Mutation scanning for sequence variation in three mitochondrial DNA regions for members of the Contracaecum osculatum (Nematoda : Ascaridoidea) complex

Anisakid nematodes of seals from different geographical origins, previously identified by multilocus enzyme electrophoresis as Contracaecum osculatum A (CoA), C. osculatum B (CoB), C. osculatum C (CoC), C. osculatum D (CoD), C. osculatum E (CoE) and C, osculatum baicalensis (Cob), were characterised genetically using a mutation scanning approach, in order to define genetic markers for their specific identification and differentiation. Three mitoc hondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit I (COI), and the small and large subunits of rRNA (ssrRNA and IsrRNA, respectively) were amplified separately from individual nematodes by polymerase chain rea ction (PCR), analysed by single-strand conformation polymorphism (SSCP), an d samples displaying sequence variability were subjected to sequencing. For ty-six haplotypes were defined for 62-66 individuals (representing the six members of C. osculatum). All taxa except Coo and CoE could be identified, or delineated from one another, by nucleotide differences in the COI, ssrRN A and/or IsrRNA sequences. For all three mtDNA regions, 4 (10.5%), 7 (18.4% ), 15 (39.5%) and 11 (28.9%) of 38 nucleotide positions were considered dia gnostic (fixed) and could thus unequivocally delineate CoA, CoB, CoC and Co b. The lack of an unequivocal nucleotide difference in any of the three mtD NA sequences between Coo and CoE was in accordance with previous ribosomal DNA sequence data but inconsistent with multilocus enzyme electrophoretic d ata. Using all fixed nucleotide positions, CoA, CoD/E and CoB were genetica lly more similar to Cob than each was to CoC, similar to previous findings. In spite of not being able to distinguish among all six taxa of C. osculat um, the present study demonstrated clearly the usefulness and attributes of the mutation scanning approach for investigating population genetic struct ures of species of parasitic nematodes.