Sex-specific effects of inbreeding in juvenile brown trout.

Inbreeding depression, that is, the reduction of health and vigour in individuals with high inbreeding coefficients, is expected to increase with environmental, social, or physiological stress. It has therefore been predicted that sexual selection and the associated stress usually lead to higher inbreeding depression in males than in females. However, sex-specific differences in life history may reverse that pattern during certain developmental stages. In some salmonids, for example, female juveniles start developing their gonads earlier than males who instead grow faster. We tested whether the sexes are differently affected by inbreeding during that time. To study the effects of inbreeding coefficients that may be typical for natural populations of brown trout (Salmo trutta), and also to control for potentially confounding maternal or paternal effects, we sampled males and females from the wild, used their gametes in a block-wise full-factorial breeding design to produce 60 full-sib families, released the offspring as yolk-sac larvae into the wild, sampled them 6 months later, identified their genetic sex, and used microsatellites to assign them to their parents. We used whole-genome resequencing to calculate the kinship coefficients for each breeding pair and hence the expected average inbreeding coefficient per family. Juvenile growth could be predicted from these expected inbreeding coefficients and the genetic sex: Females reached lower body sizes with increasing inbreeding coefficient, while no such link could be found in males. This sex-specific inbreeding depression led to the overall pattern that females were on average smaller than males by the end of their first summer.

Parental kinship coefficient but not paternal coloration predicts early offspring growth in lake char.

The 'good genes' hypotheses of sexual selection predict that females prefer males with strong ornaments because they are in good health and vigor and can afford the costs of the ornaments. A key assumption of this concept is that male health and vigor are useful predictors of genetic quality and hence offspring performance. We tested this prediction in wild-caught lake char (Salvelinus umbla) whose breeding coloration is known to reveal aspects of male health. We first reanalyzed results from sperm competition trials in which embryos of known parenthood had been raised singly in either a stress- or non-stress environment. Paternal coloration did not correlate with any measures of offspring performance. However, offspring growth was reduced with higher kinship coefficients between the parents. To test the robustness of these first observations, we collected a new sample of wild males and females, used their gametes in a full-factorial in vitro breeding experiment, and singly raised about 3000 embryos in either a stress- or non-stress environment (stress induced by microbes). Again, paternal coloration did not predict offspring performance, while offspring growth was reduced with higher kinship between the parents. We conclude that, in lake char, the genetic benefits of mate choice would be strongest if females could recognize and avoid genetically related males, while male breeding colors may be more relevant in intra-sexual selection.

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

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

Correction to: Sex-specific changes in gene expression in response to estrogen pollution around the onset of sex differentiation in grayling (Salmonidae).

An amendment to this paper has been published and can be accessed via the original article.

Effects of global warming on sex ratios in fishes.

In fishes, sex is determined by genetics, the environment or an interaction of both. Temperature is among the most important environmental factors that can affect sex determination. As a consequence, changes in temperature at critical developmental stages can induce biases in primary sex ratios in some species. However, early sex ratios can also be biased by sex-specific tolerances to environmental stresses that may, in some cases, be amplified by changes in water temperature. Sex-specific reactions to environmental stress have been observed at early larval stages before gonad formation starts. It is therefore necessary to distinguish between temperature effects on sex determination, generally acting through the stress axis or epigenetic mechanisms, and temperature effects on sex-specific mortality. Both are likely to affect sex ratios and hence population dynamics. Moreover, in cases where temperature effects on sex determination lead to genotype-phenotype mismatches, long-term effects on population dynamics are possible, for example temperature-induced masculinization potentially leading to the loss of Y chromosomes or feminization to male-biased operational sex ratios in future generations. To date, most studies under controlled conditions conclude that if temperature affects sex ratios, elevated temperatures mostly lead to a male bias. The few studies that have been performed on wild populations seem to confirm this general trend. Recent findings suggest that transgenerational plasticity could mitigate the effects of warming on sex ratios in some populations.

Low adaptive potential for tolerance to ethynylestradiol, but also low toxicity, in a grayling population (Thymallus thymallus).

