Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and extreme temperatures can cause sex reversal during early ontogeny (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here, we developed a genetic sexing method based on sex-linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex-reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress-induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North-Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female-to-male sex-reversed adults had similar body mass as normal males. We recorded no events of male-to-female sex reversal either in the laboratory or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human-induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex reversal is associated with stress and poor health in early life, but sex-reversed individuals surviving to adulthood may participate in breeding.
Defensive toxins are widespread in nature, yet we know little about how various environmental factors shape the evolution of chemical defense, especially in vertebrates. In this study we investigated the natural variation in the amount and composition of bufadienolide toxins, and the relative importance of ecological factors in predicting that variation, in larvae of the common toad, Bufo bufo, an amphibian that produces toxins de novo. We found that tadpoles' toxin content varied markedly among populations, and the number of compounds per tadpole also differed between two geographical regions. The most consistent predictor of toxicity was the strength of competition, indicating that tadpoles produced more compounds and larger amounts of toxins when coexisting with more competitors. Additionally, tadpoles tended to contain larger concentrations of bufadienolides in ponds that were less prone to desiccation, suggesting that the costs of toxin production can only be afforded by tadpoles that do not need to drastically speed up their development. Interestingly, this trade-off was not alleviated by higher food abundance, as periphyton biomass had negligible effect on chemical defense. Even more surprisingly, we found no evidence that higher predation risk enhances chemical defenses, suggesting that low predictability of predation risk and high mortality cost of low toxicity might select for constitutive expression of chemical defense irrespective of the actual level of predation risk. Our findings highlight that the variation in chemical defense may be influenced by environmental heterogeneity in both the need for, and constraints on, toxicity as predicted by optimal defense theory.
Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2′-deoxyuridine 5′-triphosphate (dUTP) into 2′-deoxyuridine 5′-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/– animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early dut −/− embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice.
Anthropogenic environmental change poses a special threat to species in which genetic sex determination can be overwritten by the thermal and chemical environment. Endocrine disrupting chemicals as well as extreme temperatures can induce sex reversal in such species, with wide-ranging consequences for fitness, demography, population viability and evolution. Despite accumulating evidence suggesting that chemical and thermal effects may interact in ecological contexts, little is known about their combined effects on sex reversal. Here we assessed the simultaneous effects of high temperature (masculinizing agent) and 17α-ethinylestradiol (EE2), a widespread xenoestrogen (feminizing agent), on sexual development and fitness-related traits in agile frogs (Rana dalmatina). We exposed tadpoles to a six-days heat wave (30°C) and/or an ecologically relevant concentration of EE2 (30 ng/L) in one of three consecutive larval periods, and diagnosed sex reversals two months after metamorphosis using species-specific markers for genetic sexing. We found that high temperature induced female-to-male sex reversal, decreased survival, delayed metamorphosis, decreased body mass at metamorphosis, and increased the proportion of animals that had no fat bodies, while EE2 had no effect on these traits. Simultaneous exposure to heat and EE2 had non-additive effects on juvenile body mass, which were dependent on treatment timing and further complicated by a negative effect of sex reversal on body mass. These results show that environmentally relevant exposure to EE2 does not diminish the masculinizing effects of high temperature. Instead, our findings on growth suggest that climate change and chemical pollution may have complex consequences for individual fitness and population persistence in species with environment-sensitive sex determination.
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