The argininosuccinate lyase gene exacerbates pyrethroid resistance in the major African vectors Anopheles funestus

Escalating pyrethroid resistance in malaria vectors is jeopardizing malaria control. Deciphering its complex evolutionary mechanisms is paramount to mitigate its impact. Here, we demonstrate that over-expression and allelic variation of the argininosuccinate lyase ( ASL) gene exacerbate pyrethroid resistance in Anopheles funestus . Multi-omics analyses revealed that ASL is upregulated Africa-wide and detected a strong signal of genetic differentiation around ASL in resistant populations exhibiting copy number variation (CNV). A predominant resistant haplotype harboring the R125H and T-277A mutations was detected in resistant mosquitoes. Transgenic expression of ASL and RNAi confirm its ability to confer pyrethroid resistance. A DNA-based assay confirmed its association with super-resistance, revealing a marked temporal increase in Uganda (2010–2023) but with a drastic reduction observed in 2024 after deployment of chlorfenapyr-based bed nets. This study elucidates a novel resistance pathway driven by ASL and introduces a new DNA-based diagnostic tool to monitor its spread and impact in Africa.