Occurrence of DNA methylation in Daphnia magna and influence of multigeneration Cd exposure.

Most of the research on the epigenetic phenomenon of DNA methylation has been performed with vertebrates and plants. Knowledge on DNA methylation in Daphnia magna, a key test organism in aquatic toxicology, is completely lacking. Through epigenetic inheritance, effects of transient chemical exposure could be transferred to non-exposed generations, which could have a major impact on ecological risk assessment procedures. In this study, we determined if CpG methylation occurs in D. magna and if this can be influenced by exposure to toxic substances. Homologs of human DNA methyltransferases DNMT1, DNMT2 and DNMT3A were found in the partially available D. magna genome. Using an optimized "Amplification of Intermethylated Sites (AIMS)" technique, two methylated fragments were discovered in D. magna DNA. No homology was found for these sequences. The methylation and the D. magna origin of the fragments were confirmed with Southern analysis. This optimized AIMS technique was then applied to DNA of D. magna which were exposed to 180 microg/L Cd for two generations. Exposure resulted in a significant decrease in reproduction. The same methylated fragments with the same band intensity were observed in DNA of both non-exposed and exposed daphnids. As such, it could not be demonstrated that Cd exposure altered DNA methylation. However, the presence of DNA methylation in D. magna shows that potentially epigenetic effects may occur in this species.