Levels of intra-host and temporal sequence variation in a large CO1 sub-units from Anisakis simplex sensu stricto (Rudolphi 1809) (Nematoda: Anisakisdae): implications for fisheries management

DNA sequence divergence at internal transcribed spacer regions (ITS-1 and ITS-2) was compared with divergence at mitochondrial cox1 or nad4 loci in pairs of congeneric nematode species. Mitochondrial sequences accumulate substitutions much more quickly than internal transcribed spacer, the difference being most striking in the most closely related species pairs. Thus, mitochondrial DNA may be the best choice for applications in which one is using sequence data on small numbers of individuals to search for potential cryptic species. On the other hand, internal transcribed spacer remains an excellent tool for DNA diagnostics (quickly distinguishing between known species) owing to its lower level of intraspecific polymorphism.

Polymerase-chain-reaction-based restriction fragment length polymorphism analysis was performed to establish genetic markers in rDNA, for the identification of the three sibling species of the Anisakis simplex complex and morphologically differentiated Anisakis species, i.e. Anisakis physeteris, Anisakis schupakovi, Anisakis typica and Anisakis ziphidarum. Different restriction patterns were found between A. simplex sensu stricto and Anisakis pegreffii with two of the restriction endonucleases used (HinfI and TaqI), between A. simplex sensu stricto and A. simplex C with one endonuclease (HhaI), and between A. simplex C and Aniskis pegreffii with three endonucleases (HhaI, HinfI and TaqI), while no variation in patterns was detected among individuals within each species. The species A. physeteris, A. schupakovi, A. typica and A. ziphidarum were found to be different from each other and different from the three sibling species of the A. simplex complex by distinct fragments using 10-12 of the endonucleases tested. The polymorphisms obtained by restriction fragment length polymorphisms have provided a new set of genetic markers for the accurate identification of sibling species and morphospecies.