Linghong Miao

A deficiency or an excess of dietary threonine level affects weight gain, enzyme activity, immune response and immune-related gene expression in juvenile blunt snout bream (Megalobrama amblycephala).

A feeding trial was conducted to investigate the impacts of deficient and excess dietary threonine levels on weight gain, plasma enzymes activities, immune responses and expressions of immune-related genes in the intestine of juvenile blunt snout bream. Triplicate groups of fish (initial weight 3.01 ± 0.01 g, 30 fish per tank) were fed with deficient (0.58%), optimum (1.58%) and excess (2.58%) threonine level diets to near satiation four times a day for 9 weeks. A mixture of l-amino acids was supplemented to simulate the whole body amino acid pattern of blunt snout bream, except for threonine. The results showed that both deficiency and excess threonine level diets significantly (P < 0.05) reduced the weight gain of blunt snout bream. Excess dietary threonine level triggered plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities (P < 0.05); whereas superoxide dismutase (SOD) activity was not significantly influenced by imbalanced-dietary threonine level (P > 0.05). Plasma complement component 3 (C3) and component 4 (C4) concentrations were significantly depressed by the deficiency of dietary threonine (P < 0.05). Dietary threonine regulated the target of rapamycin (TOR), eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2), tumour necrosis factor alpha (TNF-α) and copper-zinc superoxide dismutase (Cu/Zn-SOD) gene expressions in the intestine of blunt snout bream, which may go further to explain the adverse effects of a deficient and/or an excess dietary threonine level on growth, immunity and health of fish. Furthermore, the present study also suggests that an optimum dietary threonine could play an important role in improving growth, enhancing immune function and maintaining health of fish.

Effects of emodin and vitamin E on the growth and crowding stress of Wuchang bream (Megalobrama amblycephala).

This study aimed to investigate the effects of dietary emodin, high-dose vitamin E and their combination on the growth of Megalobrama amblycephala and its resistance to acute crowding stress. The fish were randomly divided into four groups: a control group fed with basal diet, and three treatment groups fed with basal diet supplemented with 60 mg/kg emodin (the emodin group), 500 mg/kg vitamin E (the vit E group), and 60 mg/kg emodin together with 500 mg/kg vitamin E (the combination group). After 60 days, the fish were exposed to acute crowding stress for 24 h. The results showed that the weight gain of the vit E group, specific growth rate of the vit E group, total serum protein concentration (TP) of the vit E group, serum lysozyme activity of the emodin group, serum superoxide dismutase (SOD) activity of the emodin group, hepatic heat shock protein 70 (HSP70) levels of the vit E group and the emodin group, and serum alanine aminotransferase (ALT) activity of the combination group significantly increased while the weight gain and specific growth rate of the combination group significantly decreased compared with the control group before stress. After crowding stress, the vit E group had improved serum TP 12 h post-stress, hepatic SOD activity 24 h post-stress, and hepatic HSP70 mRNA levels 12 and 24 h post-stress while the emodin group had enhanced serum SOD activity 12 and 24 h post-stress and hepatic HSP70 mRNA levels 12 and 24 h post-stress, as compared with the control. However, the serum cortisol content of the three treatment groups 12 and 24 h post-stress, ALT activity in the vit E group and emodin group 24 h post-stress, and serum alkaline phosphatase and liver catalase activity in the combination group 24 h post-stress were lower than those in the control group. The cumulative mortality was lower in the emodin, vit E, and combination group after Aeromonas hydrophila infection compared with the control group. Therefore, dietary supplementation with 60 mg/kg emodin or 500 mg/kg vitamin E can improve HSP70 mRNA levels and antioxidant capabilities, resistance to crowding stress, and growth in M. amblycephala. However, the combination of emodin and vit E does not have a synergistic effect in M. amblycephala.

