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In recent years, high-fat diet (HFD) has been widely applied in aquaculture, which reduces the intestinal health of cultured fish. The current study evaluated the protective effects of nano-selenium (nano-Se) on intestinal health of juvenile grass carp (Ctenopharyngodon idella) fed with HFD. A total of 135 experimental fish were fed with a regular diet (Con), a HFD (HFD) and a HFD containing nano-Se at 0.6 mg/kg (HSe) for 10 weeks. The results showed that dietary nano-Se significantly improved the survival rate and feed efficiency which were reduced by HFD in juvenile grass carp (P

Interleukin 8 (IL-8) is a critical chemokine regulating immune cells chemotaxis as well as their physiological or pathological activations. In fish cells, recombinant IL-8 proteins induced transcriptions of pro-inflammatory cytokines. However, the exact mechanisms underlying the function of fish IL-8 as a pro-inflammatory cytokine are still unclear. In this paper, we first prepared recombinant grass carp IL-8 (rgcIL-8) using an E. coli expression system, and later confirmed rgcIL-8 increased gene expression of il8, il1β and tumor necrosis factor alpha (tnfα) in grass carp head kidney leukocytes (HKLs). Using signaling pathway inhibitors, we demonstrated that rgcIL-8 regulated transcriptions of pro-inflammatory cytokines via MAPK and/or NF-κB signaling pathways. We cloned gcIL-8 specific receptor CXCR1 and subsequently discovered that gcIL-8 could increase the activity of NF-κB and the transcription of IL-1β via CXCR1. Simultaneously, antibody neutralization assay showed that endogenous IL-8 is partially relevant to the self-regulation of IL-1β. Moreover, rgcIL-8 led to the expression of inducible nitric oxide synthase gene, causing an accumulation of nitric oxide in the culture medium of HKLs, suggesting the potential of gcIL-8 to mediate inflammatory response. This study not only enriched the function of IL-8 in teleost, but also revealed it as a potential target for the inflammatory control in grass carp. This article is protected by copyright. All rights reserved.

The polymeric immunoglobulin receptor (pIgR) plays an important role in mediating the transcytosis of polymeric immunoglobulins (pIgs) to protect organisms against pathogen invasion. Here, a polyclonal antibody against grass carp (Ctenopharyngodon idellus) recombinant pIgR was developed by immunizing New Zealand white rabbit, and the responses of pIgR, IgM and IgZ were analyzed after bath immunization and intraperitoneal administration with Flavobacterium columnare. The results showed that pIgR transcription level was similar to IgM and IgZ, but pIgR rose much faster and peaked earlier than IgM and IgZ; the pIgR mRNA levels were higher in the skin and spleen for both immunized groups, while IgM and IgZ mRNA expression were higher in skin, gills, and intestines in bath immersion group, or spleen and head kidney in intraperitoneal immunization group. ELISA revealed that the IgM, IgZ and pIgR protein levels were up-regulated in skin mucus, gill mucus, gut mucus and bile, reaching a higher peak level earlier in skin mucus and gill mucus in bath immersion group, but a higher peak level in bile in injection group. Moreover, secretory component molecules were detected in grass carp's skin, gill and intestine mucus and bile, but not in serum, which molecular mass was near the theoretical mass obtained from the sequence of grass carp pIgR. These results demonstrated that bath and intraperitoneal immunization up-regulated pIgR and secretory Ig expression in secretions, which provided more insights into the role of pIgR in immunity and offer insight into ways of protecting teleost against pathogen invasion.

Lipid metabolism disorders are found ubiquitously in farmed fish and occur as a result of excessive fat accumulation. Previous studies have found that miR-33 is involved in lipid metabolism; however, its role in fish lipid metabolism is unclear. We sought to clarify this relationship in grass carp in vivo and in vitro. Our findings revealed the length of miR-33 to be 65 bp. Phylogenetic tree analysis showed that grass carp miR-33 was most closely related to fish miR-33 (Siganus canaliculatus). Hepatocytes transfected with miR-33 mimic displayed markedly raised TG content (P

Following the discovery of heart regeneration in zebrafish, several more species within the Cyprinidae family have been found to have the same capability, suggesting heart regeneration may be conserved within this family. Although gonad regeneration has been observed in grass carp (Ctenopharyngodon idella), one of the largest cyprinid fish, the species’ response to cardiac injury has not been characterized. Surprisingly, we found cardiomyocytes do not repopulate the injured region following cryoinjury to the ventricle, instead exhibiting unresolved fibrosis and decreased cardiac function that persists for the 8-week duration of this study. Compared to other cyprinid fish studied, infiltration of macrophages is delayed and muted in this model. Additionally, fibroblasts are depleted following injury, a phenomenon not previously described in any cardiac model. This study shows that heart regeneration is not conserved among the Cyprinidae family and suggests the important role of non-fibroblasts in chronic fibrosis. Further study of these phenomenon may reveal the underlying differences between regeneration versus unresolved fibrosis in heart disease.Summary statementGrass carp, a member of the Cyprinidae family that includes regenerative zebrafish, do not regenerate functional cardiac tissue after cryoinjury. Instead, healing progresses through collagen deposition and scar formation.

