Discovery of novel non-live bacterial bioinsecticides for mosquito control through a high-throughput screening pipeline

• High Efficacy: Three of our isolates achieved 100% larval mortality within 24 h, demonstrating their potential as highly effective biocontrol agents. • Broad-Spectrum Activity: Further bioassays confirmed a dose-dependent efficacy against Aedes aegypti , Culex quinquefasciatus , and Anopheles stephensi , indicating a broad-spectrum larvicidal activity. • Taxonomic Diversity: The active isolates represent a diverse range of genera, including Enterobacter , Bacillus , Serratia , Acinetobacter , Pseudomonas , Chromobacterium , Pantoea , Staphylococcus , and Raoultella . • Practical Advantages: Crucially, the larvicidal effect was retained in non-live, heat-inactivated bacterial biomass. This offers significant practical advantages over live biocontrol agents in terms of stability, safety, and ease of deployment. • Enhanced Formulation: We also found that delivering the bacterial biomass in a pellet form can enhance larval mortality compared to a suspended powder, likely due to improved retention and a localized concentration of the active metabolites. The growing threat of insecticide resistance and ecological harm from synthetic insecticides underscores the urgent need for new sustainable mosquito control strategies. In this study, we implemented a systematic high-throughput screening (HTS) of non-live bacterial cultures to identify novel bacterial strains with potent mosquito larvicidal activity. From 48 environmental samples collected in a tropical environment on the southeast coast of Puerto Rico, 486 bacterial isolates were obtained, of which 30 exhibited significant killing-activity as non-live desiccated cultures, against Aedes aegypti larvae. Notably, three isolates achieved 100% larval mortality within 24 h. Further bioassays confirmed dose-dependent efficacy across Aedes aegypti, Culex quinquefasciatus, and Anopheles stephensi, indicating broad-spectrum larvicidal activity. Taxonomic analysis revealed diverse genera, including Enterobacter, Bacillus, Serratia, Acinetobacter, Pseudomonas, Chromobacterium, Pantoea, Staphylococcus, and Raoultella. Crucially, the larvicidal effect was retained in non-live bacterial biomass after heat inactivation, highlighting practical advantages with regard to stability, safety, and deployment over live biocontrol agents. Comparative assays of different formulations further indicated that delivering bacterial biomass in pellet form can enhance larval mortality compared to suspended powder in the larval breeding water, likely due to improved retention and localized concentration of active metabolites. Collectively, this study establishes a robust discovery pipeline for environmentally friendly, microbe-derived non-live bioinsecticides to support integrated mosquito vector management.