High-throughput phenotypic profiling of insecticide responses in mosquito larvae

Extensive use of insecticides is increasing resistance risks that could severely reduce the control of mosquito vectors that transmit infectious agents of Neglected Tropical Diseases. Addressing resistance threats by discovering new insecticides can be challenging because of the limited throughput and translatability of existing in vitro screening pipelines. Acquiring multiple phenotypic endpoints of larvae could alleviate these restrictions by more thoroughly profiling drug effects on whole organisms, providing leads to compounds with novel mechanisms of action, and increasing screening throughput compared to adult-stage assays. Here, we establish a pair of assays for profiling motility and development traits in larval-stage mosquitoes at a higher scale than standard larvicidal screening techniques. We optimized assay parameters and developed novel image processing approaches that enable relatively high throughput screening of chemical compounds on single larvae within a screen with condition replicates. We tested the assay with an insect growth regulator (S-methoprene), a slow-acting pyrrole (chlorfenapyr), and two microbial larvicides (Spinosad and Lysinibacillus sphaericus ). These measurements aligned well with known insecticide mechanisms, and dose response curves established assay baselines for comparison in future screens. Finally, we present ways in which the assay design can be modified across different imaging technologies, showing the flexibility of the screening approach.