Anisakis pegreffii (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 A. pegreffii, 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 (p < 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 A. pegreffii 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.

The species of Anisakis constitute one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognised in the A. simplex (s. l.) complex, i.e. A. pegreffii, A. simplex (s. s.) and A. berlandi. They are distributed in populations of their intermediate/paratenic (fish and squids) and definitive (cetaceans) hosts. A panel of seven microsatellite loci (Anisl 05784, Anisl 08059, Anisl 00875, Anisl 07132, Anisl 00314, Anisl 10535 and Anisl 00185), were developed and validated on a total of N = 943 specimens of A. pegreffii and A. simplex (s. s.), collected in fish and cetacean hosts from allopatric areas within the range of distribution of these parasite species. In addition, the locus Anisl 7, previously detected in those Anisakis spp., was investigated. The parasites were first identified by sequence analysis of the EF1 α-1 nDNA. The panel of the microsatellites loci here developed have allowed to: (i) detect diagnostic microsatellite loci between the two species; (ii) identify specimens of the two species A. pegreffii, A. simplex (s. s.) in a multi-marker nuclear genotyping approach; (iii) discover two sex-linked loci in both Anisakis species and (iv) estimate levels of genetic differentiation at both the inter- and intra-specific level.

A multi-marker nuclear genotyping approach was performed on larval and adult specimens of Anisakis spp. (N = 689) collected from fish and cetaceans in allopatric and sympatric areas of the two species Anisakis pegreffii and Anisakis simplex (s. s.), in order to: (1) identify specimens belonging to the parental taxa by using nuclear markers (allozymes loci) and sequence analysis of a new diagnostic nuclear DNA locus (i.e. partial sequence of the EF1 α−1 nDNA region) and (2) recognize hybrid categories. According to the Bayesian clustering algorithms, based on those markers, most of the individuals (N = 678) were identified as the parental species [i.e. A. pegreffii or A. simplex (s. s.)], whereas a smaller portion (N = 11) were recognized as F1 hybrids. Discordant results were obtained when using the polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLPs) of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) on the same specimens, which indicated the occurrence of a large number of ‘hybrids’ both in sympatry and allopatry. These findings raise the question of possible misidentification of specimens belonging to the two parental Anisakis and their hybrid categories derived from the application of that single marker (i.e. PCR–RFLPs analysis of the ITS of rDNA). Finally, Bayesian clustering, using allozymes and EF1 α−1 nDNA markers, has demonstrated that hybridization between A. pegreffii and A. simplex (s. s.) is a contemporary phenomenon in sympatric areas, while no introgressive hybridization takes place between the two species.