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

Abstract

Large-scale surveillance and informed vector control approaches are urgently needed to ensure that national malaria programs remain effective in reducing transmission and, ultimately, achieving malaria elimination targets. In South and Central America, Anopheles darlingi is the primary malaria vector, responsible for the majority of Plasmodium species transmission. However, little is known about their molecular markers associated with insecticide resistance. Here we developed a low-cost, high throughput amplicon sequencing (“amp-seq”) panel, consisting of 11 amplicons that target genes linked to mosquito species (cox-1 and its2) and insecticide resistance (ace-1, GSTe2, vgsc and rdl). Used in tandem with dual index barcoding of amplicons, our approach permits high numbers of loci and samples to be sequenced in single runs, thereby decreasing costs and increasing efficiency. By screening 200 An. darlingi mosquitoes collected in Brazil, our amp-seq approach identified 10 point mutations leading to amino acid alterations in ace-1 (V243I, N194H, S673N, S674N/T) and GSTe2 genes (I114V, D128E, T166I, T179I, and T205A). Overall, our work has demonstrated the utility of amp-seq to provide insights into the genetic diversity of An. darlingi mosquitoes. The amp-seq approach can be applied as a wide-scale insecticide-resistance surveillance technique to better inform vector-control methods.