Improving carbohydrate utilization can contribute to sustainability of aquaculture. In order to explore the feedback mechanism of glucose homeostasis in fish, one control diet (25% carbohydrate and 40% protein), one relatively high carbohydrate diet named HG (42% carbohydrate and 40% protein), and one high dietary carbohydrate coupled with relatively low protein diet named HGP (42% carbohydrate and 25% protein) were fed to grass carp for 40 days. After the feeding trial, HG group impeded the food intake and growth performance of fish compared with the other two groups. Meanwhile, the serum glucose and insulin level were both significantly elevated under the condition of high carbohydrates intake when compared HG with control group. However, although no significant difference was observed in peripheral glucose or insulin between HG and HGP groups, fish fed with HGP diet increased growth performance and food intake compared with the HG group. Gene expression data indicated that fish selectively regulated the expressions of the cerebral anorexigenic genes (cart and pomc) to adapt to the HG and HGP intake. Therefore, the HGP diet with high carbohydrate and low protein was more suitable for grass carp feeding and growth when compared with the other two diets, possibly because the diet composition was closer to the natural food of this fish. In addition, the serum leptin level was highly consistent with changes in food intake and anorexigenic gene expressions when comparing the three experimental diets, indicating that leptin might be the key to mediate the feedback regulation of carbohydrates intake on food intake and appetite in fish.

In the present study, two new SLC34 family members, named slc34a1b and slc34a2a, were isolated and characterized from grass carp Ctenopharyngodon idella. Topology, tissue distribution, and transcriptional response to phosphorus (Pi) and pH were compared among three members of SLC34 family (slc34a1b, slc34a2a, and slc34a2b) in grass carp. The length of validated cDNAs of grass carp slc34a1b and slc34a2a was 1494 bp and 1902 bp, and these two cDNAs encoded 497 and 633 amino acid residues, respectively. The domain analysis showed that three SLC34 members of grass carp contain architecture similar to that in mammals. Moreover, the mRNA of three slc34s was widely expressed in nine tissues (heart, brain, intestine, kidney, liver, muscle, gill, spleen, and skin), but at various levels. Our results revealed that 6 mM and 9 mM Pi incubation significantly reduced the mRNA expression of three slc34s in both CIK and L8824 cell lines from grass carp. The expression of slc34a1b was decreased in the CIK cells, but not in the L8824 cells after 3 mM Pi incubation. In CIK cells, 3 mM Pi incubation downregulated the expression of slc34a1b and slc34a2a, but not slc34a2b. In addition, the expression of three slc34s was significantly reduced at acidic pH in the CIK cells. Taken together, we characterized three SLC34 family members, revealed their specific distribution among different tissues, and elucidated their transcriptional responses to Pi and pH in two cell lines from grass carp. Our findings provide an insight into the physiological function of three SLC34s in fish.

Selenium (Se), an essential component of deiodinases (DIOs), regulates the contents of thyroid hormones and thus improves animal growth. To explore the influences of selenium supplementation on fish growth metabolism, a total of 270 healthy grass carp (Ctenopharyngodon idella) were divided into three groups and feed three graded dietary selenium (0.141, 0.562, and 1.044 mg Se/kg) levels. The results showed that after 60-day feeding, dietary selenium improved the final body weight and specific growth rate (SGR) of grass carp. The hepatic DIO activities in selenium-supplemented groups were higher than those in control group. A significant increase in triiodothyronine (T3), free triiodothyronine (FT3), and thyroid-stimulating hormone (TSH) levels was accompanied by a decrease in the contents of thyroxine (T4) and free thyroxine (FT4) in selenium-supplemented groups. The histopathological observation of thyroid suggested that selenium deficiency resulted in hypertrophy of follicular epithelial cells. Moreover, the gene relative expression levels of dio1, dio2, and dio3 showed an increasing trend with the rising concentration of dietary selenium. The transcription levels of HPT axis-related genes (crh, tsh-β, ttr, tr-s, tpo, nis) and GH/IGF1-related genes (gh, ghr, igf1, igf1r) were significantly upregulated in selenium-supplemented groups. No significant differences in the above indicators were observed between 0.562 and 1.044 mg Se/kg diet group except T3 content and dio1 relative expression ratio. These results indicate that dietary selenium supplementation improves the hepatic DIO activities and thyroid hormone metabolism and regulates the transcription levels of HPT and GH/IGF axis-related genes, which may be responsible for the growth promotion in grass carp.

This dissertation was primarily focused on the immune response, inflammatory response and molecular mechanisms in the skin, head kidney and spleen of grown-up grass carp (Ctenopharyngodon idella). Six iso-nitrogen diets differing in lysine concentrations (5.6, 8.5, 11.6, 14.4, 17.5 and 20.7 g/kg) were fed to 540 grass carp (164.85 ± 0.79 g) for 60 d. After that, grass carp were challenged by Aeromonas hydrophila for 6 d. This study revealed that lysine deficiency (1) suppressed the growth performance of the fish and decreased their ability to resist skin lesion morbidity, (2) impaired the immune organ's immune response by decreasing the gene expressions of mucin, liver-expressed antimicrobial peptide (LEAP)-2B, β-defensin-1 and LEAP-2A and the production of antibacterial compounds of grown-up grass carp, and (3) aggravated the inflammatory response of immune organs in the fish by increasing the gene expressions of pro-inflammatory cytokines (interferon γ2 [IFN-γ2], tumor necrosis factor α [TNF-α], interleukin [IL]-15, IL-17D, IL-12p40, IL-6 and IL-8) and down-regulating anti-inflammatory cytokines (IL-11, transforming growth factor β1 [TGF-β1], IL-10 and IL-4/13A), which were tightly correlated with signal transducer and activator of transcription (STAT)1 and STAT3 signaling pathway, respectively. The different phenomenon in the skin, spleen and head kidney of fish may be correlated with the difference in gene subtype. In addition, using quadratic regression analysis of percent weight gain (PWG), skin lesion morbidity, and the lysozyme activities in the spleen and head kidney, the dietary lysine requirements for grown-up grass carp were estimated to be 13.58, 13.51, 14.56 and 14.18 g/kg, respectively.

