Effects of selenoprotein extracts from Cardamine hupingshanensis on growth, selenium metabolism, antioxidant capacity, immunity and intestinal health in largemouth bass Micropterus salmoides.

This study aimed to assess the impact of dietary selenoprotein extracts from Cardamine hupingshanensis (SePCH) on the growth, hematological parameters, selenium metabolism, immune responses, antioxidant capacities, inflammatory reactions and intestinal barrier functions in juvenile largemouth bass (Micropterus salmoides). The base diet was supplemented with four different concentrations of SePCH: 0.00, 0.30, 0.60 and 1.20 g/Kg (actual selenium contents: 0.37, 0.59, 0.84 and 1.30 mg/kg). These concentrations were used to formulate four isonitrogenous and isoenergetic diets for juvenile largemouth bass during a 60-day culture period. Adequate dietary SePCH (0.60 and 1.20 g/Kg) significantly increased weight gain and daily growth rate compared to the control groups (0.00 g/Kg). Furthermore, 0.60 and 1.20 g/Kg SePCH significantly enhanced amounts of white blood cells, red blood cells, platelets, lymphocytes and monocytes, and levels of hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin in the hemocytes. In addition, 0.60 and 1.20 g/Kg SePCH increased the mRNA expression levels of selenocysteine lyase, selenophosphate synthase 1, 15 kDa selenoprotein, selenoprotein T2, selenoprotein H, selenoprotein P and selenoprotein K in the fish liver and intestine compared to the controls. Adequate SePCH not only significantly elevated the activities of antioxidant enzymes (Total superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase), the levels of total antioxidant capacity and glutathione, while increased mRNA transcription levels of NF-E2-related factor 2, Cu/Zn-superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase. However, adequate SePCH significantly decreased levels of malondialdehyde and H2O2 and the mRNA expression levels of kelch-like ECH-associated protein 1a and kelch-like ECH-associated protein 1b in the fish liver and intestine compared to the controls. Meanwhile, adequate SePCH markedly enhanced the levels of immune factors (alkaline phosphatase, acid phosphatase, lysozyme, complement component 3, complement component 4 and immunoglobulin M) and innate immune-related genes (lysozyme, hepcidin, liver-expressed antimicrobial peptide 2, complement component 3 and complement component 4) in the fish liver and intestine compared to the controls. Adequate SePCH reduced the levels of pro-inflammatory cytokines (tumour necrosis factor-α, interleukin 8, interleukin 1ß and interferon γ), while increasing transforming growth factor ß1 levels at both transcriptional and protein levels in the liver and intestine. The mRNA expression levels of mitogen-activated protein kinase 13 (MAPK 13), MAPK14 and nuclear factor kappa B p65 were significantly reduced in the liver and intestine of fish fed with 0.60 and 1.20 g/Kg SePCH compared to the controls. Histological sections also demonstrated that 0.60 and 1.20 g/Kg SePCH significantly increased intestinal villus height and villus width compared to the controls. Furthermore, the mRNA expression levels of tight junction proteins (zonula occludens-1, zonula occludens-3, Claudin-1, Claudin-3, Claudin-5, Claudin-11, Claudin-23 and Claudin-34) and Mucin-17 were significantly upregulated in the intestinal epithelial cells of 0.60 and 1.20 g/Kg SePCH groups compared to the controls. In conclusion, these results found that 0.60 and 1.20 g/Kg dietary SePCH can not only improve growth, hematological parameters, selenium metabolism, antioxidant capacities, enhance immune responses and intestinal functions, but also alleviate inflammatory responses. This information can serve as a useful reference for formulating feeds for largemouth bass.

Selenium yeast improve growth, serum biochemical indices, metabolic ability, antioxidant capacity and immunity in black carp Mylopharyngodnpiceus.

