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The environment that parents experience can influence their reproductive output and their offspring’s fitness via parental effects. Perceived predation risk can affect both parent and offspring phenotype, but it remains unclear to what extent offspring behavioral traits are affected when the mother is exposed to predation risk. This is particularly unclear in live-bearing species where maternal effects could occur during embryogenesis. Here, using a half-sib design to control for paternal effects, we experimentally exposed females of a live-bearing fish, the guppy (Poecilia reticulata), to visual predator cues and conspecific alarm cues during their gestation. Females exposed to predation risk cues increased their antipredator behaviors throughout the entire treatment. Offspring of mothers exposed to the predation stimuli exhibited more pronounced exploratory behavior, but did not show any significant differences in their schooling behavior, compared to controls. Thus, while maternally perceived risk affected offspring’s exploration during early stages of life, offspring’s schooling behavior could be influenced more by direct environmental experience rather than via maternal cues. Our results suggest a rather limited role in predator-induced maternal effects on the behavior of juvenile guppies.

The perception of predation risk could affect prey phenotype both within and between generations (via parental effects). The response to predation risk could involve modifications in physiology, morphology, and behavior and can ultimately affect long‐term fitness. Among the possible modifications mediated by the exposure to predation risk, telomere length could be a proxy for investigating the response to predation risk both within and between generations, as telomeres can be significantly affected by environmental stress. Maternal exposure to the perception of predation risk can affect a variety of offspring traits but the effect on offspring telomere length has never been experimentally tested. Using a live‐bearing fish, the guppy (Poecilia reticulata), we tested if the perceived risk of predation could affect the telomere length of adult females directly and that of their offspring with a balanced experimental setup that allowed us to control for both maternal and paternal contribution. We exposed female guppies to the perception of predation risk during gestation using a combination of both visual and chemical cues and we then measured female telomere length after the exposure period. Maternal effects mediated by the exposure to predation risk were measured on offspring telomere length and body size at birth. Contrary to our predictions, we did not find a significant effect of predation‐exposure neither on female nor on offspring telomere length, but females exposed to predation risk produced smaller offspring at birth. We discuss the possible explanations for our findings and advocate for further research on telomere dynamics in ectotherms., Telomere length could be a proxy for investigating the response to predation risk both within and between generations. We tested if the perception of predation risk could affect the telomere length of adult females and their offspring using the guppy, Poecilia reticulata, a live‐bearing fish. Contrary to our predictions, we did not find a significant effect of predation‐exposure neither on female mothers nor on offspring telomere length, but female mothers exposed to predation risk produced smaller offspring at birth.

In many species, males can rapidly adjust their ejaculate performance in response to changing levels of sperm competition, an ability that is probably mediated by seminal fluid adaptive plasticity. In the black goby, Gobius niger , territorial males attach viscous ejaculate trails to the nest roof, from which sperm are slowly released into the water during the long-lasting spawning events. Sneaker males release their sperm in the vicinity of the nest, and territorial males try to keep them at a distance by patrolling their territory. We show here that territorial males' ejaculate trails released a higher proportion of their sperm in the presence of a single sneaker, but this proportion decreased when there were three sneakers, an effect that is most likely mediated by a change in the seminal fluid composition. Field observations showed that when multiple sneaking attempts occurred, territorial males spent more time outside the nest, suggesting that ejaculation rate and territory defence are traded-off. Altogether, these results suggest that the adjustment of sperm release from the ejaculate may be strategic, guaranteeing a more continuous concentration of the territorial male's sperm in the nest, although at a lower level, when he is engaged in prolonged territory defence outside the nest.

Although it is often expected that adverse environmental conditions depress the expression of condition-dependent sexually selected traits, the full consequences of environmental change for the action of sexual selection, in terms of the opportunity for total sexual selection and patterns of phenotypic selection, are unknown. Here we show that dietary stress in guppies, Poecilia reticulata, reduces the expression of several sexually selected traits and increases the opportunity for total sexual selection (standardized variance in reproductive success) in males. Furthermore, our results show that dietary stress modulates the relative importance of precopulatory (mating success) and postcopulatory (relative fertilization success) sexual selection, and that the form of multivariate sexual selection (linear vs. nonlinear) depends on dietary regime. Overall, our results are consistent with a pattern of heightened directional selection on condition-dependent sexually selected traits under environmental stress, and underscore the importance of sexual selection in shaping adaptation in a changing world., This work was funded by the University of Padova (Progetto CPDA120105 and Assegno di Ricerca BIRD 2017) to A.P. S.C. was supported by a Postgraduate Scholarship through The University of Padova, and F.G.G. by the Spanish Ministry of Economy (CGL2016-76173-P AEI/FEDER, UE). J.P.E. was supported by a sabbatical grant funded by the University of Western Australia.

Males of many species evolved the capability of adjusting their ejaculate phenotype in response to social cues to match the expected mating conditions. When females store sperm for a prolonged time, the expected fitness return of plastic adjustments of ejaculate phenotype may depend on the interval between mating and fertilization. Although prolonged female sperm storage (FSS) increases the opportunity for sperm competition, as a consequence of the longer temporal overlap of ejaculates from several males, it may also create variable selective forces on ejaculate phenotype, for example by exposing trade-offs between sperm velocity and sperm survival. We evaluated the relationship between the plasticity of ejaculate quality and FSS in the guppy, Poecilia reticulata, a polyandrous live-bearing fish in which females store sperm for several months and where stored sperm contribute significantly to a male's lifelong reproductive success. In this species, males respond to the perception of future mating opportunities by increasing the quantity (number) and quality (swimming velocity) of ready-to-use sperm (an anticipatory response called 'sperm priming'). Here we investigated (a) the effect of sperm priming on in vitro sperm viability at stripping and its temporal decline (as an estimate of sperm survival), and (b) the in vivo competitive fertilization success in relation to female sperm storage using artificial insemination. As expected, sperm-primed males produced more numerous and faster sperm, but with a reduced in vitro sperm viability at stripping and after 4 hr, compared with their counterparts. Artificial insemination revealed that the small (nonsignificant) advantage of primed sperm when fertilization immediately follows insemination is reversed when eggs are fertilized by female-stored sperm, weeks after insemination. By suggesting a plastic trade-off between sperm velocity and viability, these results demonstrate that prolonged female sperm storage generates divergent selection pressures on ejaculate phenotype.

