Resolving mechanisms of short-term competitive fertilization success in the red flour beetle

Postcopulatory sexual selection occurs when sperm from multiple males occupy a female’s reproductive tract at the same time and is expected to generate strong selection pressures on traits related to competitive fertilization success. However, knowledge of competitive fertilization success mechanisms and characters targeted by resulting selection is limited, partially due to the difficulty of discriminating among sperm from different males within the female reproductive tract. Here, we resolved mechanisms of competitive fertilization success in the promiscuous flour beetle Tribolium castaneum. Through creation of transgenic lines with fluorescent-tagged sperm heads, we followed the fate of focal male sperm in female reproductive tracts while tracking paternity across numerous rematings. Our results indicate that a given male’s sperm persist and fertilize eggs through at least seven rematings. Additionally, the proportion of a male’s sperm in the bursa (the site of spermatophore deposition), which is influenced by both timing of female’s ejecting excess sperm and male size, significantly predicted paternity share in the 24 h following a mating. Contrary to expectation, proportional representation of sperm within the female’s specialized sperm-storage organ did not significantly predict paternity, though spermathecal sperm may play a role in fertilization when females do not have access to mates for longer time periods. We address the adaptive significance of the identified reproductive mechanisms in the context of T. castaneum’s unique mating system and ecology.     

Pre‐ and postcopulatory sexual selection act in concert to determine male reproductive success in Tribolium castaneum

To capture how sexual selection shapes male reproductive success across different stages of reproduction in Tribolium castaneum (Coleoptera, Tenebrionidae), we combined sequential sperm defence (P1) and sperm offence (P2) trials with additional trials where both males were added simultaneously to the female. We found a positive correlation between the relative paternity share in simultaneous male–male competition trials and the P2 trial. This suggests that males preferred by females as sires achieve superior fertilization success during sperm competition in the second male position. In simultaneous male–male competition trials, where pre‐, peri‐ and postcopulatory sexual selection were all allowed to act, the relative paternity share of preferred males was more than 20% higher than in P2 sperm competition trials where precopulatory female choice was disabled. Additional behavioural observations revealed that mating with more attractive males resulted significantly more frequently in offspring production than mating with less attractive males. Thus, by comparing male fertilization success in trials where precopulatory choice was turned off with more inclusive estimates of fertilization success where pre‐ and pericopulatory choice could occur, we show that female mate choice may effectively inhibit sperm competition. Female mate choice and sperm competition (P2) are positively correlated, which is consistent with directional sexual selection in this species. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 67–75.     

A potential mechanism for cryptic female choice in a flour beetle

In polyandrous mating systems, events occurring during copulation can alter the fate of the mating male's sperm. These events may exert selective pressures resulting in the evolution of diverse reproductive behaviours, morphologies and physiologies. This study investigates the role of male and female copulatory behaviours on sperm fate in the red flour beetle, Tribolium castaneum. I describe and quantify copulatory behaviours for mating pairs, and examine sperm fate by quantifying sperm transfer, sperm storage and second male sperm precedence. Whereas female quiescence during copulation and male leg rubbing during copulation together account for significant variation (26%) in sperm precedence, female copulatory quiescence alone provides information about the timing and numbers of sperm transferred. These experiments show that a female copulatory behaviour predicts sperm fate, and emphasize the value of studying variation in both male and female copulatory behaviours for understanding factors affecting sperm use.     

Fecundity and MHC affects ejaculation tactics and paternity bias in sand lizards

We demonstrate that extending copulation enhances probability of paternity in sand lizards and that determinants of copulation duration depend on a males' mating order (first or second). First males, with no information on presence of rivals, extend copulation when mating with a more fecund female. Second males, however, adjust copula duration in relation to a first male's relatedness with his female, which there is reason to believe can be deduced from the MHC-related odor of the copulatory plug. Male-female relatedness negatively influences a male's probability of paternity, and when second males are in a favored role (i.e., the first male is the one more closely related to the female), second males transfer larger ejaculates, resulting in higher probability of paternity. This result corroborates predictions from recent theoretical models on sperm expenditure theory incorporating cryptic female choice and sexual conflict. More specifically, the results conform to a "random roles" model, which depicts males as being favored by some females and disfavored by others, but not to a "constant-type" model, in which a male is either favored or disfavored uniformly by all females in a population.     

