RNAi-mediated gene silencing of a 26S proteasome subunit increases mortality of the Japanese beetle Popillia japonica.

BACKGROUND The Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) is a highly polyphagous quarantine invasive species causing severe crop damages. Its management is based on broad-spectrum insecticides and sustainable alternatives are needed. Strategies based on RNA interference (RNAi) emerged in crop protection and we aimed to explore its use to control P. japonica. RESULTS Nine genes of P. japonica were selected as lethal candidates, based on previous wide-genome screenings on other coleopterans. To avoid off-target effects, genes showing over 80% identity with pollinator homologues were excluded and P. japonica double-stranded RNAs (dsRNAs) were designed in the least conserved portions according to alignments with Apis mellifera. When incubated in P. japonica midgut juice, dsRNAs were not degraded. Injection and plant-mediated feeding were used to deliver dsRNAs to larvae and adults. Five targets were tested, and two genes were selected as the most effective in increasing mortality, namely regulatory particle non-ATPase 6 subunit (RPN) and shibire_dynamin-like protein (SHI). A significant transcript reduction up to 21 days (RPN: 3-5 fold-change silencing) after dsRNA injection indicated that effective gene silencing occurred, as also supported by sequencing of small RNA libraries. In adults, RNAi-mediated depletion of RPN transcript reduced survival, either when insects were injected or mass-fed on vine leaves dsRNA-treated. CONCLUSION A subunit of the 26S proteasome was indicated as promising RNAi target for dsRNA-based insecticide against the Japanese beetle. The data pave the way for the possible use of RNAi approaches to control this pest, proactively waiting for the European Union approval of exogenously applied dsRNAs. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.