Anisakid nematode species identification in harbour porpoises (Phocoena phocoena) from the North Sea, Baltic Sea and North Atlantic using RFLP analysis

Harbour porpoises (Phocoena phocoena) are the only native cetacean species in the German North and Baltic Seas and the final host of Anisakis (A.) simplex, which infects their first and second gastric compartments and may cause chronic ulcerative gastritis. Anisakis simplex belongs to the family Anisakidae (Ascaridoidea, Rhabditida) as well as the phocine gastric nematode species Pseudoterranova (P.) decipiens and Contracaecum (C.) osculatum. These nematode species are the main causative agents for the zoonosis anisakidosis. The taxonomy of these genus with life cycles including crustaceans and commercially important fish is complex because of the formation of sibling species. Little is known about anisakid species infecting porpoises in the study area. Mature nematodes and larval stages are often identifiable only by molecular methods due to high morphological and genetic similarity. The restriction fragment length polymorphism (RFLP) method is an alternative to sequencing and was applied to identify anisakid nematodes found in harbour porpoises from the North Sea, Baltic Sea and North Atlantic to species level for the first time. In the study areas, five gastric nematodes from different harbour porpoise hosts were selected to be investigated with restriction enzymes HinfI, RsaI and HaeIII, which were able to differentiate several anisakid nematode species by characteristic banding patterns. Anisakis simplex s. s. was the dominant species found in the North Sea and Baltic porpoises, identified by all three restriction enzymes. Additionally, a hybrid of A. simplex s. s. and A. pegreffii was determined by HinfI in the North Sea samples. Within the North Atlantic specimens, A. simplex s. s., P. decipiens s. s. and Hysterothylacium (H.) aduncum were identified by all enzymes. This demonstrates the value of the RFLP method and the chosen restriction enzymes for the species identification of a broad variety of anisakid nematodes affecting the health of marine mammals.