Prometryn, a triazine pesticide product used to control weed growth, poses a high risk to aquatic organisms in the environment. Several toxicological evaluations have been performed on bony fish and shrimp exposed to prometryn. However, there have been no reports conducted on the toxic mechanism of prometryn with regard to Eriocheir sinensis. In this study, our research evaluated the toxic effects of prometryn via in vitro and in vivo toxicity tests on E. sinensis. Firstly, we estimated the exposure toxicity of prometryn to E. sinensis, and then we constructed a 6 h transcriptional profile and conducted an enrichment analysis. To further reveal the toxicity of prometryn, the hepatopancreas (hepatopancreatic cells) was analyzed for antioxidant, immune and lipid-metabolism-related enzymes, antioxidant- and apoptosis-related gene expression, histopathology and TUNEL. From the results, we determined that the 96 h-LD50 was 70.059 mg/kg, and using RNA-seq, we identified 933 differentially expressed genes (DEGs), which were mainly enriched in the amino and fatty acid metabolism and the cell-fate-determination-related signaling pathway. The results of the biochemical assays showed that prometryn could significantly decrease the activities/levels of CAT, SOD, GSH, AKP and ACP, reduce the levels of T-AOC, TG, TCH, C3 and C4, and increase the MDA content. In addition, the expression levels of Nrf2, GSTs and HO-1 were first upregulated and then downregulated with increasing time. Histopathology showed that prometryn damaged the structure of the hepatopancreas cells and induced apoptosis, suggesting that the PI3K-Akt signaling pathway may be involved in the damage process of hepatopancreas cells (PI3K, PDK and Akt were downregulated whereas Bax was upregulated), leading to their apoptosis. The above results indicated that prometryn could cause injury of the hepatopancreas through oxidative stress, induce cell apoptosis, disrupt the lipid metabolism and cause immune damage. This study provided useful data for understanding and evaluating the toxicity of prometryn to aquatic crustacea.

There is growing evidence that long-term exposure to prometryn (a widely used herbicide) can induce toxicity in bony fish and shrimp. Our previous study demonstrated its 96 h acute toxicity on the crab Eriocheir sinensis. However, studies on whether longer exposure to prometryn with a lower dose induces toxicity in E. sinensis are scarce. Therefore, we conducted a 20 d exposure experiment to investigate its effects on the hepatopancreas and intestine of E. sinensi. Prometryn reduce the activities of antioxidant enzymes, increase the level of lipid peroxidation and cause oxidative stress. Moreover, long-term exposure resulted in immune and detoxification fatigue, while short-term exposure to prometryn could upregulate the expression of genes related to immunity, inflammation and detoxification. Prometryn altered the morphological structure of the hepatopancreas (swollen lumen) and intestine (shorter intestinal villi, thinner muscle layer and thicker peritrophic membrane). In addition, prometryn changed the species composition of the intestinal flora. In particular, Bacteroidota and Proteobacteria showed a dose-dependent decrease accompanied by a dose-dependent increase in Firmicutes at the phylum level. At the genus level, all exposure groups significantly increased the abundance of Zoogloea and a Firmicutes bacterium ZOR0006, but decreased Shewanella abundance. Interestingly, Pearson correlation analysis indicated a potential association between differential flora and hepatopancreatic disorder. Phenotypic abundance analysis indicated that changes in the gut flora decreased the intestinal organ’s resistance to stress and increased the potential for opportunistic infection. In summary, our research provides new insights into the prevention and defense strategies in response to external adverse environments and contributes to the sustainable development of E. sinensis culture.

Oxidative stress represents a critical threat to aquatic animal health and aquaculture productivity. Allicin, a natural plant extract, has not been systematically investigated for its antioxidant mechanisms in aquatic crustaceans. This study established in vitro and in vivo models of tert-butyl hydroperoxide (T-BHP)-induced oxidative stress in Chinese mitten crabs (Eriocheir sinensis) to evaluate the hepatoprotective effects of allicin. Integrating biochemical, transcriptomic, and ultrastructural analyses, we found that allicin significantly alleviated T-BHP-induced cytotoxicity and oxidative damage in vitro. Mechanistically, allicin up-regulated antioxidant genes including glutathione peroxidase (gpx) and thioredoxin reductase 1 (trxr1), and down-regulated pro-inflammatory cytokines such as interleukin-1 beta (il-1β), suggesting the concomitant activation of the Nrf2 signaling pathway and inhibition of the p38-MAPK/NF-κB pathway. Transcriptomics further indicated its role in restoring proteostasis and mitochondrial function. A 35-day feeding trial validated these findings in vivo; dietary supplementation with 300 mg·kg⁻¹ allicin effectively reversed T-BHP-induced disturbances in antioxidant enzyme activities and immune-related gene expression. These consistent findings demonstrate that allicin alleviates hepatopancreatic oxidative damage through multi-pathway synergism, supporting its potential as a green and effective antioxidant feed additive in aquaculture.