PARTIAL DNA-SEQUENCES FROM THE MITOCHONDRIAL CYTOCHROME-OXIDASE SUBUNIT-I (COI) GENE CAN DIFFERENTIATE THE INTERMEDIATE SNAIL HOSTS BULINUS-GLOBOSUS AND BULINUS-NASUTUS (GASTROPODA, PLANORBIDAE)

In order to test the current taxonomic status of two East African snai l species within the Bulinus africanus group and develop molecular met hods for subsequent identification, snails from Zanzibar were investig ated and assayed for DNA sequence variation. A 450 bp region within th e mitochondrial cytochrome oxidase subunit I (COI) gene was amplified by Polymerase Chain Reaction (PCR), using evolutionarily conserved pri mers, from 143 snails representing two species within the Bulinus afri canus group: B. globosus and B. nasutus. PCR products were subjected t o restriction fragment length polymorphism (RFLP) analysis using doubl e digestion with the enzymes AluI and RsaI. RFLP was observable betwee n species. To identify further variation within this molecule, PCR pro ducts from several snails were also sequenced. A 340 bp data set was o btained from twelve snails representative of the two species. These da ta were subjected to cladistic analysis with designated outgroups Cepa ea nemoralis and Albinaria turrita. From the DNA alignment, there were 33 variable sites within the Bulinus sequences. The inferred phylogen y supported the specific status of B. globosus and B. nasutus. In orde r to find a diagnostic RFLP marker, COI sequence variation was screene d against restriction enzyme recognition sites and a species-specific restriction site was found within B. globosus with the enzyme SspI (re cognition site ATATTT). PCR products originating from 19 snails repres enting the two species and also a third species B. africanus were rest ricted with this enzyme. Only B. globosus sequences were digested sugg esting that this assay may prove useful for routine identification.