Proteomics and metabolomics analysis of American shad (Alosa sapidissima) liver responses to heat stress.

The dramatic changes in the global climate pose a major threat to the survival of many organisms, including fish. To date, the regulatory mechanisms behind the physiological responses of fish to temperature changes have been studied, and a comprehensive analysis of the regulatory mechanisms of temperature tolerance will help to propose effective strategies for fish to cope with global warming. In this study, we investigated the expression profiles of proteins and metabolites in liver tissues of American shad (Alosa sapidissima) corresponding to different water temperatures (24 °C, 27 °C and 30 °C) at various times (1-month intervals) under natural culture conditions. Proteomic analysis showed that the expression levels of the heat shock protein family (e.g. HSPE1, HSP70, HSPA5 and HSPA.1) increase significantly with temperature and that many differentially expressed proteins were highly enriched especially in pathways related to the endoplasmic reticulum, oxidative phosphorylation and glycolysis/gluconeogenesis processes. In addition, the results of conjoint metabolomics and proteomics analysis suggested that the contents of several important amino acids and chemical compounds, including L-serine, L-isoleucine, L-cystine, choline and betaine, changed significantly under high-temperature environmental stress, affecting the metabolic levels of starch, amino acid and glucose, which is thought to represent a possible energy conservation method for A. sapidissima to cope with rapid changes in external temperature. In summary, our findings demonstrate that living under high temperatures for a long period of time leads to different physiological defense responses in A. sapidissima, which provides some new ideas for analyzing the molecular regulatory patterns of adaptation to high temperature and also provides a theoretical basis for the subsequent improvement of fish culture in response to global warming.

Molecular characterization and function of hif1a and fih1 in response to acute thermal stress in American shad (Alosa sapidissima).

In order to evaluate the function of hypoxia-inducible factor-1 alpha (hif1α) and factor inhibiting hif1α (fih1) in response to thermal stress, we first conducted a functional analysis of A. sapidissima hif1α and fih1, and determined hif1α and fih1 expressions in different tissues in response to thermal stress based on identified housekeeping genes (HKGs). The results showed that hif1α and fih1 were mainly located in the nucleus and cytoplasm. The full length cDNA sequence of hif1α and fih1 was 4073 bp and 2759 bp, respectively. The cDNA sequence of hif1α includes 15 exons encoding 750 amino acid residues, and the full length cDNA sequence of fih1 contains 9 exons encoding 354 amino acid residues. During the acute thermal stress transferring from 16 ± 0.5 °C (control) to 20 ± 0.5 °C, 25 ± 0.5 °C, and 30 ± 0.5 °C for 15 min, it was found that the expression trends of hif1α and fih1 showed an inhibitory regulation in the heart, while they consistently expressed in brain, intestine, muscle, gill, kidney and liver. In conclusion, this is the first study to identify the tissue-specific HKGs in A. sapidissima and found that ef1α and ß-actin are the most suitable HKGs. Hif1α and Fih1 are mainly the nuclear and cytoplasmic proteins, respectively, having high levels in the heart and brain. Alosa sapidissima countered a temperature increase from 16 to 25 ℃ by regulating the expressions of hif1α and fih1, but their physiological regulatory functions were unable to cope with acute thermal stress when the temperature difference was 14 ℃ (from 16 to 30 ℃).

Momordica charantia saponins administration in low-protein-high-carbohydrate diet improves growth, blood biochemical, intestinal health and microflora composition of juvenile common carp (Cyprinus carpio).

