Fish populations can be threatened by distorted sex ratios that arise during sex differentiation. Here we describe sex differentiation in a wild grayling (Thymallus thymallus) population that suffers from distorted sex ratios. We verified that sex determination is linked to the sex determining locus (sdY) of salmonids. This allowed us to study sex-specific gene expression and gonadal development. Sex-specific gene expression could be observed during embryogenesis and was strong around hatching. About half of the fish showed immature testes around eleven weeks after fertilization. This phenotype was mostly replaced by the “testis-to-ovary” or “ovaries” phenotypes during development. The gonads of the remaining fish stayed undifferentiated until six months after fertilization. Genetic sexing revealed that fish with undifferentiated gonads were all males, who grew larger than the genetic females during the observational period. Only 12% of the genetic males showed testicular tissue six months after fertilization. We conclude that sex differentiation starts before hatching, goes through an all-male stage for both sexes (which represents a rare case of “undifferentiated” gonochoristic species that usually go through an all-female stage), and is delayed in males. During these juvenile stages males grow faster than females instead of developing their gonads.

One of the most common and potent pollutants of freshwater habitats is 17‐alpha‐ethynylestradiol (EE2), a synthetic component of oral contraceptives that is not completely eliminated during sewage treatment and that threatens natural populations of fish. Previous studies found additive genetic variance for the tolerance against EE2 in different salmonid fishes and concluded that rapid evolution to this type of pollution seems possible. However, these previous studies were done with fishes that are lake‐dwelling and hence typically less exposed to EE2 than river‐dwelling species. Here, we test whether there is additive genetic variance for the tolerance against EE2 also in river‐dwelling salmonid populations that have been exposed to various concentrations of EE2 over the last decades. We sampled 287 adult brown trout ( Salmo trutta ) from seven populations that show much genetic diversity within populations, are genetically differentiated, and that vary in their exposure to sewage‐treated effluent. In order to estimate their potential to evolve tolerance to EE2, we collected their gametes to produce 730 experimental families in blockwise full‐factorial in vitro fertilizations. We then raised 7,302 embryos singly in 2‐ml containers each and either exposed them to 1 ng/L EE2 (an ecologically relevant concentration, i.e., 2 pg per embryo added in a single spike to the water) or sham‐treated them. Exposure to EE2 increased embryo mortality, delayed hatching time, and decreased hatchling length. We found no population differences and no additive genetic variance for tolerance to EE2. We conclude that EE2 has detrimental effects that may adversely affect population even at a very low concentration, but that our study populations lack the potential for rapid genetic adaptation to this type of pollution. One possible explanation for the latter is that continuous selection over the last decades has depleted genetic variance for tolerance to this synthetic stressor.

Salmonids are a socioeconomically and ecologically important group of fish that are often managed by stocking. Little is known about potential sex-specific effects of stocking, but recent studies found that the sexes differ in their stress tolerances already at late embryonic stage, i.e., before hatchery-born larvae are released into the wild and long before morphological gonad formation. It has also been speculated that sex-specific life histories can affect juvenile growth and mortality, and that a resulting sex-biassed demography can reduce population growth. Here we test whether juvenile brown trout (Salmo trutta) show sex-specific life histories and whether such sex effects differ in hatchery- and wild-born fish. We modified a genetic sexing protocol to reduce false assignment rates and used it to study the timing of sex differentiation in a laboratory setting, and in a large-scale field experiment to study growth and mortality of hatchery- and wild-born fish in different environments. We found no sex-specific mortality in any of the environments we studied. However, females started sex differentiation earlier than males, and while growth rates were similar in the laboratory, they differed significantly in the field depending on location and origin of fish. Overall, hatchery-born males grew larger than hatchery-born females while wild-born fish showed the reverse pattern. Whether males or females grew larger was location-specific. We conclude that juvenile brown trout show sex-specific growth that is affected by stocking and by other environmental factors that remain to be identified.

For natural populations to adapt to anthropogenic threats, heritable variation must persist in tolerance traits. Silver nanoparticles, the most widely used engineered nanoparticles, are expected to increase in concentrations in freshwaters. Little is known about how these particles affect wild populations, and whether genetic variation persists in tolerance to permit rapid evolutionary responses. We sampled wild adult whitefish and crossed them in vitro full factorially. In total, 2896 singly raised embryos of 48 families were exposed to two concentrations (0.5 μg/L; 100 μg/L) of differently sized silver nanoparticles or ions (silver nitrate). These doses were not lethal; yet higher concentrations prompted embryos to hatch earlier and at a smaller size. The induced hatching did not vary with nanoparticle size and was stronger in the silver nitrate group. Additive genetic variation for hatching time was significant across all treatments, with no apparent environmental dependencies. No genetic variation was found for hatching plasticity. We found some treatment‐dependent heritable variation for larval length and yolk volume, and one instance of additive genetic variation for the reaction norm on length at hatching. Our assessment suggests that the effects of silver exposure on additive genetic variation vary according to trait and silver source. While the long‐term fitness consequences of low‐level silver exposure on whitefish embryos must be further investigated to determine whether it is, in fact, detrimental, our results suggest that the evolutionary potential for adaptation to these types of pollutants may be low.

27Fish can be threatened by distorted sex ratios that arise during sex differentiation. It is 28 therefore important to understand sex determination and differentiation, especially in river-29 dwelling fish that are often exposed to environmental factors that may interfere with sex 30 differentiation. However, sex differentiation is not sufficiently understood in keystone taxa 31 such as the Thymallinae, one of the three salmonid subfamilies. Here we study a wild 32 grayling (Thymallus thymallus) population that suffers from distorted sex ratios. We found 33 sex determination in the wild and in captivity to be genetic and linked to the sdY locus. We 34 therefore studied sex-specific gene expression in embryos and early larvae that were bred and 35 raised under different experimental conditions, and we studied gonadal morphology in five 36 monthly samples taken after hatching. Significant sex-specific changes in gene expression 37 (affecting about 25,000 genes) started around hatching. Gonads were still undifferentiated 38 three weeks after hatching, but about half of the fish showed immature testes around seven 39 weeks after hatching. Over the next few months, this phenotype was mostly replaced by the 40 "testis-to-ovary" or "ovaries" phenotypes. The gonads of the remaining fish, i.e. 41 approximately half of the fish in each sampling period, remained undifferentiated until six 42 months after fertilization. Genetic sexing of the last two samples revealed that fish with 43 undifferentiated gonads were all males, who, by that time, were on average larger than the 44 genetic females (verified in 8-months old juveniles raised in another experiment). Only 12% 45 of the genetic males showed testicular tissue six months after fertilization. We conclude that 46 sex differentiation starts around hatching, goes through an all-male stage for both sexes 47 (which represents a rare case of "undifferentiated" gonochoristic species that usually go 48 through an all-female stage), and is delayed in males who, instead of developing their gonads, 49 grow faster than females during these juvenile stages. 50 51 peer-reviewed)