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01.09.14 KB. Ok to add working paper to spiral, author retain copyright

Genetic variation of Contracaecum ogmorhini (sensu lato) populations from different otariid seals of the northern and southern hemisphere was studied on the basis of 18 enzyme loci as well as preliminary sequence analysis of the mitochondrial cyt b gene (260 bp). Samples were collected from Zalophus californianus in the boreal region and from Arctocephalus pusillus pusillus, A. pusillus doriferus and A. australis from the austral region. Marked genetic heterogeneity was found between C. ogmorhini (sensu lato) samples from the boreal and austral region, respectively. Two loci (Mdh-2 and NADHdh) showed fixed differences and a further three loci (Iddh, Mdh-1 and 6Pgdh) were highly differentiated between boreal and austral samples. Their average genetic distance was DNei = 0.36 at isozyme level. At mitochondrial DNA level, an average proportion of nucleotide substitution of 3.7% was observed. These findings support the existence of two distinct sibling species, for which the names C. ogmorhini (sensu stricto) and C. margolisi n. sp., respectively, for the austral and boreal taxon, are proposed. A description for C. margolisi n. sp. is provided. No diagnostic morphological characters have so far been detected; on the other hand, two enzyme loci, Mdh-2 and NADHdh, fully diagnostic between the two species, can be used for the routine identification of males, females and larval stages. Mirounga leonina was found to host C. ogmorhini (s.s.) inmixed infections with C. osculatum (s.l.) (of which C. ogmorhini (s.l.) was in the past considered to be a synonym) and C. miroungae; no hybrid genotypes were found,confirming the reproductive isolation of these three anisakid species. The hosts and geographical range so far recorded for C. margolisi n. sp. and C. ogmorhini (s.s.) are given.

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 mitochondrial 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 reaction (PCR), analysed by single-strand conformation polymorphism (SSCP), and samples displaying sequence variability were subjected to sequencing. Forty-six haplotypes were defined for 62-66 individuals (representing the six members of C. osculatum). All taxa except CoD and CoE could be identified, or delineated from one another, by nucleotide differences in the COI, ssrRNA 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 diagnostic (fixed) and could thus unequivocally delineate CoA, CoB, CoC and Cob. The lack of an unequivocal nucleotide difference in any of the three mtDNA sequences between CoD and CoE was in accordance with previous ribosomal DNA sequence data but inconsistent with multilocus enzyme electrophoretic data. Using all fixed nucleotide positions, CoA, CoD/E and CoB were genetically 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. osculatum, the present study demonstrated clearly the usefulness and attributes of the mutation scanning approach for investigating population genetic structures of species of parasitic nematodes.