Whole-genome sequencing analysis and protocol for RNA interference of the endoparasitoid wasp Asobara japonica

Abstract Asobara japonica is an endoparasitic wasp that parasitizes Drosophila flies. It synthesizes various toxic components in the venom gland and injects them into host larvae during oviposition. To identify and characterize these toxic components for enabling parasitism, we performed the whole-genome sequencing (WGS) and devised a protocol for RNA interference (RNAi) with A. japonica. Because it has a parthenogenetic lineage due to Wolbachia infection, we generated a clonal strain from a single wasp to obtain highly homogenous genomic DNA. The WGS analysis revealed that the estimated genome size was 322 Mb with a heterozygosity of 0.132%. We also performed RNA-seq analyses for gene annotation. Based on the qualified WGS platform, we cloned ebony-Aj, which encodes the enzyme N-β-alanyl dopamine synthetase, which is involved in melanin production. The microinjection of double-stranded RNA (dsRNA) targeting ebony-Aj led to body colour changes in adult wasps, phenocopying ebony-Dm mutants. Furthermore, we identified putative venom genes as a target of RNAi, confirming that dsRNA injection-based RNAi specifically suppressed the expression of the target gene in wasp adults. Taken together, our results provide a powerful genetic toolkit for studying the molecular mechanisms of parasitism.