Rapid and sensitive detection of waterfowl mycoplasmas using TaqMan assays.

Waterfowl-specific mycoplasmas cause significant economic losses worldwide. However, only limited resources are available for the specific detection of three such bacteria, Mycoplasma anatis, M. anseris and M. cloacale. We developed species-specific TaqMan assays and tested their reliability across 20 strains of the respective target species as well as 84 non-target avian bacterial strains. Furthermore, we analysed 32 clinical DNA samples and compared the results with those of previously published conventional PCRs. The TaqMan assays showed 100% specificity and very high sensitivity, enabling the detection of target DNA as low as either 10 or 100 copies/µl concentration, depending on the assay. Importantly, we found that while the here developed TaqMan assays are reliable for species-specific detection of M. anatis, the previously published conventional PCR assay may give false positive results. In conclusion, the new assays are reliable, sensitive and suitable for clinical diagnostics of the target species.

HerpSexDet: the herpetological database of sex determination and sex reversal.

Wildlife exhibits various sex-determination systems where sex chromosomes and environmental temperatures may both contribute to individual sexual development. The causes and consequences of this variability are important questions for evolutionary ecology, especially in light of ongoing environmental change. Amphibians and reptiles are emerging as a key group for studying these questions, with new data accumulating acceleratingly. We collected empirical data from earlier databases, reviews and primary literature to create the most up-to-date database on herpetological sex determination. We named our database HerpSexDet, which currently features data on genetic and temperature-dependent sex determination as well as reports on sex reversal for a total of 192 amphibian and 697 reptile species. This dataset, which we will regularly update in the future, facilitates interspecific comparative studies on the evolution of sex determination and its consequences for species-specific traits such as life history and conservation status, and may also help guiding future research by identifying species or higher taxa that are potentially most enlightening for the study of environmentally driven sex reversal.

Characterization of atypical Mycoplasma anserisalpingitidis strains.

Mycoplasma anserisalpingitidis is a waterfowl colonizing mycoplasma, mainly found in geese. In this study, we compared the whole genomes of five atypical M. anserisalpingitidis strains originating from China, Vietnam and Hungary, with the rest of the collection. Common methods used in the description of species are genomic analyses like the analysis of 16 S - intergenic transcribed spacer (ITS) - 23 S rRNA, of housekeeping genes, of the average nucleotide identity (ANI) and average amino acid identity (AAI) and phenotypic analyses like testing the growth inhibition and the growth parameters of the strains. The atypical strains showed notable genomic differences in all of the genetic analyses: on average ANI and AAI 95% (M. anserisalpingitidis ANI Minimum: 92.45, Maximum: 95.10; AAI Minimum: 93.34, Maximum: 96.37). The atypical strains formed a separate branch among the M. anserisalpingitidis strains in all phylogenetic studies. The small genome size and possibly higher mutation rate of the M. anserisalpingitidis species likely contributed to the observed genetic difference. Based on genetic analyses, the studied strains clearly represent a new genotype of M. anserisalpingitidis. The atypical strains showed slower growth in the medium containing fructose and three of the atypical strains showed diminished growth in the inhibition test. However, no definitive geno-phenotype associations were found regarding the fructose metabolism pathway in the atypical strains. The atypical strains are potentially at an early stage of speciation.

Asymmetrical sex reversal: Does the type of heterogamety predict propensity for sex reversal?

Sex reversal, a mismatch between phenotypic and genetic sex, can be induced by chemical and thermal insults in ectotherms. Therefore, climate change and environmental pollution may increase sex-reversal frequency in wild populations, with wide-ranging implications for sex ratios, population dynamics, and the evolution of sex determination. We propose that reconsidering the half-century old theory "Witschi's rule" should facilitate understanding the differences between species in sex-reversal propensity and thereby predicting their vulnerability to anthropogenic environmental change. The idea is that sex reversal should be asymmetrical: more likely to occur in the homogametic sex, assuming that sex-reversed heterogametic individuals would produce new genotypes with reduced fitness. A review of the existing evidence shows that while sex reversal can be induced in both homogametic and heterogametic individuals, the latter seem to require stronger stimuli in several cases. We provide guidelines for future studies on sex reversal to facilitate data comparability and reliability.

"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.

Evolutionary and demographic consequences of temperature-induced masculinization under climate warming: the effects of mate choice.

BACKGROUND: One of the dangers of global climate change to wildlife is distorting sex ratios by temperature-induced sex reversals in populations where sex determination is not exclusively genetic, potentially leading to population collapse and/or sex-determination system transformation. Here we introduce a new concept on how these outcomes may be altered by mate choice if sex-chromosome-linked phenotypic traits allow females to choose between normal and sex-reversed (genetically female) males. RESULTS: We developed a theoretical model to investigate if an already existing autosomal allele encoding preference for sex-reversed males would spread and affect demographic and evolutionary processes under climate warming. We found that preference for sex-reversed males (1) more likely spread in ZW/ZZ than in XX/XY sex-determination systems, (2) in populations starting with ZW/ZZ system, it significantly hastened the transitions between different sex-determination systems and maintained more balanced adult sex ratio for longer compared to populations where all females preferred normal males; and (3) in ZW/ZZ systems with low but non-zero viability of WW individuals, a widespread preference for sex-reversed males saved the populations from early extinction. CONCLUSIONS: Our results suggest that climate change may affect the evolution of mate choice, which in turn may influence the evolution of sex-determination systems, sex ratios, and thereby adaptive potential and population persistence. These findings show that preferences for sex-linked traits have special implications in species with sex reversal, highlighting the need for empirical research on the role of sex reversal in mate choice.

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.

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.

Climate-driven shifts in adult sex ratios via sex reversals: the type of sex determination matters.

Sex reversals whereby individuals of one genetic sex develop the phenotype of the opposite sex occur in ectothermic vertebrates with genetic sex-determination systems that are sensitive to extreme temperatures during sexual differentiation. Recent rises in global temperatures have led researchers to predict that sex reversals will become more common, resulting in the distortion of many populations' sex ratios. However, it is unclear whether susceptibility to climate-driven sex-ratio shifts depends on the type of sex determination that varies across species. First, we show here using individual-based theoretical models that XX/XY (male-heterogametic) and ZZ/ZW (female-heterogametic) sex-determination systems can respond differentially to temperature-induced sex reversals. Interestingly, the impacts of climate warming on adult sex ratio (ASR) depend on the effects of both genotypic and phenotypic sex on survival and reproduction. Second, we analyse the temporal changes of ASR in natural amphibian populations using data from the literature, and find that ASR shifted towards males in ZZ/ZW species over the past 60 years, but did not change significantly in XX/XY species. Our results highlight the fact that we need a better understanding of the interactions between genetic and environmental sex-determining mechanisms to predict the responses of ectotherms to climate change and the associated extinction risks.This article is part of the themed issue 'Adult sex ratios and reproductive decisions: a critical re-examination of sex differences in human and animal societies'.