應用PCR-RFLP調查花腹鯖魚和白帶魚之海獸胃線蟲感染狀況與藉熱休克蛋白的表達評估芥末醬和異硫氰酸酯的殺蟲效果

The Anisakis (Nematodes, Anisakidae) is the main causative agent of the human zoonotic disease Anisakidosis. There are nine (9) species, which are morphologically categorized into two group: Anisakis Clade I and Clade II. There are increased consumption of Sashimi and Sushi (raw fish fillet) among Taiwanese due to globalized exotic ethnic dishes and vitamins preservation. Therefore, this diets changes may increase risks of anisakiasis acquisition through consumption of raw fish fillet. Thus, it remained necessary to investigate the status of Anisakis infection to explore the potential risky fishes species.Two host fish species, Scomber australasicus and Trichiurus lepturus were investigated in the present study. Anisakis larvae collected from these fishes were morphological differentiation from other larvae using a light microscope and genetically analyzed with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using the internal transcribed spacer (ITS-1 and ITS-2) regions to identify the species. The survival rate, median lethal time (LT50) and mRNA expression of Heat Shock Protein (HSP) were determined using Kaplan Meier curves and quantitative real time PCR (qRT-PCR), respectively. A total of 1302 anisakis third stage larvae (L3) were collected from the host species S. australasicus (n=26) and T. lepturus (n=10) with a mean of 21.62 (± 27.61) and 74.00 (± 62.16) respectively. Estimated total of 40% (n= 215 and 336 respectively) larvae were randomly collected from each hosts species and analyzed with PCR-RFLP, 83.72% was identified as A. pegreffi, 6.05% as A. typica, 0.93% as A. physteris and 8.84% as hybrid genotype ( A. pegreffii + A. simplex [s.s]) in S. australasicus host and 86.31% as A. pegreffi, 2.38% as A. typica, and 11.31% for hybrid genotype ( A. pegreffii + A. simplex [s.s]) in T. lepturus host. The survival rate of A. pegreffi larva treated with wasabi paste shown a significant difference with a hazards mortality of 3.1 and 3.8 percent per minute’s treated with the 1g/ml and 2g/ml of Wasabi paste in PBS respectively. Relatively, the two groups show a significant difference (P=0.0227) and high significant difference from the control group (P<0.0001). The mean lethal time estimated for the two treatment group shown 30 and 45minutes respectively. The AITC has high toxicity to larvae, with the mean lethal time estimated to be 16, 25 and 34minutes for 10-, 20- and 30-Fold dilution respectively. Compared to the control (Oil), AITC shows significant difference 10-Fold (P=0.0001), 20-F0ld (P=0.0003) and 30-Fold (P=0.0108). Both concentration groups also shown significant difference (P<0.05). When larva treatment with WSP, the HSP90 mRNA transcription is gradually expressed and reach to the peak at 12mins post-treatments and declined (P=0.0077).The HSP70 mRNA transcribed in all the treatments time point (P<0.05) post-exposure with Wasabi Paste. The HSP90 protein does not significantly express (P=0.5633) and whereas the HSP70 protein translation gradually increased significantly in a time-dependent manner and reaches its peak at 18mins (P<0.05). When the parasite larva was treated with 10-Fold AITC, the HSP90 mRNA expression gradually decrease (P=0.01132, R2=0.7094) and significantly downregulated at 12mins compared to the control (P<0.05). The HSP70 mRNA expression basally upregulated in all the treatment time point and significantly increase in 12mins post-exposure to AITC compared to the control (P=0.0389). The HSP90 proteins significantly upregulated at 2mins (P=0.0249) and gradually downregulated (P>0.05) whereas the HSP70 proteins significantly increased in 2mins, 4mins, 6 mins and 12mins (P<0.05). Thus, wasabi and AITC are highly toxic and induced stress sensor protein to the anisakis larvae, which is evident by high expression of HSP70 mRNA at all treatments time point.