Abstract
Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels. This study was carried out to unravel the biological information in the ITS2 region of An. culicifacies and the internal relationships between the five species of Anopheles culicifacies. In achieving these objectives, twenty two ITS2 sequences (approximately 370bp) of An. culicifacies species were retrieved from GenBank and secondary structures were generated. For the refinement of the primary structures, i.e. nucleotide sequence of ITS2 sequences, generated secondary structures were used. The improved ITS2 primary structures sequences were then aligned and used for the construction of phylogenetic trees. ITS2 secondary structures of culicifacies closely resembled near universal eukaryotes secondary structure and had three helices, and the structures of helix II and distal region of helix III of ITS2 of An. culicifacies were strikingly similar to those regions of other organisms strengthening possible involvement of these regions in rRNA biogenesis. Phylogenetic analysis of improved ITS2 sequences revealed two main clades one representing sibling B, C and E and A and D in the other. Near sequence identity of ITS2 regions of the members in a particular clade indicate that this region is undergoing parallel evolution to perform clade specific RNA biogenesis. The divergence of certain isolates of An. culicifacies from main clades in phylogenetic analyses suggests the possible existence of camouflaged sub-species within the complex of culicifacies. Using the fixed nucleotide differences, we estimate that these two clades have diverged nearly 3.3 million years ago, while the sibling species in clade 2 are under less evolutionary pressure, which may have evolved much later than the members in clade 1.
Highlights
Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels
Anopheles culicifacies Giles sensu lato is a major vector of malaria in Indian subcontinent and Sri Lanka [1]
Correct identification of malaria vector is essential for targeted malaria control
Summary
Anopheles culicifacies Giles sensu lato is a major vector of malaria in Indian subcontinent and Sri Lanka [1]. The of identical sequences in fast evolving ITS2 regions of the objectives of the current study were to understand the internal isolates from different geographical locales with eco-climatic relationships between the five sibling species and the conditions somewhat intriguing and may imply this region of biological information of the ITS2 region in An. culicifacies these sibling species is under high evolutionary pressure to using the primary and secondary structures of the ITS2 rDNA perform the group-specific functions. Secondary structure of ITS is valuable to improve the (A, B, C, D, E and An. Culicifacies that are unassigned to a nucleotide alignments for the correct phylogenetic particular species) were retrieved using ITS2 sequence of An. comparison of a given group of organisms [12]. The secondary structure analyses of ITS2 regions of helix I of culicifacies clade I and 2 was nearly identical and culicifacies complex revealed insertions/deletions, transitions helix III, being longest helix in ITS2 of culicifacies as in other and transversions within the members in group 1 and 2, organism the branching of proximal region only seen in clade these changes preserve the integrity/identity of the ITS2 secondary structure of the members in a particular
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