An 8-week feeding trial was conducted to explore the feasibility of Momordica charantia saponins (MCS) administration to facilitate the protein-sparing action of high carbohydrate in diets for juvenile common carp (Cyprinus carpio) with initial mass of 5.41 ± 0.02 g. Based on our previous study, four diets with different the ratio of protein and carbohydrate (P/C ratio) were designed: 32%P/40%C, 30%P/43%C, 28%P/46%C, 28%P/46%C supplemented with 0.16% MCS (28%P/46%C + MCS). Each diet treatment was divided into 3 replicates. Results revealed that 30%P/43%C group increased growth performance and intestinal digestion, decreased intestinal inflammation, and optimized the intestinal microbiota compared to 32%P/40%C group, which presented the stronger protein-sparing action of high carbohydrate. But if the P/C ratio reduced to 28%P/46%C or less, the saving action would be restrained. However, compared to the 30%P/43%C and 28%P/46%C groups, 28%P/46%C + MCS group significantly elevated growth performance and activities of digestive enzymes and antioxidative enzymes, whilst the opposite trend occurred in the contents of glucose, triglyceride, total cholesterol, low density lipoprotein cholesterol, blood urea nitrogen, glutamic oxalacetic transaminase, glutamic-pyruvic transaminase and malondialdehyde. In addition, 28%P/46%C + MCS group markedly upregulated the expressions of GH/IGF axis genes, genes involved in protein synthesis, antioxidant genes and anti-inflammatory cytokine, whilst the opposite trend occurred in the expressions of pro-inflammatory cytokines. Moreover, 28%P/46%C + MCS group obtained the remarkably higher Enterococcus proportion and lower Lactococcus proportion compared to the 30%P/43%C and 28%P/46%C groups, whereas the opposite occurred in 30%P/43%C group, which indicated that there existed differences in the improvement mechanism on intestinal microflora composition between MCS and appropriate P/C ratio. Combined with the above mentioned changes in our research, we concluded that 0.16% MCS administration in a 28%P/46%C diet could facilitate the protein-sparing action of high carbohydrate in diets for common carp, which could decrease the 5% dosage of soybean meal and synchronously reduce the 4% crude protein of diets without affecting the growth and immune ability for common carp.

Mulberry leaf meal: A potential feed supplement for juvenile Megalobrama amblycephala "Huahai No. 1".

This study was performed to evaluate the potential application of mulberry leaf meal (ML) and fermented mulberry leaf meal (FML) as feed supplements in aquatic animals for developing varieties of practical and economical feed ingredients. Juveniles Megalobrama amblycephala were fed a basal diet (35.7% crude protein, 10.4% crude lipid; control group) supplemented with 2.22% and 4.44% mulberry leaf meals (ML2, ML4) and fermented mulberry leaf meals (FML2, FML4) for 8 weeks. Generally, the two-way ANOVA showed the supplementation level exhibited a prominent effect on the growth performance and physiological status of fish. Furthermore, the two-way ANOVA showed the supplementary fermented mulberry leaf meal increased plasma complement 4 (C4) content (P < 0.05). The weight gain rate (WGR, 145.87%) and the specific growth rate (SGR, 1.63%) were significantly increased in FML2 group compared with the control group (P < 0.05). The muscle crude lipid content and hepatosomatic index (HSI) were higher in FML2 group than that in ML2 group (P < 0.05). The hepatic GSH content in ML4 group and CAT, T-SOD activities in FML4 group were significantly increased compared with the control group (P < 0.05). The hepatic MDA content in FML4 group was significantly decreased compared with the FML2 group (P < 0.05). Total cholesterol (TC) contents showed a significant decrease in ML4 and FML4 groups compared with the control group (P < 0.05). Regarding the gene expression, sirtiun 1 (Sirt1) gene expression was elevated in FML2 group compared with the ML2 group (P < 0.05). Compare to the control group, FML2 diet significantly increased the expression of i-kappa-B alpha (IKBα) gene in liver, and decreased the expression of forkhead box O1 α (FoxO1α), toll-like receptors 4 (TLR4) and nuclear factor-kappa B (NF-κB) genes (P < 0.05). In conclusion, 2.22% FML promoted the growth performance of M. amblycephala and enhanced the anti-inflammatory responses by inhibiting TLR4/NF-κB signaling pathway. On the other hand, 4.44% FML reduced plasma lipid content (hypolipedemic effect) and improved the hepatic antioxidant capacity of M. amblycephala.

Effects of M. oleifera leaf extract on the growth, physiological response and related immune gene expression of crucian carp fingerlings under Aeromonas hydrophila infection.

