Gill Of Grass Carp Research Articles

Exploring the Potential of Isalo Scorpion Cytotoxic Peptide in Enhancing Gill Barrier Function and Immunity in Grass Carp (Ctenopharyngodon idella) Infected with Flavobacterium columnare.

The objective of this research was to investigate how dietary antimicrobial peptides (AMP), namely, Isalo scorpion cytotoxic peptide (IsCT), affect the gill physical barrier function and immune function of grass carp challenged with Flavobacterium columnare (F. columnare). Five hundred forty grass carp were randomly allocated to six groups and fed to varying levels of IsCT in the diet (0, 0.6, 1.2, 1.8, 2.4, and 3.0 mg/kg diet) for a duration of 60 days. Afterward, the grass carps faced a challenge from F. columnare. The results revealed that the use of optimal IsCT dramatically mitigated gill damage in grass carp that were infected with F. columnare. Additionally, IsCT exhibited a notable enhancement in gill antioxidant capabilities, as evidenced by a significant reduction in ROS, MDA, and PC levels, an elevation in antioxidant enzyme activities, and an upregulation of antioxidant-related genes and Nrf2 mRNA levels. Conversely, the expression of Keap1a and Keap1b mRNA was decreased. Besides, IsCT exhibited its capability to inhibit apoptosis via downregulating the mRNA levels of caspase-2, caspase-3, caspase-7, caspase-8, caspase-9, Apaf1, Fasl, Bax, and JNK while concurrently increasing the mRNA levels of Bcl-2, Mcl-1, and IAP in fish gills. Additionally, IsCT promoted the integrity of tight junction barrier by increasing the gene expression of claudin-b, claudin-c, claudin-3c, ZO-1, ZO-2b, occludin, and JAM while suppressing MLCK signaling. Additionally, optimal dietary IsCT improved antibacterial ability, as evidenced by heightened LZ, ACP activities, and elevated levels of C3, C4, and IgM. Additionally, there was an upregulation in β-defensin-1, LEAP-2A, LEAP-2B, hepcidin, and mucin-2 mRNA expression in the gills. Simultaneously, the inclusion of optimal dietary IsCT in the diet resulted in improved gill immunity barriers through the reduction of proinflammatory cytokine mRNA levels and the increase in the expression of anti-inflammatory cytokine mRNA levels. This was partly facilitated by the IκBa/NF-κB p65 signaling pathway and TOR/S6K1 signaling pathways in the gills of grass carp. Therefore, supplementing the diet with IsCT has potential advantages in enhancing gill health by improved physical barriers and immunity in grass carp. Based on LZ activity and against lipid peroxidation, optimum IsCT concentrations in on-growing grass carp (136.88 ± 0.72 g) were found to be 1.68 and 1.54 mg/kg diet, respectively.

The Bacillus velezensis CYS06 Strain Exhibits Promising Applications in Fighting Grass Carp Bacterial Diseases

Aeromonas septicemia and columnaris disease are major bacterial diseases in grass carp; however, the drugs currently used to control these diseases pose environmental and health risks. This study aimed to screen for a probiotic Bacillus strain with antagonistic activity to prevent and control bacterial diseases in grass carp and to evaluate the antimicrobial activities, biosafety, and biocontrol effects of this strain. A Bacillus strain with antagonistic activity against Aeromonas hydrophila, obtained from grass carp intestines, was screened, and the isolate CYS06 was identified by analyzing the 16S rRNA and gyrA gene sequences. The antimicrobial spectrum of the strain CYS06 was determined, and the activities of amylase, cellulase, protease, and lipase of the strain CYS06 were determined. The whole genome of the strain CYS06 was sequenced using the nanopore sequencing technology platform, followed by the analysis of the antagonistic substance synthesis gene clusters and CAZy enzyme gene families. The biosafety of the strain CYS06 was evaluated via intraperitoneal injection into healthy grass carp. After the strain CYS06 was fed to the grass carp, its biological control effect on this fish was evaluated through artificial infection experiments. The strain CYS06 was identified as Bacillus velezensis, based on molecular identification, which shows broad antimicrobial activity against various fish pathogens. The strain CYS06 secretes amylase, cellulase, protease, and lipase. The genome size of the strain CYS06 is 3,914,159 bp, and it contains eight antagonistic substance synthesis gene clusters and many CAZy enzymes. The strain CYS06 exhibits high biological safety for grass carp, based on the challenge test. Feeding grass carp with the strain CYS06 for 4 weeks significantly enhanced the resistance of the fish to A. hydrophila. Strain CYS06 could inhibit the growth of Flavobacterium columnare under co-culture and reduce the amount of F. columnare adherence on the gills of grass carp, indicating that CYS06 has good potential for the prevention and control of columnaris disease. In conclusion, we isolated an antagonistic probiotic strain, CYS06, which exhibits a biological control effect on septicemia and columnaris disease caused by Aeromonas spp. and F. columnare in grass carp, respectively. This strain contains many antagonistic substance synthesis-related gene clusters and holds the potential to degrade various types of carbohydrates. As a biological control agent, the strain CYS06 exhibits significant potential for the prevention and control of bacterial diseases in grass carp.

