Identification of Snails within the Bulinus africanus Group from East Africa by Multiplex SNaPshotä Analysis of Single Nucleotide Polymorphisms within the Cytochrome Oxidase Subunit I

Vol. 97(Suppl.): 31-36, 2002

JR Stothard, J Llewellyn-Hughes, CE Griffin, SJ Hubbard*, TK Kristensen**, D Rollinson/⁺

Wolfson Wellcome Biomedical Laboratories, Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD *Faculty of Agricultural and Environmental Sciences, McGill University, Québec, Canada **Danish Bilharziasis Laboratory, Charlottenlund, Denmark

Identification of populations of Bulinus nasutus and B. globosus from East Africa is unreliable using characters of the shell. In this paper, a molecular method of identification is presented for each species based on DNA sequence variation within the mitochondrial cytochrome oxidase subunit I (COI) as detected by a novel multiplexed SNaPshot^TM assay. In total, snails from 7 localities from coastal Kenya were typed using this assay and variation within shell morphology was compared to reference material from Zanzibar. Four locations were found to contain B. nasutus and 2 locations were found to contain B. globosus. A mixed population containing both B. nasutus and B. globosus was found at Kinango. Morphometric variation between samples was considerable and UPGMA cluster analysis failed to differentiate species. The multiplex SNaPshot^TM assay is an important development for more precise methods of identification of B. africanus group snails. The assay could be further broadened for identification of other snail intermediate host species.

Key words: Bulinus - cytochrome oxidase - single nucleotide polymorphism - schistosomiasis - Schistosoma haematobium

The freshwater pulmonate snail genus Bulinus is divided into four species groups: B. africanus group, B. forskalii group, B. reticulatus group and the B. truncatus/tropicus complex (Brown 1994). Despite limited morphological divergence within species groups, there is considerable molecular divergence (Jones et al. 2001, Stothard et al. 2001). Within the B. africanus group 10 species are recognised and distributed throughout Afro-tropical regions and Madagascar. Several B. africanus group species are known, or suspected, to act as intermediate snail hosts for Schistosoma haematobium, a trematode parasite that causes urinary schistosomiasis.

Interactions between B. africanus group species and S. haematobium can be complex. Not all snail species act as intermediate hosts e.g. B. ugandae appears refractory to infection, or only certain snail species act as hosts in specific areas (Rollinson et al. 2001). Lack of clear-cut morphological characters hinders identification of natural populations (Mandahl-Barth 1965). For accurate separation of these snail species it is necessary to use biochemical (Rollinson & Southgate 1979) or molecular DNA methods (Rollinson et al. 2001). There are many single nucleotide polymorphisms (SNPs) within the mitochondrial cytochrome oxidase subunit I (COI) gene which may be exploited for species identification (Fig. 1). Taxon specific polymerase chain reaction (PCR) primers for B. globosus and B. nasutus have been designed based on genetic variation within the COI (Stothard et al. in press).

Whilst taxon specific primers are highly discriminatory, the PCR assay is limited within the known scope of detected sequence variation; further sequence variation may lead to false negatives. SNaPshot^TM is a commercially available product from PE Biosystems, UK for genotyping SNPs using a fluorescent based, primer extension assay (Rollinson et al. 2001). Makridakis and Reichardt (2002) have taken the SNaPshot^TM assay a step further by multiplexing SNaPshot^TM primers of differing length coining the terminology `multiplex automated primer extension analysis' (MAPA). Although this multiplex assay requires a semi-automated DNA sequencer, the assay has certain key advantages; many snails can be individually typed simultaneously for several key SNPs, and detection and precise characterisation of further DNA variation is possible.

This paper reports on the development and implementation of a multiplexed SNaPshot^TM assay to type simultaneously four SNPs within the COI (Fig. 2) of Bulinus species. The assay is then used for identification of B. africanus group snails collected from 7 localities within coastal Kenya. The Kenyan shell material is compared to a selection of B. globosus and B. nasutus shells from Zanzibar to ascertain if there is any morphological divergence.

MATERIALS AND METHODS

RESULTS

DISCUSSION

REFERENCES

Fig. 1 | Fig. 2 | Fig. 3 | Fig. 4 | Fig. 5 | Table

Funded by the Wellcome Trust.

⁺Corresponding author. Fax + 44-0207-942-5518. E-mail: d.rollinson@nhm.ac.uk

Received 18 June 2002

Accepted 15 August 2002