This experiment was conducted to investigate the impacts of dietary selenium yeast (SeY) on the growth performance, fish body composition, metabolic ability, antioxidant capability, immunity and inflammatory responses in juvenile black carp (Mylopharyngodn piceus). The base diet was supplemented with 0.00, 0.30 and 0.60 g/kg SeY (0.04, 0.59 and 1.15 mg/kg of selenium) to form three isonitrogenous and isoenergetic diets for juvenile black carp with a 60-day. Adequate dietary SeY (0.30 and 0.60 g/kg) could significantly increase the weight gain (WG), special growth rate (SGR) compared to the SeY deficient groups (0.00 g/kg) (P < 0.05). Meanwhile, 0.30 and 0.60 g/kg SeY elevated the mRNA levels of selenoprotein T2 (SEPT2), selenoprotein H (SEPH), selenoprotein S (SEPS) and selenoprotein M (SEPM) in the liver and intestine compared with the SeY deficient groups (P < 0.05). Adequate dietary SeY could promote glucose catabolism and utilization through activating glucose transport (GLUT2), glycolysis (GCK, HK, PFK, PK, PDH), tricarboxylic acid cycle (ICDH and MDH), glycogen synthesis (LG, GCS and GBE) and IRS/PI3K/AKT signal pathway molecules (IRS2b, PI3Kc and AKT1) compared with the SeY deficient groups (P < 0.05). Similarly, adequate dietary SeY could improve lipid transport and triglycerides (TG) synthesis through increasing transcription amounts of CD36, GK, DGAT, ACC and FAS in the fish liver compared with the SeY deficient groups (P < 0.05). In addition, adequate SeY could markedly elevate activities of antioxidant enzymes (T-SOD, CAT, GR, GPX) and contents of T-AOC and GSH, while increased transcription amounts of Nrf2, Cu/Zn-SOD, CAT, and GPX in fish liver and intestine (P < 0.05). However, adequate SeY notably decreased contents of MDA, and the mRNA transcription levels of Keap1 in the intestine compared with the SeY deficient groups (P < 0.05). Adequate SeY markedly increased amounts or levels of the immune factors (ALP, ACP, LZM, C3, C4 and IgM) and the transcription levels of innate immune-related functional genes in the liver and intestine (LZM, C3 and C9) compared to the SeY deficient groups (P < 0.05). Moreover, adequate SeY could notably reduce levels of IL-8, IL-1ß, and IFN-γ and elevate TGF-1ß levels in fish intestine (P < 0.05). The transcription levels of MAPK13, MAPK14 and NF-κB p65 were notably reduced in fish intestine treated with 0.30 and 0.60 g/kg SeY (P < 0.05). In conclusion, these results suggested that 0.30 and 0.60 g/kg SeY could not only improve growth performance, increase Se, glucose and lipid metabolic abilities, enhance antioxidant capabilities and immune responses, but also alleviate inflammation, thereby supplying useful reference for producing artificial feeds in black carp.

Dietary Soybean Lecithin Improves Growth, Immunity, Antioxidant Capability and Intestinal Barrier Functions in Largemouth Bass Micropterus salmoides Juveniles.

This study evaluated the effects of dietary soybean lecithin (SBL) on the growth, haematological indices, immunities, antioxidant capabilities, and inflammatory and intestinal barrier functions because little information of dietary SBL could be obtained in juvenile largemouth bass (Micropterus salmoides). The fish were fed identical diets except for SBL added at 0, 2, 4 and 8%. It was found that 4 and 8% SBL significantly increased fish weight gain and daily growth rate (p < 0.05), while 4% SBL was optimal for enhancing RBC, HGB, PLT, MCV, MCH, WBC and MON in blood, and ALB and ALP in serum (p < 0.05). SBL (4%) also significantly elevated the antioxidant enzymes activities of T-SOD, CAT, GR, GPx, GST and T-AOC and GSH contents; increased mRNA transcription levels of Nrf2, Cu/Zn-SOD, CAT, GR, GST3 and GPx3; and decreased MDA contents. Keap1a and Keap1b levels were markedly down-regulated (p < 0.05). SBL (4%) significantly enhanced levels of the immune factors (ACP, LZM and C3) and the mRNA expression levels of innate immune-related genes (C3, C4, CFD, HEPC and MHC-I) compared with the control groups (0%) (p < 0.05). SBL (4%) significantly increased IgM and T-NOS in the intestine (p < 0.05) and significantly decreased levels of TNF-α, IL-8, IL-1ß and IFN-γ and increased TGF-ß1 at both transcription and protein levels in the liver and intestine (p < 0.05). The mRNA expression levels of MAPK13, MAPK14 and NF-κB P65 were significantly decreased in the intestine in the 4% SBL groups (p < 0.05). Histological sections also demonstrated that 4% SBL protected intestinal morphological structures compared with controls. This included increased intestinal villus height and muscular thickness (p < 0.05). Furthermore, the mRNA expression levels of the intestinal epithelial cell tight junction proteins (TJs) (ZO-1, claudin-3, claudin-4, claudin-5, claudin-23 and claudin-34) and mucin-5AC were significantly up-regulated in the 4% SBL groups compared with the controls (p < 0.05). In conclusion, these results suggested that 4% dietary SBL could not only improve growth, haematological indices, antioxidant capabilities, immune responses and intestinal functions, but also alleviate inflammatory responses, thereby providing reference information for the feed formulations in cultured largemouth bass.

Effects of dietary berberine on growth performance, lipid metabolism, antioxidant capacity and lipometabolism-related genes expression of AMPK signaling pathway in juvenile black carp (Mylopharyngodon piceus) fed high-fat diets.

