Optimization of dsRNA Length and Target Position for Enhanced RNA Interference Efficiency Targeting the Chitinase Gene MsChi2 in Mythimna separata

Chitinase is a key enzyme involved in insect chitin metabolism, and RNA interference (RNAi) targeting the corresponding gene represents a promising strategy for eco-friendly pest management. However, the design parameters of double-stranded RNA (dsRNA), particularly fragment length and target position, remain insufficiently optimized. To investigate the effects of these parameters on the RNAi efficiency against the MsChi2 gene in Mythimna separata, we synthesized four dsRNAs of 60, 200, 500, and 1000 bp and administered them to larvae. The results indicated that 200 and 500 bp dsRNAs significantly inhibited larval growth, reduced survival, and silenced MsChi2 expression. Among these, the 200 bp dsRNA exhibited the most pronounced effects, including a 59% inhibition rate of larval body weight, 65.9% mortality, and 62% target gene silencing efficiency, accompanied by a significant reduction in chitin content and abnormalities in molting and pupation. Using 200 bp as the optimal length, five fragments targeting different coding regions of the gene were evaluated. All except one exhibited strong activity, and their RNAi efficiencies were positively correlated with the predicted numbers of effective small interfering RNAs. Collectively, these findings demonstrate that dsRNA length and target position jointly modulate RNAi efficiency, with 200 bp representing the optimal fragment length. This study provides a rational design framework for dsRNA development and establishes both theoretical and practical foundations for RNAi-based management of M. separata.