Characterization of a dual-action adulticidal and larvicidal interfering RNA pesticide targeting the Shaker gene of multiple disease vector mosquitoes.

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 mosquito Shaker (Sh) gene, which encodes an evolutionarily conserved voltage-gated potassium channel subunit. Delivery of the IRP to Aedes aegypti adult mosquitoes in the form of siRNA that was injected or provided as an attractive toxic sugar bait (ATSB) led to Sh gene silencing that resulted in severe neural and behavioral defects and high levels of adult mortality. Likewise, when provided to A. aegypti larvae in the form of short hairpin RNA (shRNA) expressed in Saccharomyces cerevisiae (baker's yeast) that had been formulated into a dried inactivated yeast tablet, the yeast IRP induced neural defects and larval death. Although the Sh IRP lacks a known target site in humans or other non-target organisms, conservation of the target site in the Sh genes of multiple mosquito species suggested that it may function as a biorational broad-range mosquito insecticide. In support of this, the Sh IRP induced both adult and larval mortality in treated Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus mosquitoes, but was not toxic to non-target arthropods. These studies indicated that IRPs targeting Sh could 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.

Characterization of an adulticidal and larvicidal interfering RNA pesticide that targets a conserved sequence in mosquito G protein-coupled dopamine 1 receptor genes.

G protein-coupled receptors (GPCRs), key regulators of a variety of critical biological processes, are attractive targets for insecticide development. Given the importance of these receptors in many organisms, including humans, it is critical that novel pesticides directed against GPCRs are designed to be species-specific. Here, we present characterization of an interfering RNA pesticide (IRP) targeting the mosquito GPCR-encoding dopamine 1 receptor (dop1) genes. A small interfering RNA corresponding to dop1 was identified in a screen for IRPs that kill Aedes aegypti during both the adult and larval stages. The 25 bp sequence targeted by this IRP is conserved in the dop1 genes of multiple mosquito species, but not in non-target organisms, indicating that it could function as a biorational mosquito insecticide. Aedes aegypti adults treated through microinjection or attractive toxic sugar bait delivery of small interfering RNA corresponding to the target site exhibited severe neural and behavioral defects and high levels of adult mortality. Likewise, A. aegypti larval consumption of dried inactivated yeast tablets prepared from a Saccharomyces cerevisiae strain engineered to express short hairpin RNA corresponding to the dop1 target site resulted in severe neural defects and larval mortality. Aedes albopictus and Anopheles gambiae adult and larval mortality was also observed following treatment with dop1 IRPs, which were not toxic to non-target arthropods. The results of this investigation indicate that dop1 IRPs can be used for species-specific targeting of dop1 GPCRs and may represent a new biorational strategy for control of both adult and larval mosquitoes.

Lure-and-Kill Yeast Interfering RNA Larvicides Targeting Neural Genes in the Human Disease Vector Mosquito Aedes aegypti.

New mosquito control strategies are vitally needed to address established arthropod-borne infectious diseases such as dengue and yellow fever and emerging diseases such as Zika and chikungunya, all of which are transmitted by the disease vector mosquito Aedes aegypti. In this investigation, Saccharomyces cerevisiae (baker's yeast) was engineered to produce short hairpin RNAs (shRNAs) corresponding to the Aedes aegypti orthologs of fasciculation and elongation protein zeta 2 (fez2) and leukocyte receptor cluster (lrc) member, two genes identified in a recent screen for A. aegypti larval lethal genes. Feeding A. aegypti with the engineered yeasts resulted in silenced target gene expression, disrupted neural development, and highly significant larval mortality. Larvicidal activities were retained following heat inactivation and drying of the yeast into tabular formulations that induced >95% mortality and were found to attract adult females to oviposit. These ready-to-use inactivated yeast interfering RNA tablets may one day facilitate the seamless integration of this new class of lure-and-kill species-specific biorational mosquito larvicides into integrated mosquito control programs.