Aflatoxin B1 damaged structural barrier through Keap1a/Nrf2/ MLCK signaling pathways and immune barrier through NF-κB/ TOR signaling pathways in gill of grass carp (Ctenopharyngodon idella)

Aquafeeds are susceptible to contamination caused by aflatoxin B1 (AFB1). The gill of fish is an important respiratory organ. However, few studies have investigated the effects of dietary AFB1 exposure on gill. This study aimed to discuss the effects of AFB1 on the structural and immune barrier of grass carp gill. Dietary AFB1 increased reactive oxygen species (ROS) levels, protein carbonyl (PC) and malondialdehyde (MDA) contents, which consequently caused oxidative damage. In contrast, dietary AFB1 decreased antioxidant enzymes activities, relative genes expression (except MnSOD) and the contents of glutathione (GSH) (P < 0.05), which are partly regulated by NF-E2-related factor 2 (Nrf2/Keap1a). Moreover, dietary AFB1 caused DNA fragmentation. The relative genes of apoptosis (except Bcl-2, McL-1 and IAP) were significantly upregulated (P < 0.05), and apoptosis was likely upregulated through p38 mitogen-activated protein kinase (p38MAPK). The relative expressions of genes associated with tight junction complexes (TJs) (except ZO-1 and claudin-12) were significantly decreased (P < 0.05), and TJs were likely regulated by myosin light chain kinase (MLCK). Overall, dietary AFB1 disrupted the structural barrier of gill. Furthermore, AFB1 increased gill sensitivity to F. columnare, increased Columnaris disease and decreased the production of antimicrobial substances (P < 0.05) in grass carp gill, and upregulated the expression of genes involved with pro-inflammatory factors (except TNF-α and IL-8) and the pro-inflammatory response partly attributed to the regulation by nuclear factor κB (NF-κB). Meanwhile, the anti-inflammatory factors were downregulated (P < 0.05) in grass carp gill after challenge with F. columnare, which was partly attributed to the target of rapamycin (TOR). These results suggested that AFB1 aggravated the disruption of the immune barrier of grass carp gill after being challenge with F. columnare. Finally, the upper limit of safety of AFB1 for grass carp, based on Columnaris disease, was 31.10 μg/kg diet.

Myo-inositol prevents the gill rot in fish caused by Flavobacterium columnare infection

The gills are some of the most metabolically active organs in fish and are involved in gas exchange, osmotic regulation, acid-base balance, and ammonia nitrogen waste excretion. Here, we explored the actions of graded levels of dietary myo-inositol [27.0 (un-supplemented), 137.9, 286.8, 438.6, 587.7, and 737.3 mg/kg diet)] on the physical barrier functioning and signaling in the gills of grass carp (Ctenopharyngodon idella) for 10-weeks, and then infected by Flavobacterium columnare. It was found that optimal myo-inositol levels (approximately 137.9–438.6 mg myo-inositol /kg diet): (1) decreased the oxidative products such as malondialdehyde (MDA) and protein carbonyl (PC), while increased the activities and mRNA levels of SOD (CuZnSOD and MnSOD), CAT, GPx (GPx1a, GPx1b, GPx4a and GPx4b), GST (GSTO1, GSTO2, GSTP1 and GSTP2) and GR enzymes, possibly related to the Nrf2/Keap1 signaling pathway, suggesting that myo-inositol inhibited oxidative injury in fish gills; (2) inhibited cell apoptosis related to the down-regulation of caspases-2, −3, −7, −8, and-9 mRNA levels, possibly linked to the inhibition of the JNK/FasL/caspase 8 and p38MAPK/(Apaf-1, Bax, Bcl-2, Mcl-1, IAP)/caspase 9/caspase 3 signaling pathways; (3) promoted cell proliferation by up-regulating cyclins B, D, and E mRNA levels, possibly related to the E2F4 signaling; (4) maintained intercellular structural integrity by up-regulating tight junctional complexes (ZO-1, ZO-2, occludin, and claudins b, −c, f, −3c, −7a, −7b, and − 11), and down-regulating pore-forming TJs (claudins-12,-15a, and-15b) mRNA levels, possibly associated with the down-regulation of MLCK signaling. Based on the inhibition of gill rot and MDA content, the optimal myo-inositol intake was calculated as 285.8 and 305.4 mg/kg diet for grass carp (initial 221 g-final 792 g), respectively.

Microbiome of Co-cultured Fish Exhibits Host Selection and Niche Differentiation at the Organ Scale.