We evaluated the effect of dietary supplementation with Moringa oleifera leaf extract on the resistance to Aeromonas hydrophila infection in crucian carp. The fish were randomly divided into five groups: the basal diet, the basal diet supplied with 0.25% (0.25 M), 0.5% (0.5 M), 0.75% (0.75 M) and 1.0% M. oleifera leaf extract (1.0 M) for 8 weeks. The growth, antioxidant capabilities, related immune genes as well as resistance to A. hydrophila infection were determined. The results showed that compared with the control group, the weight gain, specific growth rate in the group of 0.5% M. oleifera leaf extract, serum superoxide dismutase (SOD), albumin (ALB) and glutathione peroxidase (GSH-Px), the gene expression of hepatopancreas BTB and CNC homolog 1 (Bach1), NF-E2-related factor 2 (Nrf2), peroxidases (PRX) and NADPH oxidase (NOX) in the group of 0.5%-1.0% M. oleifera leaf extract increased, while feed conversion ratio, serum cortisol, red blood cell (RBC), alanine aminotransferase (ALT), malonaldehyde (MDA) decreased in the group of 0.5%-1.0% M. oleifera leaf extract before the stress. After the infection, the group of 0.5% or 0.75% M. oleifera leaf extract also could improve the serum ALB, hepatopancreas Kelch-like-ECH-associated protein 1 (Keap1), Bach1, Nrf2, TOR, PRX and NOX and reduce cortisol compared with the control group. In summary, this study suggested that 0.5% M. oleifera leaf extract inclusion increased the growth performance, even had positive effects on physiological and immune function, and enhanced resistance against pathogenic infections in crucian carp. The optimum level of M. oleifera leaf extract for crucian carp was estimated to be 0.35%-0.48% based on polynomial comparison with FCR and SGR.

Effects of dietary threonine on growth and immune response of oriental river prawn (Macrobrachium nipponense).

A 70-day feeding trial was conducted to ascertain the effects of threonine on immune response of juvenile oriental river prawn (Macrobrachium nipponense). Six isonitrogen and isolipidic feeds were formulated according to levels of dietary threonine (0.35%, 0.79%, 1.18%, 1.67%, 2.08% and 2.48% respectively). The juvenile prawns were divided into six groups with four replicates, and stocked into 24 tanks with 50 prawns per tank (initial weight 0.20 ± 0.02 g). The results showed a significant increasing trend of final body weight, specific growth rate, protein efficiency ratio, and weight gain rate when threonine levels increased to 1.67% (P < 0.05). However, feed intake, feed conversion ratio, and whole-body lipid composition significantly decreased as threonine levels in the feed increased up to 1.67% (P < 0.05). Moreover, haemolymph N-urea content was significantly lowest at 1.67% threonine level (P < 0.05), whereas glucose was highest at 0.79% followed by 1.67% of threonine levels in the feeds. Aspartate aminotransferase (AST) enzyme activities were significantly decreased by an imbalance (except 1.67%) of threonine in the feed (P < 0.05). Activities of Alanine aminotransferase (ALT) and albumen (ALB) were not significantly affected by threonine in the feed (P > 0.05). Excessive dietary threonine level (2.48%) significantly activated haemolymph catalase (CAT) activity (P < 0.05), whereas malondialdehyde (MDA) content was significantly affected by deficient (0.35% and 0.79%) dietary threonine levels (P < 0.05). Inducible nitric oxide synthase (iNOS) activity and haemolymph complement component 4 (C4) content were significantly decreased by deficient levels of threonine in the feed (P < 0.05). Excess threonine concentration significantly down-regulated Toll, Dorsal, Relish, and heat shock protein 60 (Hsp60) gene expressions in the hepatopancreas of M. nipponense (P < 0.05), while all genes were significantly up-regulated by the optimal (1.67%) threonine level (P < 0.05). The threonine level at which maximum specific growth rate of M. nipponense occurred was estimated by second degree polynomial regression analysis as 1.65% of threonine level, equivalent to 4.44% dry weight bases of protein in the feed.

Molecular cloning and expression mechanism of Mnp65 in Megalobrama amblycephala response to Aeromonas hydrophilia challenge.

