dsRNA biodistribution and RNAi in three mosquito disease vectors

RNAi has become an integral part of the molecular entomology toolkit, yet there is inconsistency in the successful application of this tool across taxa. This inconsistency could be related to the methodological approach including the route of administration, use of transfection/wetting agents, and RNAi trigger design. Additionally, biological variation may play a role as the mechanism by which double stranded RNA (dsRNA) is trafficked and internalized in tissues and cells of different species is not well documented nor understood. Together, biological and methodological differences may ultimately contribute to the variable success of this approach. The most straightforward approach to understanding dsRNA uptake in insects, for which there is no genetic transformation system, is to track the movement of labelled RNA using fluorescence microscopy.  This technique cannot resolve the presence of trace quantities of dsRNA or determine structure. To improve upon the detection of trace amounts of dsRNA and breakdown siRNA products, we developed a reverse transcriptase & PCR (RT-PCR) approach to amplify and detect these RNA species in such a way that the structural integrity, length and sequence can be determined. Here, we combine fluorescence microscopy and RT-PCR to describe the tissue biodistribution profiles of injected, ingested or topically applied dsRNA in three species of medically significant mosquito, namely: Aedes aegypti, Anopheles gambiae, and Culex pipiens.