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.

Verena Pichler,Simone Martini,Alessandra Della Torre,Annapaola Rizzoli,Romeo Bellini,Maria Calzetta,Beniamino Caputo,Francesca Paoli,Rodolfo Veronesi,Emiliano Mancini,Joao Pinto,Martina Micocci,Daniele Arnoldi,Claudio De Liberato,Andrea Drago,Valeria Lencioni,Arianna Ermenegildi

doi:10.3390/insects12010079

Abstract

Simple SummaryThe Asian Tiger mosquito, Aedes albopictus, is an invasive species which has become a worldwide public health concern due to its colonization of all continents (except Antarctica), its aggressive biting behavior and its capacity to transmit potentially deadly human viruses, such as Dengue and Chikungunya. Insecticides currently represent the most commonly used weapon to control epidemics of mosquito-borne viruses, but their effectiveness is threatened by the fast and worldwide spread of resistant mosquito vector populations. Molecular approaches able to easily detect mosquito genetic traits associated with insecticide resistance are among the key tools to counteract this phenomenon. We developed and tested a method that makes it possible to detect the presence in Aedes albopictus of a specific genetic trait (the so-called knock-down resistance (kdr) mutation) associated with resistance to pyrethroids, the most commonly used insecticidal class. We tested this approach on mosquitoes sampled across Italy and show that the kdr mutation is widespread in the country and reaches worrying frequencies (up to 45%) in coastal areas where pyrethroids are widely exploited to reduce mosquito nuisance. These results should serve as a warning bell and encourage further studies to inform insecticide management policies with the aim of maintaining the effectiveness of pyrethroids in the long term.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.

Highlights

Vector-borne diseases account for approximately 17% of the estimated global burden of all infectious diseases and cause more than 700,000 deaths each year [1]
Validation of the resulting AS-Polymerase chain reaction (PCR) genotypes by sequencing of a ~500 bp fragment of domain II of the vssc gene, including the V1016G locus, on 39 specimens led to an estimated accuracy of 95% (Table 2)
The second incorrectly genotyped specimen was genotyped as heterozygote by sequencing, but as homozygote 1016G/1016G by the allele-specific PCR (AS-PCR) assay: this specimen carried the two last positions of the primer binding site in heterozygosis; the resulting codons did not affect the amino acidic sequence, resulting in a 1016V/1016G heterozygote, but probably caused a suboptimal binding of the primer Val1016rev

Summary

Introduction

Vector-borne diseases account for approximately 17% of the estimated global burden of all infectious diseases and cause more than 700,000 deaths each year [1]. Pyrethroids, a class of synthetic insecticides characterized by a high level of effectiveness against target species and low acute toxicity to vertebrates, remain the first choice for chemical-based vector control [15]. Due to their longstanding and widespread use, resistance to pyrethroids (PyR) in mosquito vector populations has become a great concern, with regard to species producing the highest public health impact, such as main Afrotropical malaria vectors and the most efficient tropical arbovirus vector, Aedes aegypti [16,17,18]. Pyrethroids are recommended only to control/interrupt outbreaks of exotic arboviruses, they are widely employed to reduce mosquito density and nuisance [19,20,21]

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