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

Gunnar Carlsson,Eva Tydén

doi:10.1007/s11356-018-3260-z

Abstract

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 (CdCl2), 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 CdCl2 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.

Highlights

Pollutants have been proposed as one factor in the worldwide declines of amphibian species and populations (Sparling et al 2001; Hayes et al 2010)
Wild tadpoles can potentially be exposed to many pollutants causing increases in gene expression of cyp1a since other pollutants have been shown to trigger cytochrome p4501A (CYP1A) activity or gene expression with different potencies than BNF (Smeets et al 1999). mt might be somewhat useful for biomonitoring even though the present study indicates that most individuals exposed to CdCl2 were not distinguished from the background
The hepatic gene expression of cyp1a was induced by BNF and the gene expression of mt was induced by CdCl2 when R. temporaria tadpoles were exposed for 1 week to the different chemicals

Summary

Introduction

Pollutants have been proposed as one factor in the worldwide declines of amphibian species and populations (Sparling et al 2001; Hayes et al 2010). Pollutants can enter aquatic environments from several sources, such as from pesticide application, which may occur in connection with amphibian reproduction and larval development. Amphibians may be attracted to wetlands such as stormwater ponds, potentially impacted of metals and organic compounds (Simon et al 2009; Brand and Snodgrass 2010; Pohl et al 2015) and are potentially. Biomarkers indicating pollution are promising tools to increase knowledge of ecological health and can be applied in effect-based environmental monitoring. Further development of tools that can screen multiple responses in parallel and monitor harmful effects of pollutants on ecosystem health are further requested (Baker et al 2014).

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Conclusion