This study aimed to investigate the effects of high-fat diet (HFD) supplemented with berberine on growth, lipid metabolism, antioxidant capacity and lipometabolism-related genes expression of AMPK signaling pathway in juvenile black carp (Mylopharyngodon piceus). Five hundred and forty healthy fish (4.04 ± 0.01 g) were randomly distributed into six groups, and fed six experimental diets: normal-fat diet (NFD, 5% fat), HFD (15% fat), and four HFDs supplemented with graded levels of berberine, respectively. The results showed that, compared with fish fed NFD, HFD had no effects on the growth of fish except for reducing survival rate, whereas HFD caused extensive lipid accumulation, oxidative stress injury and hepatic abnormalities. However, compared with the HFD group, fish fed HFD containing an appropriate berberine (98.26 or 196.21 mg/kg) improved the growth performance, increased hepatic lipid metabolism and antioxidant enzymes activities, and up-regulated the mRNA expression levels of ampk subunits and lipolysis genes such as pparα, cpt-1, acox, atgl and hsl (P < 0.05). Meanwhile, HFD supplemented with an appropriate berberine reduced crude lipid contents in liver and whole-body, decreased serum lipid contents, and ALT and AST activities, and down-regulated the mRNA expression levels of lipogenesis genes such as srebp-1, acc1, gpat, fas and pparγ, and lipid transporter genes such as fatp, fabp and fat/cd36 (P < 0.05). Thus, HFD supplemented with an appropriate berberine could improve growth of black carp, promote lipid metabolism and enhance antioxidant capacity. The lipid-lowering mechanism of berberine might be mediated by activating AMPK pathway, up-regulating lipolysis genes expression, and down-regulating lipogenesis and transport genes expression.

Influence of polystyrene microplastics on levofloxacin removal by microalgae from freshwater aquaculture wastewater.

Chlorella vulgaris (C. vulgaris) has attracted widespread attention because of its ability to absorb, enrich, and degrade typical endocrine-disrupting antibiotics (such as levofloxacin) in aquaculture wastewater. However, microplastic pollution in wastewater, which is becoming an increasingly severe problem, will exert a toxic effect on aquatic organisms (such as C. vulgaris and other microalgae). Polystyrene microplastics (PS-MPs), which are commonly found in freshwater aquaculture wastewater, are the most harmful. Therefore, clarifying the effects of PS-MPs on the ability of C. vulgaris to degrade typical endocrine-disrupting antibiotics in freshwater aquaculture wastewater and determining the mechanism of the effect are particularly important. The results of this study showed that under the stress of PS-MPs, the growth of C. vulgaris was significantly inhibited; the EPS-polysaccharide content per algal cell, EPS adsorption, intracellular enrichment and degradation of levofloxacin, total CYP450 content, and total CYP450 activity all decreased; and the relative expression of key genes related to the metabolic activity of algal cells, such as psbA, psaB, and rbcL, was generally downregulated. PS-MPs mainly affected the removal of a typical endocrine-disrupting antibiotic by C. vulgaris by altering adsorption, enrichment, and enzyme degradation. The results provide a reference for research on the impact of microplastic pollution on the treatment of freshwater aquaculture wastewater.

Solving post-prandial reduction in performance by adaptive regurgitation in a freshwater fish.

Foraging animals must balance benefits of food acquisition with costs induced by a post-prandial reduction in performance. Eating to satiation can lead to a reduction in locomotor and escape performance, which increases risk should a threat subsequently arises, but limiting feeding behaviour may be maladaptive if food intake is unnecessarily reduced in the prediction of threats that do not arise. The efficacy of the trade-off between continued and interrupted feeding therefore relies on information about the future risk, which is imperfect. Here, we find that black carp (Mylopharyngodon piceus) can balance this trade-off using an a posteriori strategy; by eating to satiation but regurgitating already ingested food when a threat arises. While degrees of satiation (DS) equal to or greater than 60% reduce elements of escape performance (turning angle, angular velocity, distance moved, linear velocity), at 40% DS or lower, performance in these tasks approaches levels comparable to that at 0% satiation. After experiencing a chasing event, we find that fish are able to regurgitate already ingested food, thereby changing the amount of food in their gastrointestinal tract to consistent levels that maintain high escape performance. Remarkably, regurgitation results in degrees of satiation between 40 and 60% DS, regardless of whether they had previously fed to 40, 60 or 100% DS. Using this response, fish are able to maximize food intake, but regurgitate extra food to maintain escape performance when they encounter a threat. This novel strategy may be effective for continual grazers and species with imperfect information about the level of threat in their environment.

Effects of dietary vitamin D3 on growth performance, antioxidant capacities and innate immune responses in juvenile black carp Mylopharyngodon piceus.

The aim of this experiment was used to investigate the effects of different contents of dietary vitamin D3 on the growth performance and antioxidant and innate immune responses in juvenile black carp Mylopharyngodon piceus. Black carp juveniles were fed six levels of dietary vitamin D3 (VD3) (96, 220, 412, 840, 1480, and 3008 IU/Kg) for 9 weeks. Results showed that highest weight gain (WG) and special growth ratio (SGR) were obtained at 534.2 IU/Kg dietary VD3 according to the second-order polynomial regression model. The protein efficiency ratio (PER) of black carp could be significantly increased by 412, 840, and 1480 IU/Kg dietary VD3 (p < 0.05), while the feed conversion ratio (FCR) were reduced by 412, 840, and 1480 IU/Kg dietary VD3 (p < 0.05). Adequate dietary VD3 content (412, 840, and 1480 IU/Kg) could significantly upregulate expression levels of lipoxygenase 5 (LPO 5); increase the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR); and improve GSH contents and total antioxidant capacities (T-AOC) in the liver of black carp. However, glutathione S-transferase (GST) activities and malondialdehyde (MDA) levels were significantly reduced by adequate dietary VD3 content (412, 840, and 1480 IU/Kg) in the fish liver. In addition, 412, 840, and 1480 IU/Kg dietary VD3 could significantly upregulate the mRNA expression levels of interferon-α (IFN-α), lysozyme (LYZ), hepcidin (HEPC), natural resistance-associated macrophage protein (NRAMP), and complement component 3 (C3) and C9 in the hemocytes and liver of black carp juveniles compared with the VD3-deficient diet (96 IU/Kg). Meanwhile, higher contents of dietary VD3 could increase serum LYZ and ACP activities and C3 and C4 contents in black carp juveniles compared with the groups fed VD3-deficient diet. In conclusion, these results suggest that adequate dietary VD3 could increase growth performances, improve antioxidant capacities, and then enhance innate immune parameters in black carp juveniles.

