Dataset Information

Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection.

ABSTRACT: BACKGROUND: Knockdown resistance (kdr) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L1014S and L1014F, at this residue in a major malaria vector Anopheles stephensi and describes new PCR assays for their detection. METHODS: Part of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of An. stephensi collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s). RESULTS: Analysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TCA) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of An. stephensi mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the kdr mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples. CONCLUSIONS: Two alternative kdr-like mutations, L1014S and L1014F, were detected in An. stephensi with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in An. stephensi. Two specific PCR assays were developed for detection of two kdr-like mutations in An. stephensi.

SUBMITTER: Singh OP

PROVIDER: S-EPMC3068127 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection.

Singh Om P OP Dykes Cherry L CL Lather Manila M Agrawal Om P OP Adak Tridibes T

Malaria journal 20110314

<h4>Background</h4>Knockdown resistance (kdr) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L101 ...[more]

PMID: 21401946

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Project description:Understanding the molecular basis of insecticide resistance is key to improve the surveillance and monitoring of malaria vector populations under control. In the major malaria vector Anopheles funestus, little is currently known about the role of the knockdown resistance (kdr) mechanism. Here, we investigated the presence and contribution of knockdown resistance (kdr) to pyrethroids/DDT resistance observed in Anopheles funestus across Africa.Pyrosequencing genotyping and sequencing of the voltage gated sodium channel (VGSC) gene did not detect the common L1014F mutation in field collected An. funestus across Africa. Amplification and cloning of the full-length of the sodium channel gene in pyrethroid resistant mosquitoes revealed evidences of alternative splicing events with three transcripts of 2092, 2061 and 2117 amino acids (93% average similarity to An. gambiae). Several amino acid changes were detected close to the domain II of the protein such as L928R, F938 W, I939S, L802S and T1008 M. However, all these mutations are found at low frequency and their role in pyrethroid resistance could not be established. The presence of the exclusive alternative splicing at exon 19 was not associated with resistance phenotype. Analysis of patterns of genetic diversity of the VGSC gene revealed a high polymorphism level of this gene across Africa with no evidence of directional selection suggesting a limited role for knockdown resistance in pyrethroid resistance in An. funestus. Patterns of genetic differentiation correlate with previous observations of the existence of barriers to gene flow Africa-wide with southern population significantly differentiated from other regions.Despite an apparent limited role of knockdown resistance in An. funestus, it is necessary to continue to monitor the contribution of the mutations detected here as increasing selection from insecticide-based interventions may change the dynamic in field populations as previously observed in other vectors.

Project description:Small laboratory cage trials of non-drive and gene-drive strains of the Asian malaria vector mosquito, Anopheles stephensi, were used to investigate release ratios and other strain properties for their impact on transgene spread during simulated population modification. We evaluated the effects of transgenes on survival, male contributions to next-generation populations, female reproductive success and the impact of accumulation of gene drive-resistant genomic target sites resulting from nonhomologous end-joining (NHEJ) mutagenesis during Cas9, guide RNA-mediated cleavage. Experiments with a non-drive, autosomally-linked malaria-resistance gene cassette showed 'full introduction' (100% of the insects have at least one copy of the transgene) within 8 weeks (? 3 generations) following weekly releases of 10:1 transgenic:wild-type males in an overlapping generation trial design. Male release ratios of 1:1 resulted in cages where mosquitoes with at least one copy of the transgene fluctuated around 50%. In comparison, two of three cages in which the malaria-resistance genes were linked to a gene-drive system in an overlapping generation, single 1:1 release reached full introduction in 6-8 generations with a third cage at ~80% within the same time. Release ratios of 0.1:1 failed to establish the transgenes. A non-overlapping generation, single-release trial of the same gene-drive strain resulted in two of three cages reaching 100% introduction within 6-12 generations following a 1:1 transgenic:wild-type male release. Two of three cages with 0.33:1 transgenic:wild-type male single releases achieved full introduction in 13-16 generations. All populations exhibiting full introduction went extinct within three generations due to a significant load on females having disruptions of both copies of the target gene, kynurenine hydroxylase. While repeated releases of high-ratio (10:1) non-drive constructs could achieve full introduction, results from the 1:1 release ratios across all experimental designs favor the use of gene drive, both for efficiency and anticipated cost of the control programs.

Project description:ObjectivesTo investigate the differential insecticide-susceptibility of two molecular forms of Anopheles subpictus complex (A and B) against DDT and pyrethroids, the occurrence of knockdown resistance (kdr) mutations in these forms, and the association of kdr mutations with insecticide resistance.MethodsInsecticide susceptibility tests of An. subpictus s.l., collected from coastal and inland areas of mainland India, were performed against DDT, permethrin and deltamethrin using the WHO standard insecticide susceptibility test kit. The mosquitoes were characterized for molecular forms using a diagnostic PCR developed in this study. Representative samples of An. subpictus molecular forms A and B were sequenced for a genomic region encompassing the IIS4-5 linker to the IIS6 segments of the voltage-gated sodium channel to identify kdr mutations. A common PIRA-PCR was developed for identifying L1014F-kdr mutation and used for genotyping in both molecular forms of An. subpictus.ResultsMolecular form A of An. subpictus was resistant to all three insecticides, i.e., DDT, Permethrin and deltamethrin, whereas Form B was categorized as 'possibly resistant' to these insecticides. Significantly higher mortalities in WHO insecticide susceptibility tests were recorded in Form B compared to Form A in sympatric populations. Molecular characterization of the IIS4-5 linker to IIS-6 segments of the voltage-gated sodium channel revealed the presence of two alternative nucleotide transversions at L1014 residue in Form A, both leading to the same amino acid change, i.e., Leu-to-Phe; however, such mutations could not be observed in Form B. PIRA-PCR-based kdr-genotyping of field populations revealed high frequencies of L1014F-kdr mutations in Form A and the absence of this mutation in Form B. The proportion of L1014F mutation was significantly higher in resistant mosquitoes following insecticide-bioassay with DDT (p<0.0001), permethrin (p<0.001) and deltamethrin (p<0.01) as compared to their susceptible counterparts.ConclusionsSignificant differences in insecticide susceptibility were found between two molecular forms of An. subpictus complex in sympatric populations. The L1014F-kdr mutation was observed in Form A only, which was found to be associated with DDT, permethrin and deltamethrin resistance.

Dataset Information

Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection.

Publications

Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection.

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