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 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'.

In polyandrous species males invest significant resources in producing large and high-quality ejaculates. As ejaculates are costly, males are expected to modulate their investment in response to social cues associated with the expected level of sperm competition or mating opportunity, to anticipate future mating conditions. Another consequence of ejaculate costs is that the increase in ejaculate production may be traded against traits linked to mate acquisition. In such cases, the effect of this anticipatory plasticity in ejaculate investment on a male's reproductive success will depend on the balance between postcopulatory benefits and precopulatory costs in the sociosexual context subsequently encountered. Here, we used the guppy, Poecilia reticulata, a live-bearing fish characterized by intense sperm competition, to investigate how mismatches between anticipatory phenotypes and the social conditions subsequently experienced affect male reproductive success. Male guppies kept in visual contact with females, a social stimulus predicting high mating opportunities, increase their sperm production compared to female-deprived males which foresee fewer opportunities to mate, but simultaneously reduce their courtship rate. Using a paired experimental design, we manipulated the male's perceived mating opportunities by housing them in the presence (FP) or in the absence (FA) of females. We then measured male mating and insemination success under two conditions: (1) a female-biased sex ratio, matching expected FP cues; and (2) a male-biased sex ratio, matching expected FA cues. Under a female-biased sex ratio, mating and insemination success were not affected by previous exposure to females, although FP males experienced a lower risk of sperm depletion after several copulations. However, FP males showed a small, but significant, reduction in their mating success under a male-biased sex ratio, when directly competing with an FA male. Males showed significant repeatability in mating and insemination success within a sex ratio context, suggesting that the costs of mismatching anticipatory ejaculate responses are probably low in this species.

This study considered possible sexual dimorphism in the relative lengths of the second, third and fourth digits (digit ratio), in calves. Furthermore, a different length of the bone structures of the third (3D) and of the fourth (4D) digits has been examined as an evolutionary adaptation to locomotion on soft ground. The length of the digital bones of the right fore-limb of 33 females and 15 male calves was measured in vivo using a portable X-ray machine. The vestigial structure of the second digit (2D), and 3D and 4D, from metacarpus to the third phalanx were considered in a mixed model, as well as some ratios between 2D and different parts of 3D or 4D (2D:3D and 2D:4D). A covariate for the mean finger length was considered for digit ratios to control for possible biases due to shape allometry. Shorter first phalanx and trotter were found in 3D than in 4D, and the reverse for the third phalanx. The 2D was significantly shorter in females, as well as the second phalanges of 3D and 4D. Significant sex differences in 2D:3D and 2D:4D were found for some digit parts of 3D and 4D and for the first phalanges of 3D:4D. These ratios were always shorter in females, in contrast to that found in most mammals. The asymmetry between 3D and 4D could mean a functional adaptation for locomotion. Sex differences in 2D:4D and 3D:4D were found, but with a reverse pattern than in most mammal species (malesfemales rather than malesfemales). In this regard digit ratio in calves was similar to that of Old World monkeys. This study is the first investigation of digit ratio in Ungulates, whose limbs differ from the limbs of most mammals, maintaining five digits. The reverse pattern of sex differences (digit ratios: malesfemales) could be due to the peculiar nature of the vestigial dewclaw of 2D and to the hormone patterns acting on this digit during development, but further research is required around this topic.

Sperm competition is taxonomically widespread in animals and is usually associated with large sperm production, being the number of sperm in the competing pool the prime predictor of fertilization success. Despite the strong postcopulatory selection acting directionally on sperm production, its genetic variance is often very high. This can be explained by trade-offs between sperm production and traits associated with mate acquisition or survival, that may contribute to generate an overall stabilizing selection. To investigate this hypothesis, we first artificially selected male guppies (Poecilia reticulata) for high and low sperm production for three generations, while simultaneously removing sexual selection. Then, we interrupted artificial selection and restored sexual selection. Sperm production responded to divergent selection in one generation, and when we restored sexual selection, both high and low lines converged back to the mean sperm production of the original population within two generations, indicating that sperm number is subject to strong stabilizing total sexual selection (i.e., selection acting simultaneously on all traits associated with reproductive success). We discuss the possible mechanisms responsible for the maintenance of high genetic variability in sperm production despite strong selection acting on it.

Producing sperm is costly and males have been selected to strategically adjust their sperm production and/or expenditure according to the fitness return associated with a specific mating. For example, males respond to fluctuations in the mating opportunities by adjusting the number of “ready” sperm. This phenomenon is known as “sperm priming” and is interpreted as a strategy to economize the investment in sperm. The cost and benefits of the sperm priming response, however, are expected to depend on a male’s baseline sperm production (BSP) in the absence of females, because of the different risk of sperm depletion and the nonlinearly increasing costs of sperm production. We tested this prediction in 2 replicated lines of male guppies Poecilia reticulata that were artificially selected for high and low BSP. BSP has a large genetic variance and a high sire heritability in guppies, and males respond to the perceived mating opportunities by increasing the number of “ready” sperm. We investigated whether males with a different BSP differed in their sperm priming response. We found that when the perceived mating opportunities increased, males from low-sperm lines had a stronger sperm priming response than those from high-sperm lines. This result suggests that adaptive plasticity in sperm priming has the potential to evolve in response to different levels of BSP. The comparison between guppy populations with different levels of sperm production would allow to test whether the pattern reported here is also observed at the interpopulation level.