Optimal dietary curcumin improved growth performance, and modulated innate immunity, antioxidant capacity and related genes expression of NF-κB and Nrf2 signaling pathways in grass carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila.

This study investigated the effects of dietary curcumin on growth performance, non-specific immunity, antioxidant capacity and related genes expression of NF-κB and Nrf2 signaling pathways in grass carp (Ctenopharyngodon idella). A total of 525 juvenile grass carps with mean initial body weight of (5.30 ± 0.10) g were randomly distributed into five groups with three replicates each, fed five diets containing graded levels of curcumin (0, 196.11, 393.67, 591.46 and 788.52 mg/kg diet) for 60 days. After feeding trial, fifteen fish per tank were challenged with Aeromonas hydrophila and the mortalities were recorded for 7 days. The results showed that optimal dietary curcumin (393.67 mg/kg diet) improved the weight gain (WG) and specific growth rate (SGR) of juvenile grass carp, reduced feed conversion ratio (FCR) and the mortalities after challenge (P < 0.05). Moreover, optimal dietary curcumin increased the activities of lysozyme (LYZ) and acid phosphatase (ACP), and complement 3 (C3) and C4 levels, decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum of grass carp after injection with A. hydrophila (P < 0.05). Meanwhile, optimal dietary curcumin up-regulated the mRNA levels of LYZ, C3 and antimicrobial peptides [hepcidin, liver-expressed antimicrobial peptide-2 (LEAP-2), ß-defensin], and anti-inflammatory cytokines of interleukin-10 (IL-10) and transforming growth factor ß1 (TGF-ß1), and inhibitor of κBα (IκBα), whereas down-regulated pro-inflammatory cytokines of tumor necrosis factor-α (TNF-α), IL-1ß, IL-6 and IL-8, and nuclear factor kappa B p65 (NF-κB p65), IκB kinases (IKKα, IKKß and IKKγ) mRNA levels in the liver and blood of grass carp after injection with A. hydrophila (P < 0.05). In addition, optimal dietary curcumin increased the reduced glutathione (GSH) content and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR), reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the liver of grass carp after injection with A. hydrophila (P < 0.05). Meanwhile, optimal dietary curcumin up-regulated the mRNA levels of these antioxidant enzymes and nuclear factor erythroid 2-related factor 2 (Nrf2), whereas down-regulated Kelch-like ECH-associated protein (Keap) 1a and Keap 1b mRNA levels (P < 0.05) in the liver and blood of grass carp after injection with A. hydrophila. Thus, optimal dietary curcumin supplementation could promote growth of juvenile grass carp, reduce FCR, and enhance disease resistance, innate immunity and antioxidant capacity of fish, attenuating inflammatory response. However, dietary excessive curcumin had negative effect on fish. Based on second-order regression analysis between dietary curcumin contents and weight gain, the optimum requirement of dietary curcumin in juvenile grass carp was determined to be 438.20 mg/kg diet.

Effect of the immobilized microcystin-LR-degrading enzyme MlrA on nodularin degradation and its immunotoxicity study.

In freshwater ecosystems with frequent cyanobacterial blooms, the cyanobacteria toxin pollution is becoming increasingly serious. Nodularin (NOD), which has strong biological toxicity, has emerged as a new pollutant and affects the normal growth, development and reproduction of aquatic organisms. However, little information is available regarding this toxin. In this study, a graphene oxide material modified by L-cysteine was synthesized and used to immobilize microcystin-LR (MC-LR)-degrading enzyme (MlrA) to form an immobilized enzyme nanocomposite, CysGO-MlrA. Free-MlrA was used as a control. The efficiency of NOD removal by CysGO-MlrA was investigated. Additionally, the effects of CysGO-MlrA and the NOD degradation product on zebrafish lymphocytes were detected to determine the biological toxicity of these two substances. The results showed the following: (1) There was no significant difference in the degradation efficiency of NOD between CysGO-MlrA and free-MlrA; the degradation rate of both was greater than 80% at 1 h (2) The degradation efficiency of the enzyme could retain greater than 81% of the initial degradation efficiency after the CysGO-MlrA had been reused 7 times. (3) CysGO-MlrA retained greater than 50% of its activity on the 8th day when preserved at 0 °C, while free-MlrA lost 50% of its activity on the 4th day. (4) CysGO-MlrA and the degradation product of NOD showed no obvious cytotoxicity to zebrafish lymphocytes. Therefore, CysGO-MlrA might be used as an efficient and ecologically safe degradation material for NOD.