Fish are the most widespread aquaculture species and maintain complex associations with microbial consortiums. However, the ecology of these associations present in multiple microhabitats in fish remains elusive, especially on the microbial assembly in fish external (skin and gill) and internal (stomach and intestine) niches, and the relationship with the rearing environment. To understand host dependence and niche differentiation of organ-specific microbiome signatures using a 16S rRNA gene-based sequencing technique, we systematically provided characterizations of a comparative framework relevant to the microbiome of stomach, regional intestine, skin, and gill in two important farmed fish species, herbivorous grass carp (Ctenopharyngodon idella) and carnivorous southern catfish (Silurus meridionalis), and of the rearing water. The different feeding habits of grass carp and southern catfish showed a significant separation of microbial community structure, with great compositional differences across body sites within each species. Site-driven divergences relied on host species: the same types of microhabitats between grass carp and southern catfish harbored differential microbiome. Additionally, body sites had remarkably distinct communities and displayed lower alpha diversity compared to rearing water. Unexpectedly, the stomach of southern catfish had the highest microbial diversity in the digestive tract of the two co-cultured fish species. For external sites within each species, a higher diversity occurred in gill of grass carp and in skin of southern catfish. Our results unveil different topographical microbiome signatures of the co-cultured species, indicating host selection in individual-level microbial assemblages and niche differentiation at the organ scale. This work represents a foundation for understanding the comprehensive microbial ecology of cohabiting farmed fish, suggesting potential applications associated with fish microbiome that urgently needs to be assessed in polycultured operations in aquaculture.

Saprolegnia parasitica impairs branchial phosphoryl transfer network in naturally infected grass carp (Ctenopharyngodon idella): prejudice on bioenergetic homeostasis

Precise coupling of spatially separated intracellular adenosine triphosphate (ATP) producing and ATP-consuming exerts a key role in bioenergetic balance, and the phosphoryl transfer network, catalyzed by creatine kinase (CK), adenylate kinase (AK), and pyruvate kinase (PK), is fundamental in energetic homeostasis of tissues with high-energy requirements, as the branchial tissue. This whole system is very sensitive, and our hypothesis is that it can be altered in cases of infectious diseases in fish, such as that caused by the oomycete Saprolegnia parasitica. The effects of S. parasitica infection on gills remain poorly understood and limited only to histopathological studies. Thus, the aim of this study was to evaluate whether natural infection by S. parasitica impairs the enzymes of the phosphoryl transfer network in gills of grass carp (Ctenopharyngodon idella), as well as the pathways involved in this inhibition. In this study, we used sick carp and compared to healthy carp, fish of similar age and receiving the same feed, but allocated in different tanks. Branchial CK (cytosolic and mitochondrial) activity and ATP levels decreased in infected fish compared to uninfected on day 7 post-infection (PI), while no significant difference was observed between groups regarding branchial AK and PK activities. Branchial sodium-potassium ion pump (Na+, K+-ATPase) activity decreased in infected carp compared to uninfected on day 7 PI, while reactive oxygen species (ROS) and thiobarbituric acid reactive substance (TBARS) levels were higher. Gill histopathology revealed massive necrosis, loss of branchial epithelium, and detachment of the epithelium interlayer with structural loss of secondary lamellae. Based on these data, the impairment of CK activity elicited by S. parasitica caused an impairment in branchial energetic homeostasis, reducing the ATP availability in the gills and provoking an impairment on Na+, K+-ATPase activity. Moreover, the inhibition on CK activity appears to be mediated by ROS overproduction and lipid peroxidation, which contribute to disease pathogenesis linked to branchial tissue.

Exposure to methylmercury chloride inhibits mitochondrial electron transport chain and phosphotransfer network in liver and gills of grass carp: Protective effects of diphenyl diselenide dietary supplementation as an alternative strategy for mercury toxicity

Methylmercury chloride (CH3HgCl) is an important contaminant found in the environment that exerts highly toxic effects in multiple organs of aquatic animals. Recently, some studies revealed the toxic effects of methylmercury and mercury chloride to the energetic metabolism of rodents. However, possible toxic effects of CH3HgCl related to bioenergetic homeostasis of adenosine triphosphate (ATP) in aquatic animals, such as fish, remain unknown. Moreover, recent evidence has demonstrated the protective effects of diphenyl diselenide (Ph2Se2) in a model of fish poisoning by mercury chloride, but its possible protective effects on bioenergetic homeostasis are not yet established. Thus, the aim of this study was to evaluate whether a dietary supplementation with Ph2Se2 prevents or minimizes hepatic and branchial bioenergetic dysfunction elicited by CH3HgCl via protective effects on respiratory chain complexes and phosphotransfer network pathway. Branchial cytosolic and mitochondrial creatine kinase activity and hepatic and branchial mitochondrial complexes II, II-III and IV activities were inhibited (except for mitochondrial complex IV in gills) in fish exposed to CH3HgCl when compared to control group. Moreover, the hepatic and branchial ATP content, as well as sodium-potassium pump (Na+/K+-ATPase) and proton-pump (H+-ATPase) activities were inhibited in fish exposed to CH3HgCl when compared to control group. Finally, a significant increase on reactive oxygen species levels was observed in fish exposed to CH3HgCl in comparison to control group, while non-protein thiols and protein thiols groups were decreased. Dietary supplementation with 3 mg/kg Ph2Se2 prevented or reduced all alterations elicited by CH3HgCl. In summary, Ph2Se2 can be considered a suitable approach to prevent/minimize the hepatic and branchial bioenergetic dysfunction elicited by CH3HgCl.

Identification of proteins differentially expressed in the gills of grass carp (Ctenopharyngodon idella) after hypoxic stress by two-dimensional gel electrophoresis analysis.

