POLYMERASE CHAIN-REACTION SPECIES DIAGNOSTIC ASSAY FOR ANOPHELES-QUADRIMACULATUS CRYPTIC SPECIES (DIPTERA, CULICIDAE) BASED ON RIBOSOMAL DNA ITS2 SEQUENCES

Species-specific differences in the nucleotide sequences of die 2nd in ternal transcribed spacer (ITS2) of nuclear ribosomal DNA (rDNA) were used to develop a diagnostic assay based on the polymerase chain react ion (PCR) that can distinguish 4 of the 5 cryptic sibling species in t he common malaria mosquito, Anopheles quadrimaculatus Say, complex. Th e assay requires only a small amount of tissue from an individual mosq uito and a mixture of 5 PCR primers. The plus strand universal primer is derived from a sequence in the 5.8S coding region that is identical in all members of the complex. The 4 minus strand primers were select ed from species-unique sequences within the ITS2 region. PCR amplifica tion produces a different sized fragment for each of the 4 species whi ch can be visualized readily under ultraviolet light after electrophor esis through an ethidium bromide-containing agarose gel. The assay has been developed and tested only with An. quadrimaculatus complex speci mens from Florida populations.