Grass Carp Tissues Research Articles

The effects of GCRV on various tissues of grass carp (Ctenopharyngodon idella) and identification of differential interferon-stimulating genes (ISGs) through muscle transcriptome analysis

Grass carp hemorrhagic disease is caused by the grass carp reovirus (GCRV). The disease spreads rapidly and has a high fatality rate, which seriously affects grass carp culture. Moreover, the molecular mechanisms underlying grass carp hemorrhagic disease remain unclear. To decipher the effects of GCRV on grass carp tissues, resistant grass carp A (GA) and susceptible grass carp B (GB) were selected through GCRV treatment, and control grass carp C (GC) was also established. The gill, liver, and muscle tissues exhibited different onset symptoms under the influence of GCRV by histological observation. We selected muscle samples with significant differences in symptoms for Illumina RNA sequencing. Analyses using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes revealed 3512, 3074, and 1853 differentially expressed genes between "GC vs. GB," "GC vs. GA," and "GA vs. GB," respectively. Additionally, 40 differential immune-related genes and 28 differential interferon-stimulating genes (ISGs) related to the interferon (IFN) pathway were identified. The expression of immunogene-related genes of GB and GA, such as MDA5, IL-34, NF-KB, TRIM25, SOCS3, CEBPB, and BCL2, and genes associated with the JAK-STAT signaling pathway, such as IRF4, STAT1, STAT3, JAK 1, and JAK 2, was significantly upregulated. The IFN and JAK-STAT signaling pathways were closely related to anti-GCRV infection. The transcriptome data and predicted immune genes and ISGs in this study provide novel insights into the treatment of GCRV.

Identification, Expression, Characteristic Analysis, and Immune Function of Two Akirin Genes in Grass Carp (Ctenopharyngodon idella).

Intensive aquaculture of grass carp often leads to decreased immunity and increased disease prevalence, resulting in economic losses. Improving grass carp immunity is therefore a critical strategy for addressing these challenges. Akirin reportedly participates in myogenesis, growth, and immune responses. However, its role in grass carp remains unclear. Herein, we isolated akirins from the spleen of grass carp and analyzed their tissue-specific expression. Akirin expression was detected following treatment with poly (I:C), LPS, and Aeromonas hydrophila (A. hydrophila). The immunological function of the akirin protein was evaluated in head kidney leukocytes (HKLs). The results revealed that the coding sequence (CDS) of akirin1 is 570 bp, encoding 189 amino acids. There was one predicted nuclear localization signal (NLS) and two predicted α- helix domains. The CDS of akirin2 is 558 bp, encoding 185 amino acids. There were two predicted NLSs and two predicted α-helix domains. Tissue-specific expression analysis showed that akirins are widely detected in grass carp tissues. akirin1 was highly detected in the brain, kidneys, heart, spleen, and gonads, while akirin2 was highly detected in the brain, liver, gonads, kidneys, spleen, and heart. The mRNA levels of akirins were promoted after treatment with poly (I:C), LPS, and A. hydrophila. Recombinant akirin proteins were produced in Escherichia coli (E. coli). il-1β, ifnγ, il-6, tnfα, il-4, iκbα, and nfκb were markedly increased in grass carp HKLs by treatment with the akirin protein. These results suggest that akirins play a role in the immunological regulation of grass carp.

A modified withdrawal time estimation and risk assessment of enrofloxacin in grass carp (Ctenopharyngodon idella) after ad libitum medicated feed based on statistical approaches in natural cultured environments.

Enrofloxacin (EF) is a broad-spectrum and highly efficient antibiotic commonly used for treating diseases in aquatic animals. However, its abuse in aquaculture applications often leads to excess residue in tissues of grass carp (Ctenopharyngodon idella). Hence, this study aimed to estimate the withdrawal time (WT) of EF and its metabolite of ciprofloxacin (CF) administered medicated feed in natural culture environments and conduct a risk assessment. Plasma and tissue samples were gathered at appropriate time points and detected by high-performance liquid chromatography. The data homogeneity was evaluated by Bartlett's test and Cochran's test. The linearity of the regressed line was evaluated by visual inspection and F test. Outliers were estimated on a normal probability scale by plotting the standardized residual versus their cumulative frequency distribution. Finally, the WT was calculated to be 51 days in muscle + skin based on the maximum residue limit of 100µg/kg. After 51 days, the concentration of EF and CF fell below 10µg/kg. The estimated daily intake was calculated to be 0.009µg/kg/d. Hazard quotient was computed to be 0.002, which was far below one. These results suggested that calculated WT of EF could ensure the safety of products from grass carp for humans.

A cytokine-like factor 1 homolog acts as a macrophage chemoattractant in grass carp

Cytokine-like factor 1 (CYTL1) is a small cytokine and has diverse biological functions in mammals. However, whether CYTL1 exists in lower vertebrates is not clear. In this study, we identified cytl homologs in fish and characterized the immune functions in a teleost species, grass carp (Ctenopharyngodon idella). Fish CYTL1 homologs share conserved molecular features with their mammalian counterparts, including 6 cysteine residues in the mature peptide, genomic organization and synteny. Gene expression analysis revealed that cytl1 was constitutively expressed in tissues of grass carp, with the highest expression detected in the heart. Upon infection with Aeromonas hydrophila (A. hydrophila), cytl1 was downregulated in the hindgut, head kidney, skin, and spleen. In the primary head kidney leukocytes (HKLs), stimulation with inactivated A. hydrophila, LPS, poly(I:C), IL-22, IFN-a or IFN-γrel resulted in downregulation of cytl1 expression. Recombinant grass carp CYTL1 protein produced in the HEK293-F cells was potent to induce il-10 expression, but had little effect on the expression of il-1β and il-6. In vivo experiments revealed that CYTL1 was effective to recruit macrophages to the muscle injected with cytl expression plasmids. Taken together, our results indicate that CYTL1 is a potent chemokine for recruitment of macrophages in fish.

Grass carp (Ctenopharyngodon idella) NIK up-regulates the expression of IL-8 by activating the NF-κB canonical pathway

NIK (NF-κB inducing kinase) belongs to the mitogen-activated protein kinase family, which activates NF-κB and plays a vital role in immunology, inflammation, apoptosis, and a series of pathological responses. In NF-κB noncanonical pathway, NIK and IKKα have been often studied in mammals and zebrafish. However, few have explored the relationship between NIK and other subunits of the IKK complex. As a classic kinase in the NF-κB canonical pathway, IKKβ has never been researched with NIK in fish. In this paper, the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) NIK (CiNIK) was first cloned and identified. The expression level of CiNIK in grass carp cells was increased under GCRV stimuli. Under the stimulation of GCRV, poly (I:C), and LPS, the expression of NIK in various tissues of grass carp was also increased. This suggests that CiNIK responds to viral stimuli. To study the relationship between CiNIK and CiIKKβ, we co-transfected CiNIK-FLAG and CiIKKB-GFP into grass carp cells in coimmunoprecipitation and immunofluorescence experiments. The results revealed that CiNIK interacts with CiIKKβ. Besides, the degree of autophosphorylation of CiNIK was enhanced under poly (I:C) stimulation. CiIKKβ was phosphorylated by CiNIK and then activated the activity of p65. The activity change of p65 indicates that NF-κB downstream inflammatory genes will be functioning. CiNIK or CiIKKβ up-regulated the expression of IL-8. It got higher when CiNIK and CiIKKβ coexisted. This paper revealed that NF-κB canonical pathway and noncanonical pathway are not completely separated in generating benefits.

Identification of the grass carp interleukin-23 receptor and its proinflammatory role in intestinal inflammation

The interleukin 23 receptor (IL-23R) is associated with a variety of inflammatory diseases in humans and other mammals. However, whether IL-23R is involved in inflammatory diseases in teleost fish is less understood. Thus, to investigate the potential involvement of IL-23R in fish inflammatory diseases, the full-length cDNA of IL-23R from grass carp Ctenopharyngodon idella was cloned and used to generate a recombinant protein (rgcIL-23R) containing the extracellular domain of IL-23R, against which a polyclonal antibody (rgcIL-23R pAb) was then developed. qPCR analysis revealed that IL-23R mRNA was significantly upregulated in most grass carp tissues in response to infection with Gram-negative Aeromonas hydrophila. Treatment with rgcIL-23R significantly induced IL-17A/F1 expression in C. idella kidney (CIK) cells. By contrast, knockdown of IL-23R caused significant decreases in IL-23R, STAT3, and IL-17N expression in CIK cells after lipopolysaccharide (LPS) stimulation. Similarly, rgcIL-23R pAb treatment effectively inhibited the LPS-induced increase in the expression of IL-23 subunit genes and those of the IL-23/IL-17 pathway in CIK cells. Furthermore, intestinal symptoms identical to those caused by A. hydrophila were induced by anal intubation with rgcIL-23R, but suppressed by rgcIL-23R pAb. Therefore, these results suggest that IL-23R has a crucial role in the regulation of intestinal inflammation and, thus, is a promising target for controlling inflammatory diseases in farmed fish.

GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination.

Grass carp reovirus (GCRV), particularly the highly prevalent type II GCRV (GCRV-II), causes huge losses in the aquaculture industry. However, little is known about the mechanisms by which GCRV-II invades grass carp and further disseminates among tissues. In the present study, monocytes/macrophages (Mo/Mφs) were isolated from the peripheral blood of grass carp and infected with GCRV-II. The results of indirect immunofluorescent microscopy, transmission electron microscopy, real-time quantitative RT-PCR (qRT-PCR), western blot (WB), and flow cytometry analysis collectively demonstrated that GCRV-II invaded Mo/Mφs and replicated in them. Additionally, we observed that GCRV-II induced different types (M1 and M2) of polarization of Mo/Mφs in multiple tissues, especially in the brain, head kidney, and intestine. To assess the impact of different types of polarization on GCRV-II replication, we recombinantly expressed and purified the intact cytokines CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B and successfully induced M1 and M2 type polarization of macrophages using these cytokines through in vitro experiments. qRT-PCR, WB, and flow cytometry analyses showed that M2 macrophages had higher susceptibility to GCRV-II infection than other types of Mo/Mφs. In addition, we found GCRV-II induced apoptosis of Mo/Mφs to facilitate virus replication and dissemination and also detected the presence of GCRV-II virus in plasma. Collectively, our findings indicated that GCRV-II could invade immune cells Mo/Mφs and induce apoptosis and polarization of Mo/Mφs for efficient infection and dissemination, emphasizing the crucial role of Mo/Mφs as a vector for GCRV-II infection.IMPORTANCEType II grass carp reovirus (GCRV) is a prevalent viral strain and causes huge losses in aquaculture. However, the related dissemination pathway and mechanism remain largely unclear. Here, our study focused on phagocytic immune cells, monocytes/macrophages (Mo/Mφs) in blood and tissues, and explored whether GCRV-II can invade Mo/Mφs and replicate and disseminate via Mo/Mφs with their differentiated type M1 and M2 macrophages. Our findings demonstrated that GCRV-II infected Mo/Mφs and replicated in them. Furthermore, GCRV-II infection induces an increased number of M1 and M2 macrophages in grass carp tissues and a higher viral load in M2 macrophages. Furthermore, GCRV-II induced Mo/Mφs apoptosis to release viruses, eventually infecting more cells. Our study identified Mo/Mφs as crucial components in the pathway of GCRV-II dissemination and provides a solid foundation for the development of treatment strategies for GCRV-II infection.

Phosphofructokinase family genes in grass carp: Molecular identification and tissue-specific expression in response to glucose, insulin and glucagon.

It is widely acknowledged that glucose serves as the primary energy source for organisms. However, fish exhibit persistent postprandial hyperglycemia and are thought to have low glucose tolerance. Glycolysis serves as the ubiquitous pathway for glucose catabolism, with phosphofructokinase (PFK) acting as a crucial rate-limiting enzyme in this process and playing an indispensable role. PFK is classified into three isoforms based on their major expression sites, i.e., PFKM (skeletal muscle type), PFKL (liver type) and PFKP (platelet type). In this study, grass carp (Ctenopharyngodon idella) was used as animal model and the open reading frame (ORF) sequences of six PFK genetic isoforms of grass carp were cloned. Real-time PCR was used to detect its tissue distribution, and expression changes in oral glucose tolerance test (OGTT), insulin and glucagon injection experiments. The results showed that the ORF of pfkla, pfklb, pfkma, pfkmb, pfkpa and pfkpb genes was 2343, 2340, 2355, 2331, 2364 and 2349bp in length, respectively. The results of tissue distribution showed that pfkla and pfklb, homologous to mammalian pfkl, exhibited low expression levels in the liver of grass carp, but were expressed at the highest level in the brain. Muscle-type pfkma and pfkmb mRNA were found to be highly expressed in both red and white muscle, with pfkmb also exhibiting high expression levels in the heart, while platelet type pfkpa and pfkpb showed high mRNA abundances in the brain and heart. Oral glucose administration stimulated pfkma and pfkmb mRNA expression in the red muscle, and up-regulated pfklb mRNA levels in the liver at 3h post treatment, but it suppressed liver-type and platelet-type PFK genes expression in the brain. The expression of pfkmb in white muscle and pfkmb and pfkpb in heart were promoted by insulin, whereas the expression of pfkla and pfkpb in the brain, pfkma and pfkmb in the red muscle, pfkma in the white muscle, and pfklb in the liver was suppressed by insulin. As for glucagon, it inhibited pfkma and pfkmb mRNA expression in the red muscle, as well as pfklb in the liver, but it up-regulated PFK genes expression in most tissues detected, such as brain (pfklb, pfkpa and pfkpb), white muscle (pfkma and pfkmb), liver (pfkla) and heart (pfkmb and pfkpb). Our results suggest that PFK family genes have different or even opposite expression patterns in response to glucose, insulin and glucagon stimulation in various tissues of grass carp, which may contribute to glucose intolerance in fish.