p65 is one of the important subunits of the inflammation-related transcription factor NF-κB. In the present study, we cloned and identified the p65 from Megalobrama amblycephala (Mnp65) by homologous cloning and RACE technique. The full-length Mnp65 cDNA consisted of 2331 bp, and included one open reading frame encoding a 604-amino acid putative protein. The protein sequence included a DNA binding motif, a well conserved N-terminal Rel-homology domain (RHD), and a C-terminal IG-like plexins transcription (IPT). Mnp65 was closely related with the other p65 proteins of Cypriniformes and clearly distinct from that of Perciformes and Salmoniformes in terms of sequence homology. Mnp65 homodimer may interact with IκBα in the IPT domain based on the predicted 3D structure of IκBα/Mnp65 complex. Mnp65 was ubiquitously expressed in M. amblycephala tissues, and the highest levels were detected in muscle and liver. Intragastric infection with Aeromonas hydrophila caused respiratory burst and cytokine storm from 8 h to 48 h, showing significantly higher level of respiratory burst activities and significantly high cytokines levels, such as TNF-α, IL-1ß, IL-6, IL-8 etc., compared to 0 h. In addition, the bacterial challenge downregulated the IkBα, and upregulated Mnp65 and TNF-α in the liver. IkBα-Mnp65 was regulated by the negative feedback of cytokine storm, to increase IkBα and decrease Mnp65. Then cytokine storm was relieved at 96 h. Finally, severe intestinal inflammation was observed from 24 h to 48 h after infection, characterized by extensive villous necrosis, epithelial hyperplasia and lymphocyte infiltration, all of which were relieved at 96 h. Taken together, Mnp65 plays a crucial role in the physiological response of teleost fish to bacterial infection.

Molecular cloning and functional characterization of the hypoxia-inducible factor-1α in bighead carp (Aristichthys nobilis).

HIF-l is the earliest documented and most widely studied hypoxia-inducible factor (HIF) and plays a key role in the cell hypoxia signal transduction pathway. Particularly, the HIF-1α protein is sensitive to oxygen and plays a critical role in hypoxia regulation. This study is the first to report on the molecular cloning and characterization of HIF-1α in bighead carp (Aristichthys nobilis; anHIF-1α). The full-length cDNA of anHIF-1α was 2361 bp, and encodes an estimated 674 amino acids with a predicted molecular mass of 76.10 kDa and a theoretical isoelectric point of 7.72. Moreover, the conserved basic Helix-Loop-Helix domain along with two Per-ARNT-Sim domains (A/B), and C-TAD were identified in this protein. Interestingly, the tertiary structure of the anHIF-1α protein was found to be extremely similar to that of mice. Multiple comparison and phylogenetic tree results demonstrated that anHIF-1α was highly conserved. Under normoxic conditions, anHIF-1α mRNA transcripts could be detected in all tissues examined with the highest expression level in the heart. With gradually decreasing oxygen concentrations, anHIF-1α mRNA level was upregulated significantly in the gill, liver, kidney, spleen, intestine, brain, and muscle tissues (P < 0.05). Similarly, anHIF-1α was expressed in all examined bighead carp tissues, and the results suggested that the upregulation of anHIF-1α at the transcriptional level may be an important stress response adaptation to hypoxia in bighead carp. Finally, based on the tertiary structure comparative analyses between anHIF-1α with mouse HIF-1α, we think the physiological function, and protein structure of HIF-1α could be compared between fish and mammal in the future.

HSP60 and HSP90ß from blunt snout bream, Megalobrama amblycephala: Molecular cloning, characterization, and comparative response to intermittent thermal stress and Aeromonas hydrophila infection.

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 HSP90ß 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 HSP90ß 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 HSP90ß presented higher in hindgut, liver, and brain. Significant mRNA expression differences were determined between HSP60 and HSP90ß 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 2 d and 1 d, 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 HSP90ß 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.

High glucose affected respiratory burst activity of peripheral leukocyte via G6PD and NOX inhibition in Megalobrama amblycephala.

High glucose levels are known to impair growth and immune function in fish. Here we investigated the role of glucose-6-phosphate dehydrogenase (G6PD) and NADPH oxidase (NOX) in high glucose-associated impairment of leukocyte respiratory burst activity in Megalobrama amblycephala. We cultured peripheral leukocytes isolated from M. amblycephala with media containing no glucose (non-glucose group), 11.1 mmol/L d-glucose (physiologic glucose group), 22.2 mmol/L d-glucose (high-glucose group), or 11.1 mmol/L d-glucose + 100 µmol/L dehydroepiandrosterone (DHEA) (DHEA-treated group). After 24 h, we assayed production of reactive oxygen species (ROS) as a measure of respiratory burst function as well as activity of G6PD and NOX. The high-glucose group and DHEA-treated group showed significantly reduced respiratory burst function, reduced production of ROS, and reduced G6PD and NOX activity at 24 h, compared to the non-glucose and physiologic glucose groups (P < 0.05). The degree of impairment was similar between high-glucose and DHEA-treated groups (P > 0.05). These findings suggest that reduced NADPH availability likely underlies the suppression of respiratory burst function in M. amblycephala leukocytes exposed to high glucose levels.

