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A novel mosquito species identification method based on PCR and capillary electrophoresis
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  • Estelle Chabanol,
  • Ottavia Romoli,
  • Stanislas Talaga,
  • Yanouk Epelboin,
  • Katy Heu,
  • Ghislaine Prévot,
  • Mathilde Gendrin
Estelle Chabanol
Institut Pasteur de la Guyane
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Ottavia Romoli
Institut Pasteur de la Guyane
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Stanislas Talaga
Institut Pasteur de la Guyane
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Yanouk Epelboin
Institut Pasteur de la Guyane
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Katy Heu
Institut Pasteur de la Guyane
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Ghislaine Prévot
Université de la Guyane
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Mathilde Gendrin
Institut Pasteur de la Guyane

Corresponding Author:mathilde.gendrin@pasteur.fr

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In the Anopheles genus, various mosquito species are able to transmit Plasmodium parasites responsible for malaria, while others are non-vectors. In an effort to better understand the biology of Anopheles species and to quantify transmission risk in an area, the identification of mosquito species collected on the field is an essential but problematic task. Morphological identification requires expertise, well-preserved specimens and high-quality equipment, and it does not allow any subsequent verification when samples are later used in a destructive treatment. Moreover, it involves physical manipulations that are not compatible with experiments requiring fast sampling and processing of specimens, hence species identification is often based on DNA sequencing of reference genes or region such as the Internal Transcribed Spacer 2 (ITS2) region of nuclear ribosomal DNA. Sequencing ITS2 for numerous samples is costly, but the design of species-specific PCR primers is not always possible when local species diversity is high. Here, we introduce a molecular technique of species identification based on precise determination of ITS2 length combined with a simple visual observation, the color of mosquito hindleg tip. DNA extracted from field-collected Anopheles mosquitoes was amplified with universal Anopheles ITS2 primers and analyzed with a capillary electrophoresis device, which precisely determines the size of the fragments. We defined windows of amplicon sizes combined with fifth hind tarsus color, which allow to discriminate the major Anopheles species found in our collections. We validated our parameters via Sanger sequencing of the ITS2 amplicons. This method can be particularly useful in situations with a moderate species diversity, i.e. when the number of local species is too high to define species-specific primers but low enough to avoid individual ITS2 sequencing. This tool will be of interest to evaluate local malaria transmission risk and this approach may further be implemented for other mosquito genera.
17 Mar 2023Submitted to Ecology and Evolution
21 Mar 2023Submission Checks Completed
21 Mar 2023Assigned to Editor
24 Mar 2023Reviewer(s) Assigned
18 May 2023Review(s) Completed, Editorial Evaluation Pending
22 May 2023Editorial Decision: Revise Minor
23 Aug 20231st Revision Received
25 Aug 2023Assigned to Editor
25 Aug 2023Submission Checks Completed
25 Aug 2023Review(s) Completed, Editorial Evaluation Pending
29 Aug 2023Reviewer(s) Assigned
16 Oct 2023Editorial Decision: Revise Minor
13 Nov 20232nd Revision Received
14 Nov 2023Assigned to Editor
14 Nov 2023Submission Checks Completed
14 Nov 2023Review(s) Completed, Editorial Evaluation Pending
17 Nov 2023Editorial Decision: Accept