Identifying mechanisms of reproductive isolation is key to understanding speciation. Among the putative mechanisms underlying reproductive isolation, sperm-female interactions (postmating-prezygotic barriers) are arguably the hardest to identify, not least because these are likely to operate at the cellular or molecular level. Yet sperm-female interactions offer great potential to prevent the transfer of genetic information between different populations at the initial stages of speciation. Here we provide a preliminary test for the presence of a putative postmating-prezygotic barrier operating between three populations of Trinidadian guppies (Poecilia reticulata), an internally fertilizing fish that inhabits streams with different levels of connectivity across Trinidad. We experimentally evaluate the effect of female ovarian fluid on sperm velocity (a predictor of competitive fertilization success) according to whether males and females were from the same (native) or different (foreign) populations. Our results reveal the potential for ovarian fluid to act as a postmating-prezygotic barrier between two populations from different drainages, but also that the strength of this barrier is different among populations. This result may explain the previous finding that, in some populations, sperm from native males have precedence over foreign sperm, which could eventually lead to reproductive isolation between these populations. This article is protected by copyright. All rights reserved.

Many fish species are social, and individuals spend most of their lives in shoals, but sociability can vary greatly between species, populations and individuals. Sociability has been largely studied by measuring the time spent by a focal fish in proximity to one or more conspecifics. To control for the behaviour of the stimulus fish, the conspecifics have often been substituted by a mirror, on the assumption that the subject perceives its mirror image as a conspecific. The validity of the mirror test has recently been questioned, at both the behavioural and the molecular level, because of the discrepancy in fish responses when exposed to a mirror image and to a live conspecific. In this study, we compared the sociability scores of a social fish, the guppy, Poecilia reticulata, obtained using live fish or a mirror as a stimulus, to assess the validity of the mirror test. We found that the sociability score assessed using the standard mirror test was not significantly correlated with the sociability assessed using live stimuli. Nevertheless, we observed a positive correlation between the scores of the two tests when the mirror test was performed in a more naturalistic context, controlling for the minimum distance between the stimulus fish and the mirror. Our findings provide evidence of the reliability of the mirror test as a measure of sociability compared to the test using live conspecifics when certain requirements are satisfied.

In species where females mate with more than one male during the same reproductive event, males typically increase the number of sperm produced to boost their fertilization share. Sperm is not limitless, however, and theory predicts that their production will come at the cost of other fitness-related traits, such as body growth or immunocompetence, although these evolutionary trade-offs are notoriously difficult to highlight. To this end, we combined artificial selection for sperm production with a transcriptome analysis using Poecilia reticulata, a fish characterized by intense sperm competition in which the number of sperm transferred during mating is the most important predictor of fertilization success, yet sperm production is highly variable among males. We compared the brain and testes transcriptome in male guppies of lines artificially selected for high and low sperm production by identifying pivotal differentially expressed gene sets that may regulate spermatogenesis and immune function in this species. Despite the small differences in single genes' expression, gene set enrichment analysis showed coordinated gene expression differences associated with several pathways differentially regulated in the two selection lines. High sperm production males showed an upregulation of pathways related to immunosuppression and development of spermatozoa indicating a possible immunological cost of sperm production.

Many animals, from humans to invertebrates, exhibit sexual preference for unfamiliar partners. This may reduce the risk of inbreeding and increase offspring heterozygosity. An alternative less tested hypothesis is that selection for neophilia in other contexts (e.g. exploration of unfamiliar environments) may promote mate preference for unfamiliar partners. We tested whether exploratory tendency covaries with female preference for unfamiliar mates in the guppy, Poecilia reticulata, a species in which exploratory behaviour show significant phenotypic and genetic variation. We measured the exploratory behaviour of females using an unfamiliar environment test. The same females were also tested for preference for an unfamiliar male over a male with which they had spent the previous 24 h. We found that a female's tendency to explore an unfamiliar environment was positively correlated with the strength of her preference for unfamiliar males. Our results support the hypothesis that exploration might play a role in the evolution and maintenance of preference for unfamiliar males via functional pleiotropy.

Female sperm storage (FSS) is taxonomically widespread and often associated with intense sperm competition, yet its consequences on postcopulatory sexual selection (PCSS) are poorly known. Theory predicts that FSS will reduce the strength of PCSS, because sperm characteristics favored before and after FSS may be traded-off, and opportunities for nondirectional PCSS should increase. We explored these questions in the guppy (Poecilia reticulata), by allowing females to mate multiply and by comparing the paternity pattern in two successive broods. Contrary to predictions, the variance in male fertilization success increased after FSS, driven by a change in male paternity share across broods. This change was positively associated with sperm velocity (measured before FSS) but not with the duration of FSS, indirectly suggesting that faster sperm were better in entering female storage organs, rather than in persisting within them. Other male traits, such as male size and orange color, heterozygosity, and relatedness to the female, did not influence paternity after FSS. These results indicate that processes associated with FSS tend to reinforce the strength of PCSS in guppies, rather than weaken it. Further work is necessary to test whether this pattern changes in case of more prolonged FSS.

Fifty years of research on sperm competition has led to a very good understanding of the interspecific variation in sperm production traits. The reasons why this variation is often very large within populations have been less investigated. We suggest that the interaction between fluctuating environmental conditions and polyandry is a key phenomenon explaining such variation. We focus here on imminent predation risk (IPR). IPR impacts significantly several aspects of prey behaviour and reproduction, and it is expected to influence the operation of sexual selection before and after mating. We estimated the effect of IPR on the male opportunity for pre- and postcopulatory sexual selection in guppies ( Poecilia reticulata ), a livebearing fish where females prefer colourful males and mate multiply. We used a repeated-measures design, in which males were allowed to mate with different females either under IPR or in a predator-free condition. We found that IPR increased the total opportunity for sexual selection and reduced the relative contribution of postcopulatory sexual selection to male reproductive success. IPR is inherently variable and our results suggest that interspecific reproductive interference by predators may contribute towards maintaining the variation in sperm production within populations. This article is part of the theme issue ‘Fifty years of sperm competition'.

