Novel dsRNA designs increase the stability and bioefficacy of RNAi-based pesticides for Varroa destructor

Varroa destructor is a major global pest of the European honey bee, and RNA interference (RNAi) has emerged as a promising technology for the development of effective control tools. However, the efficacy of RNAi-based approaches remains highly variable, due in part to the rapid degradation and inefficient delivery of exogenously applied double stranded RNA (dsRNA) This study investigated whether advanced dsRNA molecular designs could improve dsRNA stability, processing, and biological outcomes in the honey bee- Varroa mite pathosystem. Loop-ended dsRNA (ledRNA) constructs incorporating either G-U wobble base-pairing or asymmetric bulge modifications were designed to target two Varroa genes: the chitin-binding protein Peritrophin-A-like (Pero) and the neuropeptide crustacean hyperglycemic hormone ( CHH ). Stability assays, immersion and indirect feeding bioassays, and small RNA sequencing were used to evaluate dsRNA persistence, RNAi activity, and biological efficacy compared to conventional dsRNA. The advanced designs exhibited double the stability in sucrose feeding solutions and within adult bees compared with conventional dsRNA. In immersion and indirect feeding bioassays, these designs produced variable but enhanced effects on target gene silencing (~36-86% reduction) and mite mortality (~32-56% increase) when compared with conventional dsRNA. Small RNA sequencing revealed effective processing of the advanced dsRNA designs, with complete siRNA coverage of the target region, 22-24 nt peak size classes and antisense strand bias. Together, these results demonstrate that advanced dsRNA molecules have increased stability, effective processing, and strong efficacy in V. destructor under laboratory conditions, supporting further investigation of structurally optimized dsRNA designs for field hive applications.