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Molecular investigations of Hysterothylacium auctum from Baltic Sea fish were performed using multilocus allozyme electrophoresis (MAE) and genomic DNA analysis based on PCR technique. Nematodes from eel-pout, flounder, cod (MAE and PCR) and herring (PCR) were examined to establish whether one species or complex of sibling ones occurs within Baltic H. auctum. Multilocus allozyme electrophoresis revealed that three enzymatic loci: Got, Adk and Pep-2 were monomorphic, two loci: Mdh-1 and Pgm-2 showed slight polymorphism and one locus: Gpi showed strong polymorphism. H. auctum from eel-pout, flounder and cod caught in the southern Baltic proved to share a common gene pool; however, an incipient subdivision of this population into specimens connected with eel-pout and flounder on the one hand and with cod on the other was ascertained with locus coding for Gpi. PCR-RFLP analysis of the fragment of ribosomal DNA embracing the 3'-end of the gene coding 18S rRNA, ITS1, gene coding 5.8S rRNA, ITS2 and the 5'-end of the gene coding 28S rRNA (~1500 bp) was performed using HaeIII, HinfI and PvuII endonucleases. In the case of particular endonuclease, electrophoretic patterns of digested PCR products were similar for all individuals of nematodes independent of host species or place of fishing. On the basis of nucleotide sequence of H. auctum obtained from eel-pout, a pair of species-specific primers was designed. This pair allows amplification of DNA of Hysterothylacium isolated from eel-pout, flounder, cod and herring and did not give cross-reactions with DNA from other Baltic anisakids: Anisakis simplex sensu stricto, Contracaecum osculatum s. str. and C. rudolphii. The results obtained suggest that only one biological species of H. auctum occurs in the southern Baltic.