According to theory, in species in which male variance in reproductive success exceeds that of the females, sons are more costly to produce; females mated with high quality males or those in better condition should produce more sons. In monogamous species, however, the variance in the reproductive success of the two sexes is often similar and mate choice is often mutual, making predictions regarding sex allocation more difficult. In the rock sparrow Petronia petronia, both males and females have a sexually selected yellow patch on the breast, whose size correlates with individual body condition. We investigated whether the brood sex ratio co‐varies with the size of the yellow patch of the father and the mother in a sample of 173 broods (818 chicks) over 8 breeding seasons. While the size of the yellow patch of the mother and the father did not predict per se a deviation from the expected 1:1 sex ratio, brood sex ratios were predicted by the interaction of male and female yellow patch size. This result is surprising, as the ornament is sexually selected by both males and females as an indicator of quality in both sexes and should therefore be inherited by all offspring irrespective of their sex. It indirectly suggests that other sex‐specific traits associated with patch size (e.g. polygyny in males and fecundity in females) may explain the sex allocation bias observed in rock sparrows. Thus, female individual quality alone, as expressed through the size of the yellow patch, was not associated with the biases in sex ratios reported in this study. Our results rather suggest that sex allocation occurs in response to male attractiveness in interaction with female attractiveness. In other words, females tend to preferentially allocate towards the sex of the parent with more developed ornament within the pair., AC was funded by a Juan de la Cierva‐formación postdoctoral grant (FJCI‐2015‐23536; Ministerio de Economía, Industria y Competitividad, MINECO, Spanish Government).

Being flexible in reproductive decisions is highly beneficial in variable environments, but flexibility may also entail costs. Male guppies modify their sperm production and sexual behavior when mating opportunities vary. We investigated lifetime effects of these responses. We show that repeatedly adjusting reproductive strategies has little cost for male guppies, as it does not compromise their condition, their lifespan, or the expression of sexual traits.

In several species, males increase their mating and sperm investment in the presence of unfamiliar females, the so-called Coolidge effect. Such an elevated reproductive effort is expected to be associated with a decreased investment in other costly traits, such as somatic growth and maintenance. How precopulatory, postcopulatory, and somatic investments interact one with each other, however, has been rarely evaluated simultaneously. We used the guppy (Poecilia reticulata), a polyandrous livebearing fish with alternative male mating tactics, to compare these 3 investments between males whose encounter rate with unfamiliar females was experimentally maintained at either high (high mate encounter rate [HER]) or low rate (low mate encounter rate [LER]) for 4 months. At the end of this period, HER males showed an increased sperm production as compared with their LER counterparts. This increment was accompanied with a reduction in the time spent following the female and by a shift from costly courtship displays to gonopodial thrusting, a less expensive coercive mating tactic. This effect may indicate a trade-off between these 2 components of male reproductive investment or may reflect a change in male optimal reproductive strategy associated with different mate encounter rate. In contrast, body growth was positively correlated with the increase in sperm production, and did not differ between experimental groups. Collectively, these results suggest that males did not vary their overall reproductive investment in response to their encounter rate with unfamiliar females, but differed significantly in the amount of resources available individually.

Several factors are involved in determining the outcome of sperm competition. In addition to sperm number, sperm quality and male phenotype, insemination order is often associated with skewed paternity share. Patterns of sperm precedence can be produced by the mechanics of sperm storage and fertilization, or by active processes under male or female control. However, as males and females always interact during copulation, it is difficult to identify the mechanism responsible. The Trinidadian guppy, Poecilia reticulata, is a polyandric species characterized by last-male sperm precedence in natural matings. During such matings, females allow attractive males to inseminate more sperm by controlling copulation duration. We used artificial insemination to clarify the extent to which female control of sperm transfer influences the observed pattern of sperm precedence in this species. This technique allowed us to experimentally manipulate the number of sperm transferred and the timing of insemination. We found a significant first-male fertilization advantage. This advantage, however, declined as the time between insemination and parturition increased. Presumably, the anatomy and the physiology of the female genital tract favour egg fertilization by the first ejaculate inseminated, whereas sperm mixing is likely to be responsible for the reduction in first-male advantage associated with longer insemination?parturition intervals. Our results suggest that the last-male precedence detected after two consecutive natural matings is caused by cryptic female preference for attractive males associated with a female trading-up strategy (i.e. the second male is more frequently more attractive than the first male), rather than by insemination order per se. As the pattern of sperm precedence has important consequences for male reproductive strategies (for example mate guarding and male mate choice copying), unravelling its dynamic represents an important contribution to understanding the sexual behaviour of this model species. Fil: Magris, Martina. Università di Padova; Italia Fil: Cardozo Milanesio, Gabriela Alejandra. Università di Padova; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Santi, Francesco. Royal Holloway University Of London; . Università di Padova; Italia Fil: Devigili, Alessandro. Stockholms Universitet; Suecia. Università di Padova; Italia Fil: Pilastro, Andrea. Università di Padova; Italia

The function and maintenance of the immune system impose costs that can take the form of energy consumption and/or physiological side effects. These, in turn, can affect the expression of several traits, including traits closely related to fitness. The interaction of the immune system with other life-history traits (e.g. growth and survival) and with precopulatory sexually selected traits (e.g. male ornaments) has been widely studied, whereas little is known about its interplay with postcopulatory sexually selected traits, such as sperm characteristics. We studied the effects of an immune challenge on the expression of precopulatory and postcopulatory traits in the guppy, Poecilia reticulata. We injected two groups of males with a lipopolysaccharide (LPS) solution or a saline solution (control) and compared the effects of the immune system activation on traits’ expression. The results showed a specific negative effect of the LPS injection on sperm traits, whereas precopulatory ornaments were lowered merel...