Biomagnification characteristics and health risk assessment of the neurotoxin BMAA in freshwater aquaculture products of Taihu Lake Basin, China.

In freshwater aquaculture ecosystems with high-frequency occurrences of cyanobacteria blooms, a chronic neurotoxic cyanobacteria toxin, ß-N-methylamino-l-alanine (BMAA), is a new pollutant that affects the normal growth, development, and reproduction of aquaculture organisms. BMAA poses a great threat to the food quality and food safety of aquatic products. In this paper, high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) was used to detect the contents of BMAA in the edible portions of six representative freshwater aquaculture products (Corbicula fluminea, Anodonta arcaeformis, Macrobrachium nipponense, Eriocheir sinensis, Ctenopharyngodon idella, and Mylopharyngodon piceus) from Taihu Lake Basin in China. Noncarcinogenic health risks were assessed with reference to the model recommended by the International Environmental Modelling and Software Society and based on the biomagnification characteristics of BMAA in the various aquaculture products investigated by the stable nitrogen isotope technique. The average BMAA concentrations in the edible portions of the six freshwater culture products were from 2.05 ±â€¯1.40 to 4.21 ±â€¯1.26 µg g-1 dry weight (DW), and the difference was significant (p < 0.05), such a difference increased with the increase in the trophic level in the aquaculture products. Although a biomagnification indication was observed, the trophic magnification factor (TMF) was only 1.20 which exhibited a relatively low biomagnification efficiency. The annual health risk values of BMAA in all the measured aquatic products were within the maximum tolerable range (<1 × 10-6 a-1), and the health risk increased with the increase in the trophic level. The risk values of BMAA in the six freshwater aquaculture products for children was slightly higher than the negligible level (<1 × 10-7 a-1), thus there might have potential health risks for children's long-term consumption. Considering China's national conditions, the guidance values of BMAA based on the quality and safety of freshwater aquaculture products were proposed to be 7.2 µg g-1 DW for adults and 1.8 µg g-1 DW for children.

Growth, antioxidant capacity, intestinal morphology, and metabolomic responses of juvenile Oriental river prawn (Macrobrachium nipponense) to chronic lead exposure.

Understanding the mechanisms of metal toxicity to organisms farmed for food may suggest mitigation strategies. We determined the 24-, 48-, 72-, and 96-h median lethal concentrations of lead in juvenile oriental river prawn (Macrobrachium nipponense). The prawns were then exposed to sub-lethal concentrations (13.13 and 26.26 µg/L) of lead for 60 days and growth, antioxidant enzyme activity, intestinal morphology, and metabolite profiles were assessed. Prawns exposed to 26.26 µg/L but not to 13.13 µg/L lead exhibited lower weight gain than controls. The lead burden in muscle was 0.067 and 0.25 µg/g of dry weight exposed to 13.13 and 26.26 µg/L, respectively. Levels of glutamic oxaloacetic transaminase and glutamic-pyruvic transaminase were not altered following exposure. Exposure increased malondialdehyde activity in the hepatopancreas and decreased superoxide dismutase and glutathione peroxidase activities. Catalase activity first increased and then decreased as lead concentrations increased. Some intestinal epithelial cells disassociated from the basement membrane in prawns exposed to 13.13 µg/L lead. Intestinal epithelial cells in prawns exposed to 26.26 µg/L lead separated completely from the basement membrane. Gas chromatography-mass spectrometry metabolomics assays showed the 13.13-µg/L exposure did not elicit significant metabolic alterations. Exposure to 26.26 µg/L lead differentially up-regulated 58 metabolites and down-regulated 21 metabolites. The metabolites identified were involved in galactose, purine, glutathione, and carbon metabolism, biosynthesis of amino acids and steroids, and neuroactive ligand-receptor interaction. These data indicate that chronic lead exposure can adversely affect growth, increase accumulation in muscle, impair intestinal morphology, and induce oxidant stress or neurotoxicity-related effects in M. nipponense.

Identification, characterization, and expression analysis of adiponectin receptors in black carp Mylopharyngodon piceus in response to dietary carbohydrate.

