Morphological and Molecular Characterization of ‘var. mysorensis’ form of Anopheles Stephensi

Abstract

Background: Anopheles stephensi Listen (1901) is the major malaria vector in the Asia, and recently in some regions of Africa. This species includes three biological forms, namely “type”, “intermediate” and “mysorensis” with varying degree of vector competence for malaria parasites. To recognize these siblings of An. stephensi lab strain, we used the morphological features of eggs and several genetic markers i.e. Obp1 (odorant binding protein), mitochondrial oxidases subunit 1 and 2 (COI and COII), nuclear internal transcribed spacer 2 locus (ITS2). Methods: Eggs were collected from individual mosquito (n = 50) and observed for the number of ridges under stereomicroscope. DNA was extracted from female mosquitoes. After amplifying fragments by using different genetic markers (Obp1, COI, COII and ITS2), the PCR products were purified and sequenced using Sanger Sequence Technology. Phylogenetic analysis was performed after aligning query sequences against the submitted sequences in GenBank using bioinformatics software. Results: The range of ridges number on each egg float was 12-13 that corresponds to the mysorensis form. Sequence analysis for COI, COII and ITS2 demonstrated 100%, 99.46% and 99.29% similarity of our species with other Chinese, Indian and Iranian strains of An. stephensi. All the sequences of Obp1 intron I region matched 100% with the previously submitted sequences for An. stephensi sibling C (mysorensis form) from Iran and Afghanistan.Conclusion: The current study elaborately describes the morphological and molecular details (sequence data) of the ‘mysorensis’ form of An. stephensi that could be helpful in elucidating its classification and also in its differentiation from other biotypes of the same and other similar anophelines species. Our findings confirmed OBP1 as the only genetic marker that successfully recognized our lab strain (mysorensis form/ sibling C). Thus using OBP1 (alone) may be very phenomenal in similar studies in the future. Future studies should involve the development of appropriate and reliable molecular keys (for sibling species identification) complementary to morphological keys.