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.

Contrasting mtDNA and microsatellite data of great reed warbler Acrocephalus arundinaceus breeding populations on a small geographic scale.

The great reed warbler has two genetically distinguishable haplogroups: "Clade A" occurs in higher proportions in Western Europe and Kazakhstan, and colonised Europe and Asia from a refugium in South-West Europe; and "Clade B", which is more common in Eastern Europe, and colonised parts of Europe from a refugium in the Middle East. Our aims were (i) to analyse the rate of differentiation in Hungarian breeding populations in order to see whether European-scale pattern is visible or not on as a small scale as the territory of Hungary and (ii) to compare the results obtained with mtDNA sequencing and microsatellite markers. To analyse the genetic differentiation, the mtDNA control region II was sequenced in 68 adult breeding birds, and 51 were fingerprinted at 11 microsatellite loci, while both analyses were performed on 36 birds (a total of 83 birds). The microsatellite data gave a better resolution and represented the fine-scale pattern of the suspected recolonisation. The lack of genetic differentiation among the breeding populations based on mitochondrial data seems to support this finding, because the admixture of the clades in this particular geographic region obliterates differentiation. Accordingly, the Fst values from different branches are significantly based on microsatellite data only. The mtDNA methods only give reliable results when a geographic and ecological factor plays a role in the population subdivision, but in the case of an intermixing population larger-scale studies are needed.

"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.

Sex interacts with age-dependent change in the abundance of lice-infesting Amur Falcons (Falco amurensis).

Sex-biassed and age-biassed parasite infections are common in nature, including ectoparasites-vertebrate host systems. We investigated the effect of Amur Falcons' sex, age and body size on the abundance of their lice at a migratory stopover site, where the falcons' habitat use and behaviour are more homogeneous across sex and age categories than during the breeding season. We sampled Amur Falcons in Nagaland, India at major roosting sites in 2016. We applied generalized linear models (with negative binomial distribution and log-link) to model the abundance of their two most numerous lice (Colpocephalum subzerafae and Degeeriella rufa) using the host age category (juvenile or adult) and wing length, both in interaction with sex, as explanatory variables. The abundance of C. subzerafae was only affected by host age, being nearly four times higher on juveniles than on adults. Juveniles were also more infested with D. rufa than the adults. Additionally, the abundance of the latter species was lower on adult male Falcons as compared to adult females. A juvenile bias in ectoparasite infestations is common in nature, probably due to juveniles being immunologically naïve, more resource-limited and may be inexperienced in body maintenance behaviours like preening and grooming. On the other hand, female-biassed infestations are much rarer than male-biassed infestations. We briefly discuss the possible causes of female-biassed infestations on Amur Falcons reported here, and in the closely related Red-footed Falcon and Lesser Kestrel as reported in the literature.

Sex-dependent changes in the louse abundance of red-footed falcons (Falco vespertinus).

Permanent ectoparasites live in stable environments; thus, their population dynamics are mostly adapted to changes in the host life cycle. We aimed to investigate how static and dynamic traits of red-footed falcons interplay with the dynamics of their louse subpopulations during breeding and how they affect the colonisation of new hosts by lice. We sampled red-footed falcon (Falco vespertinus) nestlings (two breeding seasons) and adults (one breeding season) in southern Hungary. The mean abundance of Colpocephalum subzerafae and Degeeriella rufa lice on the nestlings was modelled with generalized linear mixed models using clutch size and host sex in interaction with wing length. For adults, we used wing length and the number of days after laying the first egg, both in interaction with sex. D. rufa abundances increased with the nestlings' wing length. In one year, this trend was steeper on females. In adult birds, both louse species exhibited higher abundances on females at the beginning, but it decreased subsequently through the breeding season. Contrarily, abundances were constantly low on adult males. Apparently, D. rufa postpones transmission until nestlings develop juvenile plumage and choose the more feathered individual among siblings. The sexual difference in the observed abundance could either be caused by the different plumage, or by the females' preference for less parasitized males. Moreover, females likely have more time to preen during the incubation period, lowering their louse burdens. Thus, sex-biased infestation levels likely arise due to parasite preferences in the nestlings and host behavioural processes in the adult falcons.