Two-dimensional gel electrophoresis (2-DE) was combined with liquid chromatography-mass spectrometry (LC-MS/MS) to identify the differential proteomics of grass carp gills after hypoxic stress to better understand the roles of proteins in the hypoxic response and to explore the possible molecular mechanisms. Protein spots were obtained from a hypoxia-stressed group (372 ± 11 individuals) and a control group (406 ± 14 individuals) using the lmage Master 2D Platinum 7.0 analysis software. Fifteen protein spots were expressed differentially in the hypoxia-stressed group and varied significantly after exposure to the hypoxic conditions. In addition, these differential proteins were identified by mass spectrometry and then searched in a database. We found the expression and upregulation of the toll-like receptor 4, ephx1 protein, isocitrate dehydrogenase, L-lactate dehydrogenase, GTP-binding nuclear protein Ran, and glyceraldehyde-3-phosphate dehydrogenase; however, the expression of the keratin type II cytoskeletal 8, type I cytokeratin, ARP3 actin-related protein 3 homolog, thyroid hormone receptor alpha-A, ATP synthase subunit beta, citrate synthase, tropomyosin 2, and tropomyosin 3 were downregulated. Six proteins were found in the hypoxia-inducible factor-1 (HIF-1) signaling pathway. We concluded that the grass carp gill is involved in response processes, including energy generation, metabolic processes, cellular structure, antioxidation, immunity, and signal transduction, to hypoxic stress. To our knowledge, this is the first study to conduct a proteomics analysis of expressed proteins in the gills of grass carp, and this study will help increase the understanding of the molecular mechanisms involved in hypoxic stress responses in fish at the protein level.

Impairing of gill health through decreasing immune function and structural integrity of grass carp (Ctenopharyngodon idella) fed graded levels dietary lipids after challenged with Flavobacterium columnare

The current study conducted to investigate the hypothesis that low or excess levels of lipids increased the gill rot morbidity through impairing the immune function and structural integrity in the gill of grass carp (Ctenopharyngodon idella). A total of 540 young grass carp with an average initial weight of 261.41 ± 0.53 g were fed diets containing six graded levels of lipids at 0.59%, 2.14%, 3.60%, 5.02%, 6.66% and 8.01% diets for 8 weeks. After the growth trial, fish were challenged with Flavobacterium columnare for 3 days. The results indicated that compared with optimal lipids supplementation (2.14%–8.01% lipids diets), low or excess levels of lipids impaired fish immune function through declining the activities of humoral compounds, down-regulated the mRNA levels of anti-inflammatory cytokines, inhibitor of κBα (IκBα) and ribosomal p70S6 kinase (S6K1), and up-regulated pro-inflammatory cytokines, nuclear factor κB p65 (NF-κB p65) (not p52), IκB kinase α (IKKα) (not IKKβ), IKKγ and eIF4E-binding protein (4EBP) in the gill of young grass carp. In addition, low or excess levels of lipids decreased young grass carp physical barrier function through down-regulating the mRNA levels of ZO-1 (rather than ZO-2b), Claudin b, c, 3, 12, 15a, 15b, 7b, 7a and Occludin through MAPKK 6/p38 MAPK/MLCK signaling molecules, decreasing antioxidant ability via Kelch-like ECH-associating protein 1a (Keap1a)/NF-E2-related factor 2 (Nrf2) signaling molecules, and down-regulating the mRNA levels of B-cell lymphoma-2 (Bcl-2) and inhibitor of apoptosis protein (IAP) and up-regulating the mRNA levels of apoptotic protease activating factor-1 (Apaf-1), Caspase-3, -8 and -9 and Fas ligand (FasL) in the gill of grass carp. Based on the quadratic regression analysis for the gill rot morbidity, C3 and MDA contents, the dietary lipids requirements for young grass carp have been estimated to be 5.60%, 6.01% and 4.58% diets.

Effects of moroxydine hydrochloride and ribavirin on the cellular growth and immune responses by inhibition of GCRV proliferation

Moroxydine hydrochloride (Mor) and ribavirin (Rib) are known for their multi-antiviral activities against DNA and RNA viruses but little information is available about the pharmacological impact in aquaculture. The present study was undertaken to investigate the response of host cells to antiviral compounds during the anti-GCRV treatment. The scanning electron microscope results showed that Ctenopharyngodon idella kidney (CIK) cells have a higher death rate at 72h post virus infection. At the concentration of 40μgmL−1, Mor and Rib had a significant protective effect on virus-infected cells. Moreover, the gene expressions of vp5, vp6 and NS66 were significantly inhibited by treatment with Mor or Rib, especially gene expression of the vp5. For the immunoregulatory action, no distinct induction of the expression of immune genes was observed after the addition of Mor and Rib to the virus-free cells. However, the compounds significantly decreased the virus-induced gene overexpression of Myd88, Mx1, IL-1β, IL-8, I-IFN and TNFα in CIK cells. Moreover, Mor and Rib significantly inhibited the immune genes upregulation which was induced by GCRV in kidney, liver, muscle and gill of grass carp, despite greater partial gene expressions were detected than the virus-free control group. Besides, Mor and Rib blocked cell cycle changes, cytopathic effects, cellular death and virus proliferation in CIK cells thereby maintaining normal morphological structure. Overall, Mor and Rib as antiviral compounds are effective for the control of GCRV replication and the indirectly regulation of cellular immune response.

