Silencing of the V-ATPase gene via RNA interference impairs fitness and survival of the invasive apiary pest Aethina tumida indicating potential for pest control

Abstract The small hive beetle ( Aethina tumida ) is an invasive pest of honeybee colonies and represents an increasing threat to apiculture outside its native sub-Saharan African range. Its establishment in Italy since 2014 has raised serious concerns for European beekeeping, particularly due to the limited availability of safe and effective chemical control options. RNA interference (RNAi) represents a promising species-specific alternative; however, oral delivery approaches in A. tumida have thus far produced inconsistent outcomes. In this study, we assessed lethal and sublethal effects of double-stranded RNA (dsRNA) targeting vacuolar ATPase subunit A (V-ATPase) in laboratory bioassays, using both ingestion and topical application as delivery routes. Ingestion of V-ATPase dsRNA significantly reduced larval growth and prolonged development, resulting in marked delays in adult emergence. Although overall mortality was low, both delivery methods consistently induced strong reductions in adult fecundity. Molecular analyses confirmed consistent knockdown of V-ATPase transcripts following dsRNA exposure. Importantly, bioinformatic analyses revealed no significant sequence homology between the dsRNA constructs and the Apis mellifera genome, and injection assays in honeybees showed no acute adverse effects on survival or feeding behavior. Collectively, these results provide the first consistent evidence of RNAi-induced molecular and phenotypic effects in A. tumida and underscore the potential of dsRNA-based bioinsecticides as a targeted and pollinator-safe strategy for managing this invasive pest.