A Novel Allele Specific Polymerase Chain Reaction (AS-PCR) Assay to Detect the V1016G Knockdown Resistance Mutation Confirms Its Widespread Presence in Populations from Italy.

Polymerase chain reaction (PCR)-based genotyping of mutations in the voltage-sensitive sodium channel () associated with resistance to pyrethroid insecticides is widely used and represents a potential early warning and monitoring system for insecticide resistance arising in mosquito populations, which are vectors of different human pathogens. In the secondary vector -an Asian species that has invaded and colonized the whole world, including temperate regions-sequencing of domain II of the gene is still needed to detect the V1016G mutation associated with pyrethroid resistance. In this study we developed and tested a novel allele-specific PCR (AS-PCR) assay to genotype the V1016G mutation in this species and applied it to the analysis of wild populations from Italy. The results confirm the high accuracy of the novel AS-PCR and highlight frequencies of the V1016G allele as >5% in most sampling sites, with peaks of 20-45% in coastal touristic sites where pyrethroid treatments are extensively implemented, mostly for mosquito nuisance reduction. The high frequency of this mutation observed in Italian populations should serve as a warning bell, advocating for increased monitoring and management of a phenomenon which risks neutralizing the only weapon today available to counteract (risks of) arbovirus outbreaks.