DISCRIMINATION OF ALL MEMBERS OF THE ANOPHELESPUNCTULATUS COMPLEX BY POLYMERASE CHAIN-REACTION RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM ANALYSIS

A method has been developed to identify the members of the Anopheles punctulatus complex using polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP). Members of the An. punctulatus complex are the most important vectors of malaria in the southwest Pacific and consist of 10 cryptic species, An. farauti no. 1-7, An. punctulatus, An. sp. near punctulatus, and An. koliensis. For each species, PCR amplification of the ribosomal DNA internal transcribed spacer produced a 750-basepair product. Digestion with Msp I and electrophoresis on a 3.0% agarose gel results in banding patterns unique to each species. Isolates of the same species from different locations gave an identical pattern. The technique is sensitive enough so that a PCR-RFLP can be generated from as little as a single mosquito leg, allowing the rest of the mosquito to be used for other important epidemiologic analyses such as determining host feeding source, and for parasite detection.