Parents are expected to strategically partition their limited resources among the current and future progeny in order to maximize their fitness. Since an equal investment in offspring of different reproductive value entails fitness costs, natural selection has promoted the evolution of reliable signals of offspring condition, allowing parents to invest in their progeny accordingly. In birds, mouth and skin colouration are hypothesized to be honest signals of offspring condition, because they are affected by diverse factors. Among these, ectoparasite load has been shown to affect nestling condition, but its influence on visual components of begging is poorly known. We experimentally investigated whether nest ectoparasite removal affected flange and skin reflectance of first- and second-brood European starling (Sturnus vulgaris) nestlings. We also tested whether high reflectance in visual components of begging mirrored other aspects of nestling condition, such as morphological (high stature) and physiological (high haematocrit and immune response) traits, and pre-fledging mortality. Ectoparasite removal did not affect visual components of begging in first or second broods. However, larger nestlings from both broods displayed higher ultraviolet (UV) reflectance of skin and higher flange reflectance in the visible-wavelength region (but lower flange UV reflectance) than their siblings. A higher skin UV reflectance relative to siblings also positively predicted pre-fledging survival within-brood. Therefore, visual components of begging did not mirror ectoparasite infestation in this species. However, they provide parents with reliable information about individual quality, thus affecting resource allocation and promoting survival of the most valuable offspring during the entire breeding season. In species with parental care, natural selection favours the evolution of reliable signals of offspring quality, thus allowing parents to invest in their progeny accordingly. We experimentally show that skin and beak flange colour does not mirror ectoparasite infection in European starling nestlings. However, begging visual signals predict nestling body size and survival until fledging. A seasonal variation in the strength of the association between begging visual signals and nestling condition is also shown, indicating that change ecological conditions can affect the association between different condition-dependent traits.

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.

Sexual selection can operate both before and after mating. For males, selection will favor a suite of traits involved in mate acquisition (precopulatory) and in the competition among ejaculates to fertilize eggs (postcopulatory). Sperm competition theory predicts a trade-off between investment in pre- and postcopulatory traits, thus generating negative correlations between traits that function during both episodes of selection. However, such trade-offs can be difficult to detect because they are typically sensitive to the pool of resources available for allocation to competing functions. Although theory predicts that trade-offs will be more apparent when resources are limiting, there have been few attempts to test this prediction. In our study, we used the freshwater fish, Poecilia reticulata, to examine how both pre- and postcopulatory sexually selected traits simultaneously respond to manipulations of diet quantity by comparing behavioral, ornamental, and ejaculate traits between males assigned at random to either a ad libitum or a restricted food diet. We also explore how the relationships between traits that function during pre- and postcopulatory episodes of selection are affected by these treatments. Our results reveal that diet manipulations influenced the expression of both precopulatory (sexual behavior and ornamentation) and postcopulatory sexually selected traits (sperm viability), reinforcing the importance of resource acquisition in sexual selection. However, our data do not support the hypothesis that males trade off their allocation toward these pre- and postcopulatory sexually selected traits. Instead, changes in the expression of pre- and postcopulatory sexually selected traits appear to occur independently. Key wor ds: dietar y manipulation, Poecilia reticulata, postcopulatory sexual selection, precopulatory sexual selection, trade-offs. [Behav Ecol]

While it is well established that females prefer to mate with well‐ornamented males, the influence of perceptive and cognitive processes on the expression of female mate choice is still poorly known. It has been suggested that the female perception of a male's attractiveness is not absolute, but depends on the other males with which he is compared that have been previously encountered (comparative evaluation). We investigated whether mate preference in bearded reedlings (Panurus biarmicus) is dependent on or independent of social context in relation to two different traits: beard and tail lengths. Each female had a choice between two to three males with different modifications of beard and tail. For each female, three different experiments were conducted (one binary and two trinary tests). We found that when females are presented with options that vary antagonistically with respect to two ornaments (binary test), some individuals prefer one trait while others the other trait. This indicates that in our bearded reedlings population exists a mate preference polymorphisms. Moreover, we found that the presence of a third stimulus, irrespective of the initial preference, reduced the strength of the initial preference – what we can call a “preference dilution effect.” Our results suggest that the female's choice may be constrained by her cognitive abilities when she is simultaneously presented with several options varying for two uncorrelated traits.

Theory predicts that in polyandrous species males that invest more in traits enhancing postcopulatory success should have less energy to invest in mating acquisition, leading to trade-offs between pre- and post-copulatory episodes of sexual selection. Although such trade-offs are well studied, the potential constraints that postcopulatory sexual selection imposes on alternative mating tactics are rarely considered. In guppies, Poecilia reticulata , individual males can obtain matings by performing energetically costly courtship displays or by using less costly forced copulations. In this study we manipulated a component of social environment (namely, the presence of females) to experimentally elevate sperm production in males. We found that male guppies rapidly compensated by reducing their reliance on courtship in favour of forced matings. As both tactics differ in mating and fertilization success, the consequences of this trade-off may have important ramifications for male reproductive fitness.

