Engineered Gut Symbiotic Bacterium-Mediated RNAi for Effective Control of Anopheles Mosquito Larvae.

mosquitoes are the primary vectors for the transmission of malaria parasites, which poses a devastating burden on global public health and welfare. The recent invasion of Anopheles stephensi in Africa has made malaria eradication more challenging due to its outdoor biting behavior and widespread resistance to insecticides. To address this issue, we developed a new approach for mosquito larvae control using gut microbiota-mediated RNA interference (RNAi). We engineered a mosquito symbiotic gut bacterium, Serratia fonticola, by deleting its gene to produce double-stranded RNAs (dsRNAs) in the mosquito larval gut. We found that the engineered strains can stably colonize mosquito larval guts and produce dsRNAs ds or ds to activate RNAi and effectively suppress the expression of methoprene-tolerant gene and ecdysone receptor gene , which encode receptors for juvenile hormone and ecdysone pathways in mosquitoes, respectively. Importantly, the engineered strains markedly inhibit the development of A. stephensi larvae and leads to a high mortality, providing an effective dsRNA delivery system for silencing genes in insects and a novel RNAi-mediated pest control strategy. Collectively, our symbiont-mediated RNAi (smRNAi) approach offers an innovative and sustainable method for controlling mosquito larvae and provides a promising strategy for combating malaria. Mosquitoes are vectors for various diseases, imposing a significant threat to public health globally. The recent invasion of A. stephensi in Africa has made malaria eradication more challenging due to its outdoor biting behavior and widespread resistance to insecticides. RNA interference (RNAi) is a promising approach that uses dsRNA to silence specific genes in pests. This study presents the use of a gut symbiotic bacterium, Serratia fonticola, as an efficient delivery system of dsRNA for RNAi-mediated pest control. The knockout of , a dsRNA-specific endonuclease gene, in using CRISPR-Cas9 led to efficient dsRNA production. Engineered strains of can colonize the mosquito larval gut and effectively suppress the expression of two critical genes, and , which inhibit mosquito development and cause high mortality in mosquito larvae. This study highlights the potential of exploring the mosquito microbiota as a source of dsRNA for RNAi-based pest control.