An Orphan Gene Enhances Male Reproductive Success in Plutella xylostella.

Plutella xylostella exhibits exceptional reproduction ability, yet the genetic basis underlying the high reproductive capacity remains unknown. Here, we demonstrate that an orphan gene, lushu, which encodes a sperm protein, plays a crucial role in male reproductive success. Lushu is located on the Z chromosome and is prevalent across different P. xylostella populations worldwide. We subsequently generated lushu mutants using transgenic CRISPR/Cas9 system. Knockout of Lushu results in reduced male mating efficiency and accelerated death in adult males. Furthermore, our findings highlight that the deficiency of lushu reduced the transfer of sperms from males to females, potentially resulting in hindered sperm competition. Additionally, the knockout of Lushu results in disrupted gene expression in energy-related pathways and elevated insulin levels in adult males. Our findings reveal that male reproductive performance has evolved through the birth of a newly evolved, lineage-specific gene with enormous potentiality in fecundity success. These insights hold valuable implications for identifying the target for genetic control, particularly in relation to species-specific traits that are pivotal in determining high levels of fecundity.

Transcriptomic changes in the posterior pallium of male zebra finches associated with social niche conformance.

Animals plastically adjust their physiological and behavioural phenotypes to conform to their social environment-social niche conformance. The degree of sexual competition is a critical part of the social environment to which animals adjust their phenotypes, but the underlying genetic mechanisms are poorly understood. We conducted a study to investigate how differences in sperm competition risk affect the gene expression profiles of the testes and two brain areas (posterior pallium and optic tectum) in breeding male zebra finches (Taeniopygia castanotis). In this pre-registered study, we investigated a large sample of 59 individual transcriptomes. We compared two experimental groups: males held in single breeding pairs (low sexual competition) versus those held in two pairs (elevated sexual competition) per breeding cage. Using weighted gene co-expression network analysis (WGCNA), we observed significant effects of the social treatment in all three tissues. However, only the treatment effects found in the pallium were confirmed by an additional randomisation test for statistical robustness. Likewise, the differential gene expression analysis revealed treatment effects only in the posterior pallium (ten genes) and optic tectum (six genes). No treatment effects were found in the testis at the single gene level. Thus, our experiments do not provide strong evidence for transcriptomic adjustment specific to manipulated sperm competition risk. However, we did observe transcriptomic adjustments to the manipulated social environment in the posterior pallium. These effects were polygenic rather than based on few individual genes with strong effects. Our findings are discussed in relation to an accompanying paper using the same animals, which reports behavioural results consistent with the results presented here.

Sperm competition favours intermediate sperm size in a hermaphrodite1.

Sperm competition is a potent mechanism of postcopulatory sexual selection that has been found to shape reproductive morphologies and behaviours in promiscuous animals. Especially sperm size has been argued to evolve in response to sperm competition through its effect on sperm longevity, sperm motility, the ability to displace competing sperm, and ultimately fertilization success. Additionally, sperm size has been observed to co-evolve with female reproductive morphology. Theoretical work predicts that sperm competition may select for longer sperm but may also favour shorter sperm if sperm size trades-off with number. In this study, we studied the relationship between sperm size and postmating success in the free-living flatworm, Macrostomum lignano. Specifically, we used inbred isolines of M. lignano that varied in sperm size to investigate how sperm size translated into the ability of worms to transfer and deposit sperm in a mating partner. Our results revealed a hump-shaped relationship with individuals producing sperm of intermediate size having the highest sperm competitiveness. This finding broadens our understanding of the evolution of sperm morphology by providing empirical support for stabilizing selection on sperm size under sperm competition.

Experience matters: genetic variation affects male reproductive success across sequential mating events in Drosophila melanogaster.

The cost of reproduction is well studied in females but only recently have the costs of mating been investigated in males. Research suggests that males allocate resources between subsequent mating events, resulting in differential success across mating bouts. Selection should favor allocation strategies that match the likelihood of successive matings. The complexity of the system, however, suggests that one fixed strategy is unlikely to be universally favored and thus I predict that genetic variation for different allocation strategies will be segregating in natural populations. To test this, I measured several components of reproductive performance in eight inbred genotypes of Drosophila melanogaster across three sequential mating events. As predicted, there was genetic variation for how previous experience affected a male's reproductive performance for both the proportion of matings that produced offspring and the proportion of offspring sired (P1). Some genotypes had the highest success in their first matings and declined in successive matings while other genotypes did best in later matings. Mating experience had consistent effects across genotypes on fertility and induced refractoriness to remating. On average, virgin matings produced the highest fertility and third matings most effectively induced refractoriness. Genotype also had a significant effect on fertility. These results have important implications for understanding how selection may be acting on males when there is variation in the likelihood of multiple mating events and could affect the evolution of male allocation strategies in the face of perceived competitors.

The Effect of Brief or Prolonged Bouts of Winning or Losing Male-Male Contests on Plasticity in Sexually Selected Traits.

AbstractFight outcomes often affect male fitness by determining their access to mates. Thus, "winner-loser" effects, where winners often win their next contest while losers tend to lose, can influence how males allocate resources toward pre- and postcopulatory traits. We experimentally manipulated the winning/losing experiences of pairs of size-matched male Gambusia holbrooki for 1 day, 1 week, or 3 weeks to test whether prior winning/losing experiences differentially affect the plasticity of male investment into either mating effort (precopulatory) or ejaculates (postcopulatory). When winner/loser pairs directly competed for a female, winners had better precopulatory outcomes than losers for three of the four traits we measured: mating attempts, successful attempts, and time spent with the female (but not aggression). However, winners and losers did not differ in either total sperm counts or sperm velocity. Interestingly, absolute male size, an important predictor of fighting success, mediated winner-loser effects on how long males then spent near a female. Compared with losers, smaller winners spent more time with the female than did larger winners, suggesting that how males respond to prior social experiences is size dependent. We discuss the general importance of controlling for inherent male condition when comparing male investment into condition-dependent traits.

How Female-Female Competition Affects Male-Male Competition: Insights into Postcopulatory Sexual Selection from Socially Polyandrous Species.

AbstractSexual selection is a major driver of trait variation, and the intensity of male competition for mating opportunities has been linked with sperm size across diverse taxa. Mating competition among females may also shape the evolution of sperm traits, but the effect of the interplay between female-female competition and male-male competition on sperm morphology is not well understood. We evaluated variation in sperm morphology in two species with socially polyandrous mating systems, in which females compete to mate with multiple males. Northern jacanas (Jacana spinosa) and wattled jacanas (J. jacana) vary in their degree of social polyandry and sexual dimorphism, suggesting species differences in the intensity of sexual selection. We compared mean and variance in sperm head, midpiece, and tail length between species and breeding stages because these measures have been associated with the intensity of sperm competition. We found that the species with greater polyandry, northern jacana, has sperm with longer midpieces and tails as well as marginally lower intraejaculate variation in tail length. Intraejaculate variation was also significantly lower in copulating males than in incubating males, suggesting flexibility in sperm production as males cycle between breeding stages. Our results indicate that stronger female-female competition for mating opportunities may also shape more intense male-male competition by selecting for longer and less variable sperm traits. These findings extend frameworks developed in socially monogamous species to reveal that sperm competition may be an important evolutionary force layered atop female-female competition for mates.

Female Reproductive Fluid Increases the Opportunities for Postmating Sexual Selection by Prolonging the Egg Fertilization Window.

AbstractFemale reproductive fluid (the fluid that surrounds the eggs) has attracted increasing attention for its role in fertilization and postmating sexual selection through its effects on sperm traits. Surprisingly, however, only a few studies have investigated the effects of the female reproductive fluid on the eggs. Yet these effects might offer great potential to affect fertilization dynamics by, for example, increasing the opportunities for postmating sexual selection. Here, we determined whether the female reproductive fluid, by extending the egg fertilization window (the time available for egg fertilization), could also increase the opportunities for multiple paternity. Using the zebrafish (Danio rerio), we first tested the prediction that female reproductive fluid increases the egg fertilization window; then, using a split-brood design with the sperm of two males added at different time points after egg activation, we tested whether the degree of multiple paternity varies in the presence or absence of female reproductive fluid. Our results reveal the potential of female reproductive fluid to increase multiple paternity through its effects on the egg fertilization window, thus broadening our knowledge of how female mechanisms affect postmating sexual selection in externally fertilizing species.

