Partial DNA sequences from the mitochondrial cytochrome oxidase subunit I (COI) gene can differentiate the intermediate snail hostsBulinus globosusandB. nasutus(Gastropoda: Planorbidae)

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Abstract
In order to test the current taxonomic status of two East African snail species within the Bulinus africanus group and develop molecular methods for subsequent identification, snails from Zanzibar were investigated and assayed for DNA sequence variation. A 450 bp region within the mitochondrial cytochrome oxidase subunit I (COI) gene was amplified by Polymerase Chain Reaction (PCR), using evolutionarily conserved primers, from 143 snails representing two species within the Bulinus africanus group: B. globosus and B. nasutus. PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis using double digestion with the enzymes AluI and RsaI. RFLP was observable between species. To identify further variation within this molecule, PCR products from several snails were also sequenced. A 340 bp data set was obtained from twelve snails representative of the two species. These data were subjected to cladistic analysis with designated outgroups Cepaea nemoralis and Albinaria turrita. From the DNA alignment, there were 33 variable sites within the Bulinus sequences. The inferred phylogeny supported the specific status of B. globosus and B. nasutus. In order to find a diagnostic RFLP marker, COI sequence variation was screened against restriction enzyme recognition sites and a species-specific restriction site was found within B. globosus with the enzyme SspI (recognition site ATATTT). PCR products originating from 19 snails representing the two species and also a third species B. africanus were restricted with this enzyme. Only B. globosus sequences were digested suggesting that this assay may prove useful for routine identification.

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