Implication of Random Amplified Polymorphic DNA method for differentiating Anopheles culicifacies sibling species

Efficient method for discriminating among members of Anopheline species complexes is of extreme significance for epidemiological studies and effective vector control programme. Anopheles culicifacies is a principal malaria vector in rural, periurban and tribal settings and responsible for about 65- 70% malaria cases in India. It is a complex of 5 isomorphic types A, B, C, D and E with varying biological characteristics. Presently, cytogenetic methods involving polytene chromosome and mitotic karyotyping are the only methods that could differentiate all the five sibling species but these techniques are time consuming and labour intensive. DNA based techniques using rDNA and COII such as AS-PCR and gene specific PCR-RFLP seems to be good alternatives but these techniques too have limitations and until now none of the techniques have been able to differentiate all the five sibling species in one step. In the present study we have been able to develop Random Amplified Polymorphic DNA (RAPD) markers that could differentiate all the five sibling species of An. culicifacies in India. A number of RAPD primers were screened and genetic fingerprints for all the 5 sibling species of An. culicifacies were developed using a selected primer. A total of 34 loci representing these sibling species using selected primer were polymorphic and able to differentiate the sibling species and showed 100% polymorphism. A consensus tree was also generated indicating genetic distances among these sibling species. The findings will be useful in devising a new method for identification of sibling species which will be helpful to develop successful vector management strategies.