C. transsilvanicum and C. alemannicum Verhoeff, 1910. Obviously, a mechanical isolation mechanism, as supposed by Verhoeff, does not exist between these nominal species. At the beginning of a copulation, the male forces the female into the mating position. When touched by males, the females try to evade them by rolling up. The possibilities are discussed that females thereby test the actual fitness of males, and that males use their clasper-like copulatory organs to prevent females from escaping.     

Risk of sperm competition does not influence copulatory behavior in the promiscuous meadow vole (<Emphasis Type="Italic">Microtus pennsylvanicus</Emphasis>)

It is not clear whether males in all mammalian species adjust their copulatory behavior when faced with risk of sperm competition (RSC). Previous work on meadow voles, Microtus pennsylvanicus, indicated that males increase their sperm expenditure but not the number of ejaculations in the presence of odors of a conspecific male. The present study follows up on this work and asks whether male meadow voles modify any aspect of their copulatory behavior when they face a RSC. We examined 46 variables of copulatory behavior and found that the presence of odors from a conspecific male did not affect any of these variables. Thus, male meadow voles, unlike some other species of mammals, do not adjust their copulatory behavior when exposed to cues associated with an elevated RSC.     

Female response to predation risk alters conspecific male behaviour during pre‐copulatory mate guarding

Mating behaviour often increases predation risk, but the vulnerability within mating pairs differs between the sexes. Such a sex difference is expected to lead to differences in responses to predation risk between the sexes. In the two‐spotted spider mite Tetranychus urticae, males engage in pre‐copulatory mate guarding because only the first mating results in fertilisation. We investigated (i) whether pre‐copulatory pairs are more conspicuous to the predatory mite Phytoseiulus persimilis than solitary females, (ii) whether the vulnerability to the predator differs between sexes within the pre‐copulatory pair, (iii) whether each sex of T. urticae responds to predation risk during pre‐copulatory mate guarding and (iv) whether T. urticae's response to predation risk affects predator behaviour. Because T. urticae females are immobile during pre‐copulatory mate guarding, we observed male behaviour to evaluate effects of predation risk. We found that the predators detect more pre‐copulatory pairs than solitary females and that more females than males of the pre‐copulatory pairs are preyed upon by the predators. The preference of spider mite males for pre‐copulatory pairs versus solitary females was affected by whether or not the female had been exposed to predators during development. Male T. urticae exposed to predation risk did not alter their behaviour. These results suggest that only the most vulnerable sex, that is the female, responds to predation risk, which modifies male behaviour. Regardless of T. urticae females’ experience, however, P. persimilis detected more T. urticae pre‐copulatory pairs than solitary females, suggesting that pre‐copulatory mate guarding itself is dangerous for T. urticae females when these predators are present. We discuss our results in the context of sex‐dependent differences in predation risk.     

Female crickets assess relatedness during mate guarding and bias storage of sperm towards unrelated males

Recent evidence shows that females exert a post‐copulatory fertilization bias in favour of unrelated males to avoid the genetic incompatibilities derived from inbreeding. One of the mechanisms suggested for fertilization biases in insects is female control over transport of sperm to the sperm‐storage organs. We investigated post‐copulatory inbreeding‐avoidance mechanisms in females of the cricket Teleogryllus oceanicus. We assessed the relative contribution of related and unrelated males to the sperm stores of double‐mated females. To demonstrate unequivocally that biased sperm storage results from female control rather than cryptic male choice, we manipulated the relatedness of mated males and of males performing post‐copulatory mate guarding. Our results show that when guarded by a related male, females store less sperm from their actual mate, irrespective of the relatedness of the mating male. Our data support the notion that inhibition of sperm storage by female crickets can act as a form of cryptic female choice to avoid the severe negative effects of inbreeding.     

