Bulinus species on Madagascar: molecular evolution, genetic markers and compatibility with Schistosoma haematobium

J. R.  STOTHARD  a1 c1, P.  BRÉMOND  a2, L.  ANDRIAMARO  a2, B.  SELLIN  a2, E.  SELLIN  a2 and D.  ROLLINSON  a1
a1 Wolfson Wellcome Biomedical Laboratories, Department of Zoology, The Natural History Museum, Cromwell Road, London, SW7 5BD
a2 Equipe Schistosomoses I.R.D., BP 434, 101 Antananarivo, Madagascar


Of the four species of Bulinus found on Madagascar, three species: B. obtusispira, B. liratus and B. bavayi are endemic while the fourth, B. forskalii, is probably a recent introduction from the African mainland. The evolutionary relationships of these species with Bulinus species from Africa were studied by phylogenetic analysis of DNA sequence variation at two mitochondrial loci: cytochrome oxidase subunit I (COI) and large ribosomal subunit (LSU) or 16S. The observed levels of nucleotide divergence within Bulinus were substantial but may underestimate the true levels as there was evidence of ‘saturation' of transitional substitutions at both loci. A putative secondary structure model for the sequenced segment of the 16S was developed. Subsequent phylogenetic analysis using transversional changes only for both loci, showed that there were contrasting levels of divergence within the four species groups. B. obtusispira was consistently placed within the B. africanus group, appearing ancestral to this group and was closest to the basal node within Bulinus. Together with B. bavayi, the two species appear to have been isolated on Madagascar for a long time, contrasting with both B. liratus and B. forskalii that appear more recent colonisers; however, estimate of exact times of divergence is problematic. A PCR-RFLP assay was developed to enable identification and discrimination of B. obtusispira and B. liratus using discriminatory variation within the COI. To enable population genetic analysis within B. obtusispira, microsatellite markers were developed using an enrichment method and 8 primer pairs are reported. Laboratory infection experiments using Madasgacan S. haematobium from the Mahabo area showed that certain populations of B. obtusispira, B. liratus and B. bavayi were compatible.

Key Words: Madagascar; Bulinus; Schistosoma haematobium; mitochondrial DNA; phlylogeny; microsatellites.

c1 Address for correspondence: e-mail: