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

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 , gste 2, 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.