Nematocidal activity of wasabi paste and allyl Isothiocyanate against <i>Anisakis</i> third-stage larvae and their stress response characterized by heat shock protein expression

<i>Anisakis pegreffii</i> (Nematoda: Anisakidae) is a marine parasitic nematode responsible for anisakiasis, a zoonotic disease acquired through the consumption of raw or undercooked fish. With the rising popularity of dishes such as sushi and sashimi, the risk of infection has grown, prompting the need for effective food-safety measures. This study investigates the nematocidal effects of commercial wasabi substitute paste (WSP) and its bioactive compound, allyl isothiocyanate (AITC), on third-stage larvae (L3) of <i>A. pegreffii</i>, and explores their associated stress-response mechanisms. WSP at 1 g/mL and 2 g/mL significantly reduced larval viability, with hazard rates of 3.1% and 3.8% per minute and mean lethal times of 45 and 30 minutes, respectively (<i>p</i> < 0.0001). AITC, tested at a 10-fold dilution, exhibited stronger activity, inducing 50% mortality in 16 minutes with a hazard rate of 13.45%/min. Expression analyses revealed that WSP robustly upregulated HSP70 mRNA and protein in a time-dependent manner, whereas AITC caused only a modest increase in HSP70 mRNA without a significant protein-level rise over the same exposure window. Conversely, HSP90 expression showed early, transient upregulation followed by suppression or downregulation, suggesting differential regulation of heat shock pathways under chemical stress. These findings demonstrate that WSP and AITC exert potent, rapid nematocidal effects on <i>A. pegreffii</i> larvae and elicit distinctive molecular stress responses. The data support the potential of wasabi-derived compounds as safe, natural agents for reducing anisakiasis risk in raw seafood consumption.