Development and evaluation of gene expression biomarkers for chemical pollution in common frog (Rana temporaria) tadpoles

Pollutants have been proposed as one factor in the worldwide declines of amphibian species and populations. Applying gene expression analysis of liver RNA in tadpoles would be a possible approach for biomarker measurements to increase knowledge of ecological health in amphibian populations. The major aim of this study was to explore the relevance of applying gene expression analyses of cytochrome p450 (cyp1a), metallothionein (mt), and vitellogenin (vtg) in Rana temporaria tadpoles. Therefore, tadpoles were exposed for 1 week to β-naphthoflavone (BNF), cadmium chloride (CdCl(2)), and ethinylestradiol (EE2). Primers were developed for RT-qPCR to analyze gene expression in livers. The result showed that the methods for gene expression analyses of cyp1a, mt, and vtg as well as the reference gene β-actin (bact) were successful not only in R. temporaria but also in another amphibian, Rana arvalis. The gene expression of cyp1a was induced by BNF and the gene expression of mt was induced by CdCl(2) but no significant induction was recorded in vtg expression after exposure to EE2. Gene expressions varied throughout the tadpole metamorphosis development, in particular for vtg. Overall, the use of gene expression of cyp1a and mt as biomarkers in wild tadpoles seems promising while the use of vtg seems less relevant due to high natural variation and low background expression. The study shows that variations in gene expressions between tadpoles of different genetic origin are important to consider when evaluating the data. The present study has thus increased the background knowledge about gene expression applicability as biomarker for tadpoles.