Cryptic female choice during spermatophore transfer in Tribolium castaneum (Coleoptera: Tenebrionidae)

Sexual selection in both males and females promotes traits and behaviors that allow control over paternity when female mates with multiple males. Nonetheless, mechanisms of cryptic female choice have been consistently overlooked, due to traditional focus on sperm competition as well as difficulty in distinguishing male vs. female influence over processes occurring during and after mating. The first part of this study describes morphology and transformation of Tribolium castaneum spermatophores inferred from dissecting females immediately after normal or interrupted copulations. T. castaneum males are found to transfer spermatophores as an invaginated tube that everts inside the female bursa and which is filled with sperm during copulation. This sequence of events makes it feasible for females to control the sperm quantity transferred in each spermatophore. Through manipulation of the male phenotypic quality (by starvation) and manipulation of female control over sperm transfer (by killing a subset of females), the second part of this study examines whether females use control over transferred sperm quantity as a cryptic choice mechanism. Fed males transferred significantly more sperm per spermatophore than starved males but only when mating with live females. These results suggest an active differentiation by live females against starved males and provide an evidence for the proposed cryptic female choice mechanism.     

Pre‐ and postcopulatory sexual selection favor aggressive,young males in polyandrous groups of red junglefowl

A challenge in evolutionary biology is to understand the operation of sexual selection on males in polyandrous groups, where sexual selection occurs before and after mating. Here, we combine fine‐grained behavioral information (>41,000 interactions) with molecular parentage data to study sexual selection in replicated, age‐structured groups of polyandrous red junglefowl, Gallus gallus. Male reproductive success was determined by the number of females mated (precopulatory sexual selection) and his paternity share, which was driven by the polyandry of his female partners (postcopulatory sexual selection). Pre‐ and postcopulatory components of male reproductive success covaried positively; males with high mating success also had high paternity share. Two male phenotypes affected male pre‐ and postcopulatory performance: average aggressiveness toward rival males and age. Aggressive males mated with more females and more often with individual females, resulting in higher sexual exclusivity. Similarly, younger males mated with more females and more often with individual females, suffering less intense sperm competition than older males. Older males had a lower paternity share even allowing for their limited sexual exclusivity, indicating they may produce less competitive ejaculates. These results show that—in these populations—postcopulatory sexual selection reinforces precopulatory sexual selection, consistently promoting younger and more aggressive males.     

Sperm stratification and paternity success in red flour beetles

Abstract. When females are inseminated by multiple males, male paternity success (sperm precedence) is determined by the underlying processes of sperm storage and sperm utilization. Although informative for many questions, two-male sperm competition experiments may offer limited insight into natural mating scenarios when females are likely to mate with several males. In this study, genetic markers in Tribolium castaneum are used to trace paternity for multiple sires, and to determine whether displacement of stored sperm that occurs after a third mating equally affects both previous mates, or if fertilizations are disproportionately lost by the female's most recent mate. For 20 days after triple-matings, first males retain significantly higher paternity success (relative to first male paternity in double-matings) compared with second males. These results demonstrate that when females remate before sperm mixing occurs, sperm stratification results in differential loss of sperm from the most recent mate. This study provides insight into the mechanisms underlying sperm precedence in a promiscuous mating system, and suggests that T. castaneum females could limit paternity success of particular mates by remating with more highly preferred males.     

Evolutionary reduction in testes size and competitive fertilization success in response to the experimental removal of sexual selection in dung beetles

Sexual selection is thought to favor the evolution of secondary sexual traits in males that contribute to mating success. In species where females mate with more than one male, sexual selection also continues after copulation in the form of sperm competition and cryptic female choice. Theory suggests that sperm competition should favor traits such as testes size and sperm production that increase a male's competitive fertilization success. Studies of experimental evolution offer a powerful approach for assessing evolutionary responses to variation in sexual selection pressures. Here we removed sexual selection by enforcing monogamy on replicate lines of a naturally polygamous horned beetle, Onthophagus taurus, and monitoring male investment in their testes for 21 generations. Testes size decreased in monogamous lines relative to lines in which sexual selection was allowed to continue. Differences in testes size were dependent on selection history and not breeding regime. Males from polygamous lines also had a competitive fertilization advantage when in sperm competition with males from monogamous lines. Females from polygamous lines produced sons in better condition, and those from monogamous lines increased their sons condition by mating polygamously. Rather than being costly for females, multiple mating appears to provide females with direct and/or indirect benefits. Neither body size nor horn size diverged between our monogamous and polygamous lines. Our data show that sperm competition does drive the evolution of testes size in onthophagine beetles, and provide general support for sperm competition theory.     

Sexual selection in flour beetles: the relationship between sperm precedence and male olfactory attractiveness

Sperm precedence, defined as nonrandom differential fertilizationsuccess among mating males, is an important postmating componentof sexual selection. This study examined the relationship betweenpremating and postmating components of sexual selection in malesof the flour beetle (Tribolium castanewn). Male olfactory attractivenessto females was positively correlated with a male's subsequentfertilization success: more attractive males achieved highersecond-male sperm precedence when allowed to mate with previouslyinseminated females. Attractive males may achieve compoundedgains in their reproductive success through enhanced matingopportunities as well as through greater fertilization success.Thus, the relationship between these reproductive fitness componentsmay augment differences in reproductive success among males.Female fecundity, estimated as the number of adult progeny produced,increased significantly with multiple malings. This result supportsincreased female reproductive success as a direct benefit ofmultiple mating in T. caslaneum and suggests that progeny productionis partially limited by sperm availability. Total progeny productionby doubly mated females remained constant at all levels of second-malesperm precedence. However, higher sperm precedence was associatedwith a decline in firstmale progeny and a concomitant increasein second-male progeny. This pattern of progeny production suggeststhat more attractive males may achieve higher fertilizationsuccess through a combination of displacement of previouslystored sperm, transfer of greater sperm quantities, or females'preferential use of sperm of attractive males for fertilizations.     

Mating systems and protein–protein interactions determine evolutionary rates of primate sperm proteins

To assess the relative impact of functional constraint and post-mating sexual selection on sequence evolution of reproductive proteins, we examined 169 primate sperm proteins. In order to recognize potential genome-wide trends, we additionally analysed a sample of altogether 318 non-reproductive (brain and postsynaptic) proteins. Based on cDNAs of eight primate species (Anthropoidea), we observed that pre-mating sperm proteins engaged in sperm composition and assembly show significantly lower incidence of site-specific positive selection and overall lower non-synonymous to synonymous substitution rates (d_N/d_S) across sites as compared with post-mating sperm proteins involved in capacitation, hyperactivation, acrosome reaction and fertilization. Moreover, database screening revealed overall more intracellular protein interaction partners in pre-mating than in post-mating sperm proteins. Finally, post-mating sperm proteins evolved at significantly higher evolutionary rates than pre-mating sperm and non-reproductive proteins on the branches to multi-male breeding species, while no such increase was observed on the branches to unimale and monogamous species. We conclude that less protein–protein interactions of post-mating sperm proteins account for lowered functional constraint, allowing for stronger impact of post-mating sexual selection, while the opposite holds true for pre-mating sperm proteins. This pattern is particularly strong in multi-male breeding species showing high female promiscuity.     

Sexual conflict and evolution in Trinidadian guppies

Recent investigations have highlighted the importance of sexual conflict in the evolution of reproductive isolation. Examination of the Trinidadian guppy (Poecilia reticulata) shows how geographic variation in sexual conflict can mediate the emergence of isolating mechanisms. On the basis of pre-mating behaviour guppy females appear to be winning the battle of the sexes in low risk localities whereas males are apparently ahead in high-predation environments. However, the conclusion that sexual selection (through female choice) is replaced by sexual coercion of females (as a consequence of sneaky mating) in predator rich (and productive) assemblages takes no account of post-copulatory mechanisms. Recent work on sperm competition in guppies suggests coevolution between males and females may also occur in the post-mating, pre-zygotic arena. The potential for the evolution of reproductive isolation at each stage of the mating sequence is assessed.     

Benefits of polyandry: Molecular evidence from field‐caught dung beetles

When females mate with multiple males, they set the stage for postcopulatory sexual selection via sperm competition and/or cryptic female choice. Surprisingly little is known about the rates of multiple mating by females in the wild, despite the importance of this information in understanding the potential for postcopulatory sexual selection to drive the evolution of reproductive behaviour, morphology and physiology. Dung beetles in the genus Onthophagus have become a laboratory model for studying pre‐ and postcopulatory sexual selection, yet we still lack information about the reproductive behaviour of female dung beetles in natural populations. Here, we develop microsatellite markers for Onthophagus taurus and use them to genotype the offspring of wild‐caught females and to estimate natural rates of multiple mating and patterns of sperm utilization. We found that O. taurus females are highly polyandrous: 88% of females produced clutches sired by at least two males, and 5% produced clutches with as many as five sires. Several females (23%) produced clutches with significant paternity skew, indicating the potential for strong postcopulatory sexual selection in natural populations. There were also strong positive correlations between the number of offspring produced and both number of fathers and paternity skew, which suggests that females benefit from mating polyandrously by inciting postcopulatory mechanisms that bias paternity towards males that can sire more viable offspring. This study evaluates the fitness consequences of polyandry for an insect in the wild and provides strong evidence that female dung beetles benefit from multiple mating under natural conditions.     

How multiple mating by females affects sexual selection

Multiple mating by females is widely thought to encourage post-mating sexual selection and enhance female fitness. We show that whether polyandrous mating has these effects depends on two conditions. Condition 1 is the pattern of sperm utilization by females; specifically, whether, among females, male mating number, m (i.e. the number of times a male mates with one or more females) covaries with male offspring number, o. Polyandrous mating enhances sexual selection only when males who are successful at multiple mating also sire most or all of each of their mates'' offspring, i.e. only when Cov_♂(m,o), is positive. Condition 2 is the pattern of female reproductive life-history; specifically, whether female mating number, m, covaries with female offspring number, o. Only semelparity does not erode sexual selection, whereas iteroparity (i.e. when Cov_♀(m,o), is positive) always increases the variance in offspring numbers among females, which always decreases the intensity of sexual selection on males. To document the covariance between mating number and offspring number for each sex, it is necessary to assign progeny to all parents, as well as identify mating and non-mating individuals. To document significant fitness gains by females through iteroparity, it is necessary to determine the relative magnitudes of male as well as female contributions to the total variance in relative fitness. We show how such data can be collected, how often they are collected, and we explain the circumstances in which selection favouring multiple mating by females can be strong or weak.     

Female Mating History Influences Copulation Behavior but Not Sperm Release in the Orb-Weaving Spider <Emphasis Type="Italic">Tetragnatha versicolor</Emphasis> (Araneae,Tetragnathidae)

We examined the influence of female mating history on copulation behavior and sperm release in the haplogyne spider Tetragnatha versicolor. Despite significant behavioral differences during mating, males released equivalent amounts of sperm to virgin and non-virgin females. When mating with non-virgin females, males showed twice as many pedipalp insertions and half the copulation duration as compared to virgin females; however, males were as likely to mate with non-virgin as virgin females. Even with these overt behavioral differences, males released half of the sperm contained within their pedipalps during mating, regardless of female mating history. With respect to male mating order, first or second, we suggest the numbers of sperm released would lead to an expectation of unbiased paternity. In this species, sperm release is not directly proportional to total copulationduration.     

Postmating sexual selection: the effects of male body size and recovery period on paternity and egg production rate in a water strider

The role of male body size in postmating sexual selection wasexplored in a semiaquatic insect, the water strider Gerris lateralis.To separate effects of male size per se from those due to numericsperm competition, male recovery period (shown here to be proportionalto ejaculate size) was manipulated independently of body sizein a factorial experiment where virgin females were mated firstwith sterile males and then with focal males. Both relativemale fertilization success and female reproductive rate were measured.The number of sperm transferred increased with male recoveryperiod, an effect that was mediated by longer copulation duration,but there were no effects of body size on ejaculate size. Neithermale size nor recovery period had any significant direct effectson male fertilization success. However, copulation durationinfluenced relative fertilization success, suggesting that malesable to transfer more sperm also achieved higher fertilizationsuccess. Females exercised cryptic female choice by modulatingtheir reproductive rate in a manner favoring large males andmales that were successful in terms of achieving high relativefertilization success. Thus, successful males gained a twofoldadvantage in postmating sexual selection. This study has important implicationsfor previous estimates of sexual selection in this group of insectsbecause pre- and postmating sexual selection will be antagonisticdue to limitations in male sperm production: males mating frequently(high mating success) will on average transfer fewer sperm ineach mating and will hence tend to fertilize fewer eggs permating (low fertilization success).     

