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

Abstract

Polymerase chain reaction (PCR)-based genotyping of mutations in the voltage-sensitive sodium channel (vssc) 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 Aedes albopictus—an Asian species that has invaded and colonized the whole world, including temperate regions—sequencing of domain II of the vssc 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 Ae. albopictus 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.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe

“…For a subsample of 265 specimens, a fragment of domain II of the vssc gene, including position 1016, was also sequenced to validate the PCR results. Consistently with the results reported by Pichler et al [40], the AS-PCR assay accuracy was 94%. All mismatches (N = 16) between AS-PCR and sequencing were due to specimens homozygous for the 1016V susceptible allele and PCR-genotyped as heterozygotes (Table 1).…”

“…The DNA was extracted from single legs or whole individual mosquito carcasses using the DNAzol ® [42] or CTAB [43] methods. The allele-specific PCR (AS-PCR) assay for V1016G genotyping was performed either on DNA extracted from single specimens or on pooled DNA extracted from three specimens [40]. In the case of detection of the 1016G allele in one of the pools, genotyping by PCR of each of the three specimens was performed separately.…”

“…Genotyping results were deposited in VectorBase.org (Project ID: VBP0000793). An interactive map reporting frequencies of the V1016G mutation per site (including also reports from previous publications; [29,40]) was created using the Leaflet package for R (https:// rstud io. github.…”

“…The latter has been shown to confer the highest levels of resistance to different pyrethroids [32,37] and has been reported from the species native range [34,36,37], as well as from Reunion Island in the Indian Ocean [34]. In the European region, it has only been detected in Italy, where it is widespread and reaches alarming frequencies of up to 45% in some coastal sites [29,40].…”

Optimasi Analisis Melting Curve untuk Skrining Cepat dan Sensitif Mutasi V1016G pada Aedes aegypti Resisten Sintetik Piretroid dengan Reaksi Rantai Polimerase Spesifik Alel

“…aegypti yang resisten piretroid di seluruh dunia. 8,12 Salah satu jenis mutasi yang sering terlibat dalam resistensi piretroid pada Ae. aegypti yaitu mutasi pada dua kodon V1016G atau V1016I dan F1534C yang dapat bertindak secara multiplikasi, terutama dalam kombinasi dengan mutasi tambahan S989P.…”