High-throughput genotyping of a full voltage-gated sodium channel gene via genomic DNA using target capture sequencing and analytical pipeline MoNaS to discover novel insecticide resistance mutations.

Kentaro Itokawa,Shinji Kasai,Koji Yatsu,Osamu Komagata,Yoshihide Maekawa,Tsuyoshi Sekizuka,Tomonori Sasaki,Masaaki Sugiura,Takashi Tomita,Kyoko Sawabe,Makoto Kuroda,Sassan Asgari

doi:10.1371/journal.pntd.0007818

Abstract

In insects, the voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein, which are selected under insecticide pressure, are known to confer insecticide resistance. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure, it is often challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes from CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all orthologous VGSC CDSs, except some tiny exons, from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency as a result of the high nucleotide-level conservation of VGSC. To improve efficiency of the downstream bioinformatic process, we developed an automated pipeline—MoNaS (Mosquito Na+ channel mutation Search)—which calls amino acid substitutions in the VGSC from NGS reads and compares those to known resistance mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetic studies on resistance alleles (with respect to the origin, selection, and migration etc.) in both clinically and agriculturally important insect pests.

Highlights

Important insect vectors such as mosquitoes undergo strong selective pressure from insecticides in the field where control of vector-borne diseases such as malaria, dengue and zika is applied
The Voltage Gated Sodium Channel (VGSC) in insect is targeted by pyrethroid insecticides and genetic variations in the protein are known to confer pyrethroid resistance
Since the voltage-gated sodium channel (VGSC) gene in genome consists of many exons and long introns, there is no simple method to genotype whole of coding regions from the genomic DNA of insect

Summary

Introduction

Important insect vectors such as mosquitoes undergo strong selective pressure from insecticides in the field where control of vector-borne diseases such as malaria, dengue and zika is applied. The kdr phenotype as well as another distinct phenotype, super-kdr, was eventually linked to amino acid (aa) substitutions on the two positions, L1014F and L1014F+M918T, respectively, on the gene coding VGSC protein [5,6] These and other aa substitutions have been found to be associated with resistance in many medical and agricultural insect pests [7,8]. Most studies employing polymerase chain reaction (PCR) and direct sequencing usually cover only restricted regions where the known resistance-conferring substitutions are frequently found; e.g., IIS5–6 [7,8] Such a bias may lower the chance of discovering novel resistance mutations existing outside the region investigated

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Conclusion