Molecular identification of Anopheles minimus s.l. beyond distinguishing the members of the species complex

Correct species identification is the starting point for studying the epide miological role of vectors. Identification is mostly achieved using morphol ogical criteria, but this fails when sibling species and species with overl apping morphological characters are involved. The problem with the identifi cation of Anopheles minimus s.l., one of the most widespread malaria vector s in South-East Asia, is twofold: it is a complex of at least two isomorphi c species, and based on morphology, members of the complex are difficult to distinguish from closely related species. An identification method was dev eloped for An. minimus species A and C, and four related species, Am aconit us, An. pampanai, An. varuna and An. jeyporiensis. PCR-amplified internal t ranscribed spacer 2 (ITS2) ribonuclear DNA (rDNA) fragments were digested w ith restriction endonuclease BsiZI. Clear diagnostic banding patterns for t he six species were obtained on agarose gels. Testing field-collected speci mens from different regions in South-East Asia indicated that the technique will be applicable over a wide geographical area. From this it is clear th at molecular identification has to focus not only on the species of complex es, but also on related species if they hamper the morphological identifica tion of the 'sensu lato species'.