Learning from Our Study Organisms about Sexual Selection: Lessons from the Ocellated Wrasse.

AbstractSexual selection is a powerful force shaping not only the details but also the breadth of what we see in nature. Yet so much unexplained variation remains. Organisms often solve the "problem" of how to pass on their genes in ways that do not fit our current expectations. I argue here that integrating empirical surprises will push our understanding of sexual selection forward. Such "nonmodel" organisms (i.e., species that do not do what we think they should do) challenge us to think deeply, integrate puzzling results, question our assumptions, and consider the new (and arguably better) questions these unexpected patterns pose. In this article, I share how puzzling observations from my long-term research on the ocellated wrasse (Symphodus ocellatus) have shaped my understanding of sexual selection and suggested new questions about the interplay among sexual selection, plasticity, and social interactions. My general premise, however, is not that others should study these questions. Instead, I argue for a change in the culture of our field-to consider unexpected results a welcome opportunity to generate new questions and learn new things about sexual selection. Those of us in positions of power (e.g., as editors, reviewers, and authors) need to lead the way.

Social Environment Influences the Temporal Dynamics of Sneak-Spawning in a Fish with Alternative Reproductive Tactics.

AbstractSeveral predictions of sperm competition theory are not well supported empirically. One potential reason is that most current theory and empirical research ignore how the social environment influence the temporal dynamics of mating. We propose that understanding these dynamics is key to understanding sexual selection and improving the predictive power of theory. To demonstrate the importance of these dynamics, we quantify how males' social role, interactions among males, and current social environment influence the timing of mating in Symphodus ocellatus, a species with three alternative male reproductive tactics. Nesting males spawn synchronously with females; sneakers and satellites sneak-spawn with some time delay. Satellites also cooperate with nesting males. We found that satellites have shorter sneak-spawning delays than sneakers, a benefit of their cooperation with nesting males. Sneak-spawning delays decreased with increasing nest activity for sneakers but not for satellites, suggesting that sneakers may benefit from increased sperm competition intensity. Current sperm competition models ignore this potential benefit, which may be why the prediction that males should decrease investment when sperm competition involves more than two males is not well supported. Our study provides insight into mechanisms that drive variation in the timing of spawning, which could explain mismatches between theoretical and empirical results.

A predominant role of genotypic variation in both expression of sperm competition genes and paternity success in Drosophila melanogaster.

Sperm competition is a crucial aspect of male reproductive success in many species, including Drosophila melanogaster, and seminal fluid proteins (Sfps) can influence sperm competitiveness. However, the combined effect of environmental and genotypic variation on sperm competition gene expression remains poorly understood. Here, we used Drosophila Genetic Reference Panel (DGRP) inbred lines and manipulated developmental population density (i.e. larval density) to test the effects of genotype, environment and genotype-by-environment interactions (GEI) on the expression of the known sperm competition genes Sex Peptide, Acp36DE and CG9997. High larval density resulted in reduced adult body size, but expression of sperm competition genes remained unaffected. Furthermore, we found no significant GEI but genotypic effects in the expression of SP and Acp36DE. Our results also revealed GEI for relative competitive paternity success (second male paternity; P2), with genes' expression positively correlated with P2. Given the effect of genotype on the expression of genes, we conducted a genome-wide association study (GWAS) and identified polymorphisms in putative cis-regulatory elements as predominant factors regulating the expression of SP and Acp36DE. The association of genotypic variation with sperm competition outcomes, and the resilience of sperm competition genes' expression against environmental challenges, demonstrates the importance of genome variation background in reproductive fitness.

Sperm competition increases sperm production and quality in Cataglyphis desert ants.

Sperm competition is a pervasive evolutionary force that shapes sperm traits to maximize fertilization success. Indeed, it has been shown to increase sperm production in both vertebrates and invertebrates. However, sperm production is energetically costly, which may result in trade-offs among sperm traits. In eusocial hymenopterans, such as ants, mating dynamics impose unique selective pressures on ejaculate. Males are sperm limited: they enter adulthood with a fixed amount of sperm that will not be renewed. We explored whether sperm competition intensity was associated with sperm quantity and quality (i.e. sperm viability and DNA fragmentation) in nine Cataglyphis desert ants. Our results provide phylogenetically robust evidence that sperm competition is positively correlated with sperm production and sperm viability. However, it was unrelated to sperm DNA integrity, indicating the absence of a trade-off involving this trait. These findings underscore that sperm competition may strongly mould sperm traits and drive reproductive performance in eusocial Hymenoptera.

