β-Sitosterol, which is used extensively in pharmaceuticals, nutraceuticals, and cosmetics, has high nutritional value along with immunomodulatory and anti-inflammatory properties. In this study, we investigated the antioxidant and anti-inflammatory effects of β-sitosterol in zebrafish and explored the associated molecular mechanisms. In an in vivo antioxidant experiment, zebrafish (Danio rerio) larvae were treated with different concentrations of β-sitosterol and then exposed to a nonlethal concentration of CuSO₄ to induce oxidative stress. Treatment with β-sitosterol at 70 or 100 μg/mL significantly reduced CuSO₄-induced oxidative stress in the zebrafish, demonstrating the strong antioxidant activity of β-sitosterol. Treatment with β-sitosterol protected zebrafish larvae against oxidative damage from CuSO₄ by upregulating the expressions of sod and gpx4b. In a zebrafish model of inflammation, pretreatment with β-sitosterol before CuSO4 exposure inhibited neutrophil recruitment and damage to lateral line neuromasts, indicating a potent anti-inflammatory effect derived from reductions in the expressions of il-8 and myd88. The results demonstrate the antioxidative and anti-inflammatory activities of β-sitosterol and suggest that β-sitosterol may be useful for the treatment of various inflammatory diseases.

Long-term or excessive oxidative stress can cause serious damage to fish. Squalene can be added to feed as an antioxidant to improve the body constitution of fish. In this study, the antioxidant activity was detected by 2,2-diphenyl-1-acrylhydrazyl (DPPH) test and fluorescent probe (dichloro-dihydro-fluorescein diacetate). Transgenic Tg (lyz: DsRed2) zebrafish were used to evaluate the effect of squalene on CuSO₄-induced inflammatory response. Quantitative real-time reverse transcription polymerase chain reaction was used to examine the expression of immune-related genes. The DPPH assay demonstrated that the highest free radical scavenging exerted by squalene was 32%. The fluorescence intensity of reactive oxygen species (ROS) decreased significantly after 0.7% or 1% squalene treatment, and squalene could exert an antioxidative effect in vivo. The number of migratory neutrophils in vivo was significantly reduced after treatment with different doses of squalene. Moreover, compared with CuSO₄ treatment alone, treatment with 1% squalene upregulated the expression of sod by 2.5-foldand gpx4b by 1.3-fold to protect zebrafish larvae against CuSO₄-induced oxidative damage. Moreover, treatment with 1% squalene significantly downregulated the expression of tnfa and cox2. This study showed that squalene has potential as an aquafeed additive to provide both anti-inflammatory and antioxidative properties.

Largemouth bass ranavirus (LMBV) infects largemouth bass, leading to significant mortality and economic losses. There are no safe and effective drugs against this disease. Oral vaccines that directly target the intestinal mucosal immune system play an important role in resisting pathogens. Herein, the B subunit of Escherichia coli heat-labile enterotoxin (LTB, a mucosal immune adjuvant) and the LMBV main capsid protein (MCP) were expressed using Saccharomyces cerevisiae surface display technology. The yeast-prepared oral vaccines were named EBY100-OMCP and EBY100-LTB-OMCP. The candidate vaccines could resist the acidic intestinal environment. After 7 days of continuous oral immunization, indicators of innate and adaptive immunity were measured on days 1, 7, 14, 21, 28, 35, and 42. High activities of immune enzymes (T-SOD, AKP, ACP, and LZM) in serum and intestinal mucus were detected. IgM in the head kidney was significantly upregulated (EBY100-OMCP group: 3.8-fold; BY100-LTB-OMCP group: 4.3-fold). IgT was upregulated in the intestines (EBY100-OMCP group: 5.6-fold; EBY100-LTB-OMCP group: 6.7-fold). Serum neutralizing antibody titers of the two groups reached 1:85. Oral vaccination protected against LMBV infection. The relative percent survival was 52.1% (EBY100-OMCP) and 66.7% (EBY100-LTB-OMCP). Thus, EBY100-OMCP and EBY100-LTB-OMCP are promising and effective candidate vaccines against LMBV infection.

The present study aimed to evaluate the effect of the dietary supplementation of Saccharomyces cerevisiae YFI-SC2 on the growth performance, intestinal morphology, immune parameters, intestinal microbiota, and disease resistance of crayfish (Procambarus clarkia). Crayfish were randomly assigned to six different boxes and two different groups in triplicate. The control group received a basal diet and the treatment group received a diet containing S. cerevisiae at 10⁷ CFU/g. After feeding for 28 days, crayfish of the treatment group exhibited a significantly better weight gain ratio (WGR) and a specific growth rate (SGR) (p < 0.05) than crayfish of the control group. Compared to the treatment group, the control group intestines showed an oedema connective tissue layer and a weak muscle layer. For immune-related genes, Crustin2 expression was similar between the groups, whereas Lysozyme and prophenoloxidase from treatment group expression levels were upregulated significantly (p < 0.05) after 14 and 28 days of feeding. Prophenoloxidase showed the highest expression, with 10.5- and 8.2-fold higher expression than in the control group at 14 and 28 days, respectively. The intestinal microbiota community structure was markedly different between the two groups. After 14 and 28 days of feeding, the relative abundance of Cetobacterium and Lactobacillus increased, whereas Citrobacter and Bacteroides decreased in the treatment group compared with that of the control group. The challenge test showed that crayfish of the treatment group had a significantly enhanced resistance against Citrobacter freundii (p < 0.05). Our results suggest that a S. cerevisiae-containing diet positively influenced the health status, immune parameters, intestinal microbiota composition, and disease resistance of crayfish.

Aflatoxin contamination of food and water is a serious problem worldwide. This study investigated the defensive ability of gibel carp exposed to aflatoxin B1 (AFB1) by challenging it with cyprinid herpesvirus 2 (CyHV-2) infection. The data showed that AFB1 exposure significantly increased the mortality of CyHV-2-infected gibel carp, and enhanced the viral load in the fish liver, kidney, and spleen. The oxidative-antioxidant balance suggested that AFB1 induced severe oxidative stress, including increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the AFB1 exposed group, and the reduced activity of superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase (CAT) in the AFB1 exposed group. Meanwhile, the related expression of nuclear factor erythroid 2-related factor 2 (Nrf2), interferon regulatory factor 3 (IRF3) and the type 1 interferon (IFN1) were noticeably down-regulated, but caspase-1 was up-regulated, after exposure to AFB1, demonstrating that fish are unable to avoid the virus infection. It should be noted that the intestinal microbiota diversity and richness were lower in the AFB1 exposed group, and the composition of intestinal microbiota was affected by AFB1, resulting in the higher abundance of bacteria (such as Aeromonas and Bacteroides) and the lower abundance of potentially beneficial bacteria (such as Cetobacterium and Clostridium) in the AFB1 exposed group. This research provides insight into the possibility that AFB1 may increase the susceptibility of C. gibelio to CyHV-2 infection, and thus amplify the viral outbreak to endanger ecological safety in aquatic environment.