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.

The role of male scent in female attraction in the bank vole, Myodes glareolus.

Chemical signals are frequently utilised by male mammals for intersexual communication and females are often attracted to male scent. However, the mechanism underlying female attraction has only been identified in a small number of mammalian species. Mammalian scents contain airborne volatiles, that are detected by receivers at a distance from the scent source, as well as non-volatile molecules, such as proteins, that require physical contact for detection. Lipocalin proteins, produced within the scent secretions of many terrestrial mammals, are thought to be particularly important in chemical signalling. Here, we explore if the male-specific protein, glareosin, expressed by adult male bank voles, Myodes glareolus, stimulates female attraction to male scent. We show that female bank voles are more attracted to male compared to female scent, supporting the results of previous studies. Increased investigation and attraction to male scent occurred to both airborne volatiles and non-volatile proteins when they were presented separately. However, we found no evidence that attraction to male scent was driven by glareosin. Our results differ from those previously described in house mice, where a single protein induces female attraction to male scent, suggesting the mechanism underlying female attraction to male scent differs between species.

Cryptic kin discrimination during communal lactation in mice favours cooperation between relatives.

Breeding females can cooperate by rearing their offspring communally, sharing synergistic benefits of offspring care but risking exploitation by partners. In lactating mammals, communal rearing occurs mostly among close relatives. Inclusive fitness theory predicts enhanced cooperation between related partners and greater willingness to compensate for any partner under-investment, while females are less likely to bias investment towards own offspring. We use a dual isotopic tracer approach to track individual milk allocation when familiar pairs of sisters or unrelated house mice reared offspring communally. Closely related pairs show lower energy demand and pups experience better access to non-maternal milk. Lactational investment is more skewed between sister partners but females pay greater energetic costs per own offspring reared with an unrelated partner. The choice of close kin as cooperative partners is strongly favoured by these direct as well as indirect benefits, providing a driver to maintain female kin groups for communal breeding.

Fitness Costs of Female Competition Linked to Resource Defense and Relatedness of Competitors.

AbstractFemale reproductive success is often limited by access to resources, and this can lead to social competition both within and between kin groups. Theory predicts that both resource availability and relatedness should influence the fitness consequences of social competition. However, testing key predictions requires differentiating the effects of these two factors. Here, we achieve this experimentally by manipulating the social environment of house mice, a facultative communal breeding species with known kin discrimination ability. This allows us to investigate (1) the reproductive costs of defending a limited resource in response to cues of social competition and (2) whether such costs, or their potential mitigation via cooperative behavior, are influenced by the relatedness of competitors. Our results support the hypothesis that resource defense can be costly for females, potentially trading off against maternal investment. When the availability of protected nest sites was limited, subjects (1) were more active, (2) responded more strongly to simulated territory intrusions via competitive signaling, and (3) produced smaller weaned offspring. However, we found no evidence that the propensity for kin to cooperate was influenced by the relatedness of rivals. Communal breeding between sisters occurred independently of the relatedness of competitors and communally breeding sisters weaned fewer offspring when competing with unrelated females, despite our study being designed to prevent infanticide between kin groups. Our findings thus demonstrate that female competition has fitness costs and that associating with kin is beneficial to avoid negative fitness consequences of competing with nonkin, in addition to more widely recognized kin-selected benefits.

Revealing mechanisms of mating plug function under sexual selection.

Mating plugs are produced by many sexually reproducing animals and are hypothesized to promote male fertilization success under promiscuous mating. However, tests of this hypothesis have been constrained by an inability to discriminate ejaculates of different males in direct competition. Here, we use stable isotope labeling in vivo and proteomics to achieve this in a promiscuous rodent, Myodes glareolus We show that, although the first male's plug is usually dislodged, it can be retained throughout the second male's copulation. Retained plugs did not completely block rival sperm but did significantly limit their numbers. Differences in the number of each male's sperm progressing through the female reproductive tract were also explained by natural variation in the size of mating plugs and reproductive accessory glands from which major plug proteins originate. Relative sperm numbers in turn predicted the relative fertilization success of rival males. Our application of stable isotopes to label ejaculates resolves a longstanding debate by revealing how rodent mating plugs promote fertilization success under competitive conditions. This approach opens new opportunities to reveal cryptic mechanisms of postcopulatory sexual selection among diverse animal taxa.

