Differentiation of members of the Anopheles fluviatilis species complex by an allele-specific polymerase chain reaction based on 28S ribosomal DNA sequences.

Anopheles fluviatilis, one of the major vectors of malaria in India, is a complex of at least three cryptic species provisionally designated as species S, T, and U. Identification of the cryptic species of An. fluviatilis complex is of paramount importance in disease control program due to contrasting differences in their vectorial efficiency, preference for feeding on humans, and resting behavior. Species S, T, and U are morphologically indistinguishable at any stage of their life cycle and can be identified only by the examination of species-specific fixed inversions in the polytene chromosomes. We report an allele-specific polymerase chain reaction assay for the differentiation of members of An. fluviatilis complex, which is based on differences in nucleotide sequences in D3 domain of 28S ribosomal DNA. The assay was evaluated against chromosomally examined individuals from different localities with different sympatric associations and was found to differentiate unambiguously all the members of the complex.