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 epidemiological role of vectors. Identification is mostly achieved using morphological criteria, but this fails when sibling species and species with overlapping morphological characters are involved. The problem with the identification of Anopheles minimus s. l., one of the most widespread malaria vectors in South-East Asia, is twofold: it is a complex of at least two isomorphic species, and based on morphology, members of the complex are difficult to distinguish from closely related species. An identification method was developed for An. minimus species A and C, and four related species, An. aconitus, An. pampanai, An. varuna and An. jeyporiensis. PCR-amplified internal transcribed spacer 2 (ITS2) ribonuclear DNA (rDNA) fragments were digested with restriction endonuclease BsiZI. Clear diagnostic banding patterns for the six species were obtained on agarose gels. Testing field-collected specimens from different regions in South-East Asia indicated that the technique will be applicable over a wide geographical area. From this it is clear that molecular identification has to focus not only on the species of complexes, but also on related species if they hamper the morphological identification of the 'sensu lato species'.