Population density effects on gamete traits and fertilisation dynamics under varying sperm environments in mussels.

Gamete traits can vary widely among species, populations and individuals, influencing fertilisation dynamics and overall reproductive fitness. Sexual selection can play an important role in determining the evolution of gamete traits with local environmental conditions determining the strength and direction of sexual selection. Here, we test for signatures of post-mating selection on gamete traits in relation to population density, and possible interactive effects of population density and sperm concentration on sperm motility and fertilisation rates among natural populations of mussels. Our study shows that males from high-density populations produce smaller sperm compared with males from low-density populations, but we detected no effect of population density on egg size. Our results also reveal that females from low-density populations tended to exhibit lower fertilisation rates across a range of sperm concentrations, although this became less important as sperm concentration increased. Variances in fertilisation success were higher for females than males and the effect of gamete compatibility between males and females increases as sperm concentrations increase. These results suggest that local population density can influence gamete traits and fertilisation dynamics but also highlight the importance of phenotypic plasticity in governing sperm-egg interactions in a highly dynamic selective environment.

Systematic approaches to assessing high-temperature limits to fertility in animals.

Critical thermal limits (CTLs) gauge the physiological impact of temperature on survival or critical biological function, aiding predictions of species range shifts and climatic resilience. Two recent Drosophila species studies, using similar approaches to determine temperatures that induce sterility (thermal fertility limits [TFLs]), reveal that TFLs are often lower than CTLs and that TFLs better predict both current species distributions and extinction probability. Moreover, many studies show fertility is more sensitive at less extreme temperatures than survival (thermal sensitivity of fertility [TSF]). These results present a more pessimistic outlook on the consequences of climate change. However, unlike CTLs, TFL data are limited to Drosophila, and variability in TSF methods poses challenges in predicting species responses to increasing temperature. To address these data and methodological gaps, we propose 3 standardized approaches for assessing thermal impacts on fertility. We focus on adult obligate sexual terrestrial invertebrates but also provide modifications for other animal groups and life-history stages. We first outline a "gold-standard" protocol for determining TFLs, focussing on the effects of short-term heat shocks and simulating more frequent extreme heat events predicted by climate models. As this approach may be difficult to apply to some organisms, we then provide a standardized TSF protocol. Finally, we provide a framework to quantify fertility loss in response to extreme heat events in nature, given the limitations in laboratory approaches. Applying these standardized approaches across many taxa, similar to CTLs, will allow robust tests of the impact of fertility loss on species responses to increasing temperatures.

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.

Female reproductive fluid concentrations affect sperm performance of alternative male phenotypes in an external fertilizer.

There is growing evidence that the female reproductive fluid (FRF) plays an important role in cryptic female choice through its differential effect on the performance of sperm from different males. In a natural spawning event, the male(s) may release ejaculate closer or further away from the spawning female. If the relative spatial proximity of competing males reflects the female pre-mating preference towards those males, then favoured males will encounter higher concentrations of FRF than unpreferred males. Despite this being a common situation in many external fertilizers, whether different concentrations of FRF can differentially influence the sperm performance of distinct male phenotypes (favoured and unfavoured by the female) remains to be elucidated. Here, we tested this hypothesis using the grass goby (Zosterisessor ophiocephalus), a fish with distinct territorial-sneaker reproductive tactics and female pre-mating preference towards territorial males, that consequently mate in an advantaged position and whose sperm experience higher concentrations of FRF. Our findings revealed a differential concentration-dependent effect of FRF over sneaker and territorial sperm motility only at low concentrations (i.e. at the distance where sneakers typically ejaculate), with increasing FRF concentrations (i.e. close to the eggs) similarly boosting the sperm performance of both sneaker and territorial males. The ability to release sperm close to the eggs is a prerogative of territorials, but FRF can likewise advantage the sperm of those sneakers that are able to get closer, allowing flexibility in the direction of female post-mating choice.

Female reproductive fluid attracts more and better sperm: implications for within-ejaculate cryptic female choice.