Soil samples were collected from diverse agricultural fields of Erode Taluk. There were 7 fungal genera which include 11 fungal species observed in composite soil sample in which 9 were identified and 2 of the Aspergillus species could not be identified. Among the fungal genera the genus Aspergillus was predominant in the test soil sample with five of its species. Total bacterial population in the gills and intestine of test fish were counted and recorded. The high percentage of occurrence was shown by E. coli and Aeromonas hydrophila (17.2%). There were 9 bacterial genera isolated from C. idella and identified as E. coli, K. pneumoniae, P. aeruginosa, B. cereus, E. faecalis, Salmonella, Vibrio, Aeromonas hydrophila and Proteus. The bacterial pathogens were tested against selected antibiotics. Biofilm was produced as black colonies. Trichoderma species 1 was found to exhibit high antimicrobial activity against all selected pathogens.

Fenvalerate (type II synthetic pyrethroid), widely used in agricultural practices, find its way into aquatic ecosystem through air, by runoff, or by percolation to groundwater. It is an extremely toxic insecticide for aquatic organisms especially fish. In the present study, the fenvalerate (FEN) induced toxicity and the protective efficacy of ascorbic acid (AA) against FEN in Ctenopharyngodon idella was evaluated by studying the structural alterations in scales, erythrocytes and gills. The fishes were exposed to 1.2 μg/L and 2 μg/L of FEN and orally administered with 1000 mg/kg diet of AA. The fishes were scrutinized on 15th, 30th and 60th day of experiment. Scanning electron microscopic studies (SEM) of FEN-treated fish revealed extensive morphological alterations on the microstructure of scales including deformed focus, uprooted lepidonts and tubercles, hole formation and worn out calcareous material from the surface. FEN intoxication induced severe damage on erythrocytes including formation of dacrocytes, serrated spherocytes, echinocytes with oozed out cytoplasmic content, contracted plasma membrane and appearance of lobopodial projections. Ultrastructural studies in gills declared profound lesions in the form of aneurysm, loss of secondary lamellae and destructed microstructures of pavement cells. On the other hand, supplementation of AA in diet mitigated the impairment provoked by FEN on the scales, erythrocytes and gills due to its antioxidant properties.

Relative contributions of aquaculture-origin and naturally-reproduced grass carp (Ctenopharyngodon idella) in the Laurentian Great Lakes have been unknown. We assessed occurrence and distribution of aquaculture-origin and wild grass carp in the Great Lakes using ploidy and otolith stable oxygen isotope (δ18O) data. We inferred natal river and dispersal from natal location for wild grass carp using otolith microchemistry and estimated ages of wild and aquaculture-origin fish to infer years in which natural reproduction and introductions occurred. Otolith δ18O indicated that the Great Lakes contain a mixture of wild grass carp and both diploid and triploid, aquaculture-origin grass carp. Eighty-eight percent of wild fish (n = 49 of 56) were caught in the Lake Erie basin. Otolith microchemistry indicated that most wild grass carp likely originated in the Sandusky or Maumee rivers where spawning has previously been confirmed, but results suggested recruitment from at least one other Great Lakes tributary may have occurred. Three fish showed evidence of movement between their inferred natal river in western Lake Erie and capture locations in other lakes in the Great Lakes basin. Age estimates indicated that multiple year classes of wild grass carp are present in the Lake Erie basin, recruitment to adulthood has occurred, and introductions of aquaculture-origin fish have happened over multiple years. Knowledge of sources contributing to grass carp in the Great Lakes basin will be useful for informing efforts to prevent further introductions and spread and to develop strategies to contain and control natural recruitment.

The current study investigates the toxic effects of acute dose of an endocrine disruptor atrazine on Grass carp (Ctenepharyngodon idella) using histopathological changes as biomarkers. Histopathology is promising field for research in aquatic toxicology, in this manner vital organs; brain, gills, and liver tissues were inspected histological after exposing to sublethal groupings of atrazine 0.025 and 0.03 μl/L for 3, 6, 9, 12 and 15 days individually with equal untreated control group. Against various doses, rapid movements, gulping of air and jumping of fish to scat from toxic medium were noticed. Various severe (+++) morphological modifications in tissue were documented in comparison with control group comprised of degenerated neurons, vacuolization, inflammatory cells infiltration and neural necrosis in brain tissue. The most well-known gills tissue alteration at all concentrations of atrazine was epithelial hyperplasia, desquamation, epithelial lifting and smaller aneurism while hepatic injuries were described by overcast expanding of hepatocytes esteemed as cloudy swelling of hepatocytes followed by karyolysis, karyohexis and dilation of sinusoids which shows that atrazine introduction upgraded the toxicosis impacts with the increase concentration, influenced the strength of the fish, inferable from histological inconsistencies.

The immune function of immune organs is extremely crucial for maintaining organism health status, which ultimately affects fish growth. Our previous study has found that dietary supplementation of (2-carboxyethyl)dimethylsulfonium Bromide (Br-DMPT) in non-fish meal (NFM) diet could promote the growth of grass carp (Ctenopharyngodon idella), whereas the underlying reason or mechanism for this results is largely unclear. Herein, we further explored whether dietary supplementation of Br-DMPT promoted fish growth is connected with the enhanced immune function in the immune organs (the head kidney, spleen and skin). In this study, 540 fish (216.49 ± 0.29 g) were irregularly distributed to six groups with three replicates (30 fish replicate−1) and fed corresponding diets group containing a fish meal (FM) diet group and five different NFM diets supplemented with gradational Br-DMPT (0–520.0 mg/kg level) group for 60 days. After the 60-days feeding trial, 8 fish from each replicate were selected and then conducted a challenge test with A. hydrophila for 14 days. Our results indicated that in the NFM diets, appropriate Br-DMPT: (1) significantly decreased the morbidity of skin haemorrhage and lesion after A. hydrophila infection (P 0.05). Taken together, in non-fish meal diet, appropriate supplementation of Br-DMPT could improve the disease resistance capacity, non-specific immunity and ameliorate inflammation, and simultaneously could mitigate these adverse effects induced by the non-fish meal diet in fish immune organs. Moreover, this study may be helpful to decipher the underlying mechanisms of how Br-DMPT promote fish growth by immune organs and also provide scientific theoretical evidence for the future application of Br-DMPT as a new immunopotentiator in aquaculture industry.

Aquatic vegetation can provide habitat and refuge for a variety of species in streams. However, the flow features generated by submerged patches of vegetation can also pose a challenge for fish lar...

Glycinin is a major protein and antinutritional factor of soybean. However, how dietary glycinin affect intestinal immune function of fish were largely unknown. In this study, we used juvenile grass carp as a model to investigate the impacts of glycinin on intestinal immune function of fish and involved mechanisms. We set three treatments including control, glycinin and glycinin + glutamine in this trial. For immune components, results revealed that compared with control group, glycinin group had lower acid phosphatase activities in the foregut, midgut and hindgut, lower C3 and C4 content, and lower mRNA abundances of IgM, IgZ, hepcidin, LEAP-2A, LEAP-2B and β-defensin-1 in the midgut and hindgut rather than foregut of grass carp. For pro-inflammatory cytokines and relevant signaling, glycinin elevated mRNA abundances of IL-1β, IL-8, IL-12p35, IL-12p40 and IL-17D in the midgut and IL-1β, IFN-γ2, IL-6, IL-8, IL-12p35, IL-12p40 and IL-17D in the hindgut, and increased protein abundances of PKC-ζ and nuclear NF-κB p65 in the midgut and hindgut in comparison to control. For anti-inflammatory cytokines and relevant signaling, glycinin reduced mRNA abundances of TGF-β1, TGF-β2, IL-4/13B (rather than IL-4/13A), IL-10 and IL-11 in the midgut and hindgut, and reduced p-mTOR (Ser 2448), p-S6K1 (Thr 389) and p-4EBP1 (Thr 37/46) protein abundances in the midgut and hindgut rather than foregut. Co-administration of glutamine with glycinin could partially enhance intestinal function and reduce intestinal inflammation compared with glycinin treatment. Concluded, glycinin decreased intestinal immune components and caused intestinal inflammation associated with PKC-ζ/NF-κB and mTORC1 signaling.

A 56 days feeding trial was conducted to investigate the effects of enzyme-treated soy protein (ETSP) supplementation in low protein diets on immune function of immune organs (head kidney, spleen and skin) in on-growing grass carp. A total of 540 on-growing grass carp (initial average weight: 325.72 ± 0.60 g) were fed six diets, which included a normal protein diet (28% crude protein) and five low protein diets (26% crude protein) supplemented with graded levels of ETSP (0.0, 0.8, 1.2, 1.6 and 2.0%). At the end of feeding period, a challenge test was performed by infection with Aeromonas hydrophila for two weeks. The results indicated that (1) reducing dietary protein content from 28 to 26% decreased antibacterial substances and aggravated inflammatory responses of above three immune organs; (2) under the condition of reducing protein level in diet, 0.8–1.2% ETSP supplementation reversed these above adverse effects on immune function of above three immune organs; (3) suitable ETSP supplementation-decreased inflammatory responses were partly associated with [IκB kinase β (IKKβ)/inhibitor of κBα (IκBα)/nuclear factor kappa B (NF-κB) p65 and p52 or NF-κB p65] signaling and [target of rapamycin (TOR)/(S6K1, 4E-BP)] signaling in above three immune organs. (4) On the basis of C3 content (head kidney), C4 content (spleen) and skin hemorrhage and lesion, the optimal ETSP supplementation levels in low protein diets were estimated to be 1.48%, 1.61% and 1.03%, respectively. In summary, ETSP supplementation in low protein diets improved immune function of head kidney, spleen and skin in on-growing grass carp.

This research investigates the influences of dietary mannan oligosaccharides (MOS) on the head-kidney and spleen immune function in on-growing grass carp (Ctenopharyngodon idella) and its related mechanism. Fish were fed during 60 days at different levels of MOS (0, 200, 400, 600, 800 and 1000 mg kg−1). Subsequently, 14 days after the feeding trial was injected intraperitoneally with Aeromonas hydrophila, the immune function was studied. The results are as follows: (1) appropriate MOS supplementation could increase the content of antibacterial compound and immunoglobulin (Ig), up-regulate antimicrobial peptides transcriptional levels in these two organs; (2) appropriate MOS supplementation attenuated inflammatory response in these two organs by regulating cytokines (pro- and anti-inflammatory cytokines) and related signalling pathways (NF-κB and TOR). The interesting points though, was, no differences were found in liver-expressed antimicrobial peptide (LEAP)-2A, interleukin (IL)-8, IL-4/13B, IκB kinase (IKK) α and nuclear factor kappa B (NF-κB) p52 gene expression in these two organs. Consequently, the present research suggests that MOS supplementation can enhance head-kidney and spleen immune function. Finally, we obtained these appropriate MOS dose (538.5 and 585.8 mg kg−1) by quadratic regression analysis of lysozyme activity (head-kidney) and phosphatase activity (spleen), respectively.

