Metagenomic sequencing suggests arthropod antiviral RNA interference is highly derived, and identifies novel viral small RNAs in a mollusc and a brown alga

RNA interference (RNAi)-related pathways target viruses and transposable element (TE) transcripts in plants, fungi, and ecdysozoans (nematodes and arthropods), giving protection against infection and transmission. In each case, this produces abundant TE and virus-derived 20-30nt small RNAs, which provide a characteristic signature of RNAi-mediated defence. The broad phylogenetic distribution of the Argonaute and Dicer-family genes that mediate these pathways suggests that defensive RNAi is ancient and probably shared by most metazoan (animal) phyla. Indeed, while vertebrates had been thought an exception, it has recently been suggested that mammals may also possess a functional antiviral RNAi pathway, albeit involving few small RNAs that are challenging to detect. Here we use a metagenomic approach to test for the presence of antiviral RNAi in five divergent metazoan phyla (Porifera, Cnidaria, Echinodermata, Mollusca, and Annelida), and in a brown alga. We use metagenomic RNA sequencing to identify around 80 virus-like contigs in these lineages, and small RNA sequencing to identify small RNAs derived from those viruses. Contrary to our expectations, we were unable to identify canonical (i.e. Drosophila-like) viral small RNAs in any of these organisms, despite the presence of abundant micro-RNAs and putative piwi-interacting piRNAs. Instead, we identified an apparently novel class of virus-derived small RNAs in the mollusc, which have a piRNA-like length distribution but lack key signatures of piRNA biogenesis, and a novel class of 21U virus-derived small RNAs in the brown alga. We also identified primary piRNAs derived from putatively endogenous copies of DNA viruses in the cnidarian and the echinoderm, and an RNA virus in the mollusc. This suggests either that the majority of metazoan phyla lack antiviral RNAi completely, such that antiviral RNAi has evolved independently in ecdysozoans, vertebrates, and molluscs, or that the antiviral RNAi response in most extant phyla - and the ancestral state of Metazoa - more closely resembles that of mammals than arthropods or nematodes.