Glutathione metabolism in Cryptocaryon irritans involved in defense against oxidative stress induced by zinc ions.

BACKGROUND: Cryptocaryon irritans is a fatal parasite for marine teleosts and causes severe economic loss for aquaculture. Galvanized materials have shown efficacy in controlling this parasite infestation through the release of zinc ions to induce oxidative stress. METHODS: In this study, the resistance mechanism in C. irritans against oxidative stress induced by zinc ions was investigated. Untargeted metabolomics analysis was used to determine metabolic regulation in C. irritans in response to zinc ion treatment by the immersion of protomonts in ZnSO4 solution at a sublethal dose (20 µmol). Eight differential metabolites were selected to assess the efficacy of defense against zinc ion stimulation in protomonts of C. irritans. Furthermore, the mRNA relative levels of glutathione metabolism-associated enzymes were measured in protomonts following treatment with ZnSO4 solution at sublethal dose. RESULTS: The results showed that zinc ion exposure disrupted amino acid metabolism, carbohydrate metabolism, lipid metabolism, and nucleotide metabolism in C. irritans. Four antioxidants, namely ascorbate, S-hexyl-glutathione, syringic acid, and ubiquinone-1, were significantly increased in the Zn group (P < 0.01), while the glutathione metabolism pathway was enhanced. The encystment rate of C. irritans was significantly higher in the ascorbate and methionine treatment (P < 0.05) groups. Additionally, at 24 h post-zinc ion exposure, the relative mRNA level of glutathione reductase (GR) was increased significantly (P < 0.01). On the contrary, the relative mRNA levels of glutathione S-transferase (GT) and phospholipid-hydroperoxide glutathione peroxidase (GPx) were significantly decreased (P < 0.05), thus indicating that the generation of reduced glutathione was enhanced. CONCLUSIONS: These results revealed that glutathione metabolism in C. irritans contributes to oxidative stress resistance from zinc ions, and could be a potential drug target for controlling C. irritans infection.

Quantitative detection of parasitic ciliate Cryptocaryon irritans in seawater with an optimized sample processing method.

The protozoan Cryptocaryon irritans is one of the most important ectoparasites of marine fish, causing 'white spot disease' and mass mortality in aquaculture. To accurately predict disease outbreaks and develop prevention strategies, improved detection methods are required that are sensitive, convenient and rapid. In this study, a pair of specific primers based on the C. irritans 18S rRNA gene was developed and used in a real-time PCR (qPCR) assay. This assay was able to detect five theronts in 1 L of natural seawater. Furthermore, a linear model was established to analyse the log of Ct value and parasite abundance in seawater (y = -2.9623x + 24.2930), and the coefficient of determination (R2 ) value was 0.979. A lysis buffer was optimized for theront DNA extraction and used for storage sample. This method was superior to the commercial water DNA kit, and there was no significant degradation of DNA at room temperature for 24-96 hr. A dilution method was developed to manage qPCR inhibitors and used to investigate natural seawater samples in a net cage farm with diseased fish, and the findings were consistent with the actual situation. This study provides a valuable tool for assisting in the early monitoring and control of cryptocaryoniasis in aquaculture.