BACKGROUND: The presence of a novel pollutant can induce rapid evolution if there is additive genetic variance for the tolerance to the stressor. Continuous selection over some generations can then reduce the toxicity of the pollutant but also deplete the additive genetic variance for the tolerance and thereby slow down adaptation. One common pollutant that has been ecologically relevant for some time is 17alpha-ethynylestradiol (EE2), a synthetic compound of oral contraceptives since their market launch in the 1960s. EE2 is typically found in higher concentrations in rivers than in lakes. Recent experimental work revealed significant genetic variance for the tolerance to EE2 in two lake-spawning salmonid species but no such variance in river-spawning brown trout. We used another river-spawning salmonid, the European grayling Thymallus thymallus, to study the toxicity of an ecologically relevant concentration of EE2. We also used a full-factorial in vitro breeding design and singly rearing of 1555 embryos and larvae of 40 sib groups to test whether there is additive genetic variance for the tolerance to this pollutant. RESULTS: We found that exposure to EE2 reduced larval growth after hatching, but contrary to what has been found in the other salmonids, there were no significant effects of EE2 on embryo growth and survival. We found additive genetic variance for embryo viability, i.e. heritability for fitness. However, there was no significant additive variance for the tolerance to EE2. CONCLUSIONS: Our findings support the hypothesis that continuous selection has reduced the toxicity of EE2 and depleted genetic variance for tolerance to this synthetic stressor.

Sex-specific changes in gene expression in response to estrogen pollution around the onset of sex differentiation in grayling (Salmonidae).

The synthetic 17α-ethinylestradiol (EE2) is a common estrogenic pollutant that has been suspected to affect the demography of river-dwelling salmonids. One possibility is that exposure to EE2 tips the balance during initial steps of sex differentiation, so that male genotypes show female-specific gene expression and gonad formation. Here we study EE2 effects on gene expression around the onset of sex differentiation in a population of European grayling (Thymallus thymallus) that suffers from sex ratio distortions. We exposed singly-raised embryos to one dose of 1 ng/L EE2, studied gene expression 10 days before hatching, at the day of hatching, and around the end of the yolk-sac stage, and related it to genetic sex (sdY genotype). We found that exposure to EE2 affects expression of a large number of genes, especially around hatching. These effects were strongly sex-dependent. We then raised fish for several months after hatching and found no evidence of sex reversal in the EE2-exposed fish. We conclude that ecologically relevant (i.e. low) levels of EE2 pollution do not cause sex reversal by simply tipping the balance at early stages of sex differentiation, but that they interfere with sex-specific gene expression.

Sperm cryopreservation reduces offspring growth.

Sperm cryopreservation is routinely used in reproductive medicine, livestock production and wildlife management. Its effect on offspring performance is often assumed to be negligible, but this still remains to be confirmed in well-controlled within-subject experiments. We use a vertebrate model that allows us to experimentally separate parental and environmental effects to test whether sperm cryopreservation influences offspring phenotype under stress and non-stress conditions, and whether such effects are male-specific. Wild brown trout (Salmo trutta) were stripped for their gametes, and a portion of each male's milt was cryopreserved. Then, 960 eggs were simultaneously fertilized with either non-cryopreserved or frozen-thawed semen and raised singly in the presence or absence of a pathogen. We found no significant effects of cryopreservation on fertilization rates, and no effects on growth, survival nor pathogen resistance during the embryo stage. However, fertilization by cryopreserved sperm led to significantly reduced larval growth after hatching. Males varied in genetic quality as determined from offspring performance, but effects of cryopreservation on larval growth were not male-specific. We conclude that cryopreservation causes a reduction in offspring growth that is easily overlooked because it only manifests itself at later developmental stages, when many other factors affect growth and survival too.

Maternal allocation of carotenoids increases tolerance to bacterial infection in brown trout.

Life-history theory predicts that iteroparous females allocate their resources differently among different breeding seasons depending on their residual reproductive value. In iteroparous salmonids there is typically much variation in egg size, egg number, and in the compounds that females allocate to their clutch. These compounds include various carotenoids whose functions are not sufficiently understood yet. We sampled 37 female and 35 male brown trout from natural streams, collected their gametes for in vitro fertilizations, experimentally produced 185 families in 7 full-factorial breeding blocks, raised the developing embryos singly (n = 2960), and either sham-treated or infected them with Pseudomonas fluorescens. We used female redness (as a measure of carotenoids stored in the skin) and their allocation of carotenoids to clutches to infer maternal strategies. Astaxanthin contents largely determined egg colour. Neither egg weight nor female size was correlated with the content of this carotenoid. However, astaxanthin content was positively correlated with larval growth and with tolerance against P. fluorescens. There was a negative correlation between female skin redness and the carotenoid content of their eggs. Although higher astaxanthin contents in the eggs were associated with an improvement of early fitness-related traits, some females appeared not to maximally support their current offspring as revealed by the negative correlation between female red skin colouration and egg carotenoid content. This correlation was not explained by female size and supports the prediction of a maternal trade-off between current and future reproduction.

Effects of host genetics and environment on egg-associated microbiotas in brown trout (Salmo trutta).