Social status and ejaculate composition in the house mouse.

Sperm competition theory predicts that males should tailor ejaculates according to their social status. Here, we test this in a model vertebrate, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our analyses reveal that both sperm production and the composition of proteins found in seminal vesicle secretions differ according to social status. Dominant males invested more in ejaculate production overall. Their epididymides contained more sperm than those of subordinate or control males, despite similar testes size between the groups. Dominant males also had larger seminal vesicle glands than subordinate or control males, despite similar body size. However, the seminal vesicle secretions of subordinate males had a significantly higher protein concentration than those of dominant males. Moreover, detailed proteomic analysis revealed subtle but consistent differences in the composition of secreted seminal vesicle proteins according to social status, involving multiple proteins of potential functional significance in sperm competition. These findings have significant implications for understanding the dynamics and outcome of sperm competition, and highlight the importance of social status as a factor influencing both sperm and seminal fluid investment strategies. This article is part of the theme issue 'Fifty years of sperm competition'.

Increased sperm production linked to competition in the maternal social environment.

Maternal or early life effects may prepare offspring for similar social conditions to those experienced by their mothers. For males, the ability to achieve mating and fertilization success is a key social challenge. Competitive conditions may therefore favour increased body size or ejaculate production in male offspring. We tested this experimentally by comparing reproductive traits of adult male bank voles (Myodes glareolus), whose mothers had experienced contrasting encounter regimes with female conspecifics while breeding. We found that daily sperm production rates and epididymis mass were significantly higher when dams had experienced more frequent encounters with female conspecifics. This response to maternal and early life experience was specific to sperm production and storage, with no evidence for effects on male body mass or the size of testes and accessory reproductive glands. Our findings reveal a potentially adaptive effect of maternal and early life experience on the development of sperm production, which is worthy of wider investigation.

Communal breeding affects offspring behaviours associated with a competitive social environment.

Communal breeding is characterised by shared care of offspring produced by more than one female, and can affect the behavioural development of young. The decision to care communally can vary according to local conditions, and has been hypothesised to occur more frequently when social competition is intense. However, it is unknown whether communal rearing of young influences adult behaviours likely to be adaptive under competitive conditions. Here, using a controlled experimental approach, we investigate effects of communal rearing on competitive and exploratory behaviours of adult male house mice. In tests of competitive scent marking, only communally-reared subjects discriminated between related and unrelated rivals, depositing more scent marks in close proximity to unrelated males. Communally-reared subjects also displayed higher exploratory tendencies, with an increased probability of crossing a water barrier, while not exhibiting higher activity levels in an open field test. Since exploration tendencies and discrimination between kin and non-kin are likely to be advantageous when dispersing from the natal territory or in a high density population, our findings suggest that communal rearing prepares male house mice for a competitive social environment. Our results add to growing evidence that the early social environment influences development of important behavioural competences to cope with social challenges later in life.

Female Chemical Signalling Underlying Reproduction in Mammals.

Chemical communication plays many key roles in mammalian reproduction, although attention has focused particularly on male scent signalling. Here, we review evidence that female chemical signals also play important roles in sexual attraction, in mediating reproductive competition and cooperation between females, and in maternal care, all central to female reproductive success. Female odours function not only to advertise sexual receptivity and location, they can also have important physiological priming effects on male development and sperm production. However, the extent to which female scents are used to assess the quality of females as potential mates has received little attention. Female investment in scent signalling is strongly influenced by the social structure and breeding system of the species. Although investment is typically male-biased, high competition between females can lead to a reversed pattern of female- biased investment. As among males, scent marking and counter-marking are often used to advertise territory defence and high social rank. Female odours have been implicated in the reproductive suppression of young or subordinate females across a range of social systems, with females of lower competitive ability potentially benefiting by delaying reproduction until conditions are more favourable. Further, the ability to recognise individuals, group members and kin through scent underpins group cohesion and cooperation in many social species, as well as playing an important role in mother-offspring recognition. However, despite the diversity of female scent signals, chemical communication in female mammals remains relatively understudied and poorly understood. We highlight several key areas of future research that are worthy of further investigation.