Grass carp hemorrhagic disease, caused by the grass carp reovirus (GCRV), is a major disease that hampers the development of grass carp aquaculture in China. The mechanism underlying GCRV infection is still largely unknown. Circular RNAs (circRNAs) are important regulators involved in various biological processes. In the present study, grass carp were infected with GCRV, and spleen samples were collected at 0 (control), 1, 3, 5, and 7 days post-infection (dpi). Samples were used to construct and sequence circRNA libraries, and a total of 5052 circRNAs were identified before and after GCRV infection, of which 41 exhibited differential expression compared with controls. Many parental genes of the differentially expressed circRNAs are involved in metal ion binding, protein ubiquitination, enzyme activity, and nucleotide binding. Moreover, 72 binding miRNAs were predicted from the differentially expressed circRNAs, of which eight targeted genes were predicted to be involved in immune responses, blood coagulation, hemostasis, and complement and coagulation cascades. Upregulation of these genes may lead to endothelial and blood cell damage and hemorrhagic symptoms. Our results indicate that an mRNA-miRNA-circRNA network may be present in grass carp infected with GCRV, providing new insight into the mechanism underlying grass carp reovirus infection.

In this study, we investigated the combined effects of temperatures fluencies and mercury (Hg) on glutathione-dependent antioxidant system in fish, by measuring the oxidative stress indicator (LPO, lipid peroxidation) and the parameters involved in the glutathione-related antioxidant defense system (GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione S-transferase; GSH, reduced glutathione), as well as the expression of related genes in grass carp, Ctenopharyngodon idella. Fish (45.37 ± 3.58 g) were exposed to 10 test groups, e.g., 15 °C with/without Hg, 20 °C with/without Hg, 25 °C with/without Hg, 30 °C with/without Hg, 35 °C with/without Hg for 4 weeks. Three-way ANOVA was used to analyze the correlation between the measured parameters and experimental conditions (water temperature, Hg exposure, exposure time, and their interactions.). Our results show that there is no interaction between mercury and low temperature, but the combined effect at high temperature has been confirmed, which indicated the glutathione-dependent enzyme system in grass carp has a complex regulatory mechanism with temperature fluctuations. In the actual field monitoring, it is necessary to consider the impact of extreme temperature on the toxicity of pollutants in the aquatic ecosystem.

The present study aimed to explore the effects of phytic acid (PA) on the antimicrobial activity and inflammatory response in three immune organs (head kidney, spleen and skin) of on-growing grass carp (Ctenopharyngodon idella). To achieve this goal, we first conducted a 60-day growth trial by feeding fish with graded levels of PA (0, 0.8, 1.6, 2.4, 3.2 and 4.0%). Then, the fish were challenged with Aeromonas hydrophila for 6 days. Compared with the control group, the following results were obtained regarding supplementation with certain levels of PA in the diet. (1) There was an increase in skin haemorrhage and lesion morbidity in fish. (2) There was a decrease in activities or contents of immune factors, including lysozyme (LZ), complement 3 (C3), C4 and immunoglobulin M (IgM), and there was downregulation of gene expression levels of hepcidin, liver-expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, and β-defensin-1 in immune organs. (3) There was upregulation in the gene expression of the following pro-inflammatory cytokines: tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β) (except in the spleen), interferon γ2 (IFN-γ2), IL-6 (except in the spleen), IL-8, IL-12p40, IL-15 and IL-17D. These changes were partly related to the nuclear factor kappa B (NF-κB) signalling pathway, but downregulation of mRNA levels of anti-inflammatory cytokines (transforming growth factor β1 (TGF-β1), TGF-β2, IL-413/A, IL-413/B, IL-10 (except in the skin) and IL-11) occurred in a manner partially related to the target of rapamycin (TOR) signalling pathway. Finally, based on the broken-line analysis of skin haemorrhage and lesion morbidity and IgM content in the head kidney, the maximum tolerance levels of PA for on-growing grass carp (120.56–452.00 g) were estimated to be 1.79 and 1.31% of the diet, respectively.

There are many polyphenols used for the preservation of fish, but the interaction mechanism between polyphenols and fish protein is rarely reported. In the present study, the interactions between two kinds of polyphenols (chlorogenic acid (CGA) and rosmarinic acid (RA)) and the myosin of grass carp (Ctenopharyngodon idella) were explored using multi-spectroscopic techniques. Both CGA and RA were found to be involved in reducing the intrinsic fluorescence and surface hydrophobicity of myosin and increasing the UV absorption intensity. This indicates that interactions between CGA, RA, and myosin ultimately result in the formation of polyphenol-myosin complexes. The binding process of CGA and RA for the formation of the complex was spontaneous. The main binding forces between RA and myosin are hydrogen bonding and van der Waals forces, whereas hydrophobic interactions were observed between CGA and myosin. The results of circular dichroism (CD) showed that the presence of CGA and RA increased the content of myosin alpha-helix. CGA and RA caused myosin aggregation which reduced the corresponding solution dispersibility. CGA and RA protected the myosin sulfhydryl groups and reduced the degree of their oxidation. Furthermore, the complexes formed by the combination of myosin, CGA, and RA exhibited the strongest synergistic antioxidant properties than any one of them. The findings of the present study provide insights into our understanding of the mechanism of interactions between myosin and polyphenols which could provide information on the application of polyphenols in preserving aquatic products.