Sperm production is negatively associated with muscle and sperm telomere length in a species subjected to strong sperm competition.

Life-history theory suggests that ageing is one of the costs of reproduction. Accordingly, a higher reproductive allocation is expected to increase the deterioration of both the somatic and the germinal lines through enhanced telomere attrition. In most species, males' reproductive allocation mainly regards traits that increase mating and fertilization success, that is sexually selected traits. In this study, we tested the hypothesis that a higher investment in sexually selected traits is associated with a reduced relative telomere length (RTL) in the guppy (Poecilia reticulata), an ectotherm species characterized by strong pre- and postcopulatory sexual selection. We first measured telomere length in both the soma and the sperm over guppies' lifespan to see whether there was any variation in telomere length associated with age. Second, we investigated whether a greater investment in pre- and postcopulatory sexually selected traits is linked to shorter telomere length in both the somatic and the sperm germinal lines, and in young and old males. We found that telomeres lengthened with age in the somatic tissue, but there was no age-dependent variation in telomere length in the sperm cells. Telomere length in guppies was significantly and negatively correlated with sperm production in both tissues and life stages considered in this study. Our findings indicate that telomere length in male guppies is strongly associated with their reproductive investment (sperm production), suggesting that a trade-off between reproduction and maintenance is occurring at each stage of males' life in this species.

Sperm of more colourful males are better adapted to ovarian fluids in lake char (Salmonidae).

Fish often spawn eggs with ovarian fluids that have been hypothesized to support the sperm of some males over others (cryptic female choice). Alternatively, sperm reactions to ovarian fluids could reveal male strategies. We used wild-caught lake char (Salvelinus umbla) to experimentally test whether sperm react differently to the presence of ovarian fluid, and whether any differential sperm reaction could be predicted by male breeding coloration, male inbreeding coefficients (based of 4150 SNPs) or the kinship coefficients between males and females. Male coloration was positively linked to body size and current health (based on lymphocytosis and thrombocytosis) but was a poor predictor of inbreeding or kinship coefficients. We found that sperm of more colourful males were faster in diluted ovarian fluids than in water only, while sperm of paler males were faster in water than in ovarian fluids. We then let equal numbers of sperm compete for fertilizations in the presence or absence of ovarian fluids and genetically assigned 1464 embryos (from 70 experimental trials) to their fathers. The presence of ovarian fluids significantly increased the success of the more colourful competitors. Sperm of less inbred competitors were more successful when tested in water only than in diluted ovarian fluids. The kinship coefficients had no significant effects on sperm traits or fertilization success in the presence of ovarian fluids, although parallel stress tests on embryos had revealed that females would profit more from mating with least related males rather than most coloured ones. We conclude that sperm of more colourful males are best adapted to ovarian fluids, and that the observed reaction norms suggest male strategies rather than cryptic female choice.

Trade-off between pre and post-copulatory traits depends on locomotor activity in male Tribolium castaneum beetles.

Locomotor performance is an indicator of dynamic exercise; thus, it is a central trait in many animal behaviours. Although higher locomotor endurance may increase male reproductive success (e.g., in mate searching and male-male contests), investment in other male reproductive traits (e.g., male attractiveness and sperm competition) may be decreased through energy consumption due to higher activity levels. Here, I investigated male attractiveness, mating success, and paternity success using males of the red flour beetle Tribolium castaneum selected for higher (H) and lower (L) locomotor endurance. Although there was no difference in male attractiveness between the selection regimes, H males had significantly higher mating success than L males. Conversely, L males had significantly higher paternity success than H males. Therefore, there was a trade-off between mating success and paternity success among the selection regimes, suggesting that locomotor endurance affects male reproduction in T. castaneum, and individual variation of locomotor endurance may be maintained within a population.

Social group composition modulates the role of last male sperm precedence in post-copulatory sexual selection.

In many species, the order in which males mate with a female explains much of the variation in paternity arising from post-copulatory sexual selection. Research in Drosophila suggests that mating order may account for the majority of the variance in male reproductive success. However, the effects of mating order on paternity bias might not be static but could potentially vary with social or environmental factors. To test this idea, we used an existing dataset, collated from an experiment we previously published (Morimoto et al., PLoS One, 11, 2016, e0154468), with the addition of unpublished data from the same experiment. These previous experiments manipulated larval density in Drosophila melanogaster which generated variation in male and female body size, assembled groups of individuals of different sizes, and measured the mating success and paternity share of focal males. The data presented here provides information on each focal male's mating order and the frequency in which focal males remated with same females ('repetitive matings'). We combined this information with our previously reported focal male reproductive success to partition variance in paternity into male mating order and repetitive matings across groups that differed in the body size composition of males and females. We found, as expected, that male mating order explained a considerable portion of the variance in male paternity. However, we also found that the impact of male mating order on male paternity was influenced by the body size composition of groups. Specifically, males that tended to mate last had a greater paternity advantage, and displayed lower variance, in groups containing a heterogenous mixture male body sizes than in groups with a single male body size. Repetitive mating only had a minor contribution to the variance in male paternity share across all experiments. Overall, our findings contribute to the growing body of research showing that post-copulatory sexual selection is subject to socio-ecological influences.

Sexual selection drives the coevolution of male and female reproductive traits in Peromyscus mice.

When females mate with multiple partners within a single reproductive cycle, sperm from rival males may compete for fertilization of a limited number of ova, and females may bias the fertilization of their ova by particular sperm. Over evolutionary timescales, these two forms of selection shape both male and female reproductive physiology when females mate multiply, yet in monogamous systems, post-copulatory sexual selection is weak or absent. Here, we examine how divergent mating strategies within a genus of closely related mice, Peromyscus, have shaped the evolution of reproductive traits. We show that in promiscuous species, males exhibit traits associated with increased sperm production and sperm swimming performance, and females exhibit traits that are predicted to limit sperm access to their ova including increased oviduct length and a larger cumulus cell mass surrounding the ova, compared to monogamous species. Importantly, we found that across species, oviduct length and cumulus cell density are significantly correlated with sperm velocity, but not sperm count or relative testes size, suggesting that these female traits may have coevolved with increased sperm quality rather than quantity. Taken together, our results highlight how male and female traits evolve in concert and respond to changes in the level of post-copulatory sexual selection.

Frequency-dependent viscosity of salmon ovarian fluid has biophysical implications for sperm-egg interactions.

Gamete-level sexual selection of externally fertilising species is usually achieved by modifying sperm behaviour with mechanisms that alter the chemical environment in which gametes perform. In fish, this can be accomplished through the ovarian fluid, a substance released with the eggs at spawning. While the biochemical effects of ovarian fluid in relation to sperm energetics have been investigated, the influence of the physical environment in which sperm compete remains poorly explored. Our objective was therefore to gain insights on the physical structure of this fluid and potential impacts on reproduction. Using soft-matter physics approaches of steady-state and oscillatory viscosity measurements, we subjected wild Atlantic salmon ovarian fluids to variable shear stresses and frequencies resembling those exerted by sperm swimming through the fluid near eggs. We show that this fluid, which in its relaxed state is a gel-like substance, displays a non-Newtonian viscoelastic and shear-thinning profile, where the viscosity decreases with increasing shear rates. We concurrently find that this fluid obeys the Cox-Merz rule below 7.6 Hz and infringes it above this level, thus indicating a shear-thickening phase where viscosity increases provided it is probed gently enough. This suggests the presence of a unique frequency-dependent structural network with relevant implications for sperm energetics and fertilisation dynamics. This article has an associated ECR Spotlight interview with Marco Graziano.

Male responses to sperm competition risk associated with increased macronutrient intake and reduced lifespan.