Vitamin E deficiency depressed gill immune response and physical barrier referring to NF-kB, TOR, Nrf2 and MLCK signalling in grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare

The aim of this study was to investigate the effect of graded levels of vitamin E (0–225mg/kg) on gill immune response and physical barriers, and relative mRNA levels of signalling molecules in the gill of grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin E supplementation, vitamin E deficiency (1) increased the rate of gill rot morbidity and aggravated gill histopathological injuries following F. columnare infection (P<0.05); (2) decreased LA and ACP activities, complement component 3, complement component 4 and IgM contents and down-regulated antimicrobial peptides (liver-expressed antimicrobial peptide-2A, -2B, hepcidin, β-defensin) and IgT mRNA levels (P<0.05); (3) down-regulated the mRNA levels of anti-inflammatory cytokines and the signalling molecules inhibitory protein κBα, the target of rapamycin and ribosome protein S6 kinase 1 (P<0.05) and up-regulated the mRNA levels of pro-inflammatory cytokines and the signalling molecules NF-κB p65, IκB kinase α (IKKα), IKKβ and eIF4E-binding protein 1 (P<0.05); (4) decreased activities of anti-superoxide anion, Cu/ZnSOD, MnSOD, GPx, GST and GR, and down-regulated mRNA levels of those antioxidant enzymes and signalling molecule NF-E2-related factor 2 in the gills of fish (P<0.05); (5) down-regulated the relative mRNA levels of B cell lymphoma-2 protein, and up-regulated the relative mRNA levels of caspase-2, -3, -7, -9, Bcl-2 associated X protein and apoptotic protease activating facter-1 (P<0.05); (6) down-regulated the relative mRNA levels of tight junction proteins (occludin, zonula occludens-1, ZO-2, claudin-3, -b, -c and -11a) and up-regulated signalling molecule myosin light chain kinase mRNA levels (P<0.05). In conclusion, vitamin E deficiency disrupted gill immune barriers and physical barrier function via impairing gill immunity and antioxidant capacity, inducing apoptosis and changing tight junction protein transcription abundances and the related signalling molecules in the gills of fish. The optimal vitamin E requirements for against gill rot morbidity of grass carp (266–1026g) were estimated to be 139.56mg/kg diet. Meanwhile, based on immune indicator (ACP activity) and antioxidant indicator (MDA content), the optimal vitamin E requirements for young grass carp were estimated to be 130.67 and128.53mg/kg diet, respectively.

Towards the modulation of oxidative damage, apoptosis and tight junction protein in response to dietary leucine deficiency: A likely cause of ROS-induced gill structural integrity impairment

The current study explored the protective effect of leucine on antioxidant status, apoptosis and tight junction damage in the gill of grass carp (Ctenopharyngodon idella Val.). The trial was conducted by feeding grass carp with six graded level of leucine (7.1, 8.9, 11.0, 13.3, 15.2 and 17.1 g kg−1 diet) for 8 weeks. The fish were fed to apparent satiation 4 times per day. The results indicated that compared with the leucine deficiency group, 8.9–11.3 g leucine kg−1 diet supplementations decreased protein carbonyl (PC), malondialdehyde (MDA) and ROS contents, which may be partially attributed to the improvement of antioxidant status in the gill by increasing hydroxyl radical capacity and anti-superoxide radicals, glutathione contents and the activities and mRNA levels of Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPx), catalase (CAT), glutathione-S-transferase (GST) and glutathione reductase (GR), that referring to the up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA expression. Moreover, leucine deficiency induced DNA fragmentation via the up-regulation of caspase-3, caspase-8 and caspase-9 expressions and down-regulation of target of rapamycin and ribosomal S6 protein kinase 1 expressions. Furthermore, leucine deficiency increased interleukin-1β (IL-1β), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) mRNA expression and decreased IL-10 and transforming growth factor β (TGF-β), which was partly related to nuclear factor κB (NF-κB) and its inhibitor (IκB). In contrast, the relative mRNA expression of IL-1, IL-8 and TNF-α was down-regulated with 8.9–11.3 g leucine kg−1 diet supplementations. Finally, the relative mRNA expression of tight junction protein, including occludin, zonula occludens-1, claudin b, claudin 3 and claudin 12 was up-regulated with leucine diet supplementations. Our results indicate that leucine protected the fish gill structural integrity partially because of the inhibition of apoptosis, the improvement of antioxidant status, the regulation of tight junction protein and related signalling molecules mRNA expressions in the fish gill.