Recent studies found fish egg-specific bacterial communities that changed over the course of embryogenesis, suggesting an interaction between the developing host and its microbiota. Indeed, single-strain infections demonstrated that the virulence of opportunistic bacteria is influenced by environmental factors and host immune genes. However, the interplay between a fish embryo host and its microbiota has not been studied yet at the community level. To test whether host genetics affects the assemblage of egg-associated bacteria, adult brown trout (Salmo trutta) were sampled from a natural population. Their gametes were used for full-factorial in vitro fertilizations to separate sire from dam effects. In total, 2520 embryos were singly raised under experimental conditions that differently support microbial growth. High-throughput 16S rRNA amplicon sequencing was applied to characterize bacterial communities on milt and fertilized eggs across treatments. Dam and sire identity influenced embryo mortality, time until hatching and composition of egg-associated microbiotas, but no link between bacterial communities on milt and on fertilized eggs could be found. Elevated resources increased embryo mortality and modified bacterial communities with a shift in their putative functional potential. Resource availability did not significantly affect any parental effects on embryo performance. Sire identity affected bacterial diversity that turned out to be a significant predictor of hatching time: embryos associated with high bacterial diversity hatched later. We conclude that both host genetics and the availability of resources define diversity and composition of egg-associated bacterial communities that then affect the life history of their hosts.

Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.

Fish populations surviving estrogen pollution.

Among the most common pollutants that enter the environment after passing municipal wastewater treatment are estrogens, especially the synthetic 17α-ethinylestradiol that is used in oral contraceptives. Estrogens are potent endocrine disruptors at concentrations frequently observed in surface waters. However, new genetic analyses suggest that some fish populations can be self-sustaining even in heavily polluted waters. We now need to understand the basis of this tolerance.

Growth of brown trout in the wild predicted by embryo stress reaction in the laboratory.

Laboratory studies on embryos of salmonids, such as the brown trout (Salmo trutta), have been extensively used to study environmental stress and how responses vary within and between natural populations. These studies are based on the implicit assumption that early life-history traits are relevant for stress tolerance in the wild. Here we test this assumption by combining two data sets from studies on the same 60 families. These families had been experimentally produced from wild breeders to determine, in separate samples, (1) stress tolerances of singly kept embryos in the laboratory and (2) growth of juveniles during 6 months in the wild. We found that growth in the wild was well predicted by the larval size of their full sibs in the laboratory, especially if these siblings had been experimentally exposed to a pathogen. Exposure to the pathogen had not caused elevated mortality among the embryos but induced early hatching. The strength of this stress-induced change of life history was a significant predictor of juvenile growth in the wild: the stronger the response in the laboratory, the slower the growth in the wild. We conclude that embryo performance in controlled environments can be a useful predictor of juvenile performance in the wild.

MHC class I expression dependent on bacterial infection and parental factors in whitefish embryos (Salmonidae).

Ecological conditions can influence not only the expression of a phenotype, but also the heritability of a trait. As such, heritable variation for a trait needs to be studied across environments. We have investigated how pathogen challenge affects the expression of MHC genes in embryos of the lake whitefish Coregonus palaea. In order to experimentally separate paternal (i.e. genetic) from maternal and environmental effects, and determine whether and how stress affects the heritable variation for MHC expression, embryos were produced in full-factorial in vitro fertilizations, reared singly, and exposed at 208 degree days (late-eyed stage) to either one of two strains of Pseudomonas fluorescens that differ in their virulence characteristics (one increased mortality, while both delayed hatching time). Gene expression was assessed 48 h postinoculation, and virulence effects of the bacterial infection were monitored until hatching. We found no evidence of MHC class II expression at this stage of development. MHC class I expression was markedly down-regulated in reaction to both pseudomonads. While MHC expression could not be linked to embryo survival, the less the gene was expressed, the earlier the embryos hatched within each treatment group, possibly due to trade-offs between immune function and developmental rate or further factors that affect both hatching timing and MHC expression. We found significant additive genetic variance for MHC class I expression in some treatments. That is, changes in pathogen pressures could induce rapid evolution in MHC class I expression. However, we found no additive genetic variance in reaction norms in our study population.

Persistent unequal sex ratio in a population of grayling (Salmonidae) and possible role of temperature increase.

