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

Abstract

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.

The plasticizer bisphenol A affects somatic and sexual development, but differently in pipid, hylid and bufonid anurans

“…Tamschick et al, 2016). BPA-treatments involved two non-model species, H. arborea and B. viridis, and the ecotoxicology model amphibian X. laevis.…”

“…DNAs were eluted in 200 ml AE buffer (Qiagen) and stored at À20 C. To establish genetic sex, species-specific polymerase chain reactions (PCRs) were conducted with subsequent gel electrophoresis and genotyping. Sexing of H. arborea and X. laevis was conducted as described (Tamschick et al, 2016), sexing of B. viridis involved markers C201, C223 (Tamschick et al, 2015), Cherp, DMRT1 and VLDLR (Gerchen et al, 2016). Only frogs and toads that completed metamorphosis were genetically sexed.…”

“…All tadpoles were fed with SeraMicron (Sera, Germany) according to Lutz et al (2008); Hyla and Bufo were additionally fed with TetraMin (Tetra, Germany) due to different food requirements (cf. Tamschick et al, 2016). Tadpoles were inspected daily, dead individuals were removed and mortality was recorded.…”

“…We aimed at testing whether previous results obtained in the model X. laevis (Pipidae) are applicable to the European nonmodel anurans Hyla arborea (European tree frog) and Bufo viridis (European green toad), or if we find susceptibility differences between these anuran lineages (cf. Tamschick et al, 2016). The Pipidae diverged from the Neobatrachia, including Bufonidae and Hylidae, more than 206 million years ago (www.timetree.org), and there are notable differences in their ecotoxicologically relevant genome composition (e.g.…”

Determining the optimal developmental stages of Xenopus laevis for initiating exposures to chemicals for sensitively detecting their feminizing effects on gonadal differentiation

“…For example, some EDCs with weak estrogenic activities, such as BPA and PCBs, were reported to cause significant intersexes in X. laevis (Kloas et al, 1999;Levy et al, 2004;Qin et al, 2003). Furthermore, estrogenic pharmaceuticals were demonstrated to result in complete male-to-female reversal in X. laevis; for instance, Tamschick et al (2016) reported that 50, 500, 5000 ng/L EE2 caused 31.3%, 76.5% and 100% phenotypic females in genetic males, respectively. The data have enough demonstrated high sensitivity of X. laevis gonadal differentiation to estrogenic EDCs.…”