Effects of RNAi-by-feeding on DNA methyltransferases in Daphnia pulex.

Abstract

DNA methyltransferases (DNMTs) are well-characterized epigenetic enzymes responsible for transferring methyl groups to and from DNA. Three main DNMT orthologues differ in function and methylation capability. They each are evolutionarily conserved across diverse taxa, but few studies investigate them jointly. We did so in Daphnia, freshwater microcrustaceans used extensively in research on genetic diversity, phenotypic plasticity, and maternal effects. Daphnia are capable of reproducing asexually through cyclic parthenogenesis, making the Daphnia system an ideal choice for studying epigenetic phenomena. Advances in gene expression control techniques, including RNA interference (RNAi), have increased the versatility and power of the Daphnia system. RNAi is a post-transcriptional gene silencing mechanism that operates through sequence-specific cleavage of endogenous messenger RNA (mRNA) transcripts. Here, we used an RNAi bacterial feeding regime to target the three DNA methyltransferase genes in two clones of Daphnia pulex. We observed significant genotypic differences in response to the RNAi feeding regime, namely, the mortality of one clone. In the other, DNMT expression significantly increased in five of the six experimental treatments, with the highest level observed in animals treated with the GFP double-stranded RNA bacterial vector control. However, DNMT expression was reduced in all three DNMT RNAi treatments relative to the GFP control. Furthermore, we found strong cross-reactivity, where targeting one DNMT resulted in a reduction in expression of the other two. This response may be associated with known immune pathways involving signal transduction that can be stimulated by viral and bacterial signals, or it may result from previously unknown aspects of DNMT biology.