High-throughput in vivo screening platform for rapid discovery of adult mosquitocides: Design, validation and...

The global rise of mosquito-borne diseases and widespread resistance to existing insecticides highlight the urgent need for novel, field-relevant mosquitocides. Here, we report the development and validation of a high-throughput, in vivo screening assay capable of evaluating adult mosquito toxicity across large chemical libraries. Utilizing a 96-well plate format, this assay enables simultaneous testing of hundreds of compounds per run using both net and filter paper substrates, with direct measurement of adult mosquito knockdown and mortality via tarsal contact - an exposure route highly relevant to real-world vector control tools such as long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). The workflow is compatible with both manual and automated (robotic) liquid handling, with robotic dispensing yielding LC₅₀ values within 2- to 3-fold of manual methods, thereby supporting scalability and sensitivity. Screening throughput is significantly enhanced, condensing weeks of testing into a half-day and enabling the evaluation of thousands of compounds per week. The assay rapidly identifies "hit" compounds for further formulation and field evaluation and is adaptable to multiple mosquito species. This platform represents a transformative advance for mosquitocide discovery, directly addressing key bottlenecks in vector control and resistance management.