The P4' Peptide-Carrying Bacillus subtilis in Cottonseed Meal Improves the Chinese Mitten Crab Eriocheir sinensis Innate Immunity, Redox Defense, and Growth Performance.

This study developed a recombinant Bacillus subtilis to carry the LGSPDVIVIR peptide (cmP4) isolated from the hydrolyzed products of cottonseed meal with excellent antioxidant and immune-enhancing properties in vitro. It was carried as a tandem of five cmP4 peptides (cmP4') to be stably expressed on a large scale. Then, its effectiveness was evaluated in Chinese mitten crab (Eriocheir sinensis) based on growth performance, redox defense, and innate immunity. A total of 280 crabs (mean body weight: 41.40 ± 0.14) were randomly assigned to seven diets including a control one (without B. subtilis) and six experimental ones with different doses (107,108, and 109 CFU/kg) of unmodified and recombinant B. subtilis, respectively, for 12 weeks. Each diet was tested in four tanks of crabs (10/tank). In terms of bacterial dosages, the final weight (FW), weight gain (WG), hemolymph and hepatopancreatic activities of superoxide dismutase (SOD), catalase (CAT), lysosome (LZM), acid phosphatase (ACP) and alkaline phosphatase (AKP), and hepatopancreatic transcriptions of cat, mitochondrial manganese superoxide dismutase (mtmnsod), thioredoxin-1 (trx1), and prophenoloxidase (propo) all increased significantly with increasing B. subtilis dosages, while hemolymph and hepatopancreatic malondialdehyde (MDA) content and the transcriptions of toll like receptors (tlrs), NF-κB-like transcription factor (relish), and lipopolysaccharide-induced TNF-α factor (litaf) all decreased remarkably. In terms of bacterial species, the recombinant B. subtilis group obtained significantly high values of FW, WG, hemolymph, and hepatopancreatic activities of SOD, CAT, LZM, ACP, and AKP, and the transcriptions of mtmnsod, peroxiredoxin 6 (prx6), and propo compared with the unmodified B. subtilis, while opposite results were noted in hemolymph and hepatopancreatic MDA content and the transcriptions of tlrs, relish, and litaf. These results indicated that dietary supplementation with 109 CFU/kg of recombinant B. subtilis can improve the growth performance, redox defense, and nonspecific immunity of E. sinensis.

Effects of a high-fat and high-carbohydrate diet on appetite regulation and central AMPK in the hypothalamus of blunt snout bream (Megalobrama amblycephala).

Adenosine monophosphate-activated protein kinase (AMPK) is a sensor of cellular energy changes and controls food intake. This study investigates the effect of a high-calorie diet (high fat diet [HFD], high carbohydrate diet [HCD] and high energy diet [HED]) on appetite and central AMPK in blunt snout bream. In the present study, fish (average initial weight 45.84 ± 0.07 g) were fed the control, HFD, HCD and HED in four replicates for 12 weeks. At the end of the feeding trial, the result showed that body mass index, specific growth rate, feed efficiency ratio and feed intake were not affected (p > 0.05) by dietary treatment. However, fish fed the HFD obtained a significantly higher (p < 0.05) lipid productive value, lipid gain and lipid intake than those fed the control diet, but no significant difference was attributed to others. Also, a significantly higher (p < 0.05) energy intake content was found in fish-fed HFD, HCD and HED than those given the control diet. Long-term HFD and HCD feeding significantly increased (p < 0.05) plasma glucose, glycated serum protein, advanced glycation end product, insulin and leptin content levels than the control group. Moreover, a significantly lower (p < 0.05) complex 1, 2 and 3 content was found in fish-fed HFD and HCD than in the control, but no differences (p > 0.05) were attributed to those in HED. Fish-fed HED significantly upregulated (p < 0.05) hypothalamic ampα 1 and ampα 2 expression, whereas the opposite trend was observed in the hypothalamic mammalian target of rapamycin than those in HFD and HCD compared to the control. However, hypothalamic neuropeptide y, peroxisome proliferator-activated receptor α (pparα), acetyl-coa oxidase and carnitine palmitoyltransferase 1 were significantly upregulated (p < 0.05) in the HCD group, while the opposite was seen in cholecystokinin expression compared to those in the control group. Our findings indicated that the central AMPK signal pathway and appetite were modulated according to the diet's energy level to regulate nutritional status and maintain energy homoeostasis in fish.

