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Oxidative stress plays a crucial role in ammonia nitrogen toxicity. In this study, the beneficial effects of dietary nano cerium oxide (nano CeO2) as a potent antioxidant were examined in the Chinese mitten crab (Eriocheir sinensis). Crabs were fed a diet supplemented with 0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, or 12.8 mg/kg nano CeO2 for 60 d. The optimum supplementation level of nano CeO2 that significantly increased weight gain rate and decreased feed coefficient was 0.8 mg/kg. This level also offered immune protection when crabs were kept under ammonia nitrogen stress and/or exposed to pathogen infection (Aeromonas hydrophila). Supplementation with 0.8 mg/kg of CeO2 (i) relieved pathological damage to the hepatopancreas; (ii) increased hemocyte counts, including total number of hemocytes, granulocytes, and hyalinocytes; (iii) decreased malondialdehyde content and increased antioxidant enzyme activities of superoxide dismutase and catalase in the hemolymph; (iv) increased the activities of lysozyme, acid phosphatase, and alkaline phosphatase in the hemolymph; and (v) increased gene and protein expression of cathepsin L in the hepatopancreas. Mortality increased when crabs were injected with bacteria under ammonia nitrogen stress, but dietary supplementation with 0.8 mg/kg nano CeO2 decreased the mortality rate. Thus, the results of this study suggested that dietary supplementation with nano CeO2 in crabs promoted growth and up-regulated immunity to bacterial infection under ammonia nitrogen stress.

This paper aims to share findings on nonclassical biomanipulation from a field study on eutrophic West Yangcheng (WY) Lake. In the forty years before 2009, cyanobacteria frequently bloomed in WY Lake, an event which has not occurred since 2009. The stocking density of filter-feeding carp was estimated to be more than 50 g/m3 in July after 2009, approximately double the amount observed before 2009, and the ratio of silver carp to bighead carp (SBR) increased from 0.8 to more than 1.6. Compared with the water quality between 2004 and 2007, the total nitrogen (TN) and the chlorophyll-a (Chl-a) concentration decreased while total phosphorus (TP) and transparency (SD) increased significantly after 2009 (P 0.05). A significant negative correlation was observed between fish yield and SD (P

Intestinal microbiota has increasingly been recognized as a major determinant of host health due to its critical role in the maintenance of immune homeostasis. An experimental fish model with disrupted intestinal microbiota allows us to identify potential strategies for restoring or modulating microbiota composition. The aim of this study was to develop a reliable grass carp model with disrupted intestinal microbiota by treatment with an antibiotic cocktail consisting of vancomycin, enrofloxacin, florfenicol, and metronidazole at different concentrations. To identify the appropriate concentration and exposure time of antibiotic treatment required to disrupt intestinal microbiota, five antibiotic regimens were compared in terms of their effects on intestinal microbiota composition, growth and health status, and histological changes in the intestine, hepatopancreas, and spleen. Antibiotic-induced changes in the expression of inflammation- and apoptosis-related genes in grass carp intestines were also examined. The results revealed that the highest antibiotic concentration over a 28 day period was required to disrupt both luminal and mucosal microbiota through significant expansion of Proteobacteria and suppression of Fusobacteria. Treatment with this regimen induced significant down-regulation of caspase-8, MLCK, ZO-1, IL-17N, and IL-23R, and up-regulation of IL-1β and IL-6 in the intestine, suggesting possible effects on intestinal epithelial tight junction and inflammatory conditions. This treatment did not produce substantial histopathological changes, and was not directly detrimental to growth and health. Therefore, this antibiotic regimen may provide a suitable option to disrupt intestinal microbiota in grass carp. In addition, our study also implies that fish may serve as a promising model for investigating growth-promoting effects of antibiotics, when treated with antibiotics in low doses.

