Molecular identification of Anisakis and Hysterothylacium larvae in marine fishes from the East China Sea and the Pacific coast of central Japan

Abstract

Anisakiasis is a human disease caused by the accidental ingestion of larvae belonging to the family Anisakidae. Three fish species, the small yellow croaker Pseudosciaena polyactis, the mackerel Pneumatophorus japonicus and the hairtail Trichiurus haumela are important source for food products in the East China Sea. The prevalence and the identification of Anisakidae larvae in these fishes will benefit the prevention and control of anisakiasis. In this study, fish samples were obtained from fish markers in the East China Sea and the Pacific coast of central Japan during April 2011 and July 2013. For species identification, the PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis of the entire ITS region (ITS1, 5.8 S and ITS2) of nuclear ribosomal DNA (rDNA) was performed. In total, 2004 larvae were collected from 80 hairtail fish, 20 small yellow croaker, and 27 mackerel from the East China Sea and the Pacific coast of central Japan. High prevalence of Anisakidae larvae infection (116/122, 95.1%) was detected in the East China Sea. Seven species were identified belonging to the genera Anisakis (Nematoda: Anisakidae) and Hysterothylacium (Nematoda: Anisakidae). Anisakis pegreffii was the predominant species accounting for 84.8% of all larvae examined in East China Sea, while all Anisakidae larvae isolated from Japan were identified as Anisakis simplex sensu stricto (s.s.). In the East China Sea, A. simplex s.s. and Anisakis typica were 0.6% (4/619) and 1.5% (9/619) of the identified nematodes, respectively. Interestingly, one larva was identified as a recombinant genotype of A. simplex s.s. and A. pegreffii. In addition, four species of the genus Hysterothylacium, namely, Hysterothylacium amoyense (31/619, 5.0%), Hysterothylacium aduncum (10/619, 1.6%), Hysterothylacium fabri (21/619, 3.4%) and Hysterothylacium spp. (18/619, 2.9%) were also identified in the present study. This is a comprehensive epidemiological dataset for the family Anisakidae in the East China Sea. The identification of A. typica, recombinant genotype of A. simplex s.s. and A. pegreffii, H. amoyense and H. fabri is first reported in this area. The wide diversity and substantial geographical distributions of these nematodes will provide a foundation for future studies of Anisakidae family. The high prevalence of these nematodes in marine fishes off the East China Sea may pose considerable food safety problems, which is a potential cause of human anisakiasis.

Keywords: Agarose (PubChem CID: 73557002); Anisakidae nematodes; EDTA (PubChem CID: 6049); East China Sea; Ethanol (PubChem CID: 702); Magnesium chloride (PubChem CID: 24584); Marine fishes; Molecular identification; Sodium chloride (PubChem CID: 5234); Tris–HCl (PubChem CID: 93573); dATP (PubChem CID: 15993); dCTP (PubChem CID: 65091); dGTP (PubChem CID: 65103); dTTP (PubChem CID: 64968).