Diet with optimal glutathione supplement improves growth, nonspecific immunity, intestinal microbiota, and antioxidant ability in Micropterus salmoides.

In this study, Micropterus salmoides were fed with dietary glutathione (GSH, 0, 100, 300, and 500 mg/kg) for 56 days to investigate its effects on growth performance, serum nonspecific immunity, liver antioxidant capacity, tissue morphology, and intestinal microbiota. The results showed that the survival rate, weight gain rate, and specific growth rate and condition factor increased, whereas the feed conversion ratio, hepato-somatic index, and viscerosomatic index decreased in the GSH groups. Compared with the control group, the serum total protein content significantly increased, whereas the triglyceride and total cholesterol significantly decreased in the 300-mg/kg dietary GSH group. The activities of lysozyme, alkaline phosphatase, and acid phosphatase were significantly higher in GSH-supplemented groups, peaking at 300-mg/kg GSH. GSH supplementation significantly increased total antioxidant capacity and decreased malondialdehyde content, with the most pronounced effects at 300-mg/kg GSH. Further antioxidant indicators showed that a dietary supplement of 300-mg/kg GSH significantly increased the activities of superoxide dismutase, glutathione transferase, endogenous glutathione, glutathione reductase, and catalase. At 300-mg/kg GSH, the liver exhibited improved characteristics with alleviated vacuolation and hepatocyte nuclear shift, and intestine showed enhanced structure with increased villus height and intestinal wall thickness. Additionally, a 300-mg/kg GSH supplementation improved the diversity of intestinal microbiota, increased the abundance of probiotics such as Bacillus, and inhibited the development of pathogenic bacteria such as Plesiomonas. Overall, the results suggest that the effect of GSH addition on improving growth performance, nonspecific immunity, antioxidant capacity, and intestinal microbiota of M. salmoides is best in the 300-mg/kg addition group. Based on second-degree polynomial regression analysis of weight gain, the optimum requirement of dietary GSH in M. salmoides is a 336.84-mg/kg diet.

Construction and efficacy of Aeromonas veronii mutant Δhcp as a live attenuated vaccine for the largemouth bass (Micropterus salmoides).

Aeromonas veronii is a human and animal co-pathogenic bacterium that could have a significant negative impact on both human health and aquaculture. In this study, a mutant strain of A. veronii with deletion of the hemolysin co-regulated protein (hcp) gene was constructed (Δhcp-AV). Compared with the wild strain, Δhcp-AV showed significantly reduced growth capacity and biofilm formation ability. Motility tests showed that the hcp gene had no significant effect on the swimming and swarming ability. In addition, the pathogenicity was also reduced. To evaluate the efficacy of Δhcp-AV as a live attenuated vaccine for prevention of Aeromonas veronii infection, we compared the immune response of largemouth bass (Micropterus salmoides) after immunization with 500 µL of 1.47 × 105 CFU/mL of Δhcp-AV and 4 × 108 CFU/mL of inactivated A. veronii. Obvious increases of serum immune related enzyme activity were observed in immunization groups. Expression levels of immune-related genes in Δhcp-AV group were up-regulated, and higher than those in inactivated A. veronii group. After challenging with live A. veronii, the relative percent survival (RPS) was 100% in Δhcp- AV group, whereas the RPS was 76.67% in inactivated A. veronii group. Our data suggest that the live attenuated vaccine Δhcp- AV could elicit a stronger immune response and provide a higher RPS than inactivated A. veronii. These data suggest that hcp gene is an important virulence factor of A. veronii, and the live attenuated vaccine Δhcp-AV is safe and effective for prevention A. veronii infection in M. salmoides farming.

Expression profiling and functional characterization of the duplicated Oxr1b gene in zebrafish.

Oxidation Resistance Gene 1 (OXR1) is a conserved gene family involved in protecting various species against oxidative stress. The zebrafish expresses a pair of OXR1 paralogs (i.e., oxr1a and oxr1b). Our previous work has revealed the importance of oxr1a in regulating antioxidant defenses during oxidative stress, but the role of oxr1b is remains unknown. Herein we reported the spatial-temporal expression of oxr1b and revealed its function through reverse genetics. The results showed that, as with oxr1a, oxr1b is a typical maternal-zygotic gene. Its mRNA is mainly distributed in the eye, brain and nervous system (e.g., anterior/posterior lateral line ganglion, neuromasts and spinal cord neuron). Although oxr1a and oxr1b genes have similar expression patterns during embryonic development, the latter have higher levels at the corresponding stages. Subsequently, a viable oxr1b-/- mutant was generated by the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) system. Oxr1b knockout caused multiple antioxidant genes (i.e., gpx4a, gpx4b, sod1 and sod3b) to be downregulated, resulting in hypersensitive to oxidative stress. Furthermore, by comparative transcriptome analysis, we found that oxr1b knockout inhibits multiple signal transduction pathways (e.g., MAPK signaling pathway, calcium signaling pathway, cAMP signaling pathway and ErbB signaling pathway) during oxidative stress, thereby suppressing early stress response and ultimately impairing the anti-apoptosis pathway. In conclusion, our findings demonstrate that the duplicated oxr1b gene has an important role in regulating the antioxidant defenses by modulating signaling transduction and early stress response during oxidative stress.