Sperm competition as an under-appreciated factor in domestication.

Humans created an environment that increased selective pressures on subgroups of those species that became domestic. We propose that the domestication process may in some cases have been facilitated by changes in mating behaviour and resultant sperm competition. By adapting to sperm competition, proto-domestic animals could potentially have outcompeted their wild counterparts in human-constructed niches. This could have contributed to the restriction of gene flow between the proto-domesticates and their wild counterparts, thereby promoting the fixation of other domestication characteristics. Further to this novel perspective for domestication, we emphasize the general potential of postcopulatory sexual selection in the restriction of gene flow between populations, and urge more studies.

Paternal care and litter size coevolution in mammals.

Biparental care of offspring occurs in diverse mammalian genera and is particularly common among species with socially monogamous mating systems. Despite numerous well-documented examples, however, the evolutionary causes and consequences of paternal care in mammals are not well understood. Here, we investigate the evolution of paternal care in relation to offspring production. Using comparative analyses to test for evidence of evolutionary associations between male care and life-history traits, we explore if biparental care is likely to have evolved because of the importance of male care to offspring survival, or if evolutionary increases in offspring production are likely to result from the evolution of biparental care. Overall, we find no evidence that paternal care has evolved in response to benefits of supporting females to rear particularly costly large offspring or litters. Rather, our findings suggest that increases in offspring production are more likely to follow the evolution of paternal care, specifically where males contribute depreciable investment such as provisioning young. Through coevolution with litter size, we conclude that paternal care in mammals is likely to play an important role in stabilizing monogamous mating systems and could ultimately promote the evolution of complex social behaviours.

Proteome Dynamics: Tissue Variation in the Kinetics of Proteostasis in Intact Animals.

Understanding the role of protein turnover in the maintenance of proteostasis requires accurate measurements of the rates of replacement of proteins in complex systems, such as intact animals. Moreover, any investigation of allometric scaling of protein turnover is likely to include species for which fully annotated proteomes are not available. We have used dietary administration of stable isotope labeled lysine to assess protein turnover rates for proteins from four tissues in the bank vole,Myodes glareolus The annotated genome for this species is not available, so protein identification was attained through cross-species matching to the mouse. For proteins for which confident identifications were derived, the pattern of lysine incorporation over 40 days was used to define the rate of synthesis of individual proteins in the four tissues. The data were heavily filtered to retain a very high quality dataset of turnover rates for 1088 proteins. Comparative analysis of the four tissues revealed different median rates of degradation (kidney: 0.099 days(-1); liver 0.136 days(-1); heart, 0.054 days(-1), and skeletal muscle, 0.035 days(-1)). These data were compared with protein degradation rates from other studies on intact animals or from cells in culture and indicate that both cell type and analytical methodology may contribute to variance in turnover data between different studies. These differences were not only due to tissue-specific proteins but were reflected in gene products common to all tissues. All data are available via ProteomeXchange with identifier PXD002054.

Cross-species proteomics in analysis of mammalian sperm proteins.