Establishment of an Efficient Expression and Regulation System in Streptomyces for Economical and High-Level Production of the Natural Blue Pigment Indigoidine.

Indigoidine, as a kind of natural blue pigment, is widely used in textiles, food, and pharmaceuticals and is mainly synthesized from l-glutamine via a condensation reaction by indigoidine synthetases, most of which originates from Streptomyces species. However, due to the complex metabolic switches of Streptomyces, most of the researchers choose to overexpress indigoidine synthetases in the heterologous host to achieve high-level production of indigoidine. Considering the advantages of low-cost culture medium and simple culture conditions during the large-scale culture of Streptomyces, here, an updated regulation system derived from the Streptomyces self-sustaining system, constructed in our previous study, was established for the highly efficient production of indigoidine in Streptomyces lividans TK24. The updated system was constructed via promoter mining and σhrdB expression optimization, and this system was applied to precisely and continuously regulate the expression of indigoidine synthetase IndC derived from Streptomyces albus J1704. Finally, the engineered strain was cultured with cheap industrial glycerol as a supplementary carbon source, and 14.3 and 46.27 g/L indigoidine could be achieved in a flask and a 4 L fermentor, respectively, reaching the highest level of microbial synthesis of indigoidine. This study will lay a foundation for the industrial application of Streptomyces cell factories to produce indigoidine.

Advances in regulating vitamin K2 production through metabolic engineering strategies.

Vitamin K2 (menaquinone, VK2, MK) is an essential lipid-soluble vitamin that plays critical roles in inhibiting cell ferroptosis, improving blood clotting, and preventing osteoporosis. The increased global demand for VK2 has inspired interest in novel production strategies. In this review, various novel metabolic regulation strategies, including static and dynamic metabolic regulation, are summarized and discussed. Furthermore, the advantages and disadvantages of both strategies are analyzed in-depth to highlight the bottlenecks facing microbial VK2 production on an industrial scale. Finally, advanced metabolic engineering biotechnology for future microbial VK2 production will also be discussed. In summary, this review provides in-depth information and offers an outlook on metabolic engineering strategies for VK2 production.

High-fat diet alters intestinal microbiota and induces endoplasmic reticulum stress via the activation of apoptosis and inflammation in blunt snout bream.

The primary organ for absorbing dietary fat is the gut. High dietary lipid intake negatively affects health and absorption by causing fat deposition in the intestine. This research explores the effect of a high-fat diet (HFD) on intestinal microbiota and its connections with endoplasmic reticulum stress and inflammation. 60 fish (average weight: 45.84 ± 0.07 g) were randomly fed a control diet (6% fat) and a high-fat diet (12 % fat) in four replicates for 12 weeks. From the result, hepatosomatic index (HSI), Visceralsomatic index (VSI), abdominal fat (ADF), Intestosomatic index (ISI), mesenteric fat (MFI), Triglycerides (TG), total cholesterol (TC), non-esterified fatty acid (NEFA) content were substantially greater on HFD compared to the control diet. Moreover, fish provided the HFD significantly obtained lower superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities. In contrast, an opposite result was seen in malondialdehyde (MDA) content in comparison to the control. HFD significantly altered intestinal microbiota in blunt snout bream, characterized by an increased abundance of Aeromonas, Plesiomonas proteobacteria, and firmicutes with a reduced abundance of Cetobacterium and ZOR0006. The transcriptional levels of glucose-regulated protein 78 (grp78), inositol requiring enzyme 1 (ire1), spliced X box-binding protein 1 (xbp1), DnaJ heat shock protein family (Hsp40) member B9 (dnajb9), tumor necrosis factor alpha (tnf-α), nuclear factor-kappa B (nf-κb), monocyte chemoattractant protein-1 (mcp-1), and interleukin-6 (il-6) in the intestine were markedly upregulated in fish fed HFD than the control group. Also, the outcome was similar in bax, caspases-3, and caspases-9, ZO-1, Occludin-1, and Occludin-2 expressions. In conclusion, HFD could alter microbiota and facilitate chronic inflammatory signals via activating endoplasmic reticulum stress.

Oral bovine serum albumin administration alleviates inflammatory signals and improves antioxidant capacity and immune response under thioacetamide stress in blunt snout bream fed a high-calorie diet.