How the length of genital parts affects copulation performance in a carabid beetle: implications for correlated genital evolution between the sexes

To identify factors leading to the correlated evolution of exaggerated male and female genitalia, we studied the effects of the variable dimensions of corresponding functional genital parts (male copulatory piece and female vaginal appendix) on copulatory performance in the polygamous carabid beetle Carabus (Ohomopterus) maiyasanus. We used mating pairs of individuals from two populations to increase the variances in genital dimensions and determined the copulation performance (insemination and spermatophore replacement, and copulation time) in single‐ and double‐mating situations. In single mating, insemination success was not affected by genital dimensions, although the copulation time was significantly shorter when the male aedeagus was longer. In the double‐mating experiment, insemination and replacement of spermatophores by the second male succeeded more frequently when the copulatory piece was shorter and the vaginal appendix was longer, and when the difference between the length of the copulatory piece and the vaginal appendix was smaller. Thus, a matching of the corresponding genital parts between the sexes increases the male's reproductive success in sperm competition, but elongation of the copulatory piece cannot be explained simply by the improvement in male reproductive success. We discuss possible factors for the elongation of genital parts in terms of sexual conflict and reproductive interference through interspecific copulation.     

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.     

Genetic variation but weak genetic covariation between pre‐ and post‐copulatory episodes of sexual selection in Drosophila melanogaster

When females mate polyandrously, male reproductive success depends both on the male's ability to attain matings and on his ability to outcompete rival males in the fertilization of ova post‐copulation. Increased investment in  ejaculate components may trade off with investment in precopulatory traits due to resource allocation. Alternatively, pre‐ and post‐copulatory traits could be positively related if individuals can afford to invest heavily in traits advantageous at both episodes of selection. There is empirical evidence for both positive and negative associations between pre‐ and post‐copulatory episodes, but little is known about the genetic basis of these correlations. In this study, we measured morphological, chemical and behavioural precopulatory male traits and investigated their relationship with measures of male fitness (male mating success, remating inhibition and offensive sperm competitiveness) across 40 isofemale lines of Drosophila melanogaster. We found significant variation among isofemale lines, indicating a genetic basis for most of the traits investigated. However, we found weak evidence for genetic correlations between precopulatory traits and our indices of male fitness. Moreover, pre‐ and post‐copulatory episodes of selection were uncorrelated, suggesting selection may act independently at the different episodes to maximize male reproductive success.     

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.     

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.     

Anatomical variability and secondary sexual characteristics in Pomphorhynchus laevis (Müller, 1776) (Acanthocephala)

A brief historical survey demonstrates the differences in opinion concerning the taxonomic status of species within the genus Pomphorhynchus. The anatomy of P. laevis from a single population in the River Severn, England was found to be highly variable. Much of this variability is produced as individuals grow. P. laevis possesses circumbursal and circumpenial rings of receptors on the male copulatory bursa. Specimens from Eire and the R. Severn had identical arrangements of these receptors. A preliminary appraisal of the value of secondary sexual characters as criteria of specific identity in the Acanthocephala is presented. ac]19860721     

Relationship between pre‐ and post‐copulatory traits in Salvator rufescens (Squamata: Teiidae)

Understanding pre‐ and post‐copulatory mechanisms of sexual selection can provide insights into the evolution of male reproductive strategies. The phenotype‐linked fertility hypothesis postulates that male sperm quality and secondary sexual characteristics will positively co‐vary, whereas the sperm competition hypothesis predicts a negative association between those traits. Male reproductive traits often show variation throughout the reproductive period, suggesting that the relationship between pre‐ and post‐copulatory sexual selection may vary temporally. Here, we evaluated the relationship between secondary sexual character and sperm traits and its temporal variation in Salvator rufescens, a south American lizard. We observed a negative relationship between jaw muscle and principal piece length of sperm and a variation in the relationship between pre‐ and post‐copulatory traits throughout the reproductive period. Collectively, our results evidenced a trade‐off between pre‐ and post‐copulatory traits and a strong seasonal flexibility of male reproductive strategies in this lizard species.     

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.     