Mounting evidence shows that the female reproductive fluid (FRF) can differently affect sperm performance of different males by biasing paternity share among competing males. Here, we tested for the first time the potential of 'within-ejaculate cryptic female choice' mediated by the FRF in the zebrafish (Danio rerio). Using a recently developed sperm selection chamber, we separated and collected FRF-selected from non-selected sperm to compare the two subpopulations of sperm in terms of sperm number, viability, DNA integrity and fertilizing ability. We showed that the sperm attracted by FRF are more numerous, more viable and with higher DNA integrity. In addition, FRF-selected sperm fertilized more eggs, but if this is due to fertilization ability per se or numerical advantage remains to be tested. Our results suggest that FRF can select sperm with a better phenotype, highlighting the crucial and impactful role that FRF might play in the process of fertilization and post-mating sexual selection dynamics, along with the potential implications for sperm selection in assisted reproductive techniques.

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.

Ecology of fear in highly invasive fish revealed by robots.

Invasive species threaten biodiversity and ecosystem functioning. We develop an innovative experimental approach, integrating biologically inspired robotics, time-series analysis, and computer vision, to build a detailed profile of the effects of non-lethal stress on the ecology and evolution of mosquitofish (Gambusia holbrooki)-a global pest. We reveal that brief exposures to a robotic predator alter mosquitofish behavior, increasing fear and stress responses, and mitigate the impact of mosquitofish on native tadpoles (Litoria moorei) in a cause-and-effect fashion. Effects of predation risk from the robot carry over to routine activity and feeding rate of mosquitofish weeks after exposure, resulting in weight loss, variation in body shape, and reduction in the fertility of both sexes-impairing survival, reproduction, and ecological success. We capitalize on evolved responses of mosquitofish to reduce predation risk-neglected in biological control practices-and provide scientific foundations for widespread use of state-of-the-art robotics in ecology and evolution research.

Sperm Accumulation Induced by the Female Reproductive Fluid: Putative Evidence of Chemoattraction Using a New Tool.

There is considerable evidence that female reproductive fluid (FRF) interacts intimately with sperm, affecting several sperm traits, including sperm motility and longevity, and ultimately fertilization success. One of the first documented interactions between FRF and sperm is the ability of FRF to attract and guide sperm towards the eggs. However, most of the evidence of FRF's chemoattraction proprieties comes from a limited number of taxa, specifically mammals and invertebrate broadcasting spawners. In other species, small FRF volumes and/or short sperm longevity often impose methodological difficulties resulting in this gap in chemoattraction studies in non-model species. One of the outcomes of sperm chemotaxis is sperm accumulation towards high chemoattractant concentrations, which can be easily quantified by measuring sperm concentration. Here, we tested sperm accumulation towards FRF in the zebrafish, Danio rerio, using an ad hoc developed, 3D printed, device ('sperm selection chamber'). This easy-to-use tool allows to select and collect the sperm that swim towards a chemical gradient, and accumulate in a chemoattractant-filled well thus providing putative evidence for chemoattraction. We found that sperm accumulate in FRF in zebrafish. We also found that none of the sperm quality traits we measured (sperm swimming velocity and trajectory, sperm motility, and longevity) were correlated with this response. Together with the 3D printable project, we provide a detailed protocol for using the selection chamber. The chamber is optimized for the zebrafish, but it can be easily adapted for other species. Our device lays the foundation for a standardized way to measure sperm accumulation and in general chemoattraction, stimulating future research aimed at understanding the role and the mechanisms of sperm chemoattraction by FRF.

Male sperm storage impairs sperm quality in the zebrafish.