Impairment of gill structural integrity by manganese deficiency or excess related to induction of oxidative damage, apoptosis and dysfunction of the physical barrier as regulated by NF-κB, caspase and Nrf2 signaling in fish

This study is for the first time to explore the possible effects of dietary manganese (Mn) on structural integrity and the related signaling in the gills of fish. Grass carp (Ctenopharyngodon idella) were fed with six diets containing graded levels of Mn [3.65–27.86 mg Mn/kg diet] for 8 weeks. The results firstly demonstrated that Mn deficiency aggravated inflammation indicated by up-regulation of pro-inflammatory cytokines (tumour necrosis factor α, interleukin 8, and interleukin 1β mRNA levels) and down-regulation of anti-inflammatory cytokines (interleukin 10, transforming growth factor-β1) mRNA levels, which might be partially related to the up-regulation of nuclear factor kappa B (NF-κB p65) and down-regulation of nuclear inhibitor factor κBα (iκBα) mRNA levels in the gills of fish. Meanwhile, Mn deficiency caused DNA fragmentation, which might be partially associated with the up-regulation of the apoptosis signaling (caspase-3, caspase-8 and caspase-9) in the gills of fish. Furthermore, Mn deficiency-caused apoptosis might be partly related to the increases of oxidative damage that indicated by increases of lipid peroxidation and protein oxidation, and decreases of antioxidant enzyme activities [included Mn superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST)]. However, Mn deficiency only down-regulated MnSOD and GST mRNA levels, which might be partially related to the up-regulation of NF-E2-related factor-2 (Nrf2) inhibitor (Keap1), and only down-regulated the gene expression of claudin-b and claudin-15 to disrupt the TJ in the gills of fish. Excessive Mn led to negative effects on partial parameters studied in the gills of fish. The optimal levels of Mn based on protecting against ROS, MDA and PC in the gills of grass carp were 17.04, 16.86 and 21.20 mg/kg diet, respectively. Collectively, Mn deficiency or excess could cause inflammation, apoptosis, antioxidant system disruption and change tight junction protein (claudin-b and claudin-15) transcription abundances, which might be partially related to the NF-κB p65, caspase-(3,8,9) and Nrf2 signaling, in the gills of fish.

Sequencing, characterization and phylogenomics of the complete mitochondrial genome of Dactylogyrus lamellatus (Monogenea: Dactylogyridae)

Despite the worldwide distribution and pathogenicity of monogenean parasites belonging to the largest helminth genus, Dactylogyrus, there are no complete Dactylogyrinae (subfamily) mitogenomes published to date. In order to fill this knowledge gap, we have sequenced and characterized the complete mitogenome of Dactylogyrus lamellatus, a common parasite on the gills of grass carp (Ctenopharyngodon idella). The circular mitogenome is 15,187 bp in size, containing the standard 22 tRNA genes, 2 rRNA genes, 12 protein-encoding genes and a long non-coding region (NCR). There are two highly repetitive regions in the NCR. We have used concatenated nucleotide sequences of all 36 genes to perform the phylogenetic analysis using Bayesian inference and maximum likelihood approaches. As expected, the two dactylogyrids, D. lamellatus (Dactylogyrinae) and Tetrancistrum nebulosi (Ancyrocephalinae), were closely related to each other. These two formed a sister group with Capsalidae, and this cluster finally formed a further sister group with Gyrodactylidae. Phylogenetic affinity between Dactylogyrinae and Ancyrocephalinae was further confirmed by the similarity in their gene arrangement. The sequencing of the first Dactylogyrinae, along with a more suitable selection of outgroups, has enabled us to infer a much better phylogenetic resolution than recent mitogenomic studies. However, as many lineages of the class Monogenea remain underrepresented or not represented at all, a much larger number of mitogenome sequences will have to be available in order to infer the evolutionary relationships among the monogeneans fully, and with certainty.

Dietary vitamin C deficiency depressed the gill physical barriers and immune barriers referring to Nrf2, apoptosis, MLCK, NF-κB and TOR signaling in grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare

This study explored the effects of vitamin C on the physical barriers and immune barriers, and relative mRNA levels of signaling molecules in the gill of grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) (1) increased reactive oxygen species, malondialdehyde and protein carbonyl (PC) contents (P < 0.05), decreased the copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and mRNA levels (P < 0.05), and glutathione and vitamin C contents (P < 0.05), down-regulated NF-E2-related factor 2 mRNA level (P < 0.05), and up-regulated Kelch-like ECH-associating protein (Keap) 1a (rather than Keap1b) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency induced oxidative injury in fish gill; (2) up-regulated caspase-3, -7, -8, -9, Fas ligand, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1 mRNA levels (P < 0.05), and down-regulated inhibitor of apoptosis protein and B-cell lymphoma-2 (rather than myeloid cell leukemia-1) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated cell apoptosis in fish gill; (3) up-regulated pore-forming TJs Claudin-12, 15a, -15b, and related signaling molecules myosin light chain kinase, p38 mitogen-activated protein kinase (rather than c-Jun N-terminal kinases) mRNA levels (P < 0.05), and down-regulated barrier-forming TJs Occludin, zonula occludens (ZO) 1, ZO-2, Claudin-c, -3c, -7a, -7b mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency disrupted tight junctional complexes in fish gill; (4) decreased lysozyme and acid phosphatase (ACP) activities, and complement 3 (C3), C4 and IgM contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, Hepcidin, β-defensin mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency decrease fish gill immune function; (5) down-regulated the mRNA levels of anti-inflammatory cytokines-related factors interleukin 10 (IL-10), IL-11, transforming growth factor (TGF) β1, TGF-β2, inhibitor of κBa and eIF4E-binding protein 1 (4E-BP1) (rather than 4E-BP2) (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors interferon γ2, IL-1β, IL-6, IL-8, IL-12 P35, IL-12 P40, nuclear factor κB (NF-κB) p65 (rather than NF-κB p52), IκB kinases (IKK) (only IKKα and IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated fish gill inflammation. In conclusion, vitamin C deficiency disrupted physical barriers and immune barriers, and regulated relative mRNA levels of signaling molecules in fish gill. The vitamin C requirement for against gill rot morbidity of grass carp (264–1031 g) was estimated to be 156.0 mg/kg diet. In addition, based on the gill biochemical indices (antioxidant indices MDA, PC and vitamin C contents, and immune indices LA and ACP activity) the vitamin C requirements for grass carp (264–1031 g) were estimated to be 116.8, 156.6, 110.8, 57.8 and 134.9 mg/kg diet, respectively.