In the present study, three cDNAs of AdipoRs (MpAdipoR1a, MpAdipoR1b, and MpAdipoR2) were identified from juvenile black carp Mylopharyngodon piceus. There were 375, 378, and 356 amino acids in the MpAdipoR1a, MpAdipoR1b, and MpAdipoR2, respectively. BLAST analysis reveals that MpAdipoRs share high identities with other known AdipoRs from zebrafish, rainbow trout, human, etc. And there were all seven transmembrane regions in the amino acid sequences of MpAdipoR1s and MpAdipoR2, respectively. The relative expression levels of MpAdipoR1s were higher in the liver, blood, brain, and eyes in black carp (p < 0.05). Relatively higher expression of MpAdipoR2 was detected in the liver (p < 0.05) and then in the adipose tissues and blood by real-time PCR assays. The relative expression levels of AdipoR mRNA in the liver, muscle, brain, blood, and adipose tissues were detected by real-time PCR in black carp fed with four levels of dietary carbohydrate (CHO) (10.65, 19.43, 28.84, and 37.91%) for 9 weeks, respectively. The expression levels of MpAdipoR1s in the liver, muscle, brain, and blood were induced and reached to the maximum at optimal dietary CHO (19.43 or 28.84%) and then were decreased at 37.91% dietary CHO, although there were no significant differences on the expression levels of MpAdipoR1a in the liver between 19.43, 28.84, and 37.91% dietary CHO. Similarly, MpAdipoR2 were also induced to the maximal levels in the liver, muscle, brain, and blood at optimal dietary CHO (19.43 or 28.84%) compared with that at 10.65% dietary CHO, although no significant differences were observed on the expression levels of MpAdipoR2 in the liver and muscle between groups fed 19.43 and 37.91% dietary CHO (p > 0.05). However, the expression levels of MpAdipoRs in the adipose tissues were significantly downregulated at excessive dietary CHO (37.91%) compared with other groups (p < 0.05). These results indicated that MpAdipoRs were inducible proteins and might enhance the understanding of their vital roles in the regulation of glucose metabolic homeostasis in black carp.

Structural and functional analysis of two novel somatostatin receptors identified from topmouth culter (Erythroculter ilishaeformis).

In the present study, we cloned and characterized two somatostatin (SS) receptors (SSTRs) from topmouth culter (Erythroculter ilishaeformis) designated as EISSTR6 and EISSTR7. Analysis of EISSTR6 and EISSTR7 signature motifs, 3D structures, and homology with the known members of the SSTR family indicated that the novel receptors had high similarity to the SSTRs of other vertebrates. EISSTR6 and EISSTR7 mRNA expression was detected in 17 topmouth culter tissues, and the highest level was observed in the pituitary. Luciferase reporter assay revealed that SS14 significantly inhibited forskolin-stimulated pCRE-luc promoter activity in HEK293 cells transiently expressing EISSTR6 and EISSTR7, indicating that the receptors can be activated by SS14. We also identified phosphorylation sites important for the functional activity of EISSTR6 and EISSTR7 by mutating Ser23, 43, 107, 196, 311 and Ser7, 29, 61, 222, 225 residues, respectively, to Ala, which significantly reduced the inhibitory effects of SS14 on the CRE promoter mediated by EISSTR6 and EISSTR7. Furthermore, treatment of juvenile topmouth culters with microcystin-LR or 17ß-estradiol significantly affected EISSTR6 and EISSTR7 transcription in the brain, liver and spleen, suggesting that these receptors may be involved in the pathogenic mechanisms induced by endocrine disruptors. Our findings should contribute to the understanding of the structure-function relationship and evolution of the SSTR family.

Effect of propionamide on the growth of Microcystis flos-aquae colonies and the underlying physiological mechanisms.

Reducing the formation and growth of Microcystis colonies is an important prerequisite for the effective prevention and treatment of cyanobacterial blooms. Microcystis flos-aquae colonies was selected to investigate the potential of propionamide for use in controlling cyanobacterial blooms. Propionamide, one of the major allelochemicals in the root exudates of E. crassipes, was tested using different concentrations (0, 0.2, 1, and 2mgL-1) and dosing methods (one-time addition, semi-continuous addition, and continuous addition) to assess its effect on the growth of M. flos-aquae colonies. The results showed that in the presence of different concentrations of propionamide, the growth of M. flos-aquae colonies followed a logistic growth model, with a higher degree of fit at lower propionamide concentrations. With the semi-continuous addition of 2mgL-1 propionamide, the growth of M. flos-aquae colonies was markedly inhibited; the relative inhibition ratio of algal cells reached >90% at day 7 of co-culture, and the colonial form gradually disintegrated, transforming mainly into unicellular and bicellular forms and small colonies (average diameter<50µm). Following the semi-continuous addition of 2mgL-1 propionamide, the exopolysaccharide content, the chlorophyll-a concentration, and the maximum photochemical efficiency of photosystem II (Fv/Fm) trended downward in M. flos-aquae colonies, whereas the relative expression of the microcystin (MC) biosynthetic genes, mcyA and mcyH, was upregulated overall. Importantly, the synthesis of intracellular microcystin-LR (MC-LR) was decreased after an initial increase, and the extracellular MC-LR concentration did not differ significantly from that in the control group (p>0.05). Moreover, an acute toxicity test showed that 2mgL-1 propionamide was generally non-toxic to Daphnia magna. In conclusion, appropriate use of propionamide could effectively control the expansion of M. flos-aquae colonies without potential risks to the ecological safety of aquatic environments; therefore, propionamide can actually be used to regulate cyanobacterial blooms in natural waters.

Monitoring and research of microcystins and environmental factors in a typical artificial freshwater aquaculture pond.

