A polyphasic approach with molecular phylogeny for the characterization of Anisakis pegreffii (Anisakidae: Nematoda) in fishes from Adriatic Sea

In this study we investigated the morphometric and molecular characterization of a liver encapsulated third-stage larval population of Anisakis spp. infecting Merluccius merluccius and Lophius piscatorius caught in the Adriatic Sea waters (southern Italy). A polyphasic approach based on PCR-RFLP profiles of the ITS region, mitochondrial COI (cytochrome c oxidase subunit 1), sequencing and molecular phylogeny of ITS and mitochondrial COI was used to identify Anisakis larvae collected from fish samples. PCR-RFLP analysis showed three banding pattern corresponding to the peculiar pattern of A. pegreffii. Sequence data from ribosomal ITS and mitochondrial COI were analysed by Neighbour Joining, Minimum Evolution and Maximum Parsimony methods to evaluate the phylogenetic relationships among A. simplex sensu lato. The phylogenetic trees obtained for both ITS and COI revealed the existence of three distinct clades for A. simplex sensu sricto, A. simplex C and A. pegreffii and the sequences obtained in this study clearly clustered together with A. pegreffii sequences present in the database. Histopathological observations of anisakid nematode specimens detected on the liver surface of M. merluccius are illustrated. Encapsulated specimens of the L3 stage of the nematode were similar in size and morphometry to those found into the peritoneal cavity. Anisakis larvae encapsulated on the liver surface within dense and pearl coloured envelops caused host hepatic tissue necrosis, large cavities and oedematous liver spots to the host.