Effects of dietary protein levels on the disease resistance, immune function and physical barrier function in the gill of grass carp (Ctenopharyngodon idella) after challenged with Flavobacterium columnare

The effects of dietary protein levels on the disease resistance, gill immune function and physical barrier function of grass carp (Ctenopharyngodon idella) were investigated in this study. A total of 540 grass carp (264.11 ± 0.76 g) were fed six diets containing graded levels of protein (143.1, 176.7, 217.2, 257.5, 292.2 and 322.8 g digestible protein kg−1 diet) for 8 weeks. After the growth trial, fish were challenged with Flavobacterium columnare for 3 days. The results indicated that optimal levels of dietary protein had the following effects: (1) the production of antibacterial components increased, and anti-inflammatory cytokines, inhibitor of κBα, target of rapamycin and ribosomal protein S6 kinases 1 mRNA levels were up-regulated, whereas mRNA levels of pro-inflammatory cytokines, nuclear factor kappa B (NF-κB) P65, NF-κB P52, IκB kinase (IKK) α, IKKβ, IKKγ, eIF4E-binding proteins (4E-BP) 1 and 4E-BP2 were down-regulated in the gills of grass carp (P < 0.05), indicating that fish gill immune function was enhanced at an optimal level of dietary protein; (2) the activities and mRNA levels of antioxidant enzymes and glutathione content increased, the contents of reactive oxygen species, malondialdehyde and protein carbonyl (PC) decreased, and NF-E2-related factor 2, B-cell lymphoma protein-2, inhibitor of apoptosis proteins, myeloid cell leukemia-1 and tight junction complexes mRNA levels were up-regulated, whereas Kelch-like-ECH-associated protein (Keap) 1a, Keap1b, cysteinyl aspartic acid-protease 3, 8, 9, fatty acid synthetase ligand, apoptotic protease activating factor-1, Bcl-2 associated X protein, c-Jun N-terminal protein kinase, myosin light chain kinase and p38 mitogen-activated protein kinase mRNA levels were down-regulated in the gills of grass carp (P < 0.05), indicating that the fish gill physical barrier function improved at an optimal level of dietary protein. Finally, based on the gill rot morbidity, ACP activity and PC content, the optimal levels of dietary protein for grass carp were estimated to be 286.65 g kg−1 diet (253.73 g digestible protein kg−1 diet), 290.46 g kg−1 diet (257.76 g digestible protein kg−1 diet) and 296.25 g kg−1 diet (260.69 g digestible protein kg−1 diet), respectively.

Seasonal Dynamics and Spatial Distribution of the Dactylogyrus Species on the Gills of Grass Carp (Ctenopharyngodon idellus) from a Fish Pond in Wuhan, China.

Spatial distribution and seasonal variation in mean intensity and prevalence of monogeneans on the gills of grass carp ( Ctenopharyngodon idellus ) were investigated from September 2012 to December 2014 in a fish pond in Wuhan, China. During this period, 2 species of Dactylogyrus were found, i.e., Dactylogyrus lamellatus and Dactylogyrus ctenopharyngodonis. Dactylogyrus lamellatus was present during the entire duration of the investigation, whereas D. ctenopharyngodonis was only detected after January 2014, when another batch of grass carp fingerlings was added to the pond. Prevalence of the 2 Dactylogyrus spp. was relatively high throughout the year, but significant seasonal changes were detected in the mean intensity of the 2 species (P < 0.05). Mean intensity of D. lamellatus peaked in late winter and spring, and then dropped to the lowest point in summer. Dactylogyrus ctenopharyngodonis exhibited a high mean intensity in summer and autumn, but low in winter and spring. Spatial distribution of the 2 species was similar: the highest mean intensity was found on medial and distal parts of the second gill arch. Moreover, no evident change was detected in niche preference of D. lamellatus after the infection of D. ctenopharyngodonis occurred. There was no significant positive correlation in abundance between the 2 species (P > 0.05). In addition, there were no significant differences in the percentage of mean intensity of the 2 species on each gill arch among low-, medium-, and high-intensity groups (P > 0.05). These results suggest unsaturated gill niches and the absence of interspecific competition between the 2 Dactylogyrus species.