This investigation looks at the impact of oral bovine serum albumin (BSA) on antioxidants, immune responses, and inflammation signals in blunt snout bream fed a high-calorie diet. 480 fish (average weight: 45.84 ± 0.07 g) were randomly fed a control diet, a high-fat diet (HFD), a high carbohydrate diet (HCD), and a high-energy diet (HED) in six replicates for 12 weeks. After the feeding trial, fish were orally administered with 10% BSA for 10 h, then blood and liver samples from five fish were randomly obtained after 10 h to determine plasma inflammatory markers and inorganic components. Also, the leftover fish were injected with thioacetamide, blood and liver samples were simultaneously obtained at 12, 48, and 96 h, respectively, to determine antioxidant, immune, and inflammatory signals, with survival rates recorded at the same time interval. After 10 h, plasma inflammatory markers such as tumour necrosis factors (TNF-α), interleukin 6 (IL6), nitric oxide (NO), Monocyte chemoattractant protein-1(MCP-1), and cortisol were significantly improved in fish fed HCD and HED as compared to the control. After thioacetamide stress, plasma lysozyme (LYM), complement 3, myeloperoxidase (MPO), and alkaline phosphatase activities, as well as immunoglobulin M, levels all increased significantly (P < 0.05) with increasing time with maximum value attained at 96 h, but shows no difference among dietary treatment. Similar results were observed in liver superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities and malondialdehyde (MDA) content, but tended to reduce at 96 h. nf-kb, tnf-α, and mcp-1 tend to decrease with the minimum value attained at 48 h and gradually decrease with increasing time at 96 h. After 96 h of the thioacetamide (TAA) challenge, the survival rate of blunt snout bream fed with an HFD and HCD was significantly lower (P < 0.05) at 48, and 96 h before the administration of BSA. However, no differences were observed among dietary treatments after the BSA administration. Overall, this study indicated that oral dietary administration of BSA might greatly enhance the antioxidant capability and innate immunity and mitigates inflammation signals after TAA stress in blunt snout bream fed high energy diet.

Cottonseed Meal Protein Hydrolysate Improves the Growth Performance of Chinese Mitten Crab (Eriocheir sinensis) by Promoting the Muscle Growth and Molting Performance.

Growth retardation and prolonged marketing cycle have been noticed in the practical aquaculture of Chinese mitten crab (Eriocheir sinensis) fed with artificial feed. Plant protein hydrolysates contain a large number of small peptides and free amino acids, which can improve the growth performance of aquatic animals. However, the potential mechanisms are still not well elucidated. In this research, the influences of cottonseed meal protein hydrolysate (CPH) on the growth, feed utilization, muscle growth, and molting performance were investigated in E. sinensis. A total of 240 crabs (mean body weight 37.32 ± 0.38 g) were individually randomly distributed to six diets supplemented with 0%, 0.2%, 0.4%, 0.8%, 1.6%, and 3.2% of CPH for 12 weeks. These findings indicated that the addition of CPH at 0.4% significantly increased the survival rate, body protein gain, apparent protein utilization, trypsin and pepsin activities, and the methyl farnesoate content. When the dose reached 0.8%, the weight growth ratio, meat yield, ecdysone concentration, and the transcription of the ecdysteroid receptor all significantly increased, while the transcriptions of both myostatin and molt-inhibiting hormone significantly decreased. When CPH was added at 1.6%-3.2%, the feed conversion ratio, body crude protein content, Na+/K+-ATPase activity, and the molting ratio were all significantly improved, while the opposite was true for the transcription of the transforming growth factor-ß type I receptor. The investigation results indicated that when added above 0.4%, CPH could stimulate the growth performance of E. sinensis and promote the muscle growth and molting performance.

Interactive effects of dietary leucine and isoleucine affect amino acid profile and metabolism through AKT/TOR signaling pathways in blunt snout bream (Megalobrama amblycephala).