Freshwater aquaculture ponds are important artificially regulated aquatic ecosystems which provide a large number of freshwater fish products in China. The cyanobacteria bloom and microcystin (MC) pollution caused by anthropogenic eutrophication have attracted much attention due to their toxic effects. To provide an insight into the cyanobacterial problem in the ponds, the environmental parameters and MCs of a typical artificial pond in the Yangtze River Delta region of China were monitored and studied from May to December 2015. During the monitoring period, the ponds were in serious eutrophication with total phosphorus (TP) concentrations between 0.95 and 1.80 µg/L, and total nitrogen (TN) concentrations between 1.1 and 4.86 µg/L. High feed coefficient and high fish stock were the main reasons for the eutrophication. The results showed that the water temperature was the key factor that affected the cyanobacteria blooming in the pond. The chlorophyll a concentration was significantly positively correlated with the cyanobacteria density during the blooming season. MC-LR and MC-RR existed simultaneously and showed a significant positive correlation. The peak concentrations of dissolved MC-LR and MC-RR in the pond water were 40.6 and 4.7 µg/L, respectively, which is considered highly toxic. Free MC-LR and MC-RR were also found in the aquaculture products. MC-LR concentrations in the bighead carp (Aristichthys nobilis) liver and shrimp (Macrobrachium nipponense) muscle were up to 2.64 and 4.17 µg/kg, respectively. MC-RR concentration was up to 1.89 µg/kg in the black carp (Mylopharyngodon piceus) liver. The results implied the potential health risks for citizens and pets caused by current artificial freshwater aquaculture pond systems.

Detection of free microcystins in the liver and muscle of freshwater fish by liquid chromatography-tandem mass spectrometry.

MC analysis of biological tissue is considered to be very difficult due to the lack of validated methods. This is the primary limiting factor for monitoring potential risks in both the flesh of aquatic organisms and the aquatic ecosystem. In this study, an effective method to determine free MCs (MC-LR and MC-RR) in the muscle and liver tissues of freshwater cultured fish was developed using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC/MS-MS). The extraction solvent, time of extraction, eluent and purification of the extract were optimized. Various SPE cartridges were also investigated. In this optimized analytical procedure, an 85% methanol/water solution (v/v) was selected as the extraction solvent, after which the extracts were purified by removing fats and proteins; a HLB cartridge was chosen for MCs enrichment; and 90% methanol containing 0.02% formic acid/water solution (v/v) was used as the eluent. Under the optimized pretreatment conditions and instrument parameters, good recoveries of MC-LR and MC-RR were obtained at three concentrations (0.5, 1.0 and 2.0 µg g-1 dry weight (DW)), with values ranging from 92.5 to 98.3% and 92.1 to 98.6%, respectively. The method detection limit (MDL) for muscle samples was 0.5 µg kg-1 and 0.4 µg kg-1 (DW) for MC-LR and MC-RR, respectively. The MDL for the liver samples was 0.8 µg kg-1 (DW) for both MC-LR and MC-RR. The developed procedure was successfully applied to analyze MCs in the muscle and liver of fish samples collected from a Chinese freshwater aquaculture pond during bloom seasons. The MC-LR concentrations ranged from below the MDL to 4.17 µg kg-1 and the MC-RR concentrations ranged from below the MDL to 2.64 µg kg-1.

Effect of feeding frequency on growth, body composition, antioxidant status and mRNA expression of immunodependent genes before or after ammonia-N stress in juvenile oriental river prawn, Macrobrachium nipponense.

Feeding frequency is important for the improvement of growth performance and immunity of aquatic animals. In this study, the effect of feeding frequency on growth, body composition, antioxidant status and mRNA expression of immunodependent genes before or after ammonia-N stress was examined in Macrobrachium nipponense. Prawns were randomly assigned to one of five feeding frequencies (1, 2, 3, 4 and 6 times/day) following the same ration size over an 8-week growth trial. After the feeding trial, prawns were challenged by ammonia-N. The weight gain of prawns fed with 3-6 times/day was significantly higher than that of prawns fed with 1 time/day. The best feed conversion ratio was obtained from prawns fed with 3-6 times/day. Body crude lipid with feeding frequency of 3, 4 or 6 times/day was quite lower than that with 1 time/day. High feeding frequency (6 times/day) induced significantly elevated hepatopancreas super oxide dismutase and catalase activities. The malondialdehyde level in prawns fed with 6 times/day was also significantly increased, which was higher than that of prawns fed with other feeding frequency. mRNA expression of toll like receptor 3 and myeloid differentiation primary response protein MyD88 was promoted by feeding frequency from 3 to 4 time/day but inhibited by high or low feeding frequency. Similar mRNA expression variation trends of the two genes were observed in prawns after ammonia-N stress. After ammonia-N challenge, the highest cumulative mortality was observed in prawns fed with 6 times/day, which was significantly higher than that of prawns fed with 2-4 times/day. These findings demonstrate that (1) too high feeding frequency induced oxidative stress and malondialdehyde accumulation, negatively affecting the health status of prawns and reduced its resistance to ammonia-N stress; (2) the optimal feeding frequency to improve growth and immune response of this species at juvenile stage is 3-4 times/day; (3) considering costs of labour, a feeding frequency of 3 times/day is recommended for this prawn.

The effects of dietary leucine on the growth performances, body composition, metabolic abilities and innate immune responses in black carp Mylopharyngodon piceus.