Effect of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of bighead carp, Aristichthys nobilis.

Nitrite (NO2-) can cause oxidative stress in aquatic animal when it accumulates in the organism, resulting in different toxic effects on fish. In the present study, we investigated the effects of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of Bighead carp (Aristichthys nobilis). Fish [Initial average weight: (180.05 ±â€¯0.092) g] were exposed to 48.634 mg/L nitrite for 96 h, and a subsequent 96 h for the recovery test. Fish livers were collected to assay antioxidant enzymes activity, hepatic structure and expression of genes after 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h of exposure and12 h, 24 h, 48 h, 72 h, 96 h of recovery. The results showed that the activity of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) increased significantly in the early stages of nitrite exposure. The study also showed that nitrite significantly up-regulated the mRNA levels of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) after 6, 48, and 72 h of exposure respectively. Nitrite also increased the formation of malondialdehyde (MDA), oxidized glutathione (GSSG), and the activity of catalase (CAT). Nitrite was observed to reduce the activity of superoxide dismutase (SOD) and the level of glutathione (GSH). In the recovery test, GSH and the GSSG recovered but did not return to pre-stress levels. The results suggested that the glutathione system played important roles in nitrite-induced oxidative stress in fish. The bighead carp responds to oxidative stress by enhancing the activity of GSH-Px, GST, GR and up-regulating the expression level of GSH-Px, GST, GR, a whilst simultaneously maintaining the dynamic balance of GSH/GSSG. CAT was also indispensable. They could reduce the degree of lipid peroxidation, and ultimately protect the body from oxidative damage.

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 (48.63 mg/L) for 72 h, 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.

In Vivo Analysis of miR-34a Regulated Glucose Metabolism Related Genes in Megalobrama amblycephala.

The Megalobrama amblycephala (M. amblycephala) is one of the most important economic freshwater fish in China. The molecular mechanism under the glucose intolerance responses which affects the growth performance and feed utilization is still confused. miR-34a was reported as a key regulator in the glucose metabolism, but how did the miR-34a exert its function in the metabolism of glucose/insulin in M. amblycephala was still unclear. In this study, we intraperitoneally injected the miR-34a inhibitor (80 nmol/100 g body weight) into M. amblycephala (fed with high starch diet, 45% starch) for 12 h, and then analyzed the gene expression profiling in livers by RNA-seq. The results showed that miR-34a expression in M. amblycephala livers was inhibited by injection of miR-34a inhibitor, and a total of 2212 differentially expressed genes (DEGs) were dysregulated (including 1183 up- and 1029 downregulated DEGs). Function enrichment analysis of DEGs showed that most of them were enriched in the peroxisome proliferator-activated receptor (PPAR), insulin, AMP-activated protein kinase (AMPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways, which were all associated with the glucose/lipid metabolic and biosynthetic processes. In addition, we examined and verified the differential expression levels of some genes involved in AMPK signaling pathway by qRT-PCR. These results demonstrated that the inhibition of miR-34a might regulate glucose metabolism in M. amblycephala through downstream target genes.

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

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.

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).

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 µg/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 µg/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 µg/ml, and inhibited at 25 µg/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 µg/ml, CAT, NOX2 and Nrf2 in the cells treated with 1 µg/ml were sharply increased, respectively. Whereas, high concentrations were dramatically down-regulated the gene expressions of CAT in the cells treated with 5, 25 µg/ml and NOX2 in the cells treated with 25 µg/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.

Identification of Differentially Expressed Micrornas Associate with Glucose Metabolism in Different Organs of Blunt Snout Bream (Megalobrama amblycephala).

