Aedes aegypti amplicons for insecticide resistance relevant regions

Aedes mosquito vectors transmit many viruses of global health concern, including dengue, chikungunya and Zika. These vector-borne viral diseases have a limited number of treatment options, and vaccines vary in their effectiveness. Consequently, integrated vector management is a primary strategy for their control. However, the increasing emergence and spread of insecticide resistance is threatening the efficacy of vector control methods. Identifying mutations associated with resistance in vector populations is important to monitor the occurrence and evolution of insecticide resistance and inform control strategies. Rapid and cost-effective genome sequencing approaches are urgently needed. Here we present an adaptable targeted amplicon approach for cost-effective implementation within next generation sequencing platforms. This approach can identify single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) in genes involved in insecticide resistance in Aedes aegypti mosquitoes. We designed and tested eleven amplicons, which included segments of the ace-1 (carbamate target), the Voltage-Gated Sodium Channel (vgsc; pyrethroids, DDT and organochlorines), and rdl (dieldrin) genes; thereby covering established knockdown resistance (kdr) mutations (e.g., S989P, I1011M/V, V1016G/I and F1534C), with the potential to identify novel ones. The amplicon assays were designed with internal barcodes, to facilitate multiplexing of large numbers of samples at low cost and were sequenced using an Illumina platform. Our approach was evaluated on 152 Ae. aegypti mosquitoes collected in Cabo Verde, an archipelago with a history of arbovirus outbreaks. The amplicon sequence data revealed 146 SNPs, including 4 new non-synonymous mutations in the vgsc gene, one in ace-1 and the 296S rdl mutation previously associated with resistance to organochlorines. The 296S rdl mutation was identified in 98% of samples tested, consistent with the recent intensive use of carbamate insecticides. Overall, our work shows that targeted amplicon sequencing is a rapid, robust, and cost-effective tool that can be used to perform high throughput monitoring of insecticide resistance.