In seasonally fluctuating environments, timing of reproduction is a crucial determinant of fitness. Studies of birds show that late breeding attempts generally result in offspring of lower reproductive value, with lower recruitment and long-term survival prospects. Several proximate mechanisms, including a seasonal decline of immune system functioning, may lead to a seasonal decline of offspring fitness. We investigated seasonal variation in offspring quality by subjecting first- and second-brood chicks of a sexually size dimorphic species, the European starling Sturnus vulgaris, to an immune challenge with a bacterial endotoxin (LPS), and evaluated their growth and physiological response in terms of total plasma antioxidant capacity (TAC), concentration of reactive oxygen metabolites and hematocrit. LPS challenge did not affect chick growth or oxidative status. However, hematocrit of second-brood chicks was higher in LPS chicks compared to controls. Body mass halfway through the rearing period (days 8–9 post-hatching), TAC and hematocrit were lower among second- vs. first-brood chicks. Interestingly, sexual dimorphism in body mass at days 8–9 post-hatching markedly differed between broods, first-brood males being 4.7% and second-brood males 22.7% heavier than their sisters, respectively. Pre-fledging mortality occurred among second-brood chicks only and was strongly female-biased. Our findings suggest that starling chicks, even if in poor conditions, are little affected by a bacterial challenge, at least in the short-term. Moreover, our study indicates that sex differences in body size, possibly mediated by sex-specific maternal investment in egg size, may heavily impact on pre-fledging survival in a different way in the course of the breeding season, resulting in sex-specific seasonal decline of offspring fitness. Finally, we suggest that levels of circulating antioxidants should be regarded among the proximate causes of the association between timing of fledging and long-term survival in avian species.

Sexual harassment is a common outcome of sexual conflict over mating rate. A large number of studies have identified several direct costs to females of sexual harassment including energy expenditure and reduced foraging ability. However, the fitness consequences of sexual harassment for descendants have rarely been investigated. Here, we manipulated the level of sexual harassment and mating rate in two groups of female guppies, Poecilia reticulata, a live-bearing fish in which sexual conflict over mating rate is particularly pronounced. Each female was allowed to interact with three males for one day (low sexual harassment, LSH) or for eight days (high sexual harassment, HSH) during each breeding cycle throughout their life. Female lifetime fecundity did not differ between the groups, but we found a strong effect on offspring fitness. HSH females produced (1) daughters with smaller bodies and (2) sons with shorter gonopodia, which were less attractive to females and less successful in coercive matings than their LSH counterparts. Although these results may be influenced by the indirect effects of sex ratio differences between treatments, they suggest that sexual harassment and elevated mating rate can have negative cross-generational fitness effects and more profound evolutionary consequences than currently thought.

As inbreeding is costly, it has been suggested that polyandry may evolve as a means to reduce the negative fitness consequences of mating with genetically related males. While several studies provide support for this hypothesis, evidence of pure post-copulatory mechanisms capable of biasing paternity towards genetically unrelated males is still lacking; yet these are necessary to support inbreeding avoidance models of polyandry evolution. Here we showed, by artificially inseminating a group of female guppies with an equal number of sperm from related (full-sib) and unrelated males, that sperm competition success of the former was 10 per cent lower, on average, than that of the unrelated male. The paternity bias towards unrelated males was not due to differential embryo survival, as the size of the brood produced by control females, which were artificially inseminated with the sperm of a single male, was not influenced by their relatedness with the male. Finally, we collected ovarian fluid (OF) from virgin females. Using computer-assisted sperm analysis, we found that sperm velocity, a predictor of sperm competition success in the guppy, was significantly lower when measured in a solution containing the OF from a sister as compared with that from an unrelated female. Our results suggest that sperm–OF interaction mediates sperm competition bias towards unrelated mates and highlight the role of post-copulatory mechanisms in reducing the cost of mating with relatives in polyandrous females.

Male and female gametes are often embedded in fluids that are produced by gonads and other reproductive tissues. Female reproductive fluids, usually called ovarian fluid (OF), which often constitute a relevant volumetric component of the egg mass, are rich in ions, sugars and proteins, and are involved in several functions, from protecting gametes to facilitating fertilization, and often act as mediators of post-mating sexual selection. Despite their applied and evolutionary importance, we know virtually nothing about the costs of female reproductive fluid production. We investigated the effect of nutritional condition on OF quality by experimentally manipulating the diet of two groups of female guppies (Poecilia reticulata) which were maintained for 20 days either on a restricted diet or had ad libitum access to food. In this species, OF enhances sperm swimming longevity and velocity (a predictor of sperm competition success) and mediates post-copulatory inbreeding avoidance. We found that sperm velocity was significantly lower in the OF of diet-restricted females, indicating that OF quality is dependent on female nutritional condition. Our results demonstrate that OF represents a non-trivial component of female reproductive investment and provides a tool to investigate which OF constituents are involved in modulating OF–sperm interactions and fertilization. Fil: Cardozo Milanesio, Gabriela Alejandra. Universita Di Padova. Dipartimento Di Biología; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentina Fil: Pilastro, Andrea. Universita Di Padova. Dipartimento Di Biología; Italia

Deleterious mutations can accumulate in the germline with age, decreasing the genetic quality of sperm and imposing a cost on female fitness. If these mutations also affect sperm competition ability or sperm production, then females will benefit from polyandry as it incites sperm competition and, consequently, minimizes the mutational load in the offspring. We tested this hypothesis in the guppy (Poecilia reticulata), a species characterized by polyandry and intense sperm competition, by investigating whether age affects post-copulatory male traits and sperm competition success. Females did not discriminate between old and young males in a mate choice experiment. While old males produced longer and slower sperm with larger reserves of strippable sperm, compared to young males, artificial insemination did not reveal any effect of age on sperm competition success. Altogether, these results do not support the hypothesis that polyandry evolved in response to costs associated with mating with old males in the guppy.

