SRA

Despite considerable speculation for the role of cytosine (DNA) methylation in biological and molecular processes in insects, direct functional tests are lacking. Here we provide evidence for the functional role of the maintenance DNA methyltransferase 1 (Dnmt1) in an insect using experimental manipulation. Through RNA interference (RNAi) we successfully post-transcriptionally knocked down Dnmt1 in ovarian tissue of the hemipteran Oncopeltus fasciatus (the large milkweed bug). Individuals depleted for dnmt1, and subsequently DNA methylation, failed to reproduce. Manipulating the levels of DNA methylation did not result in changes in overall gene expression. Furthermore, reductions in levels of DNA methylation at transposable elements (TEs) did not lead to large-scale reactivation of TE transcription. Despite the lack of a causal relationship between reduced DNA methylation and gene expression in the tissue we surveyed, eggs were inviable revealing an important function of DNA methylation in O. fasciatus. Our work provides direct experimental evidence for a functional role of Dnmt1 and DNA methylation in insects and presents O. fasciatus as a tractable model for further exploration of the function of DNA methylation in other tissues and life history circumstances for insects. Overall design: DNA methylation. MethylC-seq libraries for a single Oncopeltus fasciatus DNMT1 knocked down and dsRED control individual were prepared according to the protocol described in Urich et al. (2015; doi:10.1038/nprot.2014.114) using genomic DNA extracted from ovaries of post-mated females. Libraries were single-end 75 bp sequenced on an Illumina NextSeq500 machine. Un-methylated lambda phage DNA was used to as a control for sodium bisulfite conversion, and an error rate of ~0.05% was estimated. Blattella germanica was additionally sequenced to generate equal numbers of hemi- and holometabolous insects investigated in this study. However, DNA was extracted from whole-body minus gastrointestinal tract. MethylC-seq libraries were prepared and sequenced identically to O. fasciatus. An error rate of ~0.14% was estimated from un-methylated lambda phage DNA. Gene expression. RNA-seq libraries for RNA extracted from three O. fasciatus DNMT1 knocked down and three control (RED) individuals were constructed using Illumina TruSeq Stranded RNA LT Kit (Illumina, San Diego, CA) following the manufacturer's instructions with limited modifications. The starting quantity of total RNA was adjusted to 1.3 µg, and all volumes were reduced to a third of the described quantity. Libraries were single-end 75 bp sequenced on an Illumina NextSeq500 machine.