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Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the  Ace-1  target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan  Culex quinquefasciatus  display CNV for the voltage-gated sodium channel gene ( Vgsc ), target-site of pyrethroid and organochlorine insecticides. In order to develop field-applicable diagnostics for  Vgsc  CN, and as a prelude to investigating the possible association of CN with insecticide resistance, three assays were compared for their accuracy in CN estimation in this species. The gold standard method is droplet digital PCR (ddPCR), however, the hardware is prohibitively expensive for widespread utility. Here, ddPCR was compared to quantitative PCR (qPCR) and pyrosequencing. Across all platforms, CNV was detected in ≈10% of mosquitoes, corresponding to three or four copies (per diploid genome). ddPCR and qPCR-Std-curve yielded similar predictions for  Vgsc  CN, indicating that the qPCR protocol developed here can be applied as a diagnostic assay, facilitating monitoring of  Vgsc  CN in wild populations and the elucidation of association between the  Vgsc  CN and insecticide resistance.

Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus