SSCP-based identification of members within the Pseudoterranova decipiens complex (Nematoda: Ascaridoidea: Anisakidae) using genetic markers in the internal transcribed spacers of ribosomal DNA

Abstract

The anisakid nematodes morphologically corresponding with Pseudoterranova decipiens sensu lato (s.l.) (Krabbe, 1878) from different seal or sea lion hosts and geographical origins, previously identified as Pseudoterranova krabbei, P. decipiens (s.s.), P. bulbosa, P. azarasi and P. cattani by multilocus enzyme electrophoresis, were characterized using a DNA approach. Also a population of P. decipiens (s.l.) from Chaenocephalus aceratus, the blackfin icefish, from Antarctica and another from Osmerus eperlanus, the European smelt, from Germany were included in the study. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by PCR from individual nematodes and analysed by single-strand conformation polymorphism (SSCP), followed by selective sequencing. While no variation in single-stranded ITS-1 and ITS-2 profiles was detected among samples representing each of the species or populations (with the exception of slight microheterogeneity), SSCP analysis of the ITS-2 amplicons allowed the unequivocal differentiation of all of the 5 sibling species of P. decipiens (s.l.) examined, which was supported by sequence differences in ITS rDNA. Samples representing the P. decipiens (s.l.) population from O. eperlanus had the same SSCP profile as those of P. decipiens (s.s.), which was supported by a lack of nucleotide difference in the ITS between them, suggesting that the former represented P. decipiens (s.s.). Based on SSCP results and ITS sequence data, P. decipiens (s.l.) from C. aceratus was genetically most distinct with respect to all other members of Pseudoterranova examined, which indicated that it may represent P. decipiens E (based on geographical origin) or a distinct species. These findings and the molecular approach taken should have important implications for studying the life-cycles, transmission patterns, epidemiology and population genetics of these anisakid nematodes, and the diagnosis of their infections.