Does stress make males? An experiment on the role of glucocorticoids in anuran sex reversal.

Environmentally sensitive sex determination may help organisms adapt to environmental change but also makes them vulnerable to anthropogenic stressors, with diverse consequences for population dynamics and evolution. The mechanisms translating environmental stimuli to sex are controversial: although several fish experiments supported the mediator role of glucocorticoid hormones, results on some reptiles challenged it. We tested this hypothesis in amphibians by investigating the effect of corticosterone on sex determination in agile frogs (Rana dalmatina). This species is liable to environmental sex reversal whereby genetic females develop into phenotypic males. After exposing tadpoles during sex determination to waterborne corticosterone, the proportion of genetic females with testes or ovotestes increased from 11% to up to 32% at 3 out of 4 concentrations. These differences were not statistically significant except for the group treated with 10 nM corticosterone, and there was no monotonous dose-effect relationship. These findings suggest that corticosterone is unlikely to mediate sex reversal in frogs. Unexpectedly, animals originating from urban habitats had higher sex-reversal and corticosterone-release rates, reduced body mass and development speed, and lower survival compared to individuals collected from woodland habitats. Thus, anthropogenic environments may affect both sex and fitness, and the underlying mechanisms may vary across ectothermic vertebrates.

"Heat waves" experienced during larval life have species-specific consequences on life-history traits and sexual development in anuran amphibians.

Extreme temperatures during heat waves can induce mass-mortality events, but can also exert sublethal negative effects by compromising life-history traits and derailing sexual development. Ectothermic animals may, however, also benefit from increased temperatures via enhanced physiological performance and the suppression of cold-adapted pathogens. Therefore, it is crucial to address how the intensity and timing of naturally occurring or human-induced heat waves affect life-history traits and sexual development in amphibians, to predict future effects of climate change and to minimize risks arising from the application of elevated temperature in disease mitigation. We raised agile frog (Rana dalmatina) and common toad (Bufo bufo) tadpoles at 19 °C and exposed them to a simulated heat wave of 28 or 30 °C for six days during one of three ontogenetic periods (early, mid or late larval development). In agile frogs, exposure to 30 °C during early larval development increased mortality. Regardless of timing, all heat-treatments delayed metamorphosis, and exposure to 30 °C decreased body mass at metamorphosis. Furthermore, exposure to 30 °C during any period and to 28 °C late in development caused female-to-male sex reversal, skewing sex ratios strongly towards males. In common toads, high temperature only slightly decreased survival and did not influence phenotypic sex ratio, while it reduced metamorph mass and length of larval development. Juvenile body mass measured 2 months after metamorphosis was not adversely affected by temperature treatments in either species. Our results indicate that heat waves may have devastating effects on amphibian populations, and the severity of these negative consequences, and sensitivity can vary greatly between species and with the timing and intensity of heat. Finally, thermal treatments against cold-adapted pathogens have to be executed with caution, taking into account the thermo-sensitivity of the species and the life stage of animals to be treated.

Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats.

Anthropogenic environmental changes are affecting biodiversity and microevolution worldwide. Ectothermic vertebrates are especially vulnerable because environmental changes can disrupt their sexual development and cause sex reversal, a mismatch between genetic and phenotypic sex. This can potentially lead to sex-ratio distortion and population decline. Despite these implications, there is scarce empirical knowledge on the incidence of sex reversal in nature. Populations in anthropogenic environments may be exposed to sex-reversing stimuli more frequently, which may lead to higher sex-reversal rate or, alternatively, these populations may adapt to resist sex reversal. We developed PCR-based genetic sex markers for the common toad (Bufo bufo) to assess the prevalence of sex reversal in wild populations living in natural, agricultural and urban habitats, and the susceptibility of the same populations to two ubiquitous oestrogenic pollutants in a common garden experiment. We found negligible sex-reversal frequency in free-living adults despite the presence of various endocrine-disrupting pollutants in their breeding ponds. Individuals from different habitat types showed similar susceptibility to sex reversal in the laboratory: all genetic males developed female phenotype when exposed to 1 µg L-1 17α-ethinylestradiol (EE2) during larval development, whereas no sex reversal occurred in response to 1 ng L-1 EE2 and a glyphosate-based herbicide with 3 µg L-1 or 3 mg L-1  glyphosate. The latter results do not support that populations in anthropogenic habitats would have either increased propensity for or higher tolerance to chemically induced sex reversal. Thus, the extremely low sex-reversal frequency in wild toads compared to other ectothermic vertebrates studied before might indicate idiosyncratic, potentially species-specific resistance to sex reversal.

