Inducing RNA interference in the arbovirus vector, Culicoides sonorensis

Biting midges in the genus Culicoides are important vectors of arboviral diseases, Epizootic Hemorrhagic Disease and Bluetongue, causing significant economic burden worldwide. Research on this vector has been hindered by the lack of a sequenced genome, the difficulty of consistent culturing of certain species, and the absence of molecular techniques such as RNA interference (RNAi). Here, we report the establishment of RNAi as a research tool to study vector-virus interactions within the adult midge, Culicoides sonorensis. Based on previous research and transcriptome analysis, which revealed putative siRNA pathway member orthologs, we hypothesized that adult C. sonorensis midges have the molecular machinery needed to preform RNA silencing. The development of an injection protocol allowed for dsRNA microinjection into the hemocoel of adult female midges. Using CO₂ exposure as a means to immobilize adult midges, injection of control dsRNA, dsGFP, into 2-3 day old adult female midges resulted in survival curves that support virus transmission. DsRNA injection targeting the newly identified C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) ortholog, resulted in increased mortality rates (Log Rank test, P<0.0001), with median survivals shortened to less than eight days compared to 30 days in dsGFP-injected controls. Transcripts of CsIAP1 were decreased significantly by 40% five days post injection (1-way ANOVA, P<0.0001). These results demonstrate the conserved function of IAP1. Importantly, they also demonstrate the feasibility of RNAi by dsRNA injection in adult midges, which will greatly facilitate studies of the underlying mechanisms of vector competence in Culicoides sonorensis.