Growing evidence suggests that structural feather colours honestly reflect individual quality or body condition but, contrary to pigment-based colours, it is not clear what mechanism links condition to reflectance in structural feather colours. We experimentally accelerated the moult speed of a group of blue tits (Cyanistes caeruleus) by exposing them to a rapidly decreasing photoperiod and compared the spectral characteristics of their structural feather colours with those of control birds. Blue tits were sexually dimorphic on the UV ⁄blue crown and on the white cheek feathers. Moult speed, however, dramatically reduced brightness and the saturation only on the UV ⁄blue crown feathers, whereas structural white on the cheek feathers was basically unaffected by moult speed. Given that the time available for moulting is usually confined to the period between the end of the breeding season and migration or wintering, UV ⁄blue colours, but not structural white, may convey long-term information about an individual’s performance during the previous breeding season. The trade-off between fast moulting and structural colour expression may represent a previously unrecognized selective advantage for early-breeding birds.

Albeit there is growing evidence that males prefer to mate with ornamented females, it has been suggested that the production of costly ornaments may reduce female fecundity, hence favoring males with a preference for females with average ornamentation. In the rock sparrow, Petronia petronia, males and females possess a sexually selected patch of yellow feathers on the breast (a carotenoid-based trait). To test whether males prefer females with the largest ornament or average ornamented females, male rock sparrows were simultaneously faced with 3 conspecific females differing in breast patch size and a female house sparrow as a control. We found that the house sparrow and rock sparrow female with the smallest patch were least preferred, and males showed a clear proximity preference for the females with the above average‐sized patch. Our results demonstrate that, contrary to theoretical predictions, a directional preference for female ornament was observed. Directional male preference may arise as consequence of a male’s sensory bias or may be associated with indirect (genetic) benefits of choosing ornamented females, if ornament size is correlated with female genetic quality. Clearly, more work is necessary to identify the conditions under which directional preference for female ornament arises. Key words: condition-dependent trait, mutual sexual selection, ornaments, plumage reflectance. [Behav Ecol]

In the present study, the correlation between sperm number, sperm quality (speed, viability, longevity and length), sperm bundles quality (size and dissolving rate) and male body size has been tested in the eastern mosquitofish Gambusia holbrooki a poecilid species characterized by coercive mating tactics where males do not possess obvious ornaments, and the body size is the key determinant of pre-copulatory male mating success. The results do not tally with theoretical predictions. Indeed, no correlation between male body size and either sperm or sperm-bundle traits has been found, evidencing the lack of the theoretically expected trade-off between the investment in characters involved in mate acquisition and the investment in ejaculate quality. An explanation for the observed pattern comes from the extremely dynamic mating system of G. holbrooki, characterized by variable size-related male mating success and strong post-copulatory selective pressure, with all males facing a similar high level of sperm competition. In this situation, a higher investment in growth and maintenance at the expense of ejaculate quality is not expected. These results underscore the necessity to comprehend detailed information on species’ reproductive biology and reproductive environment to understand both the evolution of ejaculate characteristics and possible deviations from theoretical predictions.

The phenotype-linked fertility hypothesis (PLFH) predicts that males with elaborated sexual ornaments signal their high fertilizing efficiency to females and that female preferences for ornamented males are driven by direct fecundity benefits. Although some studies have demonstrated that attractive males produce more or higher quality sperm, there is limited experimental evidence that females derive fecundity benefits by mating with attractive males. Some of the best indirect evidence for the PLFH comes from work on guppies (Poecilia reticulata), an internally fertilizing species of freshwater fish in which phenotypically attractive males produce larger and relatively higher quality ejaculates than their less attractive counterparts. We used artificial insemination to impregnate female guppies using known numbers of sperm from a range of males with different phenotypes and related female fecundity (brood success, time from insemination to parturition, and brood size) to sperm numbers and male phenotype (body size and the relative area of color spots). We found no evidence that male phenotype or experimentally adjusted ‘‘ejaculate’’ size influenced any of our measures of female fecundity. These results highlight the importance of experimentally investigating potential fecundity benefits associated with female mating preferences before concluding that the maintenance of these preferences is driven by the pursuit of such benefits. Key words: artificial insemination, direct benefits, fecundity, female choice, male sexual secondary characters, sperm number. [Behav Ecol]

In polyandrous species, females may influence paternity by biasing sperm usage in favour of particular males. In the guppy, Poecilia reticulata, the number of sperm inseminated in a copulation depends on the female's perception of male attractiveness. We videorecorded copulations in the laboratory to test the hypothesis that there is a positive correlation between the number of sperm inseminated and the duration of the copulation. Duration was positively correlated with the number of sperm retrieved from the female's gonoduct. Once copulation duration was statistically controlled for, more sperm were retrieved from the gonoduct in courtship copulations than in coercive copulations, and in postpartum females than in virgin females. Copulation duration was positively correlated with the degree of carotenoid coloration of the male, suggesting that copulation duration might be the proximate mechanism of cryptic female choice in this species. The intensity of ‘jerking’, a postcopulatory behaviour of male guppies, was positively correlated with the number of sperm inseminated, suggesting a possible signalling function of this display.

We investigated the effect of moult speed on the expression of a sexually selected, carotenoid-based feather ornament in the rock sparrow (Petronia petronia). We experimentally accelerated the moult speed of a group of birds by exposing them to a rapidly decreasing photoperiod and compared the area and the spectral characteristics of their ornaments with those of control birds. Birds with accelerated moulting rate showed a smaller yellow patch with lower yellow reflectance compared to their slow-moulting counterparts. Considering that the time available for moulting is usually constrained between the end of the breeding season and migration or wintering, carotenoid feather ornaments, whose expression is mediated by moult speed, may convey long term information about an individual's condition, potentially encompassing the previous breeding season. Furthermore, the observed trade-off between moult speed and ornament expression may represent a previously unrecognized selective advantage for early breeding birds.

