The efficacy of RNAi in the arbovirus vector, Culicoides sonorensis

Apoptosis is the conserved cellular process of programmed cell death used as an antiviral defense in multiple insect species. This process is induced by inactivating inhibitor of apoptosis 1 (IAP1), which results in activation of the initiator caspase, DRONC. Active DRONC, initiates a caspase cascade, ultimately causing cell death. Biting midges in the genus Culicoides are important vectors of the arboviral diseases which cause significant economic burden worldwide. Research on this vector has been hindered by the lack of sequenced genome and difficulty in culturing certain species. Previously, we developed RNA interference (RNAi) in Culicoides sonorensis adults. Here, we illustrate the effectiveness of RNAi. After microinjection of double-stranded (ds)RNA specific to the Culicoides ortholog of IAP1 (CsIAP1), midge midguts were dissected. DsCsIAP1-injection resulted in severely deformed midguts, which were more fragile and diminished compared to controls. These data suggest midgut degradation as the mechanism behind dsCsIAP1-mortality.To further probe the apoptotic pathway, the Culicoides ortholog to DRONC (CsDRONC) was identified, and midges were injected with dsCsIAP1 and/or dsCsDRONC, then assessed for transcript abundance and mortality. Quantitative PCR revealed a 10-30% mRNA transcript decrease specific to the dsRNA injected. DsCsIAP1/dsCsDRONC-injection resulted in a reversal of the increased mortality phenotype observed in dsCsIAP1-injected midges. In addition, dissections revealed deformed midguts in both dsCsIAP1 control and dsCsIAP1/dsCsDRONC-injected midges. These data confirm the conserved function of CsIAP1 and CsDRONC. Importantly, they demonstrate the midgut’s ability to uptake and utilize exogenous dsRNA, revealing the effectiveness of RNAi in adult midges.