Variation in sperm traits is widely documented both at inter- and intraspecific level. However, sperm traits vary also between ejaculates of the same male, due for example, to fluctuations in female availability. Variability in the opportunities to mate can indeed have important consequences for sperm traits, as it determines how often sperm are used, and thus the rate at which they are produced and how long they are stored before the mating. While being stored within males' bodies, sperm are subjected to ageing due to oxidative stress. Sperm storage may significantly impair sperm quality, but evidence linking male sperm storage and variation in sperm traits is still scarce. Here, we tested the effect of the duration of sperm storage on within-male variation in sperm traits in the zebrafish, Danio rerio. We found that without mating opportunities, sperm number increased as storage duration increased, indicating that sperm continue to be produced and accumulate over time within males without being discharged in another way. Long sperm storage (12 days) was associated with an overall impairment in sperm quality, namely sperm motility, sperm longevity, and sperm DNA fragmentation, indicating that sperm aged, and their quality declined during storage. Our results confirm that male sperm storage may generate substantial variation in sperm phenotype, a source of variation which is usually neglected but that should be accounted for in experimental protocols aiming to assay sperm traits or maximise fertilization success.

The role of female reproductive fluid in sperm competition.

The role of non-gametic components of the ejaculate (seminal fluid) in fertility and sperm competitiveness is now well established. Surprisingly, however, we know far less about female reproductive fluid (FRF) in the context of sexual selection, and insights into male-FRF interactions in the context of sperm competition have only recently emerged. Despite this limited knowledge, evidence from taxonomically diverse species has revealed insights into the effects of FRF on sperm traits that have previously been implicated in studies of sperm competition. Specifically, through the differential effects of FRF on a range of sperm traits, including chemoattraction and alterations in sperm velocity, FRF has been shown to exert positive phenotypic effects on the sperm of males that are preferred as mating partners, or those from the most compatible or genetically diverse males. Despite these tantalizing insights into the putative sexually selected functions of FRF, we largely lack a mechanistic understanding of these processes. Taken together, the evidence presented here highlights the likely ubiquity of FRF-regulated biases in fertilization success across a diverse range of taxa, thus potentially elevating the importance of FRF to other non-gametic components that have so far been studied largely in males. This article is part of the theme issue 'Fifty years of sperm competition'.

Molecular insights into post-mating immune response in a fish with internal fertilization.

The tight connection between immunity and reproduction has been studied for decades. However, basic knowledge at the molecular level of the effect of mating on immune function is still lacking in many taxa. Determining whether and how the immune system is engaged after mating is a crucial step in understanding post-mating mechanisms of reproduction and sexual selection. Here, we study the transcriptional changes in immunity-related genes caused by the ejaculate in the female reproductive tract using a model species for sexual selection studies, the guppy Poecilia reticulata. To study changes triggered by the ejaculate only, rather than caused by mating, we used artificial inseminations to transfer ejaculate into females. We then compared gene expression in the reproductive tract (gonoduct and ovary) of females artificially inseminated either with ejaculate or with a control solution, after 1 hr and after 6 hr. Overall, contact with ejaculate caused short-term changes in the expression of immune-related genes in the female reproductive tract, with a complex pattern of up- and down-regulation of immune-related pathways, but with clear indication of a marked down-regulation of the immune system shortly after ejaculate contact. This suggests a link between immune function and processes occurring between mating and fertilization in this species.

The bold and the sperm: positive association between boldness and sperm number in the guppy.

Assessing the consequences of personality traits on reproductive success is one of the most important challenges in personality studies and critical to understand the evolutionary implications of behavioural variability among animals. Personality traits are typically associated with mating acquisition in males, and, hence, linked to variation in their reproductive success. However, in most species, sexual selection continues after mating, and sperm traits (such as sperm number and quality) become very important in determining post-mating competitive success. Here, we investigate whether variation in personality traits is associated with variation in sperm traits using the guppy (Poecilia reticulata), a species with high levels of sperm competition. We found a positive association between boldness and sperm number but not sperm velocity, suggesting that bolder males have increased post-copulatory success than shyer individuals. No association was found between exploration and sperm traits. Our work highlights the importance of considering post-copulatory traits when investigating fitness consequences of personality traits, especially in species with high levels of female multiple matings and hence sperm competition.

Sexual selection and ageing: interplay between pre- and post-copulatory traits senescence in the guppy.

