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Improved Molecular Technique for the Differentiation of Neotropical Anopheline Species

We evaluated a PCR-RFLP of the ribosomal internal transcribed spacer 2 region (ITS2) to distinguish species of Anopheles commonly reported in the Amazon and validated this method using reared F₁ offspring. The following species of Anopheles were used for molecular analysis: An. (Nys.) benarrochi, An. (Nys.) darlingi, An. (Nys.) nuneztovari, An. (Nys.) konderi, An. (Nys.) rangeli, and An. (Nys.) triannulatus sensu lato (s.l.). In addition, three species of the subgenus Anopheles, An. (Ano.) forattini, An. (Ano.) mattogrossensis, and An. (Ano.) peryassui were included for testing. Each of the nine species tested yielded diagnostic banding patterns. The PCR-RFLP method was successful in identifying all life stages including exuviae with small fractions of the sample. The assay is rapid and can be applied as an unbiased confirmatory method for identification of morphologic variants, disputed samples, imperfectly preserved specimens, and life stages from which taxonomic keys do not allow for definitive species determination.

Received September 1, 2007. Accepted for publication December 10, 2007.

Acknowledgments: The authors thank Maribel Paredes Olortegui and ST Unit for logistical support and Graciela Meza Sanchez and Carmen Montalvan for critical review and encouragement.

Financial support: The work was supported by the Malaria Research Institute from the Department of Molecular Microbiology and Immunology at Johns Hopkins (PI: Margaret Kosek). Ryan Matson was supported by T37MD001580. Margaret Kosek is supported by the Fogarty International Center K01 TW05717 and the project was further supported by R01AI067727 and K24AI068903 to Joseph M. Vinetz.

^* Address correspondence to Margaret Kosek, 615 N Wolfe St, Rm W5515, Baltimore, MD 21205. E-mail: mkosek{at}jhsph.edu

Authors’ addresses: Ryan Matson, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, Telephone: (415) 456-4538, E-mail: ryanmatson{at}gmail.com. Carlos Tong Rios and Victor Lopez Sifuentes, NAMRID-Iquitos, La Marina 451, Iquitos, Maynas, Peru, E-mails: ctong32{at}gmail.com and vlopez{at}nmrcd.med.navy.mil. Cesar Banda Chavez, Morona 452, Iquitos, Maynas, Peru, 65600082. Robert Gilman, Pablo Peñataro Yori, and Margaret Kosek, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street w5515, Baltimore, MD 21205, Telephone: (410) 614-3959, E-mails: gilmanbob{at}gmail.com, pyori{at}jhsph.edu, and mkosek{at}jhsph.edu. David Florin and Roberto Fernandez, U.S. Naval Medical Research Center Detachment American Embassy-Lima, Peru Unit 3800 APO AA 34031, Telephone: 011-511-614-4141, E-mails: florin{at}nmrcd.med.navy.mil and rfernadez{at}nmrcd.med.navy.mil. Roldan Cardenas Greffa, Telephone: 011-51-65-269605. Joseph Vinetz, University of California San Diego, 9500 Gilman Drive 0741, Palade Laboratories Rm 125, La Jolla, CA 92093-0741, Telephone: (858) 822-4469, E-mail: jvinetz{at}ucsd.edu.