Dietary choline regulates antibacterial activity, inflammatory response and barrier function in the gills of grass carp (Ctenopharyngodon idella).

An 8-week feeding trial was conducted to determine the effects of graded levels of choline (197-1795 mg/kg) on antibacterial properties, inflammatory status and barrier function in the gills of grass carp. The results showed that optimal dietary choline supplementation significantly improved lysozyme and acid phosphatase activities, complement component 3 (C3) content, and the liver expressed antimicrobial peptide 2 and Hepcidin mRNA levels in the gills of fish (P < 0.05). In addition, appropriate dietary choline significantly decreased the oxidative damage, which might be partly due to increase copper, zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) activities and increased glutathione content in the gills of fish (P < 0.05). Moreover, appropriate dietary choline significantly up-regulated the mRNA levels of interleukin 10 and transforming growth factor β1, Zonula occludens 1, Occludin, Claudin-b, c, 3 and 12, inhibitor of κBα, target of rapamycin, Cu/Zn-SOD, CAT, GR, GPx, GST and NF-E2-related factor 2 in the gills of fish (P < 0.05). Conversely, appropriate dietary choline significantly down-regulated the mRNA levels of pro-inflammatory cytokines, tumor necrosis factor α, interleukin 8, interferon γ, interleukin 1β, and related signaling factors, nuclear factor kappa B p65, IκB kinase β, IκB kinase γ, myosin light chain kinase and Kelch-like-ECH-associated protein 1a (Keap1a) in the gills of fish (P < 0.05). However, choline did not have a significant effect on the mRNA levels of IκB kinase α, Claudin-15 and Keap1b in the gills of fish. Collectively, appropriate dietary choline levels improved gill antibacterial properties and relative gene expression levels of tight junction proteins, and decreased inflammatory status, as well as up-regulated the mRNA levels of related signaling molecules in the gills of fish. Based on gill C3 content and AHR activity, the dietary choline requirements for young grass carp (266.5-787.1 g) were estimated to be 1191.0 and 1555.0 mg/kg diet, respectively.

Dietary pantothenic acid depressed the gill immune and physical barrier function via NF-κB, TOR, Nrf2, p38MAPK and MLCK signaling pathways in grass carp (Ctenopharyngodon idella).

This study explored the effects of pantothenic acid (PA) on the immune and physical barrier function, and relative mRNA levels of signaling molecules in the gill of grass carp (Ctenopharyngodon idella). The results indicated that compared with optimal PA supplementation, PA deficiency (1.31 mg/kg diet) decreased gill interleukin 10, transforming growth factor β1, inhibitor of κBα (IκBα), eIF4E-binding protein 2, Claudin b and ZO-1 mRNA levels; anti-superoxide anion activity, and activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, glutathione peroxidase, glutathione reductase and NF-E2-related factor (P < 0.05). Additionally, PA deficiency and excess (75.08 mg/kg diet) decreased gill complement 3 and glutathione contents, lysozyme and acid phosphatase, anti-hydroxy radical, catalase and glutathione S-transferases activities, and liver-expression antimicrobial peptide 2, hepcidin, Claudin 3, Claudin c and Occludin mRNA levels (P < 0.05). Conversely, PA deficiency increased gill reactive oxygen species and protein carbonyl contents, and interferon γ2, interleukin 8, nuclear factor kappa B P65, Claudin 15a, Kelch-like ECH-associating protein 1a and Kelch-like ECH-associating protein 1b mRNA levels (P<0.05). Moreover, PA deficiency and excess increased gill malondialdehyde content, and tumor necrosis factor α, interleukin 1β, IκB kinase α, IκB kinase β, IκB kinase γ, target of rapamycin and ribosomal S6 protein kinase1 p38 mitogen-activated protein kinases and myosin light-chain kinase mRNA levels (P<0.05). In conclusion, PA deficiency decreased immune and physical barrier function, and regulated relative mRNA levels of signaling molecules in fish gill. Based on the quadratic regression analysis of gill lysozyme activity, the optimal PA levels in grass carp (253.44-745.25 g) were estimated to be 36.97 mg/kg diet.

Effects of Copper Sulfate on gill histopathology of grass carp (Ctenopharyngodon idella)

The toxic impact of copper sulfate in lethal and sublethal concentrations was investigated on gill of grass carp, Ctenopharynogodon idella . In this investigation, 27 specimens were used as control group and three treatment groups were treated with 2.5 and 5mg/l of copper sulfate. Gill samples were collected from each treatment after 96hr and lesions were analyzed by light microscopy. In histopathological study of the gill tissues, hyperplasia was clearly obvious in treatment specimens. In all of the treated groups, heavy gill-mucus response was observed, which indicate a direct relation with high concentrations. Also, in histological study of the gill, epithelial cells faced to hyperplasia, which increased with high copper densities. Primary lamellar cells wrinkling and also changing in formation were observed in chloride cells. This lesion enhanced in higher densities and in concentrations of 2.5 and 5mg/l, primary and secondary lamellar epithelial cells were degenerated.