Dataset Information

Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus.

ABSTRACT: Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-site of pyrethroid and organochlorine insecticides. In order to develop field-applicable diagnostics for Vgsc CN, and as a prelude to investigating the possible association of CN with insecticide resistance, three assays were compared for their accuracy in CN estimation in this species. The gold standard method is droplet digital PCR (ddPCR), however, the hardware is prohibitively expensive for widespread utility. Here, ddPCR was compared to quantitative PCR (qPCR) and pyrosequencing. Across all platforms, CNV was detected in ?10% of mosquitoes, corresponding to three or four copies (per diploid genome). ddPCR and qPCR-Std-curve yielded similar predictions for Vgsc CN, indicating that the qPCR protocol developed here can be applied as a diagnostic assay, facilitating monitoring of Vgsc CN in wild populations and the elucidation of association between the Vgsc CN and insecticide resistance.

SUBMITTER: Martins WFS

PROVIDER: S-EPMC5517494 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus.

Martins Walter Fabricio Silva WFS Subramaniam Krishanthi K Steen Keith K Mawejje Henry H Liloglou Triantafillos T Donnelly Martin James MJ Wilding Craig Stephen CS

Scientific reports 20170719 1

Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-s ...[more]

PMID: 28725028

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Project description:Voltage-gated sodium channels are the target sites of both DDT and pyrethroid insecticides. The importance of alternative splicing as a key mechanism governing the structural and functional diversity of sodium channels and the resulting development of insecticide and acaricide resistance is widely recognized, as shown by the extensive research on characterizing alternative splicing and variants of sodium channels in medically and agriculturally important insect species. Here we present the first comparative study of multiple variants of the sodium channel transcripts in the mosquito Culex quinquefasciatus. The variants were classified into two categories, CxNa-L and CxNa-S based on their distinguishing sequence sizes of ~6.5 kb and ~4.0 kb, respectively, and generated via major extensive alternative splicing with minor small deletions/ insertions in susceptible S-Lab, low resistant HAmCq(G0), and highly resistant HAmCq(G8)Culex strains. Four alternative Cx-Na-L splice variants were identified, including three full length variants with three optional exons (2, 5, and 21i) and one with in-frame-stop codons. Large, multi-exon-alternative splices were identified in the CxNa-S category. All CxNa-S splicing variants in the S-Lab and HAmCq(G0) strains contained in-frame stop codons, suggesting that any resulting proteins would be truncated. The ~1000 to ~3000-fold lower expression of these splice variants with stop codons compared with the CxNa-L splicing variants may support the lower importance of these variants in S-Lab and HAmCq(G0). Interestingly, two alternative splicing variants of CxNa-S in HAmCq(G8) included entire ORFs but lacked exons 5 to18 and these two variants had much higher expression levels in HAmCq(G8) than in S-Lab and HAmCq(G0). These results provide a functional basis for further characterizing how alternative splicing of a voltage-gated sodium channel contributes to diversity in neuronal signaling in mosquitoes in response to pyrethroids, and possibly indicates the role of these variants in the development of pyrethroid resistance.

Dataset Information

Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus.

Publications

Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus.

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