Increased expenditure on the ejaculate is a taxonomically widespread male response to sperm competition. Increased ejaculate expenditure is assumed to come at a cost to future reproduction, otherwise males should always invest maximally. However, the life-history costs of strategic ejaculation are not well documented. Macronutrient intake is known to affect the trade-off between reproduction and lifespan. Intakes of protein and carbohydrate that maximize reproduction often differ from those that maximize lifespan. Here, we asked whether strategic expenditure on the ejaculate by male crickets, Teleogryllus oceanicus, is mediated by macronutrient intake, and whether it comes at a cost of reduced lifespan. Males were exposed to rival song throughout their lifespan or were held in a silent non-competitive environment. Males exposed to song had a higher intake of both protein and carbohydrate, they reached adulthood sooner, produced ejaculates of higher quality, and died sooner than males living in a silent environment. Our findings provide a rare example of both the mechanisms and life-history costs associated with strategic ejaculation.

Female reproductive fluid and male seminal fluid: a non-gametic conflict for post-mating control.

Growing evidence shows that non-gametic components released by both males and females can significantly drive sperm competition outcomes. Seminal fluid (SF) was shown to influence paternity success by affecting rival males' sperm performance, and, in some species with male alternative reproductive tactics, to selectively decrease the fertilization success of males of the opposite tactic. Female reproductive fluid (FRF) has been proven to differentially influence ejaculates of different males and bias fertilization towards specific partners. Whether, and with what outcome, these two processes can intersect to influence sperm competition is still unknown. Here we explore this scenario in the grass goby (Zosterisessor ophiocephalus), a fish with territorial-sneaker reproductive tactics, where sneaker males can exploit the territorials' SF while penalizing territorial sperm performance with their own fluid. To test whether FRF can rebalance the ejaculate competition in favour of territorial males, we used in vitro fertilization with a SF mixture (territorial + sneaker), using increasing concentrations of FRF, to simulate the natural conditions that ejaculates encounter towards the eggs. Our findings revealed a differential effect of FRF on the different tactics' fertilization success, favouring territorial ejaculates, possibly through an attenuation of the detrimental effects of sneaker SF, and enabling females to regain control over the fertilization process.

Mating behavior and reproductive morphology predict macroevolution of sex allocation in hermaphroditic flatworms.

BACKGROUND: Sex allocation is the distribution of resources to male or female reproduction. In hermaphrodites, this concerns an individual's resource allocation to, for example, the production of male or female gametes. Macroevolutionary studies across hermaphroditic plants have revealed that the self-pollination rate and the pollination mode are strong predictors of sex allocation. Consequently, we expect similar factors such as the selfing rate and aspects of the reproductive biology, like the mating behaviour and the intensity of postcopulatory sexual selection, to predict sex allocation in hermaphroditic animals. However, comparative work on hermaphroditic animals is limited. Here, we study sex allocation in 120 species of the hermaphroditic free-living flatworm genus Macrostomum. We ask how hypodermic insemination, a convergently evolved mating behaviour where sperm are traumatically injected through the partner's epidermis, affects the evolution of sex allocation. We also test the commonly-made assumption that investment into male and female reproduction should trade-off. Finally, we ask if morphological indicators of the intensity of postcopulatory sexual selection (female genital complexity, male copulatory organ length, and sperm length) can predict sex allocation. RESULTS: We find that the repeated evolution of hypodermic insemination predicts a more female-biased sex allocation (i.e., a relative shift towards female allocation). Moreover, transcriptome-based estimates of heterozygosity reveal reduced heterozygosity in hypodermically mating species, indicating that this mating behavior is linked to increased selfing or biparental inbreeding. Therefore, hypodermic insemination could represent a selfing syndrome. Furthermore, across the genus, allocation to male and female gametes is negatively related, and larger species have a more female-biased sex allocation. Finally, increased female genital complexity, longer sperm, and a longer male copulatory organ predict a more male-biased sex allocation. CONCLUSIONS: Selfing syndromes have repeatedly originated in plants. Remarkably, this macroevolutionary pattern is replicated in Macrostomum flatworms and linked to repeated shifts in reproductive behavior. We also find a trade-off between male and female reproduction, a fundamental assumption of most theories of sex allocation. Beyond that, no theory predicts a more female-biased allocation in larger species, suggesting avenues for future work. Finally, morphological indicators of more intense postcopulatory sexual selection appear to predict more intense sperm competition.