The genomic and coding sequences of four members of grass carp interleukin (IL)-17 cytokine family, IL-17A/F2, IL-17A/F3, IL-17C and IL-17N, were isolated using rapid amplification of cDNA ends and conventional PCR techniques. A phylogenetic analysis showed that grass carp IL-17 family genes, including previously identified IL-17A/F1 and IL-17D, clustered with their counterparts from other fish species. IL-17D had two exons and one intron, while all the other members were composed of three exons and two introns. A multiple sequence alignment revealed that grass carp IL-17 family cytokines shared a cystine-knot motif that was highly conserved in mammals. A quantitative real-time PCR analysis revealed that these genes were expressed in the different examined tissues at varying levels. Moreover, they displayed different tissue expression patterns in fish at 24 h after intraperitoneal injection (IP) or anal intubation (AI) of Aeromonas hydrophila, implying roles in inflammatory responses. Next, we compared disease activity status and histopathological changes in intestinal tissues over a 7 day period after IP or AI, and then examine the potential differential effects of different infection routes on the expression profiles of IL-17 family genes in inflamed intestinal tissues. The intraperitoneal route resulted in more pronounced pathological symptoms than the anal route in terms of the disease activity index (DAI). Changes in the DAI highly coincided with the severity of intestinal inflammation. The two infection routes resulted in differential IL-17 family gene expression patterns over time in grass carp intestine, which suggests that IL-17 family members may play differential roles in mucosal and systemic immune responses and that IL-17N and IL-17A/F1 might possibly function as pro-inflammatory cytokines that provide defense against bacterial infection in grass carp intestine.

Bacterial enteritis is a widespread inflammatory disease in most farmed fish, yet its pathogenesis is largely unknown, primarily because of the paucity of reproducible experimental models of intestinal inflammation in fish. The aim of the present study was to develop a model of intestinal inflammation in the grass carp by challenging it with a strain of Aeromonas hydrophila . To induce intestinal inflammation, the fish were challenged with A . hydrophila via anal intubation at 2.7 × 10 6 cfu/fish, an appropriate dose based on the disease activity index for inflammatory signs. The pathological changes occurring in the intestines over the 21 day experimental period were examined with light microscopy and scanning electron microscopy. Histopathological examinations showed that A . hydrophila infection caused severe intestinal lesions, including intestinal villus fusion and shedding, and induced heavy inflammatory cell infiltration on day 3 after challenge. Ultrastructural observations also revealed that bacterial infection caused microvillus effacement. The injured intestinal villi were gradually repaired during the subsequent 18 days of the experimental period. Our results also indicated that the inflammatory responses induced by A . hydrophila infection were closely associated with the expression of pro-inflammatory cytokines interleukin 1β (IL-1β), IL-8, and tumor necrosis factor α (TNF-α) and myeloperoxidase (MPO) activity. The highest mRNA levels of these cytokines and highest MPO activity in the intestine were observed on day 3 after challenge. These results show that the intestinal lesions caused by A . hydrophila infection and their repair were completely consistent with the time course of inflammation-related genes or biomarkers. Thus, we have developed an experimental model of intestinal inflammation in an intensively farmed fish. This model is expected to be useful in clarifying the pathogenesis of bacterial enteritis in the grass carp and other farmed fish, and in testing antimicrobial drugs for potential use in aquaculture.

This paper is to explore the biogas production potential of wetland aquatic biomass plants. 7 species of wetland aquatic biomass plants are used in the study, which include 4 plants with more fiber carbohydrate, Acorus calamus Linn, Typha orientalis Presl, Pontederia cordata and Canna indica , and 3 plants with more starch carbohydrate, Colocasia tonoimo Nakai, Thalia dealbata and Hydrocotyle vulgaris. In the experiment, these plants were treated by anaerobic fermentation in batch mode at 37 °C. The results show that the anaerobic biogas production potential (ABP, mL·g −1 VS) of aquatic biomass plants is different for different components content (%TS). The correlation between ABP and hemicellulose content is significant and negative ( R = −0.784, 0.01 p R = 0.767, 0.01 p y = 238.62 + 2.60 x 1 + 28.55 x 2 −2.08 x 2 x 3 + 12.67 x 3 , (Adj- R 2 = 0.962, p (intercept) = 0.034, p ( x 1 ) = 0.101, p ( x 2 ) = 0.036, p ( x 2 x 3 ) = 0.066, p ( x 3 ) = 0.031, p = 0.025, SD = 9.95), y represents ABP (mL g −1 VS), x 1 , x 2 and x 3 represents the cellulose, lignin and starch carbohydrate content (%TS) respectively.

