In central China, Anopheles anthropophagus is considered the primary malaria vector and Anopheles sinensis is a secondary vector. IdentiÞcation of these two cryptic species would 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

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... agment 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 be An. sinensis, while specimens from Liaoning Province, which were variable in their egg morphology, were found to be An. anthropophagus. The presence of An. anthropophagus in Liaoning Province extends the range of this species north to 42 o N. 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, and one member of the Nigerrimus subgroup, with the resulting molecular groupings at odds with the current morphological groupings.