Species-specific yeast insecticides for Culicoides biting midge control

PROJECT SUMMARY Culicoides spp. biting midges (no-seeums) are present throughout the Americas and require blood meals for reproduction. Their bites, which can result in annoying red itchy welts and allergic reactions, can also lead to the transmission of disease-causing viruses acquired in prior blood meals, including Oropouche virus, a pathogen that causes Oropouche fever. Oropouche is a febrile illness that can result in meningitis, encephalitis, or bleeding. Recent evidence suggests that Oropouche virus, like Zika, can be transmitted to the fetus during pregnancy and result in poor pregnancy outcomes or congenital abnormalities. Outbreaks of Oropouche occurred in South America in 2023, at which time the range of the virus began to expand. Recent travel cases in the U.S., combined with the presence of Culicoides vectors may result in Oropouche becoming endemic in the United States. The present methods of Culicoides control are proving to be insufficient for preventing the spread of these insects and the virus. New methods to combat emerging insecticide resistance and the deleterious effects of some insecticides on non-targets are urgently needed. The long-term goal of our research program is to combat vector insects through the transfer of ecofriendly RNAi-based insect gene silencing from the bench to the field. The goal of the proposed research program is to develop methods of assessing gene function in Culicoides. 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 to silence genes in Culicoides. Yeast expressing shRNA corresponding to target sequences in genes conserved among multiple Culicoides spp. will be evaluated in C. sonorensis, the only human disease vector midge species that has been successfully cultured. The genes selected in these studies were identified in larval and adult lethal screens pursued in mosquitoes, thus increasing the likelihood that in addition to developing functional gene silencing studies in Culicoides, the shRNAs expressed in yeast might also represent future insecticidal agents. The specific aims include: 1) Production of RNAi yeast strains for evaluation in Culicoides sonorensis adults, 2) Evaluation of RNAi yeast strains in C. sonorensis larvae and 3) Generation of C. sonorensis female-specific RNAi yeast larvicides. The aims will be pursued through laboratory trials that are designed to achieve the expected outcome of developing methods of assessing gene function in Culicoides. These studies may also promote the development of new RNAi yeast-based insecticides to facilitate eco-friendly Culicoides control. The proposed identification of female-specific larvicides may also facilitate the development of methodology for the scaled sorting of male insects that could one day be deployed in population-based Culicoides control programs. These innovative new RNAi yeast tools will help silence the threat of disease vector Culicoides.