Timing of female sexual unreceptivity and male adjustment of copulatory behaviour under competition risk in the wolf spider <Emphasis Type="Italic">Schizocosa malitiosa</Emphasis>

Males can change their copulatory or sperm transfer patterns in response to sperm competition risk. Schizocosa malitiosa performs long copulations, which include two consecutive patterns (Patterns 1 and 2). Virgin females are very sexually receptive, but mated females diminish their receptiveness. Female unreceptivity has been attributed to the action of receptivity-inhibiting substances, mainly transferred during Pattern 1. We tested: (1) if females who mated only with Pattern 1 were immediately unreceptive; (2) male and female behaviours when the copulating couple was exposed to another male. For (1), we interrupted mating when Pattern 1 finished and immediately exposed the female to a second male. For (2), we introduced a second male when the couple was starting (Ei) or finishing copulation (Li). Females were unreceptive immediately after finishing Pattern 1. Males from Ei and Li dismounted and approached the second males. Ei males diminished the frequencies of insertion after perceiving the presence of a second male and dismounted less frequently when copulating with heavy females. The study provides insights about the timing of sexual unreceptivity in S. malitiosa under possibilities of sperm competition, discussing male adjustment of copulatory behaviour in the presence of rival males. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Testosterone induction of male‐typical sexual behavior is associated with increased preoptic NADPH diaphorase and citrulline production in female whiptail lizards

In rodents, male‐typical copulatory behavior is generally dependent on gonadal sex steroids such as testosterone, and it is thought that the mechanism by which the hormone gates the behavior involves the gaseous neurotransmitter nitric oxide. According to one model, testosterone induces an up‐regulation of nitric oxide synthase (NOS) in the preoptic area, increasing nitric oxide synthesis following exposure to a sexual stimulus. Nitric oxide in turn, possibly through its effect on catecholamine turnover, influences the way the stimulus is processed and enables the appropriate copulatory behavioral response. In whiptail lizards (genus Cnemidophorus), administration of male‐typical levels of testosterone to females induces the display of male‐like copulatory responses to receptive females, and we hypothesized that this radical change in behavioral phenotype would be accompanied by a large change in the expression of NOS in the preoptic area. As well as comparing NOS expression using NADPH diaphorase histochemistry between testosterone‐treated females and controls, we examined citrulline immunoreactivity (a marker of recent nitric oxide production) in the two groups, following a sexual stimulus and following a nonsexual stimulus. Substantially more NADPH diaphorase‐stained cells were observed in the testosterone‐treated animals. Citrulline immunoreactivity was greater in testosterone‐implanted animals than in blank‐implanted animals, but only following exposure to a sexual stimulus. This is the first demonstration that not only is NOS up‐regulated by testosterone, but NOS thus up‐regulated is activated during male‐typical copulatory behavior. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

A redescription of Heterocotyle pastinacae Scott, 1904 (Monogenea: Monocotylidae) from Dasyatis pastinaca (Dasyatididae), with a neotype designation

Heterocotyle pastinacae Scott, 1904 is redescribed and a neotype is designated. H. pastinacae is most easily distinguished from other members of the genus by the elaborate arrangement of sclerotised spines in the vagina. The male copulatory organ also serves to differentiate it from other species in the genus. A possible function for the vaginal spines and unique form of the male copulatory organ is discussed. The use of head organ number as a generic character is considered questionable.     

Evolution of the Hominoid Semenogelin Genes,the Major Proteins of Ejaculated Semen

The hominoid primates (apes and humans) exhibit remarkable diversity in their social and sexual behavioral systems. This is reflected in many ways in their anatomy and physiology. For example, the testes and seminal vesicles are relatively large in species with high sperm competition like the chimpanzee and small in species with low or no sperm competition like the gorilla. Additionally, the chimpanzee is the only hominoid primate known to produce a firm copulatory plug, which presumably functions in sperm competition by blocking insemination of subsequent males. Here we examine the molecular evolution of the semenogelin genes (SEMG1 and SEMG2), which code for the predominant structural proteins in human semen. High molecular weight complexes of these proteins are responsible for the viscous gelatinous consistency of human semen; their rodent homologs are responsible for the formation of a firm copulatory plug. Chimpanzees have an expanded SEMG1 gene caused by duplications of tandem repeats, each encoding 60 amino acids, resulting in a protein nearly twice as long as that of humans. In contrast, at both SEMG1 and SEMG2 we observed several gorilla haplotypes that contain at least one premature stop codon. We suggest that these structural changes in the semenogelin proteins that have arisen since the human–chimpanzee–gorilla split may be responsible for the physiological differences between these species ejaculated semen that correlate with their sociosexual behavior. Present address: (Jensen-Seaman) Human and Molecular Genetics Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA