A next generation targeted amplicon sequencing method to screen for insecticide resistance mutations in Aedes aegypti populations reveals a rdl mutation in mosquitoes from Cabo Verde

Abstract

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 disease 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 mosquitoes 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 four non-synonymous polymorphisms 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 mosquitoes screened, consistent with the past use of an organochlorine compound (e.g., DDT). 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.

Towards environmental detection, quantification, and molecular characterization of Anopheles stephensi and Aedes aegypti from experimental larval breeding sites

“…stephensi 28 and Ae. aegypti 29 , fragments of key insecticide resistance genes (including portions of the voltage-gated sodium channel and acetylcholinesterase) were able to be amplified from eDNA shed by as few as 16–32 s instar larvae in 50 ml of water; as expected, no insecticide resistance mutations were identified in our insecticide-susceptible insectary colonies. The detection sensitivity of insecticide resistance genes in natural breeding sites may be greater than in our laboratory study, particularly as larval densities can be higher and vector populations will deposit eDNA throughout their ~ 2 week lifecycle in breeding sites.…”

“…Similarly, eDNA from Ae. aegypt i 50 ml artificial breeding sites containing 4 and 8 larvae did not amplify using amplicon-seq primers, targeting the vgsc and ace-1 genes 29 . PCR reactions using eDNA from Ae.…”

“…aegypti , briefly, sequences for portions of the voltage-gated sodium channel ( vgsc ; AAEL023266-RL) and acetylcholinesterase-1 ( Ace-1 ; AAEL000511-RJ) were extracted from publicly available assemblies for Ae. aegypti (LVP AGWG) 29 . Forward and reverse primers were designed using PrimerBLAST software to amplify regions of 450–500 bp that contain known SNP loci or regions of interest, with 6 bp inline barcodes and partial Illumina tails that allow the samples to be sequenced on an Illumina sequencing platform.…”

“…Samples were demultiplexed based on the in-line barcodes in each forward and reverse primer using an in-house python script 29 . Sequences were trimmed, aligned to reference ( Ae.…”

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.…”