MORPHOLOGY, SYSTEMATICS, EVOLUTION Molecular Identification of the Malaria Vectors Anopheles anthropophagus and Anopheles sinensis (Diptera: Culicidae) in Central China Using Polymerase Chain Reaction and Appraisal of Their Position Within the Hyrcanus Group

In central China, Anopheles anthropophagus is considered the primary malaria vector andAnopheles sinensis is a secondary vector. IdentiÞcation of these twocryptic specieswould facilitate studies on malaria transmission and the application of control measures. At present, the only reliable morphological markers occur in the egg stage, making this approach impractical for any large scale Þeld studies. In this study, we report on the development of a polymerase chain reaction (PCR)restriction fragment length polymorphism procedure involving the ribosomal DNA ITS2 region for discrimination of these species. The PCR-ampliÞed product size of the ITS2 was 574 bp for An. anthropophagus and 594 bp for An. sinensis. Diagnostic restriction fragment length polymorphisms appeared with the restriction enzymes RsaI or HinfI. This diagnostic PCR was tested on mosquitoes collected from different locations throughout China. Specimens identiÞed morphologically as An. anthropophagus in the adult and egg stage from one location in Quangdong Province were found to beAn. sinensis,while specimens fromLiaoningProvince,whichwerevariable in their eggmorphology, were found to be An. anthropophagus. The presence of An. anthropophagus in Liaoning Province extends the range of this species north to 42N. The ITS2 spacer sequence was used in a maximum parsimony phylogenetic reconstruction of six members of the Hyrcanus group, two members of the Lesteri subgroup, andonememberof theNigerrimus subgroup,with the resultingmoleculargroupings at odds with the current morphological groupings.