The purpose of this research is to explore the interaction between dietary leucine and isoleucine levels on whole-body composition, plasma and liver biochemical indexes, amino acids deposition in the liver, and amino acid metabolism of blunt snout bream (Megalobrama amblycephala). The test fish (average weight: 56.00 ± 0.55 g) were fed one of six diets at random containing two leucine levels (1.70% and 2.50%) and three isoleucine levels (1.00%, 1.20%, and 1.40%) for 8 weeks. The results showed that the final weight and weight gain rate were the highest in the fish fed low-level leucine and high-level isoleucine diets (P > 0.05). Furthermore, the crude lipid content was significantly adjusted by diets with diverse levels of leucine and isoleucine (P < 0.05). In addition, interactive effects of these two branched-chain amino acids (BCAAs) were found on plasma total protein, blood ammonia, and blood urea nitrogen of test fish (P < 0.05). Additionally, the liver amino acid profiles were significantly influenced by the interactive effects of the two BCAAs (P < 0.05). Moreover, interactive effects of dietary leucine and isoleucine were significantly observed in the expressions of amino acid metabolism-related genes (P < 0.05). These findings suggested that dietary leucine and isoleucine had interaction. Meanwhile, the interaction between them was more conducive to the growth and quality improvement of blunt snout bream when the dietary leucine level was 1.70% and isoleucine level was 1.40%.

Feed types affect the growth, nutrient utilization, digestive capabilities, and endocrine functions of Megalobrama amblycephala: a comparative study between pelleted and extruded feed.

Nowadays, both pelleted feed (PF) and extruded feed (EF) have been widely adopted in the aquaculture industry. However, limited information is available comparing their utilization efficiencies and meanwhile interpreting the underlying mechanisms. This study aimed to compare the utilization efficiencies of both PF and EF by blunt snout bream (Megalobrama amblycephala) based on growth performance, digestive capacities, and endocrine functions. Two feeds with identical formulas were prepared and named PF and EF. Fish were randomly distributed into two groups, including one that fed the PF continuously, and one that offered the EF continuously. The whole feeding trail lasted 8 weeks. The results showed that the protein efficiency (PER), retention of nitrogen and energy (NRE and ERE), viscera index (VSI), apparent digestibility of dry matter, protein, carbohydrate, and gross energy, whole-body crude protein and energy contents, intestinal enzymatic activities of protease, amylase, and Na+,K+-ATPase, intestinal villi length, crypt depth, muscular layer thickness, and the transcriptions of leptin (LEP) and cholecystokinin (CCK) of the EF group were all significantly higher than those of the PF group, while the opposite was true for feed intake and feed conversion ratio. These findings suggested that compared with PF, EF could improve the feed utilization and nutrient retention of blunt snout bream by enhancing the intestinal digestive and absorptive functions but reduce the feed intake through the stimulation of both LEP and CCK.

Lipid metabolism, immune and apoptosis transcriptomic responses of the hepatopancreas of Chinese mitten crab to the exposure to microcystin-LR.

Global warming is favouring the incidence, intensity and duration of harmful cyanobacterial blooms. Microcystin-LR (MC-LR), a hepatotoxic agent, is produced during cyanobacterial blooms. To understand the molecular mechanisms of acute hepatotoxic effect of low doses of MC-LR in crab, we examined differentially expressed genes in samples of the hepatopancreas of Chinese mitten crab (Eriocheir sinensis) collected in 48 h after injections of MC-LR at doses of 0, 25, 50, and 75 µg/kg. The results revealed that MC-LR induced changes in corresponding gene led to the accumulation of triglycerides. MC-LR exposure affected sterol metabolism. Apoptosis-related genes such as Fas-L, Bcl-XL, Cytc, AiF, p53, PERK, calpain, CASP2, CASP7, α-tubulin, PARP, GF, G12, and PKC were upregulated. Conversely, expression levels of CASP10 and ASK1 were downregulated. Genes related to the regulation of actin cytoskeleton (Rho, ROCK, MLCP, MLC, PAK, and PFN) were upregulated. Further, expression levels of genes encoding fatty acid elongation-related enzymes were upregulated, but the expression of genes related to fatty acid synthesis was slightly down regulated. Taken together, these results demonstrated the hepatic toxicity and molecular mechanisms of changes in lipid metabolism, immune and apoptosis in Chinese mitten crab under the MC-LR-induced stress, which is the first report on crabs and performs a comprehensive analysis and a new insight of the molecular toxicological responses in crabs.

The Mechanism of Lipopolysaccharide Escaping the Intestinal Barrier in Megalobrama amblycephala Fed a High-Fat Diet.