Sex reversal and ontogeny under climate change and chemical pollution: are there interactions between the effects of elevated temperature and a xenoestrogen on early development in agile frogs?

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 potentially 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 (female-to-male sex-reversing agent) and 17α-ethinylestradiol (EE2), a widespread xenoestrogen (male-to-female sex-reversing 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 female-to-male sex-reversing 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.

Stressed tadpoles mount more efficient glucocorticoid negative feedback in anthropogenic habitats due to phenotypic plasticity.

Coping with anthropogenic environmental change is among the greatest challenges faced by wildlife, and endocrine flexibility is a potentially crucial coping mechanism. Animals may adapt to anthropogenic environments by dampening their glucocorticoid stress response, but empirical tests of this hypothesis have provided mixed evidence. An alternative hypothesis is that a non-attenuated stress response and efficient negative feedback are favored in anthropogenic habitats. To test this idea, we non-invasively sampled corticosterone release rates of common toad (Bufo bufo) tadpoles in agricultural, urban, and natural habitats, and quantified their stress response and negative feedback by a standardized stress-and-recovery protocol. We repeated the same sampling with tadpoles raised from eggs from the same ponds in a common-garden experiment to infer if the differences observed between populations in different habitats were due to individual phenotypic plasticity rather than microevolution or transgenerational effects. We found that, compared to tadpoles in natural ponds, urban tadpoles had higher baseline and stressed corticosterone release rates, and tadpoles in agricultural ponds had similar corticosterone release rates but greater stress-induced change, indicating stronger stress responses in both types of anthropogenic habitats. As predicted, tadpoles in both agricultural and urban ponds showed more efficient negative feedback than did tadpoles in natural ponds. Water pollution levels, as indicated by the concentrations of carbamazepine and corticoid-disrupting compounds in pond water, contributed to elevating the stress response regardless of land use. Infection by neither Batrachochytrium dendrobatidis nor Ranavirus was detected in free-living tadpoles. No habitat-related glucocorticoid differences persisted in the common-garden experiment. These results suggest that toad tadpoles in anthropogenic habitats increased their glucocorticoid flexibility via phenotypic plasticity. The coupling of stronger stress response and stronger negative feedback in these habitats supports the importance of rapidly "turning on and off" the stress response as a mechanism for coping with anthropogenic environmental change.

Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog (Rana dalmatina) associated with anthropogenic land use.

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.

Effects of two little-studied environmental pollutants on early development in anurans.