Effect of High Dietary Carbohydrate on the Growth Performance and Physiological Responses of Juvenile Wuchang Bream, Megalobrama amblycephala

An optimum dietary carbohydrate content is important for maximum fish growth. In this study, we fed Wuchang bream (Megalobrama amblycephala) with either control diet (30.42%) or high carbohydrate diet (52.92%) for 90 d. Fish were fed to apparent satiation three times daily in an aquarium with automatic temperature control and circulated water. Growth performance, plasma biochemical parameters, hepatic morphology and enzyme activities were determined. It was shown that compared to fish fed control diet, fish fed high carbohydrate diet had higher plasma triglyceride and cortisol levels for d 90, and lower alkaline phosphatase level for d 45, lower hepatic superoxide dismutase and total antioxidative capacity for d 90, higher malondialdehyde for d 45 and glycogen content for d 45 and 90 (p<0.05). Histological and transmission electron microscopy studies showed that hepatocytes of fish fed high carbohydrate diet contained large lipid droplets, causing displacement of cellular organelles to periphery of hepatocytes. The relative level of hepatic heat shock protein 70 (HSP70) mRNA of Wuchang bream fed high carbohydrate diet was significantly higher than that of fish fed the control diet for 90 d (p<0.05). These changes led to decreased specific growth rate and increased feed conversion ratio (p<0.05). Upon hypoxia challenge, fish fed high carbohydrate diet had higher cumulative mortality than those fed the control diet (p<0.05). These results suggested that high dietary carbohydrate (52.92%) was detrimental to the growth performance and health of Wuchang bream.

Comparative proteomic analysis of liver antioxidant mechanisms in Megalobrama amblycephala stimulated with dietary emodin

Oxidative stress is a toxicological endpoint that correlates with the nutrition status of fish through cellular damage, inflammation, and apoptosis. In order to understand the antioxidant mechanism induced by dietary emodin in Megalobrama amblycephala liver, a comparative proteomic analysis was performed to investigate the proteome alteration under emodin administration. 27 altered protein spots were separated under 30 mg kg−1 emodin stimulation based on 2-DE, and were all successfully identified using MALDI-TOF/TOF, representing 17 unique proteins. These proteins were functionally classified into antioxidant, metabolism, cytoskeleton, chaperone, signal transduction and cofactor groups. Network interaction and Gene Ontology annotation indicated 10 unique proteins were closely related to antioxidation and directly regulated by each other. Compared with the control group, administration of 30 mg kg−1 emodin significantly increased the antioxidant-related mRNA expressions of GPx1, GSTm and HSP70, but decreased the mRNA expressions of GAPDH and Sord, which was consistent with the protein expression. Nevertheless, Pgk1 and Aldh8a1 were up- and down-regulated, and ALDOB was down- and up-regulated at the mRNA and protein levels, respectively. These results revealed that the altered proteins enhanced antioxidation via complex regulatory mechanisms, and 30 mg kg−1 emodin was a suitable immunostimulant for M. amblycephala.

Dietary arginine affects the insulin signaling pathway, glucose metabolism and lipogenesis in juvenile blunt snout bream Megalobrama amblycephala

This study evaluated the mechanisms governing insulin resistance, glucose metabolism and lipogenesis in juvenile fish fed with graded levels of dietary arginine. The results showed that, compared with the control group (0.87%), 2.31% dietary arginine level resulted in the upregulation of the relative gene expression of IRS-1, PI3K and Akt in the insulin signaling pathway, while 2.70% dietary arginine level led to inhibition of these genes. 1.62% dietary arginine level upregulated glycolysis by increasing GK mRNA level; 2.70% dietary arginine level upregulated gluconeogenesis and resulted in high plasma glucose content by increasing PEPCK and G6P mRNA level. Furthermore, 2.70% dietary arginine level significantly lowered GLUT2 and increased PK mRNA levels. 1.62% dietary arginine level significantly upregulated ACC, FAS and G6PDH mRNA levels in the fat synthesis pathway and resulted in high plasma TG content. These results indicate that 1.62% dietary arginine level improves glycolysis and fatty acid synthesis in juvenile blunt snout bream. However, 2.70% dietary arginine level results in high plasma glucose, which could lead to negative feedback of insulin resistance, including inhibition of IRS-1 mRNA levels and activation of gluconeogenesis-related gene expression. This mechanism seems to be different from mammals at the molecular level.

The effects of emodin on cell viability, respiratory burst and gene expression of Nrf2-Keap1 signaling molecules in the peripheral blood leukocytes of blunt snout bream (Megalobrama amblycephala)

ABSTRACT We determined the effects of emodin on the cell viability, respiratory burst activity, mRNA levels of antioxidative enzymes (Cu‐Zn SOD, CAT and NOX2), and gene expressions of the Nrf2‐Keap1 signaling molecules in the peripheral blood leukocytes of blunt snout bream. Triplicate groups of cultured cells were treated with different concentrations of emodin (0.04–25 &mgr;g/ml) for 24 h. Results showed that the emodin caused a dramatic loss in cell viability, and occurred in a dose‐dependent manner. Emodin exposure (1–25 &mgr;g/ml) were significantly induced the ROS generation compared to the control. The respiratory burst and NADPH oxidase activities were significantly induced at a concentration of 0.20 &mgr;g/ml, and inhibited at 25 &mgr;g/ml. Besides, mRNA levels of antioxidant enzyme genes were dramatically regulated by emodin exposure for 24 h. During low concentrations of exposure, mRNA levels of Cu‐Zn SOD in the cells treated with 0.04, 0.20 &mgr;g/ml, CAT, NOX2 and Nrf2 in the cells treated with 1 &mgr;g/ml were sharply increased, respectively. Whereas, high concentrations were dramatically down‐regulated the gene expressions of CAT in the cells treated with 5, 25 &mgr;g/ml and NOX2 in the cells treated with 25 &mgr;g/ml. Furthermore, sharp increase in Keap1and Bach1 expression levels were observed a dose‐dependent manner. In conclusion, this study demonstrated that emodin could induce antioxidant defenses which were involved in cytotoxic activities, respiratory burst and the transcriptional regulation levels of antioxidant enzymes and Nrf2‐Keap1 signaling molecules. HighlightsHigh concentrations of emodin could induce cytotoxic effects in peripheral blood leukocyte.Emodin could mediated or induced oxidative stress damage of the peripheral blood leukocyte.Nrf2‐Keap1 signaling pathway might play a crucial role in defending against oxidative damage in peripheral blood leukocyte.

Comparative transcriptome analysis reveals the gene expression profiling in bighead carp (Aristichthys nobilis) in response to acute nitrite toxicity

Objective: Nitrite exposure induces growth inhibition, metabolic disturbance, oxidative stress, organic damage, and infection‐mediated mortality of aquatic organism. This study aimed to investigate the mechanism in responses to acute nitrite toxicity in bighead carp (Aristichthys nobilis, A. nobilis) by RNA‐seq analysis. Methods: Bighead carps were exposed to water with high nitrite content (48.63mg/L) for 72h, and fish livers and gills were separated for RNA‐seq analysis. De novo assembly was performed, and differentially expressed genes (DEGs) between control and nitrite‐exposed fishes were identified. Furthermore, enrichment analysis was performed for DEGs to annotate the molecular functions. Results: A total of 406,135 transcripts and 352,730 unigenes were tagged after de novo assembly. Accordingly, 4108 and 928 DEGs were respectively identified in gill and liver in responses to nitrite exposure. Most of these DEGs were up‐regulated DEGs. Enrichment analysis showed these DEGs were mainly associated with immune responses and nitrogen metabolism. Conclusions: We suggested that the nitrite toxicity‐induced DEGs were probably related to dysregulation of nitrogen metabolism and immune responses in A. nobilis, particularly in gill. HIGHLIGHTSNitrite exposure caused DEGs in bighead carp gill and liver.DEGs in gill and liver responded to nitrite stress were associated with immune response.Nitrite exposure‐induced pathomechanism associated with immune responses and infections.Gills of bighead carp are more susceptible to nitrite stress than liver.

