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.

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.

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.

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.

Study on the release routes of allelochemicals from Pistia stratiotes Linn., and its anti-cyanobacteria mechanisms on Microcystis aeruginosa.

Allelochemicals in Pistia stratiotes Linn. have a strong anti-cyanobacteria effect on Microcystis aeruginosa. To further determine the release routes of allelochemicals in P. stratiotes and understand their anti-cyanobacteria mechanisms, we aimed to systematically investigate the allelopathic effects of leaf leachates, leaf volatilization, root exudates, and residue decomposition of P. stratiotes on M. aeruginosa. The influences of P. stratiotes allelochemicals on the physiological properties of M. aeruginosa were also studied. Root exudates of P. stratiotes exhibited the strongest inhibitory effect on M. aeruginosa growth. The residue decomposition and leaf leachates exhibited a relatively strong inhibitory effect on M. aeruginosa growth. By contrast, the leaf volatilization stimulated M. aeruginosa growth. Therefore, root exudation was determined to be the main release route of allelochemicals from P. stratiotes. The mixed culture experiment of P. stratiotes root exudates and M. aeruginosa showed that the allelochemicals released from root exudation had no effect on the electron transfer of M. aeruginosa photosynthetic system II. However, it reduced the phycocyanin (PC) content and phycocyanin to allophycocyanin (PC/APC) ratio in the photosynthetic system. As the root exudates concentration increased, the electrical conductivity (EC) and superoxide anion radical (O2(*-)) values in the M. aeruginosa culture fluid increased significantly, indicating that the allelochemicals released from the root of P. stratiotes inhibited algae growth by affecting the PC and PC/APC levels in photosynthesis, destroying the cell membrane, and increasing O2(*-) content to result in oxidative damage of M. aeruginosa.

Purification effects of two eco-ditch systems on Chinese soft-shelled turtle greenhouse culture wastewater pollution.

The present study used an eco-ditch system that employed Eichhornia crassipes, Bacillus subtilis, and Bellamya aeruginosa (E-B-B) during the summer and fall (high temperature) seasons and a second eco-ditch system that employed Elodea nuttallii, a compound microbial preparation called "EM bacteria", and Hypophthalmichthys molitrix (E-E-H) during the winter and spring (low temperature) seasons successively to purify the discharged wastewater produced by Chinese soft-shelled turtle greenhouse cultivation. The wastewater was sampled, and the dynamic changes in the major nutrient pollutant indicators over several months were analysed. After the E-B-B and E-E-H eco-ditch purification systems were operated for nearly 140 days each, the following results were observed: the total nitrogen (TN) removal rates in the wastewater were 75 % and 69 %, respectively; the total phosphorus (TP) removal rates were 82 % and 86 %, respectively; the NH4 (+)-N removal rates were 91 % and 75 %, respectively; the chemical oxygen demand (CODcr) decreased 54 % and 44 %, respectively; the dissolved oxygen (DO) contents increased nearly 3 to 4 times; and the wastewater was maintained at neutral or alkaline pH values. The wastewater physical traits gradually changed from being yellow, brown, and muddy to being pale yellow, slightly turbid, and odourless. Both eco-ditch systems were observed to have a relatively favourable effect on the purification of Chinese soft-shelled turtle aquaculture wastewater. The continuous use of both eco-ditch systems could result in a year-round purification effect on Chinese soft-shelled turtle greenhouse aquaculture wastewater; therefore, this method has good prospects for promotion and application.

Effects of allelochemical extracted from water lettuce (Pistia stratiotes Linn.) on the growth, microcystin production and release of Microcystis aeruginosa.

This study explored the optimisation of a method of extracting allelochemicals from Pistia stratiotes Linn., identified the optimal dose range for the allelochemicals' anti-algal effect and investigated their impact on the growth of Microcystis aeruginosa, as well as the production and release of microcystin-LR (MC-LR). Based on measured changes in algal cell density and chlorophyll a (Chl-a) content, the allelochemicals were confirmed to have the strongest anti-algal effect with the lowest half-effect concentration of 65 mg L(-1) when they were extracted using ethyl acetate as the extraction solvent, 1:20 g mL(-1) as the extraction ratio and 1 h as the extraction time. The allelochemicals extracted from P. stratiotes using this optimal method exhibited the strongest inhibitory effect on the growth of algae when used within a dose range of 60-100 mg L(-1); the relative inhibitory ratio reached 50-90%, and Chl-a content reduced 50-75% in algae cell cultures within 3-7 days. In addition, the extracted allelochemical compounds demonstrated no significant impact on the extracellular release of MC-LR during the culturing period. The amount of intracellular MC-LR per 10(6) algal cells increased depending on the increasing dose of allelochemicals from P. stratiotes after 7 days of culturing and maintained stability after 16 days. There was no increase in the total amount of MC-LR in the algal cell culture medium. Therefore, the application of allelochemicals from P. stratiotes to inhibit M. aeruginosa has a high degree of ecological safety and can be adopted in practical applications for treating water subjected to algae blooms because the treatment can effectively inhibit the proliferation of algal cells without increasing the release of cyanotoxin.

