Multiplexed amplicon sequencing reveals the heterogeneous spatial distribution of pyrethroid resistance mutations in Aedes albopictus mosquito populations in southern France

Abstract

Background: The risk of mosquito-borne disease transmission is increasing in temperate climates with the colonization and proliferation of the Asian tiger mosquito vector Aedes albopictus and the rapid mass transport of passengers returning from tropical regions where viruses are endemic. The prevention of major Aedes-borne viruses heavily relies on the use of insecticides for vector control, mainly pyrethroids. In Europe, only deltamethrin is authorized.

Methods: High-throughput molecular assays can provide a cost-effective surrogate to phenotypic insecticide resistance assays when mutations have been previously linked to a resistance phenotype. Here, we screened for the spatial distribution of knockdown resistance (kdr) mutations at a large scale using a two-step approach based on multiplexed amplicon sequencing and an unprecedented collection of field-derived mosquitoes from 95 sites in 61 municipalities, alongside a west-to-east transect in the south of France, from June to September 2021.

Results: We identified the presence of the V1016G allele in 14 sites. The V1016G allele was predominantly found in southeast France close to the Italian border, with two additional isolated sites close to Bordeaux and Marmande. All mosquitoes were heterozygous for this mutation and should not be phenotypically resistant to pyrethroid insecticide. Four other mutations were identified in our targeted genomic sequence: I1532T, M1006L, M1586L, M995L. Sequencing a section of maternally inherited mitochondrial genome confirmed that the spread of Ae. albopictus in France originated from founders within haplogroup A1.

Conclusions: These findings contribute to the broader understanding of resistance dynamics in Europe and can inform targeted approaches to mitigate the impact of resistance on vector control.

Keywords: Aedes albopictus; Amplicon sequencing; Arbovirus; Knock-down resistance; Molecular surveillance; Pool DNA-sequencing; Pyrethroid resistance.

Fig. 1

Geographic location of alleles confirmed in knockdown resistance (kdr) in Aedes albopictus in the south of France. A Location and frequencies of kdr alleles as revealed by amplicon sequencing based on sequencing of DNA from pooled mosquito heads. Allele frequencies are represented in brackets for each locality. B Location and prevalence of kdr alleles as revealed by amplicon sequencing on single mosquitoes from each locality. The identity and prevalence of mosquitoes carrying the mutations are represented for each locality. Mosquitoes are identified on the basis of their original pool number and a unique number. Gray points represent localities where no confirmed kdr alleles were identified

Fig. 2

Phylogenetic relationships among a subset of Aedes albopictus mosquitoes analyzed in this study were based on a curated alignment of 3243 bp nucleotide region of the mitochondrial genome. The mitochondrial genome of Ae. aegypti (NC_035159) was used as an outgroup in the phylogenetic tree. The best-scoring maximum-likelihood (ML) tree was generated with 120 bootstrap replicates. Only bootstrap scores > 60 are represented in dark red. Mosquitoes are identified on the basis of their original pool number and a unique identifier. Pool localities are represented on the map with a color code representing the longitude (west-to-east transect gradient is represented with a blue-to-red color gradient). Localities with at least one mosquito carrying a kdr allele are represented in red on both the map and the phylogenetic tree