High-throughput barcoding method for the genetic surveillance of insecticide resistance and species identification in Anopheles gambiae complex malaria vectors

Abstract

Surveillance of malaria vector species and the monitoring of insecticide resistance are essential to inform malaria control strategies and support the reduction of infections and disease. Genetic barcoding of mosquitoes is a useful tool to assist the high-throughput surveillance of insecticide resistance, discriminate between sibling species and to detect the presence of Plasmodium infections. In this study, we combined multiplex PCR, custom designed dual indexing, and Illumina next generation sequencing for high throughput single nucleotide polymorphism (SNP)-profiling of four species from the Anopheles (An.) gambiae complex (An. gambiae sensu stricto, An. coluzzii, An. arabiensis and An. melas). By amplifying and sequencing only 14 genetic fragments (500 bp each), we were able to simultaneously detect Plasmodium infection; insecticide resistance-conferring SNPs in ace1, gste2, vgsc and rdl genes; the partial sequences of nuclear ribosomal internal transcribed spacers (ITS1 and ITS2) and intergenic spacers (IGS), Short INterspersed Elements (SINE), as well as mitochondrial genes (cox1 and nd4) for species identification and genetic diversity. Using this amplicon sequencing approach with the four selected An. gambiae complex species, we identified a total of 15 non-synonymous mutations in the insecticide target genes, including previously described mutations associated with resistance and two new mutations (F1525L in vgsc and D148E in gste2). Overall, we present a reliable and cost-effective high-throughput panel for surveillance of An. gambiae complex mosquitoes in malaria endemic regions.

Multi-centre discriminating concentration determination of broflanilide and potential for cross-resistance to other public health insecticides in Anopheles vector populations

“…Screening of the Ag1000 genome data has failed to identify any naturally-occurring mutations in these amino acids in Anopheles field populations 28 . These genetic regions warrant inclusion in newly developed amplicon-sequencing panels 53 , which are being rolled out to monitor insecticide resistance across Anopheles vector populations, in conjunction with standard insecticide susceptibility monitoring.…”

Identification of two insecticide resistance markers in Ethiopian Anopheles stephensi mosquitoes using a multiplex amplicon sequencing assay

“…Two genomic regions were targeted for species identification and/or phylogenetic analyses: the nuclear ribosomal internal transcribed spacer 2 (ITS2) and the mitochondrially encoded cytochrome c oxidase I (cox1), (Supplementary Table 3). The primer sequences were concatenated with unique 5' tag barcode (6 bp long) to discriminate individual samples and enable multiplexing, along with sequences complementary to Illumina adapters for sequencing as described previously 38 . Prior to PCR amplification, each sample was assigned one unique barcode in each forward and reverse primer to be used for amplification of all loci.…”

“…Variant annotation was carried out using the SnpEff tool, combined with a database based on the UCI_V1 reference genome 62,63 . The percentage of alternative allele to total depth coverage was used to classify genotypes to homozygous reference (< 25% alternate allele reads), homozygous alternate (> 75% alternate allele reads) or heterozygous (25-75% alternate allele), as described previously 38 .…”

“…gambiae s.l. populations 37,38 . Here we designed and validated a targeted Amp-seq assay, combined with dual indexing barcodes and Illumina sequencing, for profiling An.…”

Application of a targeted amplicon sequencing panel to screen for insecticide resistance mutations in Anopheles darlingi populations from Brazil

“…darlingi mosquitoes. The amp-seq approach can be applied as a wide-scale insecticide-resistance surveillance technique to better inform vector-control methods.high-throughput and low-cost screening method for insecticide or drug resistance mutations in target loci [34][35][36][37] . Targeting several candidate genes in many samples permits the tracking of emerging resistance and spread of known mutations in the population.…”