Molecular cloning, characterization and mRNA expression of copper-binding protein hemocyanin subunit in Chinese mitten crab, Eriocheir sinensis.

Hemocyanin is a copper-binding protein and plays a crucial role in the physiological processes in crustacean. In this study, the cDNA encoding hemocyanin subunit from Chinese mitten crab Eriocheir sinensis (EsHc) was cloned by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of EsHc was 2573 bp, consisting of a 5' untranslated region of 51 bp, a 3' untranslated region of 458 bp, and an open reading frame of 2064 bp. The deduced protein had 688 amino acid residues with molecular mass of 77,997.31 Da. Quantitative real-time RT-PCR analysis showed that the EsHc gene was expressed in haemocytes, hepatopancreas, muscles, gills, and intestines with the highest level of expression in the hepatopancreas and the lowest in the muscles. After Aeromonas hydrophila challenge, the relative expression level of EsHc in hemolymph was up-regulated at 3 h post-injection of bacteria followed by a gradual recovery from 12 to 24 h. In the second set of transcriptional studies, the mRNA expression patterns of EsHc in haemocytes and hepatopancreas were measured by quantitative real-time RT-PCR after the Chinese mitten crab were fed six diets containing different levels of copper (0, 10, 20, 40, 80 and 400 mg kg(-1)) for 8 weeks, respectively. The feeding trial showed that the expression levels of EsHc mRNA significantly increased at the copper levels of 20-40 mg kg(-1). This study implies that the expression levels of EsHc could be affected by dietary copper in the hepatopancreas and haemocytes, and hemocyanin may be potentially involved in the immune responses of the Chinese mitten crab.

Effects of emodin and vitamin C on growth performance, biochemical parameters and two HSP70s mRNA expression of Wuchang bream (Megalobrama amblycephala Yih) under high temperature stress.

In order to study the effects of dietary emodin, high-dose vitamin C (Vc) and their combination on growth of Wuchang bream (Megalobrama amblycephala Y.) and its resistance to high temperature stress, 1200 healthy Wuchang bream with initial body weight of 133.44 ± 2.11 g were randomly divided into four groups: a control group fed with basal diet (containing 50.3 mg/kg Vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin, 700 mg/kg Vc, and the combination of 60 mg/kg emodin + 700 mg/kg Vc, respectively. After feeding for 60 days, the growth performance of Wuchang bream was measured. Then 25 fish per tank were exposed to heat stress of 34 °C. The biochemical parameters of blood and liver, and expression levels of liver two HSP70s mRNA before and after heat stress were determined and the cumulative mortality of each group under heat stress was counted. The results showed that before stress, compared with the control, the weight gain (WG) and specific growth rate (SGR), serum total protein (TP), lysozyme (LSZ), and alkaline phosphatase (ALP) levels, liver superoxide dismutase (SOD) activity and expression level of HSP70 mRNA significantly increased in emodin and Vc groups while feed conversion rate (FCR), serum cortisol (COR), triglyceride (TG) and liver malondialdehyde (MDA) contents decreased (P < 0.05); liver catalase (CAT) activity also significantly increased in emodin group (P < 0.05). Although serum TP, LSZ, and liver HSP70 mRNA levels significantly increased and liver MDA level decreased in combination group (P < 0.05), no synergism was observed. After heat stress, compared with the control, the serum TP, LSZ, ALP levels, liver SOD, CAT activities, and expression levels of HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees and serum COR, glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), TG and liver MDA levels decreased to some extent. Although these parameters had similar changing trend as above ones in combination group, it did not show any synergism either. Statistics showed that under heat stress, the cumulative mortalities of emodin and Vc groups, except at 6 h in emodin group, were significantly lower than that of the control (P < 0.05) while the difference between the combination and control groups was not significant (P > 0.05). Thus, the basal diet supplemented with 60 mg/kg emodin or 700 mg/kg Vc could promote the growth of Wuchang bream, reduce FCR, increase non-specific immunity of fish, antioxidant capacity, and two HSP70s mRNA expression levels, and enhance resistance to heat stress in fish. However, the combination of emodin and high-dose Vc showed no better effect.