The present study was focused on the growth, body composition, metabolic abilities and innate immune responses in juvenile black carp Mylopharyngodon piceus fed with six levels of dietary leucine (7.3, 12.4, 16.2, 21.9, 28.3 and 34.5 g kg-1) for 9 weeks. Results showed that the highest weight gain (WG) and the lowest feed conversion ratio (FCR) was obtained at 23.5 and 23.9 g kg-1 dietary leucine using second-order polynomial model, respectively. Adequate dietary leucine content (21.9 and 28.3 g kg-1) could significantly up-regulate the expression levels of neuropeptide Y (NPY) and ghrelin (GRL) in the brain of black carp juveniles. The protein efficiency ratio (PER), feed efficiency ratio (FER) and protein deposition ratio (PDR) were also significantly increased by adequate dietary leucine content (21.9 and 28.3 g kg-1) (p < 0.05). Adequate dietary leucine content (21.9 and 28.3 g kg-1) could significantly up-regulate the activities of metabolic enzymes, such as α amylase, trypsin, chymotrypsin and elastase in the liver of Black carp (p < 0.05). However, the activities of alanine transaminase (ALT), aspartate aminotransferase (AST) and leucine aminopeptidase (LAP) were significantly reduced in the fish serum by adequate dietary leucine content (21.9 and 28.3 g kg-1) compared with leucine-deficient diet (7.3 and 12.4 g kg-1). In addition, 21.9 and 28.3 g kg-1 dietary leucine could significantly increase complement component 3 (C3) and C4 contents, lysozyme (LYZ) activities in the serum compared with the leucine-deficient diet (7.3 and 12.4 g kg-1) (p < 0.05). Furthermore, optimal dietary leucine could also significantly up-regulate the mRNA expression levels of LYZ, interferon α (IFN-α), hepcidin (HEPC), natural resistance-associated macrophage protein (NRAMP), C3 and C9 in the blood of juvenile black carp compared with the leucine-deficient diets (7.3 and 12.4 g kg-1) (p < 0.05). In conclusion, these results suggest that adequate dietary leucine (21.9 and 28.3 g kg-1) could increase growth performances, improve metabolic abilities and then enhance non-specific immunities in black carp juveniles.

Identification, characterization of selenoprotein W and its mRNA expression patterns in response to somatostatin 14, cysteamine hydrochloride, 17ß-estradiol and a binary mixture of 17ß-estradiol and cysteamine hydrochloride in topmouth culter (Erythroculter ilishaeformis).

In this study, a selenoprotein W cDNA was cloned from topmouth culter (Erythroculter ilishaeformis), and it was designated as EISelW. The EISelW open reading frame was composed of 261 base pairs (bp), encoding 86-amino-acid protein. The 5' untranslated region (UTR) consisted of 104 bp, and the 3'-UTR was composed of 365 bp. A selenocysteine insertion sequence (SECIS) element was found in the 3'-UTR of EISelW mRNA. The SECIS element was classified as form II because of a small additional apical loop presented in SECIS element of EISelW mRNA. Bioinformatic approaches showed that the secondary structure of EISelW was a ß1-α1-ß2-ß3-ß4-α2 pattern from amino-terminal to carboxy-terminal. Real-time PCR analysis of EISelW mRNAs expression in 17 tissues showed that the EISelW mRNA was predominantly expressed in liver, ovary, pituitary, various regions of the brain, spinal cord and head kidney. Study of intraperitoneal injection showed that the levels of EISelW mRNA in brain, liver, ovary and spleen were regulated by somatostatin 14 (SS14), 17ß-estradiol (E2), cysteamine hydrochloride (CSH) and a binary mixture of E2 and CSH, dependent on the dosage. These results suggest that E2, SS14 and CSH status may affect tissues of selenium metabolism by regulating the expression of SelW mRNA, as SelW plays a central role in selenium metabolism.

Scavenger Receptor Class B, Type I, a CD36 Related Protein in Macrobrachium nipponense: Characterization, RNA Interference, and Expression Analysis with Different Dietary Lipid Sources.

The scavenger receptor class B, type I (SR-BI), is a member of the CD36 superfamily comprising transmembrane proteins involved in mammalian and fish lipid homeostasis regulation. We hypothesize that this receptor plays an important role in Macrobrachium nipponense lipid metabolism. However, little attention has been paid to SR-BI in commercial crustaceans. In the present study, we report a cDNA encoding M. nipponense scavenger receptor class B, type I (designated as MnSR-BI), obtained from a hepatopancreas cDNA library. The complete MnSR-BI coding sequence was 1545 bp, encoding 514 amino acid peptides. The MnSR-BI primary structure consisted of a CD36 domain that contained two transmembrane regions at the N- and C-terminals of the protein. SR-BI mRNA expression was specifically detected in muscle, gill, ovum, intestine, hepatopancreas, stomach, and ovary tissues. Furthermore, its expression in the hepatopancreas was regulated by dietary lipid sources, with prawns fed soybean and linseed oils exhibiting higher expression levels. RNAi-based SR-BI silencing resulted in the suppression of its expression in the hepatopancreas and variation in the expression of lipid metabolism-related genes. This is the first report of SR-BI in freshwater prawns and provides the basis for further studies on SR-BI in crustaceans.