Target gene screening and validation for RNAi-based suppression of the microsporidian parasite Vairimorpha (=Nosema) ceranae in honeybees

Abstract The microsporidian Vairimorpha ( = Nosema) ceranae is a major pathogen affecting honeybee health worldwide, yet safe and effective control strategies remain elusive. RNA interference (RNAi) using double-stranded RNA (dsRNA) has emerged as a promising strategy for species-specific suppression with minimal environmental impact, but effective target selection and mechanistic validation remain key challenges. Here, we screened 11 dsRNAs targeting V. ceranae genes and identified three potent targets ( swp25 , metap2 , and spp ). Among them, dsRNA- swp25 exhibited the most pronounced suppression efficacy, outperforming previously validated targets. qRT-PCR analysis confirmed significant post-transcriptional gene silencing, while spore load and midgut infection area were markedly reduced in bees treated with dsRNA- swp25 . Furthermore, fluorescence imaging demonstrated that Cy3-labeled dsRNA was directly internalized by V. ceranae spores in a time-dependent manner without host mediation. Collectively, these findings highlight swp25 as a high-impact RNAi target and offer mechanistic insights into pathogen-directed RNAi responsiveness. This work advances the development of practical, environmentally sustainable RNAi-based strategies for apicultural disease management. Graphical Abstract