Whole-genome sequencing analysis and protocol for RNA interference of the endoparasitoid waspAsobara 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 (NBAD), which is involved in melanin production. The microinjection of double-stranded RNA targeting ebony-Aj led to body color 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.

Parasitoid wasp venoms degradeDrosophilaimaginal discs for successful parasitism

“…Therefore, we can efficiently monitor developmental processes of koinobionts and host larvae. Moreover, we have previously performed whole-genome sequencing analysis and established a protocol for RNAi methods in A. japonica , facilitating the functional analysis of venom genes ( 9 ).…”

“…To identify A. japonica venom components crucial for IDD-inducing activity, we conducted RNA-sequencing (RNA-seq) analysis based on our gene annotation database ( 9 ). We compared expression levels of 12,508 annotated genes between the venom gland and other parts of the wasp body, which led to identification of 807 genes that were significantly enriched in venom glands (Fig.…”

“…We then conducted functional screening of IDD-related A. japonica genes using a previously established RNAi technique ( 9 ). In the first screen, 63 genes were divided into two to four gene groups according to their high false discovery rates in the transcriptome analysis.…”

Construction of a Banker Plant System via the Host Switch Trait of a Natural Enemy Aenasius bambawalei

“…This synchronization plays a crucial role in population development and pest control of BCAs [6], and that can lead to a reduction in the use of pesticide control. Parasitic wasps, being important BCAs, have been valued for their use as biological pest control in agriculture and forestry [7][8][9][10][11][12]. Research on host switching in parasitic wasps has primarily focused on documenting new host records [13,14], studying the phylogenetic distance between hosts [15], exploring bacterial communities [16] in generalist parasitic wasps [17,18], and so on.…”