The viral hemorrhage disease caused by grass carp reovirus (GCRV) is a serious contagious disease of grass carp that mainly infects fingerlings and yearlings. Epidemiological studies have shown that GCRV genotype II is currently the prominent genotype. However, little is known about the histopathological characteristics, virus distribution, and expression of immunity-related genes in grass carp infected by GCRV genotype II. In this study, we found that grass carp infected by GCRV genotype II lost appetite, swam alone, and rolled, and their fins, eyes, operculum, oral cavity, abdomen, intestine, and muscles showed pronounced punctate hemorrhage. Congestion, swelling, deformation, thinning of membranes, dilatation and darkened color of nucleoli, cathepsis, erythrocyte infiltration, and vacuole formation were observed in some infected tissues. A qRT-PCR test showed that the 11 genome segments of GCRV had similar expression patterns in different tissues. The S8 segment, with unknown function and no homologous sequences, had the highest expression level, while the most conserved segment, L2, had the lowest expression level. GCRV particles were distributed in different tissues, especially in the intestine. In the infected intestine, the expression of various receptors and adaptor molecules was modulated at different levels. Pro-inflammatory cytokine interleukin-1β (IL-1β) expression was 2160.9 times higher than that in the control group. The upregulation of immunity-related genes activated the antiviral immunity pathways. Therefore, the intestine might play a dual role in mediating GCRV infection and the antiviral immune response. This study provides detailed information about the pathogenicity of GCRV and expression of immunity-related genes, laying the foundation for further research on virus control and treatment.

The GH (growth hormone)/IGFs (insulin-like growth factors) system has an important function in the regulation of growth. In this system, IGFBPs play a crucial regulatory role in IGF functions. As a member of the IGFBP family, IGFBP2 can bind to IGF and regulate IGF functions to regulate development and growth. In addition, IGFBP2 shows key regulatory functions in cell proliferation and metabolism. In this study, the igfbp2 gene was cloned from grass carp (Ctenopharyngodon idellus) liver. The ORF of grass carp igfbp2 is 834 bp long and encodes 277 amino acids. The tissue distribution results showed that igfbp2 is expressed in multiple tissues in grass carp and has a high expression level in the liver. In the OGTT, igfbp2 expression was significantly decreased in the liver and brain after 6 h of treatment with glucose. In vitro, igfbp2 expression in grass carp's primary hepatocytes was significantly suppressed by insulin after treatment for 6 and 12 h. Moreover, igfbp2 expression was markedly increased in a dose-dependent manner with glucagon incubation in grass carp's primary hepatocytes. To the best of our knowledge, this is the first report about Igfbp2 in grass carp. These results will provide a basis for the in-depth study of grass carp Igfbp2.

The present research was designed to investigate the growth promoting and immunostimulating properties of Moringa oleferia leaf meal (MLM) in grass carp. Juvenile grass carp (22.03 g ± 1.164) were fed with diets supplemented with 0, 50, 100 and 150 g/kg MLM for 48 days. At the end of feeding trial, skin mucus was used for analysis of lysozyme, protease, antiprotease and peroxidase activity. Head kidney was used for expression analysis of tumour necrosis factor‐alpha, interleukin‐8 and interferon‐γ. The obtained results showed that fish fed with 100 and 150 g/kg MLM had significant increase in weight gain and specific growth rate (p .05). In conclusion, supplementing juvenile grass carp feed with MLM up to 150 g/kg has growth promoting and immunostimulating effects.

Grass carp reovirus (GCRV) is an efficient pathogen causing high mortality in grass carp, meanwhile, fish interferon (IFN) is a powerful cytokine enabling host cells to establish an antiviral state; therefore, the strategies used by GCRV to escape the cellular IFN response need to be investigated. Here, we report that GCRV VP56 inhibits host IFN production by degrading the transcription factor IFN regulatory factor 7 (IRF7). First, overexpression of VP56 inhibited the IFN production induced by the polyinosinic-polycytidylic acid (poly I:C) and mitochondrial antiviral signaling protein (MAVS), while the capacity of IRF7 on IFN induction was unaffected. Second, VP56 interacted with RLRs but did not affect the stabilization of the proteins in the normal state, while the phosphorylated IRF7 activated by TBK1 was degraded by VP56 through K48-linked ubiquitination. Finally, overexpression of VP56 remarkably reduced the host cellular ifn transcription and facilitated viral proliferation. Taken together, our results demonstrate that GCRV VP56 suppresses the host IFN response by targeting phosphorylated IRF7 for ubiquitination and degradation., Highlights • VP56 inhibits IFN activation induced by MAVS. • VP56 induces the K48-linked ubiquitination and degradation of phosphorylated IRF7. • VP56 blocks the cellular IFN response and facilitates viral replication.

