PROJECT SUMMARY Anopheles stephensi, a highly competent vector of urban malaria parasites, is rapidly spreading across the globe. The ability of this mosquito to thrive in urban environments distinguishes it from other primary malaria vectors that inhabit rural areas, making it a significant threat to millions of people who live in cities. The long-term goal of our research program is to combat mosquitoes through the transfer of eco- friendly RNAi-based mosquito gene silencing from the bench to the field. The objective of the proposed research program is to evaluate the potential for RNAi yeast-based technologies to facilitate the control of A. stephensi. These innovative new yeast insecticides have not yet been assessed in this dangerous mosquito. The proposed investigation will test the hypothesis, which is supported by strong preliminary data, that interfering RNA produced and delivered in Saccharomyces cerevisiae can be utilized as insecticidal agents targeting A. stephensi. The specific aims are to 1) evaluate RNAi-based yeast attractive targeted sugar baits (ATSBs) for control of A. stephensi, 2) identify RNAi yeast formulations that can be used to target A. stephensi larvae, and 3) develop and assess A. stephensi female-specific RNAi yeast larvicides to facilitate male sex-sorting. The aims will be pursued through a combination of laboratory and semi-field insecticide trials that are designed to achieve the expected outcome of identifying a new class of RNAi yeast-based insecticides to facilitate A. stephensi control. Moreover, the identification of female-specific larvicides will permit the design of an RNAi yeast-based mass rearing diet for the production of fit adult males that can be deployed in emerging population-based A. stephensi control programs. These innovative new RNAi tools will help silence the threat of deadly A. stephensi mosquitoes.