Despite intensive ecotoxicological research, we still know relatively little about the ecological impacts of many environmental contaminants. Filling these knowledge gaps is particularly important regarding amphibians, because they play significant roles in freshwater and terrestrial ecosystems, and their populations are declining worldwide. In this study, we investigated two pollutants that have been poorly studied in ecotoxicology despite their widespread occurrence in surface waters: the herbicide terbuthylazine and the pharmaceutical drug carbamazepine. We exposed two anuran species throughout their larval development to each of two environmentally relevant concentrations of each pollutant, and recorded mortality and 17 sub-lethal endpoints up to several months after exposure. Mortality was low and unrelated to treatment. In agile frogs (Rana dalmatina), we found that treatment with 0.3 µg/L terbuthylazine decreased tadpole activity and reduced fat bodies in juveniles, whereas treatment with 50 µg/L carbamazepine decreased spleen size and increased spleen pigmentation. In common toads (Bufo bufo), treatment with 0.003 µg/L terbuthylazine increased body mass at metamorphosis, treatment with 0.3 µg/L terbuthylazine increased the size of optic tecta, and treatment with 0.5 µg/L carbamazepine decreased hypothalamus size. Treatment with 50 µg/L carbamazepine reduced the feeding activity of toad tadpoles, decreased their production of anti-predatory bufadienolide toxins, and increased their body mass at metamorphosis; juvenile toads in this treatment group had reduced spleen pigmentation. Neither treatments affected the time to metamorphosis, post-metamorphic body mass, or sex ratios significantly. These results show that environmental levels of both terbuthylazine and carbamazepine can have several sub-lethal effects on anurans, which may be detrimental to individual fitness and population persistence in natural conditions. Our findings further highlight that toxic effects cannot be generalized between chemicals of similar structure, because the terbuthylazine effects we found do not conform with previously reported effects of atrazine, a related and extensively studied herbicide.

Toads phenotypically adjust their chemical defences to anthropogenic habitat change.

Despite the well-documented effects of human-induced environmental changes on the morphology, physiology, behaviour and life history of wild animals, next to nothing is known about how anthropogenic habitats influence anti-predatory chemical defence, a crucial fitness component of many species. We investigated the amount and composition of defensive toxins in adult common toads (Bufo bufo) captured in natural, agricultural and urban habitats, and in their offspring raised in a common-garden experiment. We found that, compared to toads captured from natural habitats, adults from both types of anthropogenic habitats had larger toxin glands (parotoids) and their toxin secretion contained higher concentrations of bufagenins, the more potent class of bufadienolide toxins. Furthermore, urban toads had lower concentrations of bufotoxins, the compounds with lower toxicity. None of these differences were present in the captive-raised juveniles; instead, toadlets originating from agricultural habitats had smaller parotoids and lower bufotoxin concentrations. These results suggest that toads' chemical defences respond to the challenges of anthropogenic environments via phenotypic plasticity. These responses may constitute non-adaptive consequences of pollution by endocrine-disrupting chemicals as well as adaptive adjustments to the altered predator assemblages of urban and agricultural habitats.

Endocrine disruptors in breeding ponds and reproductive health of toads in agricultural, urban and natural landscapes.

Many chemical pollutants have endocrine disrupting effects which can cause lifelong reproductive abnormalities in animals. Amphibians are the most threatened group of vertebrates, but there is little information on the nature and quantity of pollutants occurring in typical amphibian breeding habitats and on the reproductive capacities of amphibian populations inhabiting polluted areas. In this study we investigated the occurrence and concentrations of endocrine disrupting chemicals in the water and sediment of under-studied amphibian breeding habitats in natural, agricultural and urbanized landscapes. Also, we captured reproductively active common toads (Bufo bufo) from these habitats and let them spawn in a 'common garden' to assess among-population differences in reproductive capacity. Across 12 ponds, we detected 41 out of the 133 contaminants we screened for, with unusually high concentrations of glyphosate and carbamazepine. Levels of polycyclic aromatic hydrocarbons, nonylphenol and bisphenol-A increased with urban land use, whereas levels of organochlorine and triazine pesticides and sex hormones increased with agricultural land use. Toads from all habitats had high fecundity, fertilization rate and offspring viability, but the F1 generation originating from agricultural and urban ponds had reduced development rates and lower body mass both as larvae and as juveniles. Females with small clutch mass produced thicker jelly coat around their eggs if they originated from agricultural and urban ponds compared with natural ponds. These results suggest that the observed pollution levels did not compromise reproductive potential in toads, but individual fitness and population viability may be reduced in anthropogenically influenced habitats, perhaps due to transgenerational effects and/or costs of tolerance to chemical contaminants.