Hematological and Histopathological Changes of Juvenile Grass Carp (Ctenopharyngodon idella) Exposed to Lethal and Sublethal Concentrations of Roundup (Glyphosate 41% SL)

The presence of pesticides and their potential toxic effects on fish can pose a threat to aquatic ecosystems and human health. The present study aimed to evaluate the effect of commercial formulations of glyphosate (Roundup) on the survival rate, hematological parameters, and tissues (gills and liver) of juvenile grass carp (Ctenopharyngodon idella). For these purposes, we exposed the fish to 0, 50, 100, and 150 mL L−1 of Roundup for 96 hr. Results showed a significant correlation between the fish’s mortality rate and pesticide concentrations ( p < 0.01 ); the LC50 96 hr of Roundup was 75.838 mL L−1 in the present study. The concentrations above 50 mL L−1 induced significant tissue lesions seen as lamellar aneurism, leukocyte infiltration, distal hyperplasia, cloudy swelling, macrophage aggregates, and necrosis in the gills and liver. We also reported a significant correlation between the severity of tissue damage and Roundup concentration. The hematocrit, hemoglobin, white, and red blood cell count significantly reduced after 96 hr of exposure to 100 and 150 mL L−1. The opposite trend was reported for concentrations of glucose, albumin, cholesterol, total protein, and triglycerides. Finally, the fish exposed to 100 and 150 mL L−1 of Roundup displayed some clinical signs, such as increasing operculum movement, darkening the skin, and swimming near the surface during the movement test. Overall, our results showed that concentrations of Roundup higher than 50 mL L−1 can induce various toxic effects and significantly reduce the survival chance of juvenile grass carp. Roundup altered fish behavior, tissue functioning, and biochemical processes. In this study, we provided some basic knowledge about the effects of a glyphosate-based herbicide on aquatic organisms and possible environmental management.

Establishment and characterization of a skeletal myoblast cell line of grass carp (Ctenopharyngodon idellus).

Skeletal muscle myoblastic cell lines can provide a valuable new in vitro model for the exploration of the mechanisms that control skeletal muscle development and its associated molecular regulation. In this study, the skeletal muscle tissues of grass carp were digested with trypsin and collagenase I to obtain the primary myoblast cell culture. Myoblast cells were obtained by differential adherence purification and further analyzed by cryopreservation and resuscitation, chromosome analysis, immunohistochemistry, and immunofluorescence. A continuous grass carp myoblast cell line (named CIM) was established from grass carp (Ctenopharyngodon idellus) muscle and has been subcultured > 100 passages in a year and more. The CIM cells revived at 79.78-95.06% viability after 1-6 months of cryopreservation, and shared a population doubling time of 27.24 h. The number of modal chromosomes of CIM cells was 48, and the mitochondrial 12S rRNA sequence of the CIM cell line shared 99% identity with those of grass carp registered in GenBank. No microorganisms (bacteria, fungi, or mycoplasma) were detected during the whole study. The cell type of CIM cells was proven to be myoblast by immunohistochemistry of specific myogenic protein markers, including CD34, desmin, MyoD, and MyHC, as well as relative expression of key genes. And the myogenic rate and fusion index of this cell line after 10 days of induced differentiation were 8.96 ~ 9.42% and 3-24%, respectively. The telomerase activity and transfection efficiency of CIM cell line were 0.027 IU/mgprot and 23 ~ 24%, respectively. These results suggest that a myoblast cell line named CIM with normal biological function has been successfully established, which may provide a valuable tool for related in vitro studies.

Pro-inflammatory regulatory mechanism of C5a/C5aR pathway in GCRV-infected grass carp (Ctenopharyngodon idella)

Death of grass carp (Ctenopharyngodon idella) caused by grass carp reovirus (GCRV) infection severely restricts the development of the grass carp industry. The interaction of C5a and C5a receptor (C5aR) in the complement system promotes inflammation and aggravates bleeding in grass carps. To reveal the regulatory mechanism of the C5a/C5aR pro-inflammatory response after GCRV infection, the expression of C5 in the liver and muscle tissues of grass carp after GCRV infection was analyzed using immunofluorescence. The effects of over expression of C5a and the C5aR antagonist PMX205 in grass carp kidney cell line (CIK) on the phosphorylation of p38 and ERK and the gene and protein expression of IL-6 and TNF-α were also analyzed using RT-qPCR and western blot. The results showed that the expression of the C5a protein in the liver and muscle of grass carp infected with GCRV first increased and then decreased, and the C5a protein level peaked on the 5th day after infection. In CIK cells, over expression of C5a significantly increased C5aR expression, phosphorylation of p38 and ERK, and overexpression of IL-6 and TNF-α. Treatment of CIK cells with PMX205 significantly decreased the expression of C5a and C5aR, phosphorylation of p38 and ERK proteins, and expression of IL-6 and TNF-α after GCRV infection. These results implied that the interaction of C5a and C5aR in grass carp resulted in the phosphorylation of p38