UNLABELLED: Many proteomics studies are conducted in model organisms for which fully annotated, detailed, high quality proteomes are available. By contrast, many studies in ecology and evolution are conducted in species which lack high quality proteome data, limiting the perceived value of a proteomic approach for protein discovery and quantification. This is particularly true of rapidly evolving proteins in the reproductive system, such as those that have an immune function or are under sexual selection, and can compromise the potential for cross-species proteomics to yield confident identification. In this investigation we analysed the sperm proteome, from a range of ungulates and rodents, and explored the potential of routine proteomic workflows to yield characterisation and quantification in non-model organisms. We report that database searching is robust to cross-species matching for a mammalian core sperm proteome, comprising 623 proteins that were common to most of the 19 species studied here, suggesting that these proteins are likely to be present and identifiable across many mammalian sperm. Further, label-free quantification reveals a consistent pattern of expression level. Functional analysis of this core proteome suggests consistency with previous studies limited to model organisms and has value as a quantitative reference for analysis of species-specific protein characterisation. SIGNIFICANCE: From analysis of the sperm proteome for diverse species (rodents and ungulates) using LC-MS/MS workflows and standard data processing, we show that it is feasible to obtain cross-species matches for a large number of proteins that can be filtered stringently to yield a highly expressed mammalian sperm core proteome, for which label-free quantitative data are also used to inform protein function and abundance.

Sperm competition risk drives plasticity in seminal fluid composition.

BACKGROUND: Ejaculates contain a diverse mixture of sperm and seminal fluid proteins, the combination of which is crucial to male reproductive success under competitive conditions. Males should therefore tailor the production of different ejaculate components according to their social environment, with particular sensitivity to cues of sperm competition risk (i.e. how likely it is that females will mate promiscuously). Here we test this hypothesis using an established vertebrate model system, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our study tests for the first time how both sperm and seminal fluid components of the ejaculate are tailored to the social environment. RESULTS: Our quantitative proteomics analysis reveals that the relative production of different proteins found in seminal fluid--i.e. seminal fluid proteome composition--differs significantly according to cues of sperm competition risk. Using a conservative analytical approach to identify differential expression of individual seminal fluid components, at least seven of 31 secreted seminal fluid proteins examined showed consistent differences in relative abundance under high versus low sperm competition conditions. Notably three important proteins with potential roles in sperm competition--SVS 6, SVS 5 and CEACAM 10--were more abundant in the high competition treatment groups. Total investment in both sperm and seminal fluid production also increased with cues of heightened sperm competition risk in the social environment. By contrast, relative investment in different ejaculate components was unaffected by cues of mating opportunities. CONCLUSIONS: Our study reveals significant plasticity in different ejaculate components, with the production of both sperm and non-sperm fractions of the ejaculate strongly influenced by the social environment. Sperm competition risk is thus shown to be a key factor in male ejaculate production decisions, including driving plasticity in seminal fluid composition.

The Genetic Basis of Kin Recognition in a Cooperatively Breeding Mammal.

Cooperation between relatives yields important fitness benefits, but genetic loci that allow recognition of unfamiliar kin have proven elusive. Sharing of kinship markers must correlate strongly with genome-wide similarity, creating a special challenge to identify specific loci used independently of other shared loci. Two highly polymorphic gene complexes, detected through scent, have been implicated in vertebrates: the major histocompatibility complex (MHC), which could be vertebrate wide, and the major urinary protein (MUP) cluster, which is species specific. Here we use a new approach to independently manipulate sharing of putative genetic kin recognition markers, with the animal itself or known family members, while genome-wide relatedness is controlled. This was applied to wild-stock outbred female house mice, which nest socially and often rear offspring cooperatively with preferred nest partners. Females preferred to nest with sisters, regardless of prior familiarity, confirming the use of phenotype matching. Among unfamiliar relatives, females strongly preferred nest partners that shared their own MUP genotype, though not those with only a partial (single-haplotype) MUP match to themselves or known family. In the absence of MUP sharing, females preferred related partners that shared multiple loci across the genome to unrelated females. However, MHC sharing was not used, even when MHC type completely matched their own or that of known relatives. Our study provides empirical evidence that highly polymorphic species-specific kinship markers can evolve where reliable recognition of close relatives is an advantage. This highlights the potential for identifying other genetic kinship markers in cooperative species and calls for better evidence that MHC can play this role.

Sexual conflict and sperm competition.