Traits associated with mating and fertilization success are expected to senesce with age, but limited information is available on their relative rates of senescence. In polyandrous species, male reproductive fitness depends on both mating and fertilization success. Because successful mating is a prerequisite for post-copulatory sexual selection, ejaculate traits are expected to senesce faster than pre-copulatory traits, as pre-copulatory sexual selection is often deemed to be stronger than post-copulatory sexual selection. This pattern has generally been found in the few empirical studies conducted so far. We tested this prediction in the guppy ( Poecilia reticulata), a livebearing fish characterized by intense sperm competition, by comparing the expression of male sexual traits at two ages (four and nine months). Contrary to prediction, we found that post-copulatory traits senesced at a significantly slower rate than pre-copulatory traits. We also looked at whether early investment in those sexual traits affects longevity, and the interaction between sperm age (duration of sperm storage inside the male) and male age. Our results suggest that the relative senescence rate of pre- and post-copulatory sexual traits may vary among species with different mating systems and ecology.

Female control over multiple matings increases the opportunity for postcopulatory sexual selection.

It is widely acknowledged that in most species sexual selection continues after mating. Although it is generally accepted that females play an important role in generating paternity biases (i.e. cryptic female choice, CFC), we lack a quantitative understanding of the relative importance of female-controlled processes in influencing variance in male reproductive fitness. Here, we address this question experimentally using the guppy Poecilia reticulata, a polyandrous fish in which pre- and postcopulatory sexual selection jointly determine male reproductive fitness. We used a paired design to quantify patterns of paternity for pairs of rival males across two mating contexts, one in which the female retained full control over double (natural) matings and one where sperm from the same two males were artificially inseminated into the female. We then compared the relative paternity share for a given pair of males across both contexts, enabling us to test the key prediction that patterns of paternity will depend on the extent to which females retain behavioural control over matings. As predicted, we found stronger paternity biases when females retained full control over mating compared with when artificial insemination (AI) was used. Concomitantly, we show that the opportunity for postcopulatory sexual selection (standardized variance in male reproductive success) was greater when females retained control over double matings compared with when AI was used. Finally, we show that the paternity success of individual males exhibited higher repeatability across successive brood cycles when females retained behavioural control of matings compared with when AI was used. Collectively, these findings underscore the critical role that females play in determining the outcome of sexual selection and to our knowledge provide the first experimental evidence that behaviourally moderated components of CFC increase the opportunity for sexual selection.

Extreme fertilization bias towards freshly inseminated sperm in a species exhibiting prolonged female sperm storage.

The storage of sperm by females across successive reproductive cycles is well documented in internal fertilizers, yet the fate of stored sperm when they compete with 'new' sperm to fertilize a female's eggs has rarely been considered. This gap in our understanding is likely due to the logistical difficulties of controlling behavioural interactions during or after mating, which in turn may influence how many sperm are inseminated and how stored sperm are ultimately used during successive bouts of sperm competition with freshly inseminated sperm. Here, we use artificial insemination (AI) in guppies (Poecilia reticulata), a polyandrous live-bearing poeciliid fish exhibiting prolonged sperm storage by females, to overcome these challenges. The use of AI enables us to control potential differential maternal effects (e.g. behaviourally mediated cryptic female choice) and specifically test for post-copulatory paternity biases that favour either stored or fresh sperm when they compete to fertilize eggs. Our paternity analyses revealed the almost complete dominance of freshly inseminated sperm over stored sperm, supporting previous studies reporting similar patterns following natural matings across successive brood cycles. However, our use of AI, which excluded behavioural interactions between males and females, most likely generated a far stronger pattern of fresh sperm precedence compared with those reported in previous studies, possibly implicating 'cryptic' forms of selection by females that may sometimes bolster the success of stored sperm.

Population demography and heterozygosity-fitness correlations in natural guppy populations: An examination using sexually selected fitness traits.

Heterozygosity-fitness correlations (HFCs) have been examined in a wide diversity of contexts, and the results are often used to infer the role of inbreeding in natural populations. Although population demography, reflected in population-level genetic parameters such as allelic diversity or identity disequilibrium, is expected to play a role in the emergence and detectability of HFCs, direct comparisons of variation in HFCs across many populations of the same species, with different genetic histories, are rare. Here, we examined the relationship between individual microsatellite heterozygosity and a range of sexually selected traits in 660 male guppies from 22 natural populations in Trinidad. Similar to previous studies, observed HFCs were weak overall. However, variation in HFCs among populations was high for some traits (although these variances were not statistically different from zero). Population-level genetic parameters, specifically genetic diversity levels (number of alleles, observed/expected heterozygosity) and measures of identity disequilibrium (g2 and heterozygosity-heterozygosity correlations), were not associated with variation in population-level HFCs. This latter result indicates that these metrics do not necessarily provide a reliable predictor of HFC effect sizes across populations. Importantly, diversity and identity disequilibrium statistics were not correlated, providing empirical evidence that these metrics capture different essential characteristics of populations. A complex genetic architecture likely underpins multiple fitness traits, including those associated with male fitness, which may have reduced our ability to detect HFCs in guppy populations. Further advances in this field would benefit from additional research to determine the demographic contexts in which HFCs are most likely to occur.

Sperm as moderators of environmentally induced paternal effects in a livebearing fish.

Until recently, paternal effects-the influence of fathers on their offspring due to environmental factors rather than genes-were largely discarded or assumed to be confined to species exhibiting paternal care. It is now recognized that paternal effects can be transmitted through the ejaculate, but unambiguous evidence for them is scarce, because it is difficult to isolate effects operating via changes to the ejaculate from maternal effects driven by female mate assessment. Here, we use artificial insemination to disentangle mate assessment from fertilization in guppies, and show that paternal effects can be transmitted to offspring exclusively via ejaculates. We show that males fed reduced diets produce poor-quality sperm and that offspring sired by such males (via artificial insemination) exhibit reduced body size at birth. These findings may have important implications for the many mating systems in which environmentally induced changes in ejaculate quality have been reported.

Postmating Female Control: 20 Years of Cryptic Female Choice.

Cryptic female choice (CFC) represents postmating intersexual selection arising from female-driven mechanisms at or after mating that bias sperm use and impact male paternity share. Although biologists began to study CFC relatively late, largely spurred by Eberhard's book published 20 years ago, the field has grown rapidly since then. Here, we review empirical progress to show that numerous female processes offer potential for CFC, from mating through to fertilization, although seldom has CFC been clearly demonstrated. We then evaluate functional implications, and argue that, under some conditions, CFC might have repercussions for female fitness, sexual conflict, and intersexual coevolution, with ramifications for related evolutionary phenomena, such as speciation. We conclude by identifying directions for future research in this rapidly growing field.

Sperm storage by males causes changes in sperm phenotype and influences the reproductive fitness of males and their sons.

Recent studies suggest that environmentally induced effects on sperm phenotype can influence offspring phenotype beyond the classic Mendelian inheritance mechanism. However, establishing whether such effects are conveyed purely through ejaculates, independently of maternal environmental effects, remains a significant challenge. Here, we assess whether environmentally induced effects on sperm phenotype affects male reproductive success and offspring fitness. We experimentally manipulated the duration of sperm storage by males, and thus sperm age, in the internally fertilizing fish Poecilia reticulata. We first confirm that sperm ageing influences sperm quality and consequently males reproductive success. Specifically, we show that aged sperm exhibit impaired velocity and are competitively inferior to fresh sperm when ejaculates compete to fertilize eggs. We then used homospermic (noncompetitive) artificial insemination to inseminate females with old or fresh sperm and found that male offspring arising from fertilizations by experimentally aged sperm suffered consistently impaired sperm quality when just sexually mature (four months old) and subsequently as adults (13 months old). Although we have yet to determine whether these effects have a genetic or epigenetic basis, our analyses provide evidence that environmentally induced variation in sperm phenotype constitutes an important source of variation in male reproductive fitness that has far reaching implications for offspring fitness.