Yeast Interfering RNA Pesticide-Based Attractive Toxic Sugar Baits for Biorational Mosquito Control

Mosquito-borne illnesses kill hundreds of thousands of people worldwide each year. These diseases pose significant threats to deployed military members who are stationed across the globe and often have prolonged outdoor exposure. Due to a lack of progress in vaccine and drug development, mosquito control is the primary means of disease prevention. However, a lack of compliance with existing control programs, rising insecticide resistance, and concerns for the negative impacts of pesticides on non-target organisms threaten efforts to control mosquito-borne diseases (cdc.gov). Although it is attracting attention in agricultural biotechnology communities, the use of RNA interference (RNAi) is an innovative and still largely unexplored approach for mosquito control. The proposed research program, which relates to the emerging viral diseases topic area, aims to develop a new class of environmentally-friendly, consumer-accepted, interfering RNA-based pesticides (IRPs) as a novel mosquito control intervention, a FY20 PRMRP area of encouragement. Our recent high-throughput screens identified hundreds of IRPs, each which silences a mosquito gene that is complementary in sequence and required for mosquito survival. A subset of these IRPs recognize target sites that are conserved in multiple mosquito species, but which are not found in non-target organisms. These IRPs kill Aedes (dengue, Zika, chikungunya, and yellow fever virus vectors), Culex (West Nile and lymphatic filariasis vector), and Anopheles (malaria vector) mosquitoes, yet are expected to have little if any impact on non-target organisms. Our initial research program examined the use of an inexpensive, scalable yeast system for production and delivery of IRPs to Aedes mosquito larvae. Preliminary data indicate that a subset of these yeast pesticides can also be deployed as attractive toxic sugar baits (ATSBs) for control of adult mosquitoes. The proposed expansion program will examine the efficacy of using yeast IRPs to target multiple species of adult arboviral mosquitoes. Commercial-ready formulations with extended shelf lives and residual activities will be developed for deployment as ATSBs. Following lab testing and optimization, the IRP-ATSBs will be evaluated in disease-endemic countries in Southeast Asia (Thailand) and the Caribbean (Trinidad and Tobago), where stakeholder acceptance will be assessed. Specific aims of this program, which evaluates the hypothesis that yeast IRP-ATSBs can be used for effective biorational mosquito control, include: Aim 1: Development and laboratory testing of yeast formulations that function as long-lasting biorational ATSBs in multiple species of disease vector mosquitoes: Long-lasting yeast IRP formulations will be designed and evaluated in laboratory and local U.S. field strains of Aedes and Culex mosquitoes. Formulations that can be delivered through ATSB bait stations and as foliar sprays will be evaluated in simulated field trials. Toxicity will also be assessed in select non-target arthropods. In these assays and throughout the proposed investigation, mosquitoes fed with (i) sugar baits alone (negative control) or sugar baits prepared with (ii) yeast that expresses shRNA with no known target in mosquitoes (negative control), (iii) experimental yeast IRPs, or (iv) boric acid (a non-specific insecticidal positive control) will be compared. Aim 2: Field evaluation of these ATSB formulations in Trinidad and Thailand: Top-performing yeast IRP formulations will be evaluated in semi-field trials conducted in Trinidad and Thailand, where both lab and field strains of Aedes and Culex disease vector mosquitoes will be evaluated. Aim 3: Assessment of stakeholder acceptance of this intervention: Community forums, paper surveys, and focus group interviews will be used to assess stakeholder acceptance of yeast IRP-ATSB technology. The proposed operationally relevant trials will facilitate rapid decision-making regarding the efficacy and feasibility of introducing yeast IRP-ATSBs into integrated mosquito control programs in the Caribbean, the Greater Mekong Subregion, and other priority geographies. Stakeholder engagement will evaluate consumer acceptance of this intervention and build interactions that can support the development of successful mosquito control programs that involve key community stakeholders. It is anticipated that yeast IRP-ATSBs can be rapidly deployed and seamlessly incorporated into existing integrated mosquito control programs, while enhancing current ASTB strategies through inclusion of a novel class of mosquito-specific pesticides that do not impact non-target organisms. Commercial-ready formulations will be developed in a multi-use capacity for global deployment by the U.S. military, mosquito control programs, or individual homeowners. Direct involvement of military entomologists will help to ensure that the novel mosquito control agents will benefit U.S. military members, Veterans, their beneficiaries, as well as millions of civilians worldwide. Pursuit of the proposed studies will accelerate progress toward incorporation of IRPs into integrated mosquito control programs with the goal of eradicating mosquito-borne illnesses. Less