With the popularity of western food characterized by excessive fat and sugars, obesity has currently been a public health issue. Low-grade chronic inflammation accompanied by obesity increases the risk of multiple epidemics such as diabetes, cancer and cardiovascular diseases. Here, we show that feeding Megalobrama amblycephala with a high-fat diet (HFD) drives obesity-related chronic inflammation and the penetration of lipopolysaccharide (LPS). Interference with antibiotics inhibits the produce of LPS and this alleviates the sustained release of pro-inflammatory factors induced by HFD. LPS penetration is attributed to weakened intestinal mucus barrier after high-fat exposure. Mechanically, the consumption of HFD inhibits the secretion of mucin 2 (MUC2) due to the induction of endoplasmic reticulum stress mediated by the inositol-requiring enzyme 1 (IRE1) /X box-binding protein 1 (XBP1) pathway in goblet cells. Furthermore, excessive lipid exacerbates the leakage of LPS across the intestinal epithelial cell barrier via the transcellular pathway. Mechanically, lipid increases the internalization of LPS in intestinal epithelial cells depending on the activation of fatty acid translocase (FAT/CD36). These results demonstrate that HFD causes the penetration of LPS due to the weakened intestinal mucosal barrier and the assistance of CD36.

Molecular characterization of farnesoid X receptor alpha in Megalobrama amblycephala and its potential roles in high-carbohydrate diet-induced alterations of bile acid metabolism.

Farnesoid X receptorα (FXRα) plays a central role in maintaining the bile acid homeostasis in mammals, while relevant processes are still poorly interpreted in aquatic species. This study was conducted to characterize the fxrα gene in a cyprinidae species: blunt snout bream (Megalobrama amblycephala), and investigate its potential roles in bile acid metabolism. The Fxrα protein contains one DNA binding domain, one ligand binding domain, one His-Try "switch" and two modifies residues. A high degree of conservation (53.18-100.00 %) was observed in the Fxrα protein among most aquatic species and higher vertebrates. The transcription of fxrα was mainly observed in intestine, liver and kidney. Then fish (35.0 ± 0.15 g) were fed two diets containing 33 % and 45 % carbohydrate levels for 12weeks. High-carbohydrate diet significantly elevated the total cholesterol concentrations in plasma, liver and hindgut as well as the triglyceride concentrations in both liver and hindgut, but decreased the total bile acid concentrations in plasma, liver and hindgut. High dietary carbohydrate levels also significantly enhanced hepatic transcriptions of 3-hydroxy-3-methylglutaryl-CoA reductase (the rate-limiting enzyme in cholesterol synthesis), and those of fxrα (a bile acid receptor) and multidrug resistance associated protein 2 (a bile acid transporter) in hindgut. Furthermore, high dietary carbohydrate levels significantly decreased the transcriptions of cholesterol 7α-hydroxylase (the rate-limiting enzyme in bile acid synthesis) and organic anion-transporting polypeptides (a bile acid transporter) in liver as well as that of takeda G-protein-coupled bile acid receptor in hindgut. The results demonstrated that the fxrα gene of blunt snout bream is highly conserved compared with other vertebrates. Besides, high dietary carbohydrate levels increased total cholesterol concentrations, and up-regulated the transcription of fxrα, thus decreasing the biosynthesis and reabsorption of bile acids by mediating various target genes.

Interactions between dietary carbohydrate and thiamine: implications on the growth performance and intestinal mitochondrial biogenesis and function of Megalobrama amblycephala.

A12-week experiment was conducted to evaluate the influences of thiamine ongrowth performance, and intestinal mitochondrial biogenesis and function of Megalobramaamblycephala fed a high-carbohydrate (HC) diet. Fish (24·73 (sem 0·45) g) were randomly assigned to one of four diets: two carbohydrate (CHO) levels (30 and 45 %) and two thiamine levels (0 and 1·5 mg/kg). HC diets significantly decreased DGC, GRMBW, FIMBW, intestinal activities of amylase, lipase, Na+, K+-ATPase, CK, complexes I, III and IV, intestinal ML, number of mitochondrial per field, ΔΨm, the P-AMPK: T-AMPK ratio, PGC-1ß protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1ß, mitochondrial transcription factor A, Opa-1, ND-1 and COX-1 and 2, while the opposite was true for ATP, AMP and reactive oxygen species, and the transcriptions of dynamin-related protein-1, fission-1 and mitochondrial fission factor. Dietarythiamine concentrations significantly increased DGC, GRMBW, intestinal activities of amylase, Na+, K+-ATPase, CK, complexes I and IV, intestinal ML, number of mitochondrial per field, ΔΨm, the P-AMPK:T-AMPK ratio, PGC-1ß protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1ß, Opa-1, ND-1, COX-1 and 2, SGLT-1 and GLUT-2. Furthermore, a significant interaction between dietary CHO and thiamine was observed in DGC, GRMBW, intestinal activities of amylase, CK, complexes I and IV, ΔΨm, the AMP:ATP ratio, the P-AMPK:T-AMPK ratio, PGC-1ß protein expression as well as the transcriptions of AMPKα1, AMPKα2, PGC-1ß, Opa-1, COX-1 and 2, SGLT-1 and GLUT-2. Overall, thiamine supplementation improved growth performance, and intestinal mitochondrial biogenesis and function of M. amblycephala fed HC diets.

