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Difenoconazole (DFZ) is a classical triazole fungicide that causes immunosuppression in non-target organisms. Ferulic acid (FA) is a polyphenolic molecule found in nature that has antioxidant and anti-inflammatory activities. The purpose of this investigation was to see if FA could prevent DFZ-induced immunosuppression and to identify the potential mechanisms. Carp were exposed to 1/10 LC50 of DFZ as well as fed normal feed or feed containing dietary additive FA for 30 d. It was found that DFZ-induced immunosuppression could be improved by FA, as evidenced by upregulation of Hb, C3 and IgM and downregulation of LDH. It was then investigated that FA could ameliorate DFZ-induced splenic injury through p53-mediated apoptosis. At the same time, enhancing the levels of CAT, GSH and T-AOC in spleen and transcription levels Nrf2 signaling pathway related genes indicated that FA reduced oxidative damage caused by DFZ by blocking the Nrf2 signaling pathway. In addition, FA inhibited the inflammatory response triggered by TRAF/TAK1/NF-κB signaling pathway, downregulated the transcript levels of pro-inflammatory factors (il-1β, tnf-α, il-6) and the level of NLRP3 inflammasome (NRLP3, ASC, Caspase 1), and upregulated the transcript levels of anti-inflammatory factors (tgf-β1, il-10). In conclusion, the above results suggested that FA mediated TRAF/TAK1/NF-κB, Nrf2, and p53 pathways to attenuate DFZ-induced inflammation, oxidative stress, and apoptosis thereby enhancing the immune capacity of carp.

Excessive use of hard-to-degrade pesticides threatens the ecological health of aquatic systems. This study aimed to investigate difenoconazole (DFZ) residues in the environment induced neurotoxicity in carp and the underlying mechanisms. A total of thirty-six carps were divided into three groups and exposed to 0, 0.5, and 2.0 mg/L DFZ for 96 h, respectively. The alterations in behavior and blood-brain barrier (BBB) were examined, and potential mechanisms were explored using immunological assays and biochemical methods. The results showed that DFZ exposure caused behavioral freezing, reduced feeding, and neuronal necrosis in carp. Mechanistically, DFZ triggered ROS accumulation and destroyed the balance between oxidation and antioxidation with increased lipid peroxidation product MDA contents and reduced antioxidant enzymes SOD and CAT activities in the carp brain by inhibiting the NF-E2-related factor 2 (Nrf2) pathway. The activation of oxidative stress further reduced tight junction proteins and MMP levels, thereby destroying BBB and leading to DFZ leakage into the brain. Increased BBB permeability additionally led to DFZ activation of nuclear factor kappa-B signaling-mediated inflammatory cytokine storm, exacerbating neuroinflammation. Meanwhile, DFZ exposure activated mitochondria-associated apoptosis in the carp’s brain by up-regulating Bcl-2 associated X protein, cleaved-caspase3, and cytochrome C and decreasing B-cell lymphoma-2 levels. Interestingly, the carp’s brain initiated a protective autophagic response via the PI3K/AKT/TOR pathway intending to counteract the neurotoxicity of DFZ. Overall, we concluded that accumulation of DFZ at high concentrations in the aquatic systems disrupted the BBB and resulted in neurotoxicity in carp through inhibition of Nrf2 pathway-mediated ROS accumulation. This study provides a reference for monitoring DFZ residues in the environment and a new target for the treatment of DFZ-induced neurotoxicity in carp.

Avermectin, a ''low toxicity insecticide'', has been widely used in recent years, but its non-target toxicity, especially to aquatic organisms, has been neglected. In this study, we evaluated the neurotoxic effects of avermectin on carp by establishing a 96 h avermectin acute toxicity test, and its possible mechanism was discussed. The 96 h LC50 of avermectin in carp was found to be 24.04 μg/L. Therefore, 3.005 μg/L and 12.02 μg/L were used as the low-dose and high-dose groups, respectively, to investigate the neurotoxic effects of avermectin on carp. The results of high-performance liquid chromatography (HPLC) analysis showed that avermectin accumulated in the carp brain. Histopathological observation and immunohistochemical analysis (IHC) of TNF-α and Bax showed that avermectin exposure led to inflammatory cell infiltration and neuronal necrosis. The mRNA levels of tight junction genes and the IHC results of ZO-1 and Occludin showed that the structure of the blood-brain barrier (BBB) was destroyed. Biochemical analysis showed that avermectin induced the accumulation of MDA in the brain and decreased the activity of antioxidant enzymes CAT and SOD, leading to oxidative stress. In addition, avermectin induces brain inflammation by activating NF-κB pathway and releasing inflammatory factors IL-1β, IL-6, TNF-α and iNOS. TEM and TUNEL assays showed that exposure to avermectin induced apoptosis in brain. what is more, the expression of apoptosis-related genes and proteins suggested that avermectin-induced apoptosis may be associated with inhibition of the PI3K/Akt signaling pathway. This study also showed that avermectin-induced NF-κB signaling activation was partially dependent on its upstream PI3K/Akt signaling pathway. Therefore, this study concludes that avermectin can induce neurotoxicity in carp by disrupting the blood-brain barrier structure and generating oxidative stress, inflammation, and apoptosis and that NF-κB and PI3K/Akt signaling pathways are involved in this process.

As the use of pesticides increases year after year, so does the level of residual pesticides in the aquatic environment, posing a serious threat to non-target organisms. Difenoconazole (DFZ), a class of long-lasting fungicides and residues in the marine environment, has been shown to cause damaging effects on different organs of aquatic organisms. However, there is no research on the damage of DFZ to carp spleen tissue. This study aimed to investigate the acute toxic effects of DFZ on the spleen tissue of carp (Cyprinus carpio) by exposing juvenile carp to environmentally relevant concentrations of DFZ. We randomly selected 30 carp, divided them into the Control, Low, and High groups, and then exposed the three groups to 0, 0.488 mg/L DFZ, and 1.953 mg/L DFZ for 96 h respectively. We then investigated the toxic effects caused by DFZ on carp and spleen tissues by detecting changes in spleen histopathologic damage, apoptosis, oxidative stress, inflammation, and blood biochemical parameters. We found that DFZ causes severe histopathology in spleen tissue, including ballooning, structural relaxation, and giant mitochondria. In addition, we found that DFZ caused excessive apoptosis in spleen tissue by TUNEL staining and expression levels of apoptosis-related genes (caspase3, caspase8, caspase9, fas, bax, bcl-2, and p53). The activities and transcript levels of the antioxidant enzymes SOD, CAT, and GSH-Px were significantly down-regulated. In addition, DFZ led to a significant increase in activation of the NF-κB signaling pathway and mRNA levels of pro-inflammatory cytokines il-6, il-1β, and tnf-α, and a substantial decrease in mRNA levels of anti-inflammatory cytokines il-10 and tgf-β1 in spleen tissue. Blood biochemical parameters showed that DFZ exposure significantly reduced erythrocyte, leukocyte, hemoglobin, C3, and IgM levels. Collectively, DFZ exposure induced apoptosis, immunosuppression, oxidative stress, and inflammatory responses in the spleen tissue of carp, resulting in spleen tissue damage.