Hepcidin links iron metabolism with innate immunity during the inhibition of bacterial infection. Our previous studies had shown that recombinant hepcidin can significantly reduce the mortality rate of Ctenopharyngodon idella infected with Flavobacterium columnare under laboratory conditions. Here, we studied the preventive and therapeutic effects of feed supplemented with different doses of recombinant hepcidin on F. columnare-challenged C. idella reared in a cage culture environment. The results showed that in the prevention groups, 30 and 90 mg/kg of added purified and unpurified hepcidin respectively resulted in a higher survival rate in the early post-infection period, while 60 mg/kg of purified hepcidin significantly improved the survival rate in the therapy group (all compared to the control group). In the hepatopancreas, the expression of hepcidin and ferritin was significantly up-regulated, and the levels of ferroportin and serum iron were significantly decreased, especially in the therapy group. In addition, the expression of iron-related genes in spleen and intestine exhibited a similar trend to that in hepatopancreas. Meanwhile, immune genes were up-regulated to varying degrees, and the therapy group exhibited a significantly improved expression of pro-inflammatory cytokines and specific immunity. In summary, our study shows that different doses of recombinant hepcidin had protective effects against bacterial infection by regulating the iron distribution and immune gene expression, which provides a strong foundation for the application of recombinant hepcidin in aquaculture.

The current study was conducted to evaluate the effect of dietary soy isoflavones (SI) on growth performance, antioxidant status, immune response and resistance to Aeromonas hydrophila in juvenile grass carp (Ctenopharyngodon idella). Six diets were formulated to contain 0 (control), 10, 50, 100, 500 or 1,000 mg SI per kg feed. Each diet was randomly allotted to triplicate net cages, and each net cage was stocked with 30 fish. The fish were fed one of the experimental diets to satiation twice per day for 60 days. The results showed that the WGR and DGC of the 500 mg/kg SI‐supplemented group were significantly higher than those of the non‐SI‐supplemented group (p

This study investigated the effect of xylanase supplementation of a plant protein‐enriched diet on the growth performance and intestinal immune function of grass carp grow‐out (Ctenopharyngodon idella). Five hundred forty healthy grass carp grow‐out (232.74 ± 0.12 g) were fed diets containing graded levels of xylanase for 60 days. The fish were then challenged with Aeromonas hydrophila for 14 days. The results indicated that xylanase supplementation (a) improved fish growth performance and optimized the intestinal microflora; (b) increased lysozyme and acid phosphatase activities, complement and immunoglobulin M levels and antibacterial peptide hepcidin mRNA levels, thereby enhancing fish intestinal immune function; and (c) attenuated intestinal inflammation associated with the inhibition of the NF‐κB signalling pathway and activation of the TOR signalling pathway, resulting in the down‐regulation of pro‐inflammatory cytokines [except IL‐1β and IL‐12p40 in the distal intestine (DI) and IL‐6 in the proximal intestine (PI)] and up‐regulation of anti‐inflammatory cytokines [except IL‐4/13B and TGF‐β1 in the mid intestine (MI) and DI]. Finally, on the premise of a basal diet containing 45.8 g/kg araboxylans (AXs), the appropriate supplementation levels based on the ability to improve per cent weight gain (PWG) and prevent enteritis were estimated to be 1,527 and 1,608 U/kg, respectively.

To investigate the effect of dietary folic acid on fish flesh quality, muscle antioxidant status and the potential mechanism, young grass carp (Ctenopharyngodon idella) were fed diets containing 0.10–3.12 mg/kg diet of folic acid for 8 weeks. The results showed that optimum folic acid increased muscle contents of protein, lipid, pH, shear force, amino acids, unsaturated fatty acids, hydroxyproline and glutathione, whereas contents of moisture, cooking loss, lactate and saturated fatty acids and cathepsin L activity showed an opposite trend (p

Cadmium (Cd), a substance with one of the most critical health hazard indices, can cause damage to both the blood and kidneys and accumulates in the body at last. The present work studied the toxicological effects of Cd and the therapeutic effects of metallothionein (MT) and vitamin E (VE) on the trunk kidney and blood of freshwater grass carp (Ctenopharyngodon idellus). Grass carp were divided into three groups: Cd + phosphate-buffered saline (PBS) group, Cd + VE group, and the Cd + MT group. Fish were injected with CdCl2 on the first day and then VE, MT, or PBS was administered 4 days post-injection. Fish not injected with Cd were used as a negative control. The blood and trunk kidney amassed Cd and suffered severe damage in the forms of organ toxicity cytotoxicity, and immunotoxicity. However, the MT reduced the Cd content in the trunk kidney and blood and partially stabilized the damaged organs. Treatment with VE, however, only demonstrated weaker protection against on Cd-induced toxicity. The results indicate that exogenous MT may play an essential role in restoring homeostasis of the Cd-poisoned urinary and circulatory system and that it may help eliminate Cd in aquatic animals.

Ctenopharyngodon idellagrowth arrest and DNA damage-inducible 45aa (CiGadd45aa) is a member of the Gadd45 family of immune-related proteins in grass carp. There is increasing evidence that microRNAs (miRNAs) are involved in the regulation of inflammatory responses and apoptosis. However, little is known about the regulatory effects of miRNAs on CiGadd45aa expression in grass carp. In the present study, CiGadd45aa was identified as a target gene of miR-731 based on miRNA expression profiling and dual-luciferase reporter assay. Our study revealed that miR-731 targets CiGadd45aa and regulates the expression of proinflammatory factors, thereby regulating immune response in grass carp. In addition, CiGadd45aa and miR-731 were both found induced apoptosis. Hence, our findings provide a theoretical basis for exploring the molecular mechanism by which miR-731 regulates inflammation and apoptosis in grass carp.

