Intragenomic sequence variations in the second internal transcribed spacer (ITS2) ribosomal DNA of the malaria vector Anopheles stephensi

Abstract

Second Internal Transcribed Spacer (ITS2) ribosomal DNA (rDNA) sequence is a widely used molecular marker for species-identification or -delimitation due to observed concerted evolution which is believed to homogenize rDNA copies in an interbreeding population. However, intra-specific differences in ITS2 of Anopheles stephensi have been reported. This study reports the presence of intragenomic sequence variation in the ITS2-rDNA of An. stephensi and hypothesizes that observed intra-specific differences in this species may have resulted due to ambiguous DNA sequence-chromatogram resulting from intragenomic heterogeneity. Anopheles stephensi collected from different parts of India were sequenced for complete ITS2 and the variable region of 28S-rDNA (d1-d3 domains). Intragenomic variations were found in ITS2 region of all An. stephensi sequenced, but no such variation was observed in d1 to d3 domains of 28S-rDNA. Cloning and sequencing of ITS2 through the d3 domain of the 28S region of rDNA from representative samples from northern, central, and southern India confirmed the presence of intragenomic variation in ITS2 due to transitions at three loci and two bp indel in a di-nucleotide microsatellite locus. Multiple haplotypes were observed in ITS2 raised from such variations. Due to the absence of detectable intragenomic sequence variation in the d1 to d3 domain of 28S rDNA of An. stephensi, this region can serve as an ideal reference sequence for taxonomic and phylogenetic studies. The presence of intragenomic variation in rDNA should be carefully examined before using this as a molecular marker for species delimitation or phylogenetic analyses.

Identification of a biological form in the Anopheles stephensi laboratory colony using the odorant-binding protein 1 intron I sequence

“…Regarding the suitability of ITS2, COI and COII for distinguishing the biotypes of An . stephensi , our current observations and previous studies [ 17 , 18 ] confirm that these markers are not the suitable markers (based on high sequence similarity). In contrast, a new study reports both COI and COII (gene variation) as suitable markers to recognize the complexes of An .…”

“…But none of these methods could clearly differentiate and give species status to the biological forms of An . stephensi [ 17 , 18 , 26 ].…”

“…Information regarding population genetics of An . stephensi is still limited [ 16 , 17 ]. The mitochondrial oxidases subunit 1 and 2 (COI and COII), ribosomal internal transcribed spacer 2 (rDNA-ITS2) and domain-3 (D3) loci are the common molecular markers used, but none of them have distinguished accurately the biological forms of An .…”

“…The mitochondrial oxidases subunit 1 and 2 (COI and COII), ribosomal internal transcribed spacer 2 (rDNA-ITS2) and domain-3 (D3) loci are the common molecular markers used, but none of them have distinguished accurately the biological forms of An . stephensi [ 15 , 17 , 18 ]. Alternatively, intron I sequences of Odorant-binding protein 1 has been recently shown to be potential genetic marker to differentiate members of the An .…”

Evaluation of intron-1 of odorant-binding protein-1 of Anopheles stephensi as a marker for the identification of biological forms or putative sibling species

“…However, the ITS2 sequence of An. stephensi from Saudi Arabia (Munawar et al, 2020) is no different from Indian, Sri Lankan and African populations (Mishra et al, 2021, Surendran et al, 2018; Tadesse et al, 2019), exhibiting mito-nuclear discordance (Singh et al, 2021). Recent data on field populations of An.…”