Polyandry and female control: the red flour beetle Tribolium castaneum as a case study

Females of many animal species are polyandrous, and there is evidence that they can control pre- and post-mating events. There has been a growing interest in consequences of polyandry for male and female reproductive success and offspring fitness, and its evolutionary significance. In several taxa, females exhibit mate choice both before and after mating and can influence the paternity of their offspring, enhancing offspring number and quality, but potentially countering male interests. Studying female mating biology and in particular post-copulatory female control mechanisms thus promises to yield insights into sexual selection and the potential of male-female coevolution. Here, we highlight the red flour beetle Tribolium castaneum (Herbst), a storage pest, as a model system to study polyandry, and review studies addressing the effects of polyandry on male sperm competitive ability and female control of post-mating events. These studies show that the outcome of sperm competition in the red flour beetle is influenced by both male and female traits. Furthermore, recent advances suggest that sexual conflict may have shaped reproductive traits in this species.     

Postcopulatory female choice increases the fertilization success of novel males in the field cricket, Gryllus vocalis

Although recent studies have demonstrated that female crickets prefer novel males to previous mates, the relative contribution of pre- and postcopulatory behaviors to this advantage remain unknown, as do the reproductive consequences to males. I paired females either with previous or novel mates, and recorded the latency to mating and the time after mating at which the female removed the male's spermatophore, terminating sperm transfer. Females that mated with familiar males removed their spermatophores sooner than females that mated with novel males. Females paired with novel males also mated more quickly than females paired with familiar males, but this difference was not statistically significant. A molecular-based paternity analysis was used to determine whether the postcopulatory preference of females for novel males influences a male's fertilization success. Females were assigned to either mate three times with the same male and then once with a novel male, or four times with four different males. The paternity of the last male was higher when the female previously had mated repeatedly with the same male than when she had mated previously with different males. These results suggest that female spermatophore removal behavior influences male paternity such that novel males receive a fertility benefit.     

Polyandry and postcopulatory sexual selection in a wild population

When females mate multiply, postcopulatory sexual selection can occur via sperm competition and cryptic female choice. Although postcopulatory selection has the potential to be a major force in driving evolution, few studies have estimated its strength in natural populations. Likewise, although polyandry is widespread across taxa and is the focus of a growing body of research, estimates of natural female mating rates are still limited in number. Microsatellites can be used to estimate the number of mates represented in females' sperm stores and the number of sires contributing to their offspring, enabling comparisons both of polyandry and of two components of postcopulatory selection: the proportion of males that mate but fail to sire offspring, and the degree of paternity skew among the males that do sire offspring. Here, we estimate the number of mates and sires among wild females in the Hawaiian swordtail cricket Laupala cerasina. We compare these estimates to the actual mating rates and paternity shares we observed in a semi‐natural population. Our results show that postcopulatory sexual selection operates strongly in this species: wild females mated with an average minimum of 3.6 males but used the sperm from only 58% of them. Furthermore, among the males that did sire offspring, paternity was significantly skewed. These patterns were similar to those observed in the field enclosure, where females mated with an average of 5.7 males and used the sperm from 62% of their mates, with paternity significantly skewed among the sires.     

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.     

The benefits of male ejaculate sex peptide transfer in Drosophila melanogaster

The accessory gland protein (Acp) ejaculate molecules of male Drosophila melanogaster mediate sexual selection and sexual conflict at the molecular level. However, to date no studies have comprehensively measured the timing and magnitude of fitness benefits to males of transferring specific Acps. This is an important omission because without this information it is not possible to fully understand the strength and form of selection acting on adaptations such as Acps. Here, we measured the fitness benefits to males of ejaculate sex peptide (SP) transfer. SP is of interest because it is a candidate for mediating sexual conflict: its frequent receipt reduces female fitness. In single matings with virgin females SP is known to increase egg laying and decrease receptivity. Hence, we predicted that SP could: (i) boost a male’s absolute paternity by increasing offspring production and delaying female remating and/or (ii) boost relative paternity share. We tested these predictions using two different lines of SP‐lacking males, in both two‐mating and free‐mating assay conditions. SP transfer conferred higher absolute, but not relative, male reproductive success. In matings with virgin females, SP transfer increased mating productivity and delayed remating and hence the onset of sperm competition. In already mated females, SP transfer did not elevate absolute progeny production, but did increase intermating intervals and hence the period over which a male could gain paternity. Consistent with this, under free‐mating conditions over an extended period, we detected a ‘per‐mating’ fitness benefit for males transferring SP. These benefits are consistent with a role for SP in mediating conflict, with SP acting to maximize short‐term fitness benefits for males.