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Background   Anopheles fluviatilis  is a species-complex comprising of four cryptic species provisionally designated as species S, T, U and V. Earlier, a 28S-rDNA based allele-specific polymerase chain reaction (ASPCR) assay was developed for the differentiation of the then known three members of the  An. fluviatilis  complex, i.e., species S, T, and U. This assay was modified in consequence of the discovery of a new cryptic member, species V, in the Fluviatilis Complex to include identification of new species.    Methods  In the modified procedure, the ASPCR assay was performed first, followed by restriction digestion of PCR product with an enzyme  Bam H I, which cleaves specifically PCR amplicon of species V and the resultant PCR–RFLP products can differentiate all the four cryptic members of the complex. Morphologically identified  An. fluviatilis  samples were subjected to sibling species identification by modified PCR-based assay and standard cytotaxonomy. The result of PCR-based assay was validated through cytotaxonomy as well as DNA sequencing of some representative samples.    Results  The modified PCR-based assay differentiates all four sibling species. The result of modified PCR-based assay tested on field samples was in agreement with results of cytotaxonomy as well as DNA sequencing of representative samples.    Conclusions  The modified PCR-based assay unambiguously differentiates all four known members of the  An. fluviatilis  species complex. This assay will be useful in studies related to bionomics of members of the Fluviatilis Complex in their role in malaria transmission.

malaria journal

Modified PCR-based assay for the differentiation of members of Anopheles fluviatilis complex in consequence of the discovery of a new cryptic species (species V)