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.

Keywords: Asobara japonica; RNA interference; endoparasitoid wasp; parasitism; whole-genome sequencing.

Figure 1

Generation of a clonal strain ‘Genome #3’ from a single female wasp. To obtain the high-quality homogenous genome sequence, we generated a clonal strain from a single female wasp derived from the Wolbachia-infected parthenogenetic strain ‘Tokyo’. After the clonal strain ‘Genome #3’ was established, we removed Wolbachia from these wasp clones by rearing host fly larvae on the standard food supplemented with tetracycline. 200 Wolbachia-free wasps were collected for genome DNA extraction. At the same time, we confirmed the removal of Wolbachia by the appearance of male wasp offspring in the next generation.

Figure 2

Phylogenetic analysis of A. japonica and other Hymenopteran species. (A) The phylogenetic tree was generated with 2,381 pairs of single-copy orthologues between A. japonica and 14 related Hymenoptera species. (B) The phylogenetic tree was generated with 3,263 pairs of single-copy orthologues between A. japonica and 8 related species in the family Braconidae. Apis mellifera was used as an out group. All nodes have 100% bootstrap support after 1,000 replications.

Figure 3

Schematic representation of the RNAi protocol during A. japonica development. (A) A standard setup of a bulk infection arena for wasp parasitism. (B) Representative images of developing wasps on an agar plate. dpi, days post-infection. Scale bar, 1 mm.

Figure 4

RNAi effects of RNAi wasps were evaluated by phenotype analysis and qPCR. (A) Representative images of wasp adults being injected with GFP dsRNA as a control. A square indicates the region of interest (ROI) for RGB value measurements. (B) Representative images of wasp adults being injected with ebony-Aj dsRNA as ebony RNAi. (C) Quantitative evaluation of wasp body colour. The Y-axis indicates the redness of the ROI. ***P < 0.005 from Student’s t-test. n = 18 (GFP dsRNA), 21 (ebony-Aj dsRNA). (D, E) The relative expression levels of gene003054 (D) and gene010975 (E) in GFP-RNAi or gene003054-RNAi wasps were quantified using the delta-delta C_t method. The expression of RpL32-Aj was used to normalize the values. All values represent the means ± SD with all data points (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.005 from Student’s t-test. n.s., non-significant (P > 0.05).