Evolutionary dynamics of diversification of brain neuronal cell types that have underlain behavioral evolution remain largely unknown. Here, we compared transcriptomes and functions of Kenyon cell (KC) types that compose the mushroom bodies between the honey bee and sawfly, a primitive hymenopteran insect whose KCs likely have the ancestral properties. Transcriptome analyses show that the sawfly KC type shares some of the gene expression profile with each honey bee KC type, although unique gene expression profiles have also been acquired in each honey bee KC type. In addition, functional analysis of two sawfly genes suggested that the functions in learning and memory of the ancestral KC type were heterogeneously inherited among the KC types in the honey bee. Our findings strongly suggest that the functional evolution of KCs in Hymenoptera involved two previously hypothesized processes for evolution of cell function: functional segregation and divergence.

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.