Molecular Identification Of Anisakis Larva And Hysterothylacium Aduncum From Marine Fishes From Yellow Sea, China

The larvae specimens of the genus Anisakis and adult specimens of Hysterothylacium aduncum from marine fishes in Yellow Sea were studied in molecular identification during May 2005 to October 2006. After identification based on morphological characters, a total of 785 Anisakis larvae nematodes from 203 fishes (belonging to 29 species) were analysed by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). The member of 48 adult Hysterothylacium aduncum with morphological differences belong to 8 different representative fish hosts were also analysed with rDNA ITS sequence. The main results are as follows:The rDNA (ribosomal DNA) ITS( internal transcribed spacers) region of Anisakis larvae nematodes were examined by PCR-RFLP, and then ITS-1 and ITS-2 of the selective samples were amplified and sequenced.The RFLP patterns of 785 samples by HinfI and analysis of the ITS sequence for selective samples showed there are three species of the genus Anisakis in the Yellow Sea. They are Anisakis pegrefii, Anisakis typica and hybrid genotype between Anisakis pegrefii and A. simplex sensu stricto. Anisakis pegrefii is the dominant species in Yellow Sea, 770 out of 785 Anisakis larvae nematodes are Anisakis pegrefii, accounts for 98% of the total, and the hosts including 29 fish species such as Agrammus agrammus (Temminck & Schlegel), Argyrosomus argentatus (Houttuyn), Auxis thazard(lacépède) and so on.The number of Anisakis typica were very few, only 3 of 785 Anisakis larvae nematodes were found, and its hosts are Auxis thazard(lacépède), Chelidonichthys kumu. Twelve hybrid genotype were also identified,and its hosts are Argyrosomus argentatus (Houttuyn),Auxis thazard (lacépède), Cleisthenes herzensteini,Gadus mac-rocephalus Tilesius,Harengula zunasi Bleeker, Lateola- brax japonicus (Cuvier&Valenciennes),Muraenesox cinereus (Forskal), Trichiurus haumela (Forskal), Zeus japonicus Cuvier &Valencienn. This hybrid genotype is first reported in China.Forty-eight adult Hysterothylacium aduncum were selected according to different morphological index value such as lateral alae width, ratio of the intestinal caecum to ventricular appendix, spicule length and morphology of tail tip.The adult Hysterothylacium aduncum were collected from 8 fish species, they are Lophius litulon (Jordan), Gadus macrocephalus Tilesius, Cleisthenes herzensteini (Schmidt), Hexagrammos otakii Jordan & Starks,Sebastodes fuscescens (Houttuyn),Scomberomorus niphonius (Guvie& Valenciennes), Inimicus japonicus (Cuvier & Valenciennes), Zoarces elongatus Kner. The result of analysis of rDNA ITS-1 and ITS-2 sequences indicated all sequences from 48 samples are identical although the samples from different host showed morphological differences. Then we can conclude that these certain metrical and morphological differences are probably associated with the state of development of these parasites and are also influenced by different numbers of the specimens examined from individual fish host species.