ITS2 characterization and Anopheles species identification of the subgenus Cellia

In Mizoram, the origin and molecular nature of Anopheles species is poorly understood, despite the region having high malarial incidence and rich biodiversity. A diagnostic PCR assay for distinguishing the Cellia subgenera members of Anopheles species was developed based on the interspecific ITS2 variation. No intraspecific variation was found and the size (362-604bp) and GC content (48.8-58.9%) of the ITS2 were highly variable among Anophelines. The ITS2 of A. vagus is significantly longer than those of other Anopheles species. Significant relationship was observed among repeats, minimum free energy and RNA secondary structures. Different types of microsatellites were identified and among them dinucleotide, pentanucleotide and polynucleotide microsatellites were predominant. Variation in the length of the ITS2 between species was due to indels in simple repeats. Four domain types of RNA secondary structures were identified and the lowest free energy values were predicted using the computer software, RNAfold. Types I and II were observed only in Neocellia and Myzomyia series and Types III and IV were common in Neocellia and Pyretophorus series. ITS2-based PCR protocol provides a means for vector ecologists, malaria epidemiologists and control personnel to accurately identify members of the subgenera Cellia and a better understanding of their genomic status in Mizoram.