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The African Anopheles funestus and the Oriental An. minimus groups are closely related and composed of major malaria vectors in Africa and Southeast Asia, respectively. None of the species of either the An. funestus or the An. minimus group can be identified with absolute certainty using the adult morphology. Polymorphisms present on the internal transcribed spacer 2 (ITS2) of ribosomal DNA allowed the development of 10 primers that combined with an universal forward primer lead to a simple and sensitive multiplex allele-specific polymerase chain reaction (AS-PCR). Moreover, the possible additional amplification of the entire ITS2 allows one to detect other anopheline species in sympatry with members of both groups not included in this assay and serves as a control band. This universal PCR method permits the discrimination of 10 species within the subgenus Cellia, among which figure three major malaria vectors, and constitutes a very efficient and powerful tool to improve our knowledge on these species distribution and biology. Not only restricted to anophelines, this AS-PCR could also be developed and applied to other insect groups.
Received July 28, 2003. Accepted for publication September 12, 2003.
Acknowledgments: This study was made possible because of the assistance of a number of people who helped with mosquito sampling on both continents. We are especially grateful to D. Fontenille (Institute of Research for Development, Montpellier, France), S. Laventure (Institut Pasteur de Madagascar, Antananarivo, Madagascar), G. Le Goff (Institute of Research for Development, Antananarivo, Madagascar), J. Mwangi and G. Otsyula (Kenya Medical Research Institute, Nairobi, Kenya), J. La Grange (Department of Health, Nelspruit, Mpumalanga, South Africa), T. Furemele (Department of Health, Tzaneen, Northern Province, South Africa), K. Hargreaves (Department of Health, Jozini, KwaZulu/Natal, South Africa), G. Kloke (Foray Consultants, Maputo, Mozambique), B. Ntomwa (Malaria Control Program, Oshakati, Namibia), T. Byembabazi (Malaria Control Program, Kampala, Uganda), and the entomology team of the National Institute of Malarialogy, Parasitology and Entomology (Hanoi, Vietnam) for assistance with field collections or supply of specimens. We also thank V. Baimai and M. Savanasoojarit (Mahidol University, Bangkok, Thailand) for specimens from Thailand.
Financial support: This work was partially supported by an EC grant, INCO-DC research project ERBIC 18CT970211, and a VIHPAL 2000 grant from the French Ministry of Research to Sylvie Manguin.
Authors’ addresses: Claire Garros and Sylvie Manguin, Center of Biology and Management of Populations, Campus International de Baillarguet, CS 30016, 34988 Montferrier sur Lez, France, Lizette L. Koekemoer and Maureen Coetzee, Vector Control Reference Unit, National Institute for Communicable Diseases, PO Box 1038, Johannesburg, South Africa, Marc Coosemans, Department of Parasitology, Prince Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium.
Reprint requests: Claire Garros or Sylvie Manguin, Center of Biology and Management of Populations, Campus International de Baillarguet, CS 30016, 34988 Montferrier sur Lez, France, Telephone: 33-4-99-62-33-28/27, Fax: 33-4-99-62-33-45, E-mails: garros{at}mpl.ird.fr and manguin{at}mpl.ird.fr.
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