Characterization of a dual-action adulticidal and larvicidal interfering RNA pesticide targeting theShakergene of multiple disease vector mosquitoes

Author summary New classes of environmentally-safe pesticides are vitally needed to address established and emerging mosquito-borne infectious diseases. In this investigation, we describe characterization of an interfering RNA pesticide corresponding to the mosquitoShakergene. Although the pesticide recognizes a conserved target site in theShakergenes of multiple species of disease vector mosquitoes, it lacks a known target site in humans or other non-target organisms. The pesticide killed adult mosquitoes when it was microinjected or provided to adults as an attractive toxic sugar bait. The pesticide also induced high mortality rates when fed to larvae using a yeast-based expression and delivery system. These studies demonstrated that interfering RNA pesticides targeting the mosquitoShakergene could one day be used for the biorational control of mosquitoes and the prevention of multiple mosquito-borne illnesses. The existing mosquito pesticide repertoire faces great challenges to sustainability, and new classes of pesticides are vitally needed to address established and emerging mosquito-borne infectious diseases. RNA interference- (RNAi-) based pesticides are emerging as a promising new biorational mosquito control strategy. In this investigation, we describe characterization of an interfering RNA pesticide (IRP) corresponding to the mosquitoShaker (Sh)gene, which encodes an evolutionarily conserved voltage-gated potassium channel subunit. Delivery of the IRP toAedes aegyptiadult mosquitoes in the form of siRNA that was injected or provided as an attractive toxic sugar bait (ATSB) led toShgene silencing that resulted in severe neural and behavioral defects and high levels of adult mortality. Likewise, when provided toA.aegyptilarvae in the form of short hairpin RNA (shRNA) expressed inSaccharomyces cerevisiae(baker's yeast) that had been formulated into a dried inactivated yeast tablet, the yeast IRP induced neural defects and larval death. Although theShIRP lacks a known target site in humans or other non-target organisms, conservation of the target site in theShgenes of multiple mosquito species suggested that it may function as a biorational broad-range mosquito insecticide. In support of this, theShIRP induced both adult and larval mortality in treatedAedes albopictus,Anopheles gambiae, andCulex quinquefasciatusmosquitoes, but was not toxic to non-target arthropods. These studies indicated that IRPs targetingShcould one day be used in integrated biorational mosquito control programs for the prevention of multiple mosquito-borne illnesses. The results of this investigation also suggest that the species-specificity of ATSB technology, a new paradigm for vector control, could be enhanced through the use of RNAi-based pesticides.