Chitosan-dsRNA improves tissue stability and delivery for RNAi-mediated Varroa destructor control.

Varroa destructor is an ectoparasitic mite and a major threat to honey-bee colony health worldwide. RNA interference (RNAi) offers a potentially species-specific approach for mite control, but practical application is limited by double-stranded RNA (dsRNA) degradation and inefficient delivery to mites. This study evaluated coatomer protein I (COPI) complex subunits as RNAi targets and tested whether chitosan-based dsRNA formulation could improve dsRNA stability, tissue uptake, and delivery from honey-bees to mites. Direct microinjection of dsRNAs targeting COPB, COPD, and COPE significantly reduced target-gene expression and mite survival compared with the double-stranded green fluorescent protein (dsGFP) control, with 72-h survival rates of 8.0%, 12.7%, and 5.3%, respectively, compared with 40.7% in the control group (all log-rank P < 0.0001). Chitosan-conjugated dsRNA remained detectable for longer periods than naked dsRNA in honey-bee tissue fluids, and CNP-Cy3-dsGFP was detected in the honey-bee midgut and fat body. A qualitative fluorescence observation in V. destructor was consistent with host-to-mite dsRNA transfer. Ingestion of COP-targeted chitosan-dsRNAs reduced mite survival, whereas honey-bee survival and expression of honey-bee COP orthologs were not affected. In silico analysis detected no contiguous ≥19-nt matches between Varroa COP dsRNAs and the honey-bee transcriptome or genome. COPI subunits are promising RNAi targets in V. destructor, and chitosan formulation may improve dsRNA persistence and uptake while supporting honey-bee-mediated delivery to mites. These laboratory findings support further evaluation of chitosan-formulated dsRNA as a potentially species-selective strategy for Varroa management, while broader safety assessment and field validation remain necessary. © 2026 Society of Chemical Industry.