Evaluation of RNAi and larvicide enhancements for the biting midge, Culicoides sonorensis (Diptera: Ceratopogonidae)

Abstract

Culicoides biting midges are important livestock pests that transmit viruses that are detrimental to animal welfare and economically impact producers’ livelihoods. Current measures to protect livestock are often expensive or ineffective, and traditional chemical insecticides are often harmful to non-target organisms, underlining a need to develop new bio-rational control methods. RNA interference (RNAi) is a promising technique for insect control that uses double- stranded RNA (dsRNA) to suppress physiologically important genes in target insects. This mechanism is not equally effective in all insects, and this research examined RNAi in Culicoides sonorensis, a model species for this genus. Cultured insect cells had a robust RNAi response, whereas larval stages only weakly responded to the treatment dsRNA. Several mechanisms contribute to RNAi inefficiency in other insects, including the instability of dsRNA. Accordingly, dsRNA-degrading enzymes (dsRNases) were characterized as potential barriers to RNAi efficiency. Seven putative enzymes were found in C. sonorensis, and the transcript expression profile of six of these enzymes shows they are present in many developmental stages, which was confirmed by ex vivo dsRNA digest in larval midge gut fluid. Three RNAi enhancers (secondary RNA structures, chitosan nanoparticles, and E. coli expressing dsRNA) were evaluated in larval midges, and only chitosan nanoparticles increased RNAi efficacy above baseline treatments. In addition to RNAi, we tested the effectiveness of Bacillus thuringiensis subsp. israelensis (Bti) for C. sonorensis larval control. Larvae were tolerant to Bti treatments compared to Aedes aegypti but increasing the alkalinity of the rearing environment significantly enhanced Bti insecticidal activity. These studies identify factors limiting the efficacy of RNAi and Bti-based insecticides and provide foundational knowledge for pursuing the development of new larval control methodologies in C. sonorensis.