Identification of two insecticide resistance markers in Ethiopian Anopheles stephensi mosquitoes using a multiplex amplicon sequencing assay targeting vgsc, rdl, gste2 and ace1 loci

Abstract

Since its first detection in 2012 in Djibouti, Anopheles stephensi has invaded and established itself in the Horn of Africa and most recently in Nigeria and Yemen. The expansion of this vector poses a significant threat to malaria control and eliminations efforts. Integrated vector management is the primary strategy used to interrupt disease transmission; however, growing insecticide resistance is threatening to reverse gains in global malaria control. We present a next-generation amplicon-sequencing approach, for high-throughput monitoring of insecticide resistance genes (ace1, gste2, vgsc and rdl), species identification and characterization of genetic diversity (its2 and cox1) in An. stephensi. Ninety-five An. stephensi mosquitoes, collected in Ethiopia, were screened, identifying 104 SNPs, including the knock-down mutation L958F (L1014F in Musca domestica), and for the first time in this vector species, the A296S substitution (A301S in Drosophila melanogaster) in the rdl locus. Two other amino acid substitutions (ace1-N177D, GSTe2-V189L) were also identified but have not been previously implicated in insecticide resistance. Genetic diversity in the mitochondrial cox1 gene revealed shared haplotypes between Ethiopian An. stephensi with samples from Pakistan, Sudan, and Djibouti. Overall, we present a reliable, cost-effective strategy using amplicon-sequencing to monitor known insecticide resistance mutations, with the potential to identify new genetic variants, to assist high-throughput surveillance of An. stephensi populations.

Genome-wide population genetics and molecular surveillance of insecticide resistance in Anopheles stephensi mosquitoes from Ethiopia

“…The low proportion of Ethiopian isolates with this SNP, along with its presence as a heterozygous genotype, implies it has recently arisen in this population. The kdr L1014F mutation has been previously identi ed in Ethiopian samples at a similar frequency to our study 21 . The low allelic frequency of the resistance marker, in the context of phenotypic resistance in the sampling location, indicates mechanisms other than target site modi cations are responsible for this observed phenotype.…”

“…The target site mutationA296S in the GABA receptor has been reported in An. stephensi from Ethiopia, and is associated with resistance to dieldrin (rdl), an organochlorine banned in the 1990s due to concerns about its environmental persistence and potential impact on human health [20][21][22] . The L1014 knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) is linked to cross resistance to DDT and pyrethroids and has been reported in An.…”

“…The L1014 knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) is linked to cross resistance to DDT and pyrethroids and has been reported in An. stephensi from Ethiopia, Afghanistan, and India 21,[23][24][25][26] . However, there have been examples of pyrethroid resistance in An.…”

Profiling Insecticide Resistance Phenotypes and Genotypes in Aedes Aegypti Populations Across Four Regions in Puerto Rico

“…Molecular methodologies have become a cost-effective approach for the monitoring of insecticide resistance, particularly when using multiplex assays that target many loci in parallel. Targeted amplicon next generation sequencing (Amp-seq) offers the possibility to analyse large number of candidate genetic regions across many samples using next generation sequencing platforms [10][11][12][13] . It offers increased sensitivity compared to PCR-RFLP and real-time PCR, which in general only target a few markers, and decreased costs in comparison to whole genome sequencing (WGS).…”

First detection ofAnopheles stephensiin Ghana using molecular surveillance

“…stephensi into these receptive areas will currently put about 126 million people at risk of malaria (2,4). Also, this invasive vector has been found to be resistant to insecticides further increasing the risk of malaria transmission in combination with limiting intervention efficacy (11)(12)(13). An.…”