Traits that increase a male's fertilization success during sperm competition can be harmful to females and therefore represent a source of sexual conflict. In this review, we consider the variety of male adaptations to sperm competition (MASC) that may give rise to sexual conflict-including mate guarding, prolonged copulations, the transfer of large numbers of sperm, and the manipulation of females through nonsperm components of the ejaculate. We then reflect on the fitness economics influencing the escalation of these sexual conflicts, considering the likelihood of females evolving traits to offset the negative effects of MASC when compared with the strong selection on males that lead to MASC. We conclude by discussing the potential evolutionary outcomes of sexual conflict arising from MASC, including the opportunities for females to mitigate conflict costs and the prospects for conflict resolution.

Sequential male mate choice under sperm competition risk.

Male eagerness to mate is a central paradigm of sexual selection theory. However, limited sperm supplies mean that male sexual restraint might sometimes be favored under promiscuous mating. Here, we demonstrate dynamic plasticity in male mating effort when females are encountered sequentially under varying sperm competition risk. Rather than showing consistent eagerness to mate, male house mice (Mus musculus domesticus) instead tailor their mating effort according to likely reproductive payoffs. They are significantly less likely to mate when sperm competition is certain and potential reproductive payoffs low, but dramatically increase investment if they do choose to mate under such circumstances. By contrast, male mice are significantly more likely to mate in situations simulating extra-territorial copulations, where future risk of competition is high but so too are potential reproductive rewards. Differential mating propensity appears to be the primary mechanism by which male house mice allocate sperm adaptively under sperm competition risk because we find no evidence for facultative adjustment of sperm numbers per ejaculate or ejaculation frequency in response to female-related cues. We conclude that sequential male mate choice under sperm competition risk could be a widespread but often unappreciated mechanism of strategic sperm allocation.

Female competition and aggression: interdisciplinary perspectives.

This paper introduces a Theme Issue combining interdisciplinary perspectives in the study of female competition and aggression. Despite a history of being largely overlooked, evidence is now accumulating for the widespread evolutionary significance of female competition. Here, we provide a synthesis of contributions to this Theme Issue on humans and other vertebrates, and highlight directions for future research. Females compete for resources needed to survive and reproduce, and for preferred mates. Although female aggression takes diverse forms, under most circumstances relatively low-risk competitive strategies are favoured, most probably due to constraints of offspring production and care. In social species, dominance relationships and threats of punishment can resolve social conflict without resort to direct aggression, and coalitions or alliances may reduce risk of retaliation. Consistent with these trends, indirect aggression is a low cost but effective form of competition among young women. Costs are also minimized by flexibility in expression of competitive traits, with aggressive behaviour and competitive signalling tailored to social and ecological conditions. Future research on female competition and the proximate mediators of female aggression will be greatly enhanced by opportunities for interdisciplinary exchange, as evidenced by contributions to this Theme Issue.

Wake up and smell the conflict: odour signals in female competition.

Odour signals used in competitive and aggressive interactions between males are well studied in the context of sexual selection. By contrast, relatively little is known about comparable signals used by females, despite current interest in the evolution of female ornaments and weaponry. Available evidence suggests that odour signals are important in competitive interactions between female mammals, with reductions or reversals of male-biased sexual dimorphism in signalling where female competition is intense. Scent marking is often associated with conflict between females over access to resources or reproductive opportunities. Female scent marks may therefore provide reliable signals of competitive ability that could be used both by competitors and potential mates. Consistent with this hypothesis, we report that aggressive behaviour of female house mice is correlated with the amount of major urinary protein (MUP) excreted in their urine, a polymorphic set of proteins that are used in scent mark signalling. Under semi-natural conditions, females with high MUP output are more likely to produce offspring sired by males that have high reproductive success, and less likely to produce offspring by multiple different sires, suggesting that females with strong MUP signals are monopolized by males of particularly high quality. We conclude that odour signals are worthy of more detailed investigation as mediators of female competition.