In some fishes, water chemistry or temperature affects sex determination or creates sex-specific selection pressures. The resulting population sex ratios are hard to predict from laboratory studies if the environmental triggers interact with other factors, whereas in field studies, singular observations of unusual sex ratios may be particularly prone to selective reporting. Long-term monitoring largely avoids these problems. We studied a population of grayling (Thymallus thymallus) in Lake Thun, Switzerland, that has been monitored since 1948. Samples of spawning fish have been caught about 3 times/week around spawning season, and water temperature at the spawning site has been continuously recorded since 1970. We used scale samples collected in different years to determine the average age of spawners (for life-stage specific analyses) and to identify the cohort born in 2003 (an extraordinarily warm year). Recent tissue samples were genotyped on microsatellite markers to test for genetic bottlenecks in the past and to estimate the genetically effective population size (N(e)). Operational sex ratios changed from approximately 65% males before 1993 to approximately 85% males from 1993 to 2011. Sex ratios correlated with the water temperatures the fish experienced in their first year of life. Sex ratios were best explained by the average temperature juvenile fish experienced during their first summer. Grayling abundance is declining, but we found no evidence of a strong genetic bottleneck that would explain the apparent lack of evolutionary response to the unequal sex ratio. Results of other studies show no evidence of endocrine disruptors in the study area. Our findings suggest temperature affects population sex ratio and thereby contributes to population decline.

Testing for local adaptation in brown trout using reciprocal transplants.

BACKGROUND: Local adaptation can drive the divergence of populations but identification of the traits under selection remains a major challenge in evolutionary biology. Reciprocal transplant experiments are ideal tests of local adaptation, yet rarely used for higher vertebrates because of the mobility and potential invasiveness of non-native organisms. Here, we reciprocally transplanted 2500 brown trout (Salmo trutta) embryos from five populations to investigate local adaptation in early life history traits. Embryos were bred in a full-factorial design and raised in natural riverbeds until emergence. Customized egg capsules were used to simulate the natural redd environment and allowed tracking the fate of every individual until retrieval. We predicted that 1) within sites, native populations would outperform non-natives, and 2) across sites, populations would show higher performance at 'home' compared to 'away' sites. RESULTS: There was no evidence for local adaptation but we found large differences in survival and hatching rates between sites, indicative of considerable variation in habitat quality. Survival was generally high across all populations (55% ± 3%), but ranged from 4% to 89% between sites. Average hatching rate was 25% ± 3% across populations ranging from 0% to 62% between sites. CONCLUSION: This study provides rare empirical data on variation in early life history traits in a population network of a salmonid, and large-scale breeding and transplantation experiments like ours provide powerful tests for local adaptation. Despite the recently reported genetic and morphological differences between the populations in our study area, local adaptation at the embryo level is small, non-existent, or confined to ecological conditions that our experiment could not capture.

Genetic and phenotypic population divergence on a microgeographic scale in brown trout.

Salmonid populations of many rivers are rapidly declining. One possible explanation is that habitat fragmentation increases genetic drift and reduces the populations' potential to adapt to changing environmental conditions. We measured the genetic and eco-morphological diversity of brown trout (Salmo trutta) in a Swiss stream system, using multivariate statistics and Bayesian clustering. We found large genetic and phenotypic variation within only 40 km of stream length. Eighty-eight percent of all pairwise F(ST) comparisons and 50% of the population comparisons in body shape were significant. High success rates of population assignment tests confirmed the distinctiveness of populations in both genotype and phenotype. Spatial analysis revealed that divergence increased with waterway distance, the number of weirs, and stretches of poor habitat between sampling locations, but effects of isolation-by-distance and habitat fragmentation could not be fully disentangled. Stocking intensity varied between streams but did not appear to erode genetic diversity within populations. A lack of association between phenotypic and genetic divergence points to a role of local adaptation or phenotypically plastic responses to habitat heterogeneity. Indeed, body shape could be largely explained by topographic stream slope, and variation in overall phenotype matched the flow regimes of the respective habitats.

Examining punishment at different explanatory levels.

Experimental studies on punishment have sometimes been over-interpreted not only for the reasons Guala lists, but also because of a frequent conflation of proximate and ultimate explanatory levels that Guala's review perpetuates. Moreover, for future analyses we may need a clearer classification of different kinds of punishment.

Adult sex ratios: causes of variation and implications for animal and human societies.

Converging lines of inquiry from across the social and biological sciences target the adult sex ratio (ASR; the proportion of males in the adult population) as a fundamental population-level determinant of behavior. The ASR, which indicates the relative number of potential mates to competitors in a population, frames the selective arena for competition, mate choice, and social interactions. Here we review a growing literature, focusing on methodological developments that sharpen knowledge of the demographic variables underlying ASR variation, experiments that enhance understanding of the consequences of ASR imbalance across societies, and phylogenetic analyses that provide novel insights into social evolution. We additionally highlight areas where research advances are expected to make accelerating contributions across the social sciences, evolutionary biology, and biodiversity conservation.