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

Abstract

Background: Anopheles stephensi, an invasive malaria vector, has been reported to have three biological forms identifiable based on the number of ridges present on the egg floats and the dimension of eggs. Recently, these forms have been designated as sibling species based on the fixed differences in the DNA sequence of the first intron of the odorant-binding protein-1 (AsteObp1). In this study, we evaluated the utility of this neutral marker in designating sibling species or identifying biological forms. Methods: Field collected and laboratory-reared An. stephensi were characterized for biological forms based on the number of floats on egg-ridge. DNA sequencing of the partial AsteObp1 gene of An. stephensi individuals were performed by Sanger method, either directly or after cloning with a plasmid vector. Results: AsteObp1 intron-1 in Indian An. stephensi populations are highly polymorphic with the presence of more than 12 haplotypes exhibiting nucleotide- as well as length-polymorphism (90-to-121 bp). A majority of the field samples were heterozygous (up to 89% in the field populations). The phasing of haplotypes in heterozygotes through Sanger sequencing was challenging due to indels (1-to-24 bp) at multiple loci. No specific haplotype or monophyletic clade of intron-1 was found associated with a specific biological form. The inbreeding coefficient for this marker was close to zero in field and laboratory populations which refute the existence of sibling species based on the AsteObp1 marker. Conclusions: AsteObp1 cannot serve as a marker for the identification of biological forms of An. stephensi. The probable existence of sibling species in An. stephensi based on the AsteObp1 intron-1 is refuted.

Enrichment of phenotype among biological forms ofAnopheles stephensiListon through establishment of isofemale lines

“…stephensi populations (insectary-colonized and wild populations) that were collected from 5 different locations, we found that most of the individuals from the wild had the SNP (G) in AsteObp1 (intron I Position 91bp) reported to be diagnostic of the type form, 18/105 individuals had an A at this same position, 18/105 had an A/G and 1/105 had an A/C in that position, likely representing population-level variations that have not been reported earlier (unpublished data). Also, many of the other markers are not universal and may vary from region to region and cannot resolve the biological forms accurately [56]. Here, we clearly demonstrate that the number of egg ridges is the gold standard and applicable marker to develop isofemale lines.…”