Effects of dietary xylooligosaccharide prebiotic supplementation on growth, antioxidant and intestinal immune-related genes expression in common carp Cyprinus carpio fed a high-fat diet.

This study investigated the effects of xylooligosaccharide (XOS) supplementation on growth, intestinal enzyme, antioxidant and immune-related genes in common carp Cyprinus carpio fed a high-fat diet (HFD). One hundred and ninety two fish with an initial weight of 19.61 ± 0.96 g were allocated into 24 tanks (eight fish per tank in four replicate) and were fed the control diet, HFD, HFD with 0.5%, 1%, 2% and 3% XOS supplementation. From the result, fish offered HFD with 1% XOS supplementation significantly obtained a higher body mass index and feed efficiency ratio, whereas condition factor was higher in fish fed HFD supplemented with 2% XOS but no difference was attributed to other supplemented group compared to control group. Also, fish fed HFD supplemented with 1%-2% XOS significantly improved protease, lipase, creatine kinase and sodium/potassium ATPase activities compared to other groups. Fish offered HFD were significantly lower in superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), myeloperoxidase, acid phosphatase, lysozyme activities and immunoglobulin content, but the opposite result was found for aspartate transaminase, alanine transaminase activities, malondialdehyde, protein carbonyl and cortisol content as compared with the control. However, this effect was reversed with HFD supplemented with XOS. Also, interleukin 1ß, interleukin 8, tumour necrosis factors, interferons, caspase-3 and caspase-9 in the intestine were all up-regulated in the HFD group, while the reverse pattern was found in SOD, GPX, lysozyme-C, complement 3 and mucin 5b (muc5b), than the control group. These effects were all enhanced by feeding the XOS diet, especially those fed 1%-3% supplementation. In conclusion, XOS inclusion can improve the growth, digestive enzymes, antioxidants and immune response of common carp fed HFD.

The ß-1,3-glucan synthase gene GFGLS2 plays major roles in mycelial growth and polysaccharide synthesis in Grifola frondosa.

ß-1,3-Glucans are well-known biological and health-promoting compounds in edible fungi. Our previous results characterized a glucan synthase gene (GFGLS) of Grifola frondosa for the first time to understand its role in mycelial growth and glucan biosynthesis. In the present study, we identified and functionally reannotated another glucan synthase gene, GFGLS2, based on our previous results. GFGLS2 had a full sequence of 5944 bp including 11 introns and 12 exons and a coding information for 1713 amino acids of a lower molecular weight (195.2 kDa) protein with different conserved domain sites than GFGLS (5927 bp with also 11 introns and a coding information for 1781 aa). Three dual-promoter RNA-silencing vectors, pAN7-iGFGLS-dual, pAN7-iGFGLS2-dual, and pAN7-CiGFGLS-dual, were constructed to downregulate GFGLS, GFGLS2, and GFGLS/GFGLS2 expression by targeting their unique exon sequence or conserved functional sequences. Silencing GFGLS2 resulted in higher downregulation efficiency than silencing GFGLS. Cosilencing GFGLS and GFGLS2 had a synergistic downregulation effect, with slower mycelial growth and glucan production by G. frondosa. These findings indicated that GFGLS2 plays major roles in mycelial growth and polysaccharide synthesis and provides a reference to understand the biosynthesis pathway of mushroom polysaccharides. KEY POINTS: • The 5944-bp glucan synthase gene GFGLS2 of G. frondosa was cloned and reannotated • GFGLS2 showed identity and significant differences with the previously identified GFGLS • GFGLS2 played a major role in fermentation and glucan biosynthesis.

Dietary Leucine Supplementation Improves Muscle Fiber Growth and Development by Activating AMPK/Sirt1 Pathway in Blunt Snout Bream (Megalobrama amblycephala).