In many species, females mate with multiple partners, meaning that sexual selection on male traits operates across a spectrum that encompasses the competition for mates (that is, before mating) and fertilizations (after mating). Despite being inextricably linked, pre- and postcopulatory sexual selection are typically studied independently, and we know almost nothing about how sexual selection operates across this divide. Here we bridge this knowledge gap using the livebearing fish Poecilia reticulata. We show that both selective episodes, as well as their covariance, explain a significant component of variance in male reproductive fitness. Moreover, linear and nonlinear selection simultaneously act on pre- and postcopulatory traits, and interact to generate multiple phenotypes with similar fitness., In species in which females mate with multiple partners, sexual selection acts on male traits involved in mating and fertilization. Here, the authors show that selection acting before and after mating explains a significant component of variance in male reproductive fitness in a livebearing fish.

Females that mate multiply have the possibility to exert postcopulatory choice and select more compatible sperm to fertilize eggs. Prior work suggests that dissimilarity in major histocompatibility complex (MHC) plays an important role in determining genetic compatibility between partners. Favouring a partner with dissimilar MHC alleles would result in offspring with high MHC diversity and therefore with enhanced survival thanks to increased resistance to pathogens and parasites. The high variability of MHC genes may further allow discrimination against the sperm from related males, reducing offspring homozygosity and inbreeding risk. Despite the large body of work conducted at precopulatory level, the role of MHC similarity between partners at postcopulatory level has been rarely investigated. We used an internal fertilizing fish with high level of multiple matings (Poecilia reticulata) to study whether MHC similarity plays a role in determining the outcome of fertilization when sperm from two males compete for the same set of eggs. We also controlled for genomewide similarity by determining similarity at 10 microsatellite loci. Contrary to prediction, we found that the more MHC-similar male sired more offspring while similarity at the microsatellite loci did not predict the outcome of sperm competition. Our results suggest that MHC discrimination may be involved in avoidance of hybridization or outbreeding rather than inbreeding avoidance. This, coupled with similar findings in salmon, suggests that the preference for MHC-dissimilar mates is far from being unanimous and that pre- and postcopulatory episodes of sexual selection can indeed act in opposite directions.

Nest-dwelling ectoparasites represent an early stressor for birds as they impair the development of nestlings, which can adaptively respond by adjusting their growth rate to current conditions. While nest ectoparasites have long-term effects on nesting adults, no study has examined if they also have an impact on the moult patterns of juveniles. Herein, we investigated whether the presence of ectoparasites in the nest influences the start and duration of the first pre-basic moult in the European starling. To do so, we experimentally removed nest-dwelling ectoparasites from a group of nests and used another group of unmanipulated (i.e. naturally infested) nests as the control. The moult began at an earlier age and lasted longer in birds from the ectoparasite-free nests compared to their control counterparts. The timing of the moult was also affected by the hatching date (i.e. birds that fledged later had shorter moults) and the brood size (i.e. birds in larger broods started their moult at an older age). We also found evidence that the removal of nest ectoparasites influenced the condition of individuals during the course of the moult. In the control birds, we observed a decrease in hematocrit levels, but these were unaltered in the birds fledged from the ectoparasite-free nests. Our study shows that nest-dwelling ectoparasites adversely affected the timing of the moult and the individual condition of juvenile starlings, with possible major consequences for their subsequent life-history events.

The introduction of a few individuals to new, isolated habitats (founder effect) is expected to reduce the genetic variability of a population. At the beginning of the last century a few eastern mosquitofish (Gambusia holbrooki) individuals were introduced to Southern Europe from North America to control malaria‐carrying mosquitoes. We studied the effect of this severe bottleneck on genetic variability in four populations of eastern mosquitofish introduced to Spain and Italy in the early 1900s and compared them to a native population in North America. Using amplified polymorphic DNA–polymerase chain reaction (RADP–PCR) we found a strong reduction of genetic diversity, in terms of both number of polymorphic loci and heterozygosity, in all four European populations. Despite this great reduction in genetic diversity, eastern mosquitofish successfully invaded European internal waters causing eutrophication problems and threatening the extinction of local populations of native fish.

We investigated whether mating behavior (sperm expenditure, courtship rate, and nest guarding) varied according to different levels of sperm competition in territorial males of two goby species, the grass and the black gobies. We measured sperm expenditure (sperm released after 30 min from the beginning of the spawning), male courtship rate, and nest-guarding behavior in territorial males of both species during simulated spawnings, in which we varied the number of attending sneakers. Our results showed that, in both species, territorial males adjusted their effort in nest guarding to the presence of rival sneakers by increasing the time spent patrolling the territory and attacking the sneakers. In contrast, sperm expenditure and male courtship rate were not influenced by the number of attending sneakers. These results are in agreement with those reported for other fish with alternative mating tactics and help to interpret previous inconsistencies between theoretical predictions and measured levels of sperm released at different levels of sperm competition by sneakers of the two gobiids studied here. Key words: alternative male mating tactics, fish, sperm expenditure. [Behav Ecol]

Recent evidence suggests that female ornaments in birds not only are genetic correlates of selection on males but may also have evolved by selection acting directly on females. However, most evidence comes from studies in captivity and it is therefore still debated whether male choice is important under natural conditions. The rock sparrow, Petronia petronia, is a monomorphic passerine in which both sexes show a yellow breast patch, although females have a smaller patch than males. Breast patch in females is positively correlated with body mass and fecundity (number of broods per year). We tested whether males preferentially mate with more ornamented females in this species. We examined male mate choice by reducing the size of the yellow breast patch of free-ranging females during the pair formation period in a colony in southeast Spain. We found that females with an experimentally reduced breast patch were courted less intensely and chased less frequently, than control females were. Furthermore, females with an experimentally reduced ornament were less likely to mate and, when mated, did so at a significantly later date than controls. These results are supported by correlational observations from another rock sparrow population, for which a larger data set was available, showing that pairs mated assortatively with respect to breast patch size. Altogether, these results therefore suggest that female ornamentation may be sexually selected in this species.