Abstract
New mosquito control strategies are vitally needed to address established and emerging arthropod-borne infectious diseases. Here we describe the characterization of a yeast interfering RNA larvicide that was developed through the genetic engineering of Saccharomyces cerevisiae (baker’s yeast) to express a short hairpin RNA targeting the Aedes aegypti synaptotagmin (Aae syt) gene. The larvicide effectively silences the Aae syt gene, causes defects at the larval neural synapse, and induces high rates of A. aegypti larval mortality in laboratory, simulated-field, and semi-field trials. Conservation of the interfering RNA target site in multiple mosquito species, but not in humans or other non-target species, suggested that it may function as a broad-range mosquito larvicide. In support of this, consumption of the yeast interfering RNA larvicide was also found to induce high rates of larval mortality in Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus mosquito larvae. The results of these studies suggest that this biorational yeast interfering RNA larvicide may represent a new intervention that can be used to combat multiple mosquito vectors of human diseases.
Highlights
Larviciding, the application of microbial or chemical agents to kill mosquito larvae in aquatic habitats, is a key component of integrated mosquito control and disease prevention strategies
Larval consumption of interfering RNA targeting Aedes aegypti synaptotagmin (Aae syt) induces high levels of mortality small interfering RNAs (siRNAs) #427, which corresponds to a target sequence in the third exon of Aae syt (S1 Table and S1 Fig), was identified in a larval siRNA soaking screen conducted on L1 larvae in which it induced 32.5±1.3% larval death (Fig 1A; P = 0.00008 vs. control siRNA treatment)
S. cerevisiae was transformed with a non-integrating multi-copy yeast shuttle plasmid from which short hairpin RNA (shRNA) corresponding to the #427 target sequence was placed under control of a constitutive promoter. #427 yeast, as well as control yeast expressing shRNA with no known target in mosquitoes, was heat-killed and prepared into an inactivated dry tablet formulation
Summary
Larviciding, the application of microbial or chemical agents to kill mosquito larvae in aquatic habitats, is a key component of integrated mosquito control and disease prevention strategies. Likewise, larviciding is a priority for control of Culex pipiens complex mosquitoes [2], the principle vectors of lymphatic filariasis [3] and West Nile virus [2]. The 1999 introduction and subsequent spread of West Nile virus across the continental United States [2] has sparked high interest in the development of new environmentally-friendly products for controlling Culex mosquitoes. Larvicide treatments are often targeted toward catch basins, major sources of Culex mosquitoes in urban areas and a primary focus of many mosquito abatement districts in the U.S [4]. Due to the increased observation of insecticide resistance to existing larvicides and escalating concerns for adverse effects of pesticides on non-target species, new larvicidal agents are vitally needed to address existing as well as emerging mosquito-borne diseases [5]. Tools that can be used to target multiple vector mosquito species are of particular interest
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