This research is aimed at evaluating the effects of leucine supplementation on muscle fibers growth and development of blunt snout bream through a feeding trial and a primary muscle cells treatment. An 8-week trial with diets containing 1.61% leucine (LL) or 2.15% leucine (HL) was conducted in blunt snout bream (mean initial weight = 56.56 ± 0.83 g). Results demonstrated that the specific gain rate and the condition factor of fish in the HL group were the highest. The essential amino acids content of fish fed HL diets was significantly higher than that fed LL diets. The texture (hardness, springiness, resilience, and chewiness), the small-sized fiber ratio, fibers density, and sarcomere lengths in fish all obtained the highest in the HL group. Additionally, the proteins expression related with the activation of the AMPK pathway (p-Ampk, Ampk, p-Ampk/Ampk, and Sirt1) and the expression of genes (myogenin (myog), myogenic regulatory factor 4 (mrf4) and myoblast determination protein (myod), and protein (Pax7) related to muscle fiber formation were significantly upregulated with increasing level of dietary leucine. In vitro, the muscle cells were treated with 0, 40 and 160 mg/L leucine for 24 h. The results showed that treated with 40 mg/L leucine significantly raised the protein expressions of BCKDHA, Ampk, p-Ampk, p-Ampk/Ampk, Sirt1, and Pax7 and the gene expressions of myog, mrf4, and myogenic factor 5 (myf5) in muscle cells. In summary, leucine supplementation promoted muscle fibers growth and development, which may be related to the activation of BCKDH and AMPK.

Optimal Niacin Requirement of Oriental River Prawn Macrobrachium nipponense as Determined by Growth, Energy Sensing, and Glycolipid Metabolism.

Niacin is indispensable for the growth and development of aquatic animals. However, the correlations between dietary niacin supplementations and the intermediary metabolism of crustaceans are still poorly elucidated. This study explored the effects of different dietary niacin levels on the growth, feed utilization, energy sensing, and glycolipid metabolism of oriental river prawn Macrobrachium nipponense. Prawns were fed with different experimental diets containing graded niacin levels (15.75, 37.62, 56.62, 97.78, 176.32, and 339.28 mg/kg, respectively) for 8 weeks. Weight gain, protein efficiency, feed intake, and hepatopancreas niacin contents all maximized in the 176.32 mg/kg group with significance noted with the control group (P <0.05), whereas the opposite was true for feed conversion ratio. Hepatopancreas niacin concentrations increased significantly (P < 0.05) as dietary niacin levels increased, and peaked at the 339.28 mg/kg group. Hemolymph glucose, total cholesterol, and triglyceride concentrations all maximized in the 37.62 mg/kg group, while total protein concentration reached the highest value in the 176.32 mg/kg group. The hepatopancreas mRNA expression of AMP-activated protein kinase α and sirtuin 1 peaked at the 97.78 and 56.62 mg/kg group, respectively, and then both decreased as dietary niacin levels increased furtherly (P < 0.05). Hepatopancreas transcriptions of the genes related to glucose transportation, glycolysis, glycogenesis, and lipogenesis all increased with increasing niacin levels up to 176.32 mg/kg, but decreased significantly (P < 0.05) as dietary niacin levels increased furtherly. However, the transcriptions of the genes related to gluconeogenesis and fatty acid ß-oxidation all decreased significantly (P < 0.05) as dietary niacin levels increased. Collectively, the optimum dietary niacin demand of oriental river prawn is 168.01-169.08 mg/kg. In addition, appropriate doses of niacin promoted the energy-sensing capability and glycolipid metabolism of this species.

High-Carbohydrate Diet Alleviates the Oxidative Stress, Inflammation and Apoptosis of Megalobrama amblycephala Following Dietary Exposure to Silver Nanoparticles.