Interleukin (IL)-2 belongs to the four-helix bundle cytokine family and plays key roles in growth, survival, activation-induced cell death and differentiation of the immune cells. In cyprinid fish, only common carp interleukin-2 (il2) has been cloned because of relatively low sequence homology between carp Il-2 and its homologs in other fish species. In the present study, the coding sequence of grass carp Il-2 (gcIl-2) was cloned and its identity was verified via bioinformatic analysis. Tissue distribution study showed that grass carp il2 (gcil2) mRNA was expressed in thymus, head kidney and gill with relatively high levels. Recombinant gcIl-2 (rgcIl-2) protein was subsequently prepared by using a prokaryotic expression system followed by a refolding method. The purified rgcIl-2 displayed an ability to stimulate the cell proliferation along with an increased mRNA expression of cd4l but not cd8a, igm or mcsfr in grass carp head kidney leukocytes (HKLs), suggesting the possible involvement of gcIl-2 in T helper (Th) cell proliferation. In the same cell model, rgcIl-2 significantly enhanced mRNA expression of some cytotoxic molecules including perforin-like protein 2, granzyme B-like and Fas ligand, indicating the modulation of cytotoxic cells by gcIl-2 in grass carp HKLs. Besides, gene expression of regulatory T (Treg) cell- and Th1/2 cell-related cytokines or transcription factors was detected in grass carp HKLs treated by rgcIl-2. Results showed that rgcIL2 treatment increased the mRNA expression of foxp3, cd25l, ifng2, il12p35, tbet, tnfa, il2, il4/13a, il4/13b and gata3l in HKLs, implying the regulatory roles of Il-2 in the expression of these immune genes and its possible involvement in differentiation of Treg and Th1/2 cells. These observations together with the related studies in other fishes suggest the existence of cytotoxic cells, Treg and Th1/2 subpopulations in fish species and the functional roles of Il-2 in these cells.

Functionally active components of faba bean (Vicia faba, FB) are known to improve the muscle texture and growth performance of grass carp (Ctenopharyngodon idellus), but this has not yet been proven by feeding trials using isonitrogenous and isoenergetic diets. Here, we formulated isonitrogenous and isoenergetic diets containing four types of FB extracts and examined their effects on the weight gain, textural quality, intestinal microbiota, metabolic enzymes and immune response of grass carp by a 100-day feeding trial. An isonitrogenous and isoenergetic diet with no FB extracts (control) and whole FB were also used. Compared to the control, the FB water extract diet increased weight gain, muscle hardness and chewiness, intestinal microbiota diversity, the activity of metabolic enzymes including lipase (LP), glucose-6-phosphatase (G-6-P), acetyl-CoA and succinate dehydrogenase (SDH) (P

The occurrence of immuno-compromised status in animals with zearalenone (ZEA) exposure may be a critical contributor to associated mucosal (gastrointestinal tract) diseases. However, it is difficult to assess the associated risks with limited reference data. This study comprehensively discussed the effects of ZEA on intestinal immune components, cytokines and molecular mechanism of juvenile grass carp infected with Aeromonas hydrophila. Specifically, the fish were fed six graded levels of dietary ZEA (0–2507 μg kg−1 diet) for 70 d. The results pointed out that the average residual amount of ZEA in the intestines increased with dose level after ZEA feeding. We further performed an infection assay using A. hydrophila. After 14 d, ZEA groups increased enteritis morbidity rate compared with controls. The acid phosphatase (ACP), lysozyme (LZ) activities and immunoglobulin M (IgM) content were significantly decreased in three intestinal segments. Furthermore, ZEA could reduce the transcription of β-defensin-1, Hepcidin, liver expressed antimicrobial peptide 2A/2B (LEAP-2A/2B) and Mucin-2. We next confirmed the loss of these immune components accompanied by the invasion of the intestinal barrier by bacteria, as indicated by activation of the nuclear factor κB (NF-κB) and the expression of downstream cytokines. Notably, the phosphorylated target of rapamycin (TOR) plays an important role in regulating these genes, thus indicating a possible target caused by ZEA. In summary, the extensive inhibition of immune components by ZEA promotes the spread of pathogens, which may increase the possibility of intestinal mucosa exposure and the risk of transforming disease.

Prolactin-releasing peptide (PrRP), a sort of vital hypothalamic neuropeptide, has been found to exert an enormous function on the food intake of mammals. However, little is known about the functional role of PrRP in teleost. In the present study, two PrRP isoforms and four PrRP receptors were isolated from grass carp. Ligand-receptor selectivity displayed that PrRP1 preferentially binds with PrRP-R1a and PrRP-R1b, while PrRP-R2a and PrRP-R2b were special receptors for PrRP2. Tissue distribution indicated that both PrRPs and PrRP-Rs were highly expressed in the hypothalamus-pituitary-gonad axis and intestine, suggesting a latent function on food intake and reproduction. Using grass carp as a model, we found that food intake could significantly induce hypothalamus PrRP mRNA expression, which suggested that PrRP should be also an anorexigenic peptide in teleost. Interestingly, intraperitoneal (IP) injection of PrRPs could significantly induce serum luteinizing hormone (LH) secretion and pituitary LHβ and GtHα mRNA expression in grass carp. Moreover, using primary culture grass carp pituitary cells as a model, we further found that PrRPs could directly induce pituitary LH secretion and synthesis mediated by AC/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-II pathways. Finally, estrogen treatment of prepubertal fish elicited increases in PrRPs and PrPR receptors expression in primary cultured grass carp hypothalamus cells, which further confirmed that the PrRP/PrRPR system may participate in the neuroendocrine control of fish reproduction. These results, taken together, suggest that PrRPs might act as a coupling factor in feeding metabolism and reproductive activities in teleost.