A dielectric barrier discharge system with a discharging zone apart from a degrading zone is developed to remove 4-chlorophenol from water. Oxygen is fed through the discharging zone where it dissociates to form a plasma stream. The plasma stream is introduced into the degrading zone to make 4-chlorophenol degraded effectively. The removal of 4-chlorophenol was influenced by the processing parameters such as gas flow rate, pH and applied voltage. Increasing the peak voltage or gas flow rate among a certain range, the removal efficiency was enhanced. More than 80% of 4-chlorophenol was removed in 6 min at pH 9.00, resulting in a high energy efficiency of 46.34 × 10 −9 mol J −1 (21.46 g/kW h). Discharging in initial 4-chlorophenol solution, the removal percent was about 90% at 30 min with peak voltage of 14.64 kV and oxygen flow rate of 200 L h −1 . The corresponding energy efficiency was 12.35 × 10 −9 mol J −1 (5.72 g/kW h). During the degradation, aromatic products and organic acids were produced. The main aromatic intermediates were different at different pH, suggesting different pathways of the target contaminant. After the benzene ring was opened, organic acid such as formic acid, oxalic acid, d -(+)-malic acid and maleic acid were formed.

Interleukin-8 (IL-8) is a CXC chemokine that plays key regulatory roles in the immune and inflammatory responses implicated in many human diseases. In this study, we identified and characterized an IL-8 homologue from the grass carp, Ctenopharyngodon idellus . A sequence alignment of the full-length cDNA and genomic DNA showed that the exon/intron organization of grass carp IL-8 (gcIL-8) is identical to those of other known CXC chemokine genes. A multiple alignment analysis showed that gcIL-8 is an ELR – CXC chemokine, and its deduced amino acid sequence shares 81% and 36% identity with common carp IL-8s L1 (GenBank ID: ABE47600 ) and L2 (GenBank ID: AB470924 ), respectively, suggesting that it belongs to the lineage 1 group of fish IL-8 proteins. On a phylogenetic tree, gcIL-8 clustered with other teleost IL-8 proteins to form a fish-specific clade, clearly distinct from those of bird, mammal, and amphibian proteins. Real-time quantitative PCR analysis indicated that gcIL-8 is differentially expressed in various tissues under normal conditions and that the expression of gcIL-8 mRNA in immune-related tissues is clearly upregulated by Aeromonas hydrophila infection. To explore the biological effects of gcIL-8, we produced a recombinant protein, rgcIL-8, in a prokaryotic expression system. Purified rgcIL-8 was confirmed to be chemoattractive for head kidney neutrophils and mononuclear leukocytes in vitro . Our histopathological study also revealed that rgcIL-8 exerts proinflammatory effects by inducing neutrophil infiltration and erythrocyte extravasation. Overall, these results suggest that IL-8 is crucially involved in the inflammatory responses of fish.

Air is often used as bubbling gas with the purpose of improving the liquid discharge performance in some pulsed high-voltage discharge systems. Nitrogen in air could dissociate into active species that might participate in a series of liquid chemical reactions. In the discharge system with 4-chlorophenol as target contaminant, some more toxic intermediates were formed and the energy efficiency was decreased by bubbling air and nitrogen. The G50 (energy cost at 50% conversion) is about 0.91 g (KW h)−1, 0.70 g (KW h)−1 and 0.49 g (KW h)−1 respectively by bubbling oxygen, air and nitrogen. Nitro- compounds such as 4-chloro-2-nitrophenol and 4-nitrocatechol were identified, providing evidence for the formation of nitro group. Chloride ion (Cl−) dropped in the degradation process could be activated and react with non-degradable 4-chlorophenol to form 2,6-dichlorophenol. Phenol was also identified for higher yield of reductive radicals. Comparing 4-chlorophenol degradation by bubbling air or nitrogen with that by oxygen, the degradation rates of original contaminants and intermediate products were lower under same energy input. By discharging for 60 min with applied input energy density of 175 W L−1, almost all of aromatic substances were removed with bubbling oxygen but they still remained at about 8.83% with bubbling air and 12.79% with nitrogen. Furthermore, nitrogen in the bubbling gas was transformed into inorganic nitrogen such as NO2- and NO3- that increased linearly with discharge time and achieved a formation rate twice as the degradation rate of original target contaminant.

By applying pulsed high voltage discharge to a needle-mesh reactor that using seven acupuncture needles as discharge electrode and stainless steel wire mesh as ground electrode, nitrogen from bubbling gas could be fixed into NO 2 − and NO 3 − with equivalent mol H + produced in the liquid phase and a small amount of NO and NO 2 yielded in the gas phase. The HNO 2 was originally formed and then converted into HNO 3 . The ·OH and H 2 O 2 stimulated the conversion reaction from HNO 2 to HNO 3 , which caused HNO 2 concentration increased in the first 12 min and then decreased until lower than its detection limit. The concentration of HNO 3 still increased with discharge time. After 36 min, HNO 3 was the only and ultimate product in the liquid. The total yield of HNO 2 and HNO 3 could be affected by processing parameters such as electric factors of peak voltage and frequency, mesh size of ground electrode and content of nitrogen in N 2 /O 2 bubbling. Increasing peak voltage or frequency, the total yield of HNO 2 and HNO 3 increased. Gas composition had a heavy impact on the fixation efficiency that obtained its maximum value at an oxygen content of 66.7% with bubbling O 2 /N 2 gas. At the end of the 36 min discharge, the HNO 3 concentration with bubbling air was 2.215 mmol L −1 at an applied voltage of 25 kV, pulse repetition frequency of 140 Hz and ground electrode mesh of 20 × 20. The energy yield was about 1.22 g (HNO 3 )/kWh.

Degradation of 4-chlorophenol by pulsed high voltage discharge is an intricate process involving a series of complex chemical reactions. Hydroxylation of 4-chlorophenol to form hydroquinone, 4-chlororesorcinol and 4-chlorocatechol is the first step, though a very small amount of direct cleavage products of the C1–C2 or C5–C6 bond are observed. The yield of 4-chlorocatechol is about twice as much as that of hydroquinone. Less 4-chloresorcinol is produced. The free chloride ions dropped from the 4-chlorophenol degradation can obtain reactivity again from the discharge, and react with undegraded 4-chlorophenol to form 2,4-dichlorophenol. Some ring-opened products have also been identified and their possible reaction routes are proposed. Several compounds are verified by use of authentic samples. The more stable ring-opened products are low molecular weight (LMW) acids such as formic, acetic, oxalic, malonate, maleic and malic acid. By discharging 4-chlorophenol aqueous solution for 36 min, the amount of carbons obtained from organic acids is more than 50% while that of carbons from aromatic products less than 20% in the carbons of degraded 4-chlorophenol, which is about 94% of initial carbons. After 60 min of discharge, all the 4-chlorophenol and its aromatic intermediates have been removed completely and the organic carbons are mainly presented as organic acid such as acetic and oxalate acid. At the end of the 120 min discharge, the amount of the remaining organic carbons is not more than 14% of the initial carbons.

Polyethylene glycol (PEG) coupled r-hirudin mutein is determined by biological methods-the coagulation system. In the present study, a competitive enzyme-linked immunosorbent assay (ELISA) is described that permits the measurement of PEG r-hirudin. The ELISA system adopts a rabbit IgG antibody to quantitatively detect PEG-hirudin in human plasma. A PEG-hirudin calibration curve ranged from 50 to 7000 ng/mL. The limit of detection was 87 ng/mL. The intraassay coefficients of variation (CV) ranged between 16 and 21%, and interassay CV between 8 and 22% for low and high PEG-hirudin concentrations, respectively. The recovery of the compound in plasma was between 96 and 111.5%. The interindividual differences between 100 and 5000 ng/mL PEG-hirudin were between 12 and 22%. The correlation of the concentration of PEG-hirudin determined with the ELISA and the ecarin clotting time was r = 0.902. No interactions between unfractionated heparin, low molecular-weight heparin, or phenprocoumon and PEG-hirudin were observed in the ELISA. Deficiencies of thrombin or antithrombin as well as low, normal, and high fibrinogen levels did not interfere with the assay. It is concluded that the ELISA determines the concentration of PEG-hirudin and is not influenced by other major anticoagulants or by plasma levels of some coagulation proteins.

A fluorescence-linked immunofiltration assay (FLIFA) was developed for the determination of heparin-induced IgG in heparin-induced thrombocytopenia (HIT) type II patients. Protein A was immobilized on a nitrocellulose membrane to bind heparin-induced IgG of HIT type II patients. Fluorescein-5-isothiocynate (FITC)-heparin was added to platelet factor 4 present in normal serum to form the neo-antigen which was captured by heparin-induced IgG. The heparin-induced IgG was quantified by the relative fluorescence intensity (RFI) of bound FITC-heparin. Values were expressed as a RFI ratio (RFI patient / RFI normal) and were 1.965+/-0.413 in HIT type II patients (n = 36) and 1.064+/-0.162 in healthy controls (n = 50, p