HSP60 and HSP90β from blunt snout bream, Megalobrama amblycephala : Molecular cloning, characterization, and comparative response to intermittent thermal stress and Aeromonas hydrophila infection

ABSTRACT Heat shock proteins (HSPs) play critical roles in the process of anti‐stress and immunity and are implicated in autoimmune diseases. In order to understand the comparative stress responses of HSP60 and HSP90&bgr; under intermittent thermal stress and Aeromonas hydrophila infection, we cloned their full‐length cDNAs from Megalobrama amblycephala liver, predicted their secondary and tertiary structure, and examined their tissue‐specific expression patterns. The full length of HSP60 and HSP90&bgr; cDNAs indicated that they included all signature sequences of corresponding protein families. They showed high homology to their counterparts in other species, and were consistent with the known classification of fishes based on phylogenetic analysis. HSP60 showed the highest expression in head‐kidney, brain, and gill, while HSP90&bgr; presented higher in hindgut, liver, and brain. Significant mRNA expression differences were determined between HSP60 and HSP90&bgr; in tissues of bladder, liver, heart, and gill. During thermal stress and recovery phase, the highest expression of them were observed at the first recovery for 2d and 1d, respectively. The expression between them were extremely significant difference during the first recovery and second stress period. After A. hydrophila infection, their expressions were extremely significantly upregulated. The significant upregulation and rapid response indicated that they were sensitive to thermal stress and bacterial challenge. This study demonstrated that HSP60 and HSP90&bgr; might participate in innate immune and environmental responses of M. amblycephala. It indicated that they could be used as biomarkers to test the stress caused by local aquaculture environment. HIGHLIGHTSHSP60 and HSP90&bgr; were conversed in revolution and shared similar functions with other known HSP60 and HSP90.HSP60 and HSP90&bgr; exhibited tissue‐specific expression among 9 different tissues.HSP60 and HSP90&bgr; were sensitive to intermittent thermal stress and Aeromonas hydrophila infection.HSP60 and HSP90&bgr; exerted antioxidant and immune effect against various adverse stresses.

Regulation mechanism of oxidative stress induced by high glucose through PI3K/Akt/Nrf2 pathway in juvenile blunt snout bream (Megalobrama amblycephala)

ABSTRACT This study was conducted to investigate the effects of oral administration of a high concentration of glucose on the respiratory burst, antioxidant status, and hepatic gene expression of heme oxygenase‐1 (ho1) and PI3K/Akt/Nrf2‐related signaling molecules in juvenile blunt snout bream (Megalobrama amblycephala). Blunt snout bream juveniles with an initial body weight of 19.94 ± 0.58 g were orally fed with a high concentration of glucose (3 g/kg body weight). The results indicated that plasma glucose exhibited a biphasic response. Acute and persistent hyperglycemia due to the oral glucose administration significantly reduced (P < 0.05) the white blood cell count, red blood cell count, and hemoglobin content and caused oxidative stress (significantly increased alanine aminotransferase, aspartate transaminase, alkaline phosphatase, and glucose levels) and early apoptosis of hepatocytes in the fish. Hepatic superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities increased rapidly (P < 0.05) as protection from oxidative stress and were downregulated (P < 0.05) because of persistent hyperglycemia. Blood respiratory burst was significantly reduced (P < 0.05) because of hyperglycemia and showed a trend that was opposite to that of plasma glucose. Slight upregulation of nrf2 mRNA and antioxidants acts as a compensative protection mechanism, and the downregulated PI3K/Akt pathway blocked this function of Nrf2. In conclusion, the PI3K/Akt pathway and Nrf2 mediated the antioxidative mechanism independently in the blunt snout bream juveniles subjected to the oral administration of a high glucose concentration. HighlightsOral glucose administration affected blood respiratory burst in M. amblycephala.Oral glucose administration induced blood glucose fluctuation and oxidative stress.Upregulation of Nrf2 against oxidative stress injury due to hyperglycemia.Inhibition of PI3K, Akt, and HO‐1 induced inflammation due to hyperglycemia.

Publisher Correction: Dietary arginine affects the insulin signaling pathway, glucose metabolism and lipogenesis in juvenile blunt snout bream Megalobrama amblycephala

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.