A 12-week feeding trial was performed to evaluate the effects of high-carbohydrate diet on oxidative stress, inflammation and apoptosis induced by silver nanoparticles (Ag-NPs) in M. amblycephala. Fish (20.12 ± 0.85 g) were randomly fed four diets (one control diet (C, 30% carbohydrate), one control diet supplemented with 100 mg kg-1 Ag-NPs (CS), one high-carbohydrate diet (HC, 45% carbohydrate) and one HC diet supplemented with 100 mg kg-1 Ag-NPs (HCS)). The results indicated that weight gain rate (WGR), specific growth rate (SGR), antioxidant enzyme (SOD and CAT) activities and expression of Trx, Cu/Zn-SOD, Mn-SOD, CAT and GPx1 of fish fed CS diet were all remarkably lower than those of other groups, whereas the opposite was true for plasma IL 1ß and IL 6 levels, liver ROS contents, hepatocytes apoptotic rate, AMP/ATP ratio, AMPKα, P 53 and caspase 3 protein contents and mRNA levels of AMPKα 1, AMPKα 2, TXNIP, NF-κB, TNFα, IL 1ß, IL 6, P 53, Bax and caspase 3. However, high-carbohydrate diet remarkably increased WGR, SGR, liver SOD and CAT activities, AMPKα protein content and mRNA levels of antioxidant genes (Cu/Zn-SOD, Mn-SOD, CAT and GPx1), anti-inflammatory cytokines (IL 10) and anti-apoptotic genes (Bcl 2) of fish facing Ag-NPs compared with the CS group, while the opposite was true for liver ROS contents, hepatocytes apoptotic rate, P 53 and caspase 3 protein contents, as well as mRNA levels of TXNIP, NF-κB, TNFα, IL 1ß, IL 6, P 53, Bax and caspase 3. Overall, high-carbohydrate diet could attenuate Ag-NPs-induced hepatic oxidative stress, inflammation and apoptosis of M. amblycephala through AMPK activation.

Molecular characterization of thioredoxin-interacting protein (TXNIP) from Megalobrama amblycephala and its potential roles in high glucose-induced inflammatory response.

This study aimed to characterize the full-length cDNA of thioredoxin-interacting protein (TXNIP) from Megalobrama amblycephala, and investigate its roles in high glucose (HC)-induced inflammatory response. The cDNA obtained covered 2706-bp with an open reading frame of 1203-bp encoding 400 amino acids, compared to Cyprinus carpio, it showed 89.96% homology. The highest expression of txnip was observed in head kidney followed by spleen and liver. After a 12-week feeding trial, high-carbohydrate diet remarkably increased txnip expression in liver and white muscle. Glucose administration resulted in a remarkably increased liver txnip expression, which peaked at 1 h. Thereafter, the expression decreased remarkably to the basal value at 12 h. However, insulin injection resulted in a significant decrease in txnip expression with minimum values attained at 2 h. Subsequently, it gradually increased to the normal values. Moreover, in the in-vitro study, over-expression of txnip along with remarkably increased il-1ß and il-6 expression in hepatocytes, and its knockdown led to remarkably reduced il-1ß expression. Furthermore, metformin treatment remarkably increased the cell viability and trx expression of hepatocytes under high glucose, while the opposite was true for ROS levels, LDH activity, the ALT/AST ratio, Txnip protein content and the transcriptions of txnip, tnfα and il-1ß.

Utilization of pelleted and extruded feed by blunt snout bream Megalobrama amblycephala: Insights from growth performance, health status and feed cost.

The present study was conducted to evaluate the utilization of both pelleted feed (PF) and extruded feed (EF) by blunt snout bream Megalobrama amblycephala based on growth performance, stress responses, innate immunity and disease resistance. Both the PF and EF were prepared with the same formula. Fish were divided randomly into 2 groups, including one fed the PF continuously and one offered the EF continuously. The whole feeding trial lasted 8 weeks, after which fish were subjected to Aeromonas hydrophila infection. The results showed that the feed intake, feed conversion ratio, hepatic total superoxide dismutase activity and glutathione content, plasma complement 3 and complement 4 levels as well as myeloperoxidase activity of the EF group were all significantly lower than those of the PF group, while the opposite was true for the condition factor, the viscera index, the abdominal fat percentage, nitrogen and energy retention, hepatic malondialdehyde content, plasma levels of cortisol, glucose, lactate, total protein and globulin as well as the activities of plasma alanine aminotransferase and aspartate aminotransferase. In addition, the EF group also obtained relatively low activities of hepatic glutathione peroxidase and plasma acid phosphatase as well as high cumulative mortality rates at 24-96 h after Aeromonas hydrophila challenge. Furthermore, the feed cost of culturing this species with EF is lower than that with PF. These findings indicated that compared with PF, EF could increase the feed utilization and economic benefits of blunt snout bream, but reduce its anti-stress ability, non-specific immunity, A. hydrophila resistance and feed cost.