Blunt snout bream (Megalobrama amblycephala) is a widely favored herbivorous fish species and is a frequentlyused fish model for studying the metabolism physiology. This study aimed to provide a comprehensive illustration of the mechanisms of a high-starch diet (HSD) induced lipid metabolic disorder by identifying microRNAs (miRNAs) controlled pathways in glucose and lipid metabolism in fish using high-throughput sequencing technologies. Small RNA libraries derived from intestines, livers, and brains of HSD and normal-starch diet (NSD) treated M. amblycephala were sequenced and 79, 124 and 77 differentially expressed miRNAs (DEMs) in intestines, livers, and brains of HSD treated fish were identified, respectively. Bioinformatics analyses showed that these DEMs targeted hundreds of predicted genes were enriched into metabolic pathways and biosynthetic processes, including peroxisome proliferator-activated receptor (PPAR), glycolysis/gluconeogenesis, and insulin signaling pathway. These analyses confirmed that miRNAs play crucial roles in glucose and lipid metabolism related to high wheat starch treatment. These results provide information on further investigation of a DEM-related mechanism dysregulated by a high carbohydrate diet.

The effect of hyperthermia on liver histology, oxidative stress and disease resistance of the Wuchang bream, Megalobrama amblycephala.

This study aimed to investigate the effects of hyperthermia on serum hormones, hepatic oxidization indices, hepatic heat shock protein (HSP60, 70, and 90) mRNA expression levels and liver cell ultrastructure in Megalobrama amblycephala before and after high temperature stress. Fish were exposed to the optimal temperature (25 ± 1 °C) or high temperature (32 ± 1 °C) and then challenged with Aeromonas hydrophila. The results showed that hyperthermic stress significantly increased serum adrenocorticotropic hormone (ACTH) at 0.5 and 2 d, serum cortisol (COR) at 0.5, 14, and 21 d and serum 3,5,3'-triiodothyronine (T3) at 1, 14, and 21 d after stress. Additionally, hyperthermia led to oxidative stress, as evidenced by a significant decrease in the hepatic anti-superoxide anion free radical concentration (ASAFER) at 1, 2, 7, and 21 d and in hepatic superoxide dismutase (SOD) activity at 1, 2, 14 and 21 d after stress; however, hepatic malondialdehyde content (MDA) increased at 1, 2, and 7 d after stress. Moreover, the expression of HSP60 at 1 d, HSP70 at 1 and 2 d, and HSP90 at 0.25, 0.5, 1 and 2 d after stress was higher in the stress group compared with the control group. The histological results clearly showed that hyperthermia resulted in fat and glycogen accumulation and structural alterations of the hepatocytes, mitochondria, and nuclei. The cumulative mortality increased in the high temperature stress group at 1 d after acute stress and at 2 and 7 d after chronic stress compared with the control group. Overall, 1 d or 2 d after hyperthermia stress damaged the hepatic ultrastructure and impaired mitochondrial bioenergetics. Dysfunction of the mitochondria subsequently mediated oxidative stress and improved HSP expression modulated the cellular anti-stress response, which in turn led to reduced efficacy of the immune system and increased mortality from Aeromonas hydrophila infection in Megalobrama amblycephala.

Chronic stress effects of high doses of vitamin D3 on Megalobrama amblycephala.

Dietary vitamin D3 plays an important role in the growth of aquatic animals, but long-term excessive feeding has potential hazards. In this study, Megalobrama amblycephala specimens were fed different experimental diets with 2000 IU/kg or 200,000 IU/kg of vitamin D3 for 90 days, in order to evaluate chronic stress effects of high doses of vitamin D3 on growth, immunity, and structural damage to enterohepatic tissues. The results showed that high doses of vitamin D3 did not have a significant influence on the growth performance of M. amblycephala (P > 0.05), but it significantly reduced the survival rate after infection by Aeromonas hydrophila (P < 0.05). Serum albumin, alkaline phosphatase, and insulin levels, as well as hepatic total antioxidant capacity, were also significantly reduced (P < 0.05). Serum cortisol levels and hepatic heat stress protein 70 expression in M. amblycephala showed that high doses of vitamin D3 significantly inhibit the anti-stress ability of M. amblycephala (P < 0.05). Paraffin tissue sections and electron microscopy showed that high doses of vitamin D3 could cause different degrees of structural damage to enterohepatic tissues of M. amblycephala. Our results indicate that, although M. amblycephala can tolerate high doses of dietary vitamin D3 over a long period, its glycolipid metabolism, immune function, anti-stress function, and resistance to pathogenic infections are adversely affected.

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.