Dataset Information

Rapid high throughput SYBR green assay for identifying the malaria vectors Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae s.s. Giles.

ABSTRACT: The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding. Several molecular diagnostic techniques for distinguishing between An. coluzzii and An. gambiae are still under improvement. Although, the current SINE method for identification between An. coluzzii and An. gambiae works reliably, this study describes a refinement of the SINE method to increase sensitivity for identification of An. coluzzii, An. gambiae and An. arabiensis based on amplicon dissociation curve characteristics. Field-collected samples, laboratory-reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were sampled from Ghana and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye. The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72°C for An. arabiensis, 75°C for An. gambiae and 86°C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.

SUBMITTER: Chabi J

PROVIDER: S-EPMC6474623 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Rapid high throughput SYBR green assay for identifying the malaria vectors Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae s.s. Giles.

Chabi Joseph J Van't Hof Arjen A N'dri Louis K LK Datsomor Alex A Okyere Dora D Njoroge Harun H Pipini Dimitra D Hadi Melinda P MP de Souza Dziedzom K DK Suzuki Takashi T Dadzie Samuel K SK Jamet Helen P HP

PloS one 20190419 4

The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding. Several molecular diagnostic techniques for distinguishing between An. coluzzii and An. gambiae are still under improvement. Although, the current SINE method for identification between An. coluzzii and An. gambiae works reliably, this study describes a refinement of the SINE method to incre ...[more]

PMID: 31002694

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Project description:Insecticide resistance threatens recent progress on malaria control in Africa. To characterize pyrethroid resistance in Uganda, Anopheles gambiae (s.s.) and Anopheles arabiensis were analyzed from 11 sites with varied vector control strategies. Mosquito larvae were collected between May 2018 and December 2020. Sites were categorized as receiving no indoor-residual spraying ('no IRS', n = 3); where IRS was delivered from 2009 to 2014 and in 2017 and then discontinued ('IRS stopped', n = 4); and where IRS had been sustained since 2014 ('IRS active', n = 4). IRS included bendiocarb, pirimiphos methyl and clothianidin. All sites received long-lasting insecticidal nets (LLINs) in 2017. Adult mosquitoes were exposed to pyrethroids; with or without piperonyl butoxide (PBO). Anopheles gambiae (s.s.) and An. arabiensis were identified using PCR. Anopheles gambiae (s.s.) were genotyped for Vgsc-995S/F, Cyp6aa1, Cyp6p4-I236M, ZZB-TE, Cyp4j5-L43F and Coeae1d, while An. arabiensis were examined for Vgsc-1014S/F. Overall, 2753 An. gambiae (s.l.), including 1105 An. gambiae (s.s.) and 1648 An. arabiensis were evaluated. Species composition varied by site; only nine An. gambiae (s.s.) were collected from 'IRS active' sites, precluding species-specific comparisons. Overall, mortality following exposure to permethrin and deltamethrin was 18.8% (148/788) in An. gambiae (s.s.) and 74.6% (912/1222) in An. arabiensis. Mortality was significantly lower in An. gambiae (s.s.) than in An. arabiensis in 'no IRS' sites (permethrin: 16.1 vs 67.7%, P < 0.001; deltamethrin: 24.6 vs 83.7%, P < 0.001) and in 'IRS stopped' sites (permethrin: 11.3 vs 63.6%, P < 0.001; deltamethrin: 25.6 vs 88.9%, P < 0.001). When PBO was added, mortality increased for An. gambiae (s.s.) and An. arabiensis. Most An. gambiae (s.s.) had the Vgsc-995S/F mutation (95% frequency) and the Cyp6p4-I236M resistance allele (87%), while the frequency of Cyp4j5 and Coeae1d were lower (52% and 55%, respectively). Resistance to pyrethroids was widespread and higher in An. gambiae (s.s.). Where IRS was active, An. arabiensis dominated. Addition of PBO to pyrethroids increased mortality, supporting deployment of PBO LLINs. Further surveillance of insecticide resistance and assessment of associations between genotypic markers and phenotypic outcomes are needed to better understand mechanisms of pyrethroid resistance and to guide vector control.

Dataset Information

Rapid high throughput SYBR green assay for identifying the malaria vectors Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae s.s. Giles.

Publications

Rapid high throughput SYBR green assay for identifying the malaria vectors Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae s.s. Giles.

Similar Datasets

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