Developing new genomic tools and rapid, automated resistance detection in mosquito larvae

Insecticide resistance is a looming threat to malaria and arbovirus control programmes targeting the mosquito vector. Larval resistance is an important phenotype to assess, however, the existing WHO recommended mortality-based larval resistance assay is low-throughput and subject to investigator bias. To address these issues, a novel assay was developed using the Invertebrate Automated Phenotyping Platform (INVAPP) and analysis algorithm (Vectorgon) which provides automated quantification of larval motility after insecticide exposure. Several molecular changes have been observed in mosquitoes displaying resistance to common larvicides. However, their phenotypic characterisation in the absence of other potential resistance mechanisms is largely unexplored. Understanding the potential impact on mosquito control programmes of each mechanism is vital. In susceptible laboratory mosquitoes, upregulation of metabolic enzymes was mimicked using a bipartite GAL4-UAS system. The INVAPP assay was used to assess insecticide susceptibility of transgenic larvae overexpressing individual enzymes - GSTe2, CYP6M2, CYP6P3 and SAP2 - which are implicated in adulticide resistance. A transgenic line expressing CCEae3a (a carboxylesterase which has been identified in Aedes mosquitoes displaying organophosphate (OP) resistance), was found to display resistance to three common OPs in larvae, four-insecticide classes in adults and significant, though moderate, reductions in adult longevity, egg laying, and larval hatch-rate. Using CRISPR-Cas9, a single nucleotide polymorphism (G119S) was introduced in the Acetylcholine esterase 1 (ACE1) gene - the target for OP and carbamate insecticides of susceptible mosquitoes and the resulting phenotypes assessed. The results of this work, highlight important links between adulticide and larvicide resistance mechanisms and the necessity to assess mechanisms individually.