Ecology and genetic structure of zoonotic Anisakis spp. from adriatic commercial fish species

Abstract

Consumption of raw or thermally inadequately treated fishery products represents a public health risk, with the possibility of propagation of live Anisakis larvae, the causative agent of the zoonotic disease anisakidosis, or anisakiasis. We investigated the population dynamics of Anisakis spp. in commercially important fish-anchovies (Anisakis), sardines (Sardina pilchardus), European hake (Merluccius merluccius), whiting (Merlangius merlangus), chub mackerel (Scomber japonicus), and Atlantic bluefin tuna (Thunnus thynnus)-captured in the main Adriatic Sea fishing ground. We observed a significant difference in the numbers of parasite larvae (1 to 32) in individual hosts and between species, with most fish showing high or very high Anisakis population indices. Phylogenetic analysis confirmed that commercial fish in the Adriatic Sea are parasitized by Anisakis pegreffii (95.95%) and Anisakis simplex sensu stricto (4.05%). The genetic structure of A. pegreffii in demersal, pelagic, and top predator hosts was unstructured, and the highest frequency of haplotype sharing (n = 10) was between demersal and pelagic fish.

FIG 1

Graphical representation of genetic diversity and population structures of three A. pegreffii populations isolated from hosts from different feeding habitats inferred from the mtDNA cox2 locus. (A) Frequencies of haplotypes in top predator, demersal, and pelagic fish. (B) Molecular diversity indices: θ_k, average number of nucleotide differences; θ_H, haplotype diversity; θ_S, number of polymorphic sites; θ_π, nucleotide diversity. (C) Haplotype distance matrix inferred from the number of pairwise differences (diff.). (D to F) Observed and expected mismatch distributions with 90 to 99% confidence intervals for A. pegreffii populations parasitizing pelagic (D), demersal (E), and top predator (F) fish.

FIG 2

Rooted phylogenetic tree inferred by Bayesian analysis of mtDNA cox2 locus fragments from Anisakis spp., with posterior probabilities shown in different colors (thickest [red] line, 0.9 to 1; thick [orange] line, 0.8 to 0.9; thin [coral] line, 0.7 to 0.8; thinnest [yellow] line, 0.6 to 0.7). A. pegreffii isolated from fish species in the Adriatic Sea is represented by 74 isolates forming a sister clade with A. pegreffii (DQ116428), while 3 isolates (M. merluccius 1, T. thynnus 3, and T. thynnus 4) branched from A. simplex sensu stricto (DQ116426), apart from the A. pegreffii group. (Inset) Magnification of part of the tree with clades composed of A. simplex sensu stricto and A. pegreffii taxa.

FIG 3

Haplotype network showing a star-like phylogeny, with most of the unique haplotypes closely related to the common central haplotype (H6). The sizes of the circles match the numbers of sequences belonging to the specific haplotypes. The circle colors represent the three A. pegreffii populations: black, demersal; white, pelagic; gray, top predator. The smallest dark-gray polygonal nodes represent hypothetical haplotypes that were required for the establishment of the existing (sampled) haplotypes.