Sex-linked molecular markers and their application to endocrine disruption research in amphibians

Die weltweit mehr als 7500 Amphibienarten sind durch anthropogene Ursachen wie Habitatzerstörung, Krankheitsverbreitung, Klimawandel und Umweltverschmutzung in ihrem Bestand bedroht. Einige der Ursachen sind kaum erforscht, so die Verschmutzung aquatischer Ökosysteme durch endokrine Disruptoren (EDs), Substanzen, die mit dem Hormonsystem interagieren. Ausgehend von neuen molekularen Markern, welche die Ermittlung des genetischen Geschlechts erstmals bei einigen Hyliden und Bufoniden erlauben, wurde in der vorliegenden Arbeit auf die Wirkung des synthetischen Östrogens 17α- Ethinylestradiol (EE2)und des Weichmachers Bisphenol A (BPA) fokussiert. Für drei Bufonidenarten wurde zunächst die Geschlechtsgebundenheit von Mikrosatelliten getestet und ein XX/XY-System nachgewiesen. Diese und bereits etablierte Marker wurden anschließend in ein neu entwickeltes Versuchsdesign für ED-Studien integriert: Nach gleichzeitiger Aufzucht von Modell- (Xenopus laevis) und Nicht-Modell-Arten (Hyla arborea, Bufo viridis) unter EE2- bzw. BPA- Exposition wurde das genetische Geschlecht bestimmt und mit dem anatomisch und histologisch ermittelten phänotypischen Geschlecht erglichen. Die drei Anuren zeigten starke Empfindlichkeitsunterschiede gegenüber beiden EDs. Umweltrelevante Konzentrationen beeinflussten die somatische Entwicklung und führten zu artspezifischen Gonaden-Fehlbildungen. EE2 bewirkte zahlreiche partielle und komplette Geschlechtsumwandlungen, mit stärkeren Effekten bei X. laevis. Diese Arbeit zeigt somit, dass bereits niedrige EE2- und BPA-Konzentrationen zu starken Schädigungen führen können und die Substanzen aufgrund ihrer erheblichen aquatischen Präsenz als ernstzunehmende Faktoren der Amphibienkrise anzusehen sind. Die Ermittlung des genetischen Geschlechts wird als wichtig eingestuft, um verlässliche Aussagen über ED-Effekte zu treffen. Zudem sollten an der Modell-Art X. laevis gewonnene Erkenntnisse nicht vorbehaltlos auf andere Amphibienarten extrapoliert werden.The more than 7500 known amphibian species are globally threatened, mainly due to anthropogenic causes like habitat destruction, dispersing diseases, climate change and environmental pollution. Some of the causes are barely investigated, e.g. the pollution of aquatic ecosystems with endocrine disrupting compounds (EDCs), substances that interfere with the hormone system. Based on new molecular markers, for the first time allowing genetic sexing in some hylids and bufonids, this thesis focused on the effects of the synthetic estrogen 7α-ethinylestradiol(EE2) and the plasticizer bisphenol A (BPA). Initially, several microsatellite markers were tested for sex-linkage in three bufonid species, and an XX/XY system could be revealed. Subsequently, these and other established markers were integrated into a newly developed experimental design for EDC-research: after simultaneous exposure of model (Xenopus laevis) and non-model species (Hyla arborea, Bufo viridis) to EE2 or BPA, metamorphs were genetically sexed. Anatomically and histologically determined phenotypic sexes were directly compared with the genetic sex of each individual. The three anurans showed striking differences in their susceptibilities in both EDCexperiments. Environmentally relevant concentrations affected the somatic development and led to species-specific gonadal anomalies. In addition, EE2 provoked high numbers of mixed sex and completely sex-reversed individuals, with more pronounced effects in X. laevis than in the two non-model species. This work shows that low concentrations of EE2 and BPA lead to severe damages. Due to their widespread presence in the aquatic environment, these substances might contribute to the worldwide amphibian crisis. To produce reliable results in EDC-studies, genetic sexing is considered important. Furthermore, findings gained with the model species X. laevis should not unreservedly be extrapolated to other amphibian species

Sex chromosome conservation, DMRT1 phylogeny and gonad morphology in diploid Palearctic green toads (Bufo viridis subgroup).

Due to the prevailing sex chromosome homomorphy and large genome size, the knowledge on sex determination systems, sex chromosomes and sex-determining genes in amphibians remains scarce. Using 3 cross-amplifying sex-linked microsatellite markers, we uncover sex determination systems and sex chromosomes in purebred, diploid Palearctic green toads (Bufo viridis subgroup), which had so far only been characterized in laboratory-bred hybrids. Our data support an XY system in B. balearicus, B. viridis and B. variabilis. While females show recombination, it is strongly suppressed (or not detectable) in males. Markers corroborate the largest chromosome pair 1 (homologous to linkage group 1 of Xenopus tropicalis) to represent the sex chromosomes in diploid species of the B. viridis subgroup (B. siculus, B. shaartusiensis, B. balearicus, B. turanensis, B. variabilis, B. viridis, and probably B. boulengeri). This chromosome harbors DMRT1, a key gene of the sexual pathway in deeply divergent animal taxa. However, our phylogenetic analysis of a 600-bp fragment of that gene in diploid green toad taxa reveals that X and Y alleles cluster by species and not by gametolog. This suggests that XY-sequence similarity stems from occasional XY recombination within DMRT1, and we preliminarily reject its role as the master sex determination gene, pending future extension of this evidence to the entire DMRT1 gene. We further create a chain of evidence, which supports the hypothesis that linkage group 1 of X. tropicalis appears to be maintained as the largest chromosome (1), and thus is homologous in anuran karyotype evolution from pipid to hylid, bufonid and ranid anurans

Sex reversal assessments reveal different vulnerability to endocrine disruption between deeply diverged anuran lineages

Multiple anthropogenic stressors cause worldwide amphibian declines. Among several poorly investigated causes is global pollution of aquatic ecosystems with endocrine disrupting compounds (EDCs). These substances interfere with the endocrine system and can affect the sexual development of vertebrates including amphibians. We test the susceptibility to an environmentally relevant contraceptive, the artificial estrogen 17α-ethinylestradiol (EE2), simultaneously in three deeply divergent systematic anuran families, a model-species, Xenopus laevis (Pipidae), and two non-models, Hyla arborea (Hylidae) and Bufo viridis (Bufonidae). Our new approach combines synchronized tadpole exposure to three EE2-concentrations (50, 500, 5,000 ng/L) in a flow-through-system and pioneers genetic and histological sexing of metamorphs in non-model anurans for EDC-studies. This novel methodology reveals striking quantitative differences in genetic-male-to-phenotypic-female sex reversal in non-model vs. model species. Our findings qualify molecular sexing in EDC-analyses as requirement to identify sex reversals and state-of-the-art approaches as mandatory to detect species-specific vulnerabilities to EDCs in amphibians

Impacts of the synthetic androgen Trenbolone on gonad differentiation and development – comparisons between three deeply diverged anuran families

Using a recently developed approach for testing endocrine disruptive chemicals (EDCs) in amphibians, comprising synchronized tadpole exposure plus genetic and histological sexing of metamorphs in a flow-through-system, we tested the effects of 17β-Trenbolone (Tb), a widely used growth promoter in cattle farming, in three deeply diverged anuran families: the amphibian model species Xenopus laevis (Pipidae) and the non-models Bufo(tes) viridis (Bufonidae) and Hyla arborea (Hylidae). Trenbolone was applied in thhttps://www.tandfonline.com/doi/full/10.1080/22221751.2020.1760144?scroll=top&needAccess=trueree environmentally and/or physiologically relevant concentrations (0.027 µg/L (10−10 M), 0.27 µg/L (10−9 M), 2.7 µg/L (10−8 M)). In none of the species, Tb caused sex reversals or masculinization of gonads but had negative species-specific impacts on gonad morphology and differentiation after the completion of metamorphosis, independently of genetic sex. In H. arborea and B. viridis, mounting Tb-concentration correlated positively with anatomical abnormalities at 27 µg/L (10−9 M) and 2.7 µg/L (10−8 M), occurring in X. laevis only at the highest Tb concentration. Despite anatomical aberrations, histologically all gonadal tissues differentiated seemingly normally when examined at the histological level but at various rates. Tb-concentration caused various species-specific mortalities (low in Xenopus, uncertain in Bufo). Our data suggest that deep phylogenetic divergence modifies EDC-vulnerability, as previously demonstrated for Bisphenol A (BPA) and Ethinylestradiol (EE2)

Supplementary Material for: Sex Chromosome Conservation, <b><i>DMRT1</i></b> Phylogeny and Gonad Morphology in Diploid Palearctic Green Toads (<b><i>Bufo viridis </i></b>Subgroup)

Due to the prevailing sex chromosome homomorphy and large genome size, the knowledge on sex determination systems, sex chromosomes and sex-determining genes in amphibians remains scarce. Using 3 cross-amplifying sex-linked microsatellite markers, we uncover sex determination systems and sex chromosomes in purebred, diploid Palearctic green toads (<i>Bufo viridis</i> subgroup), which had so far only been characterized in laboratory-bred hybrids. Our data support an XY system in <i>B. balearicus</i>, <i>B. viridis</i> and <i>B. variabilis</i>. While females show recombination, it is strongly suppressed (or not detectable) in males. Markers corroborate the largest chromosome pair 1 (homologous to linkage group 1 of <i>Xenopus tropicalis</i>) to represent the sex chromosomes in diploid species of the <i>B. viridis</i> subgroup <i>(B. siculus, B. shaartusiensis, B. balearicus, B. turanensis, B. variabilis, B. viridis</i>, and probably <i>B. boulengeri)</i>. This chromosome harbors <i>DMRT1</i>, a key gene of the sexual pathway in deeply divergent animal taxa. However, our phylogenetic analysis of a 600-bp fragment of that gene in diploid green toad taxa reveals that X and Y alleles cluster by species and not by gametolog. This suggests that XY-sequence similarity stems from occasional XY recombination within <i>DMRT1</i>, and we preliminarily reject its role as the master sex determination gene, pending future extension of this evidence to the entire <i>DMRT1 </i>gene. We further create a chain of evidence, which supports the hypothesis that linkage group 1 of <i>X. tropicalis</i> appears to be maintained as the largest chromosome (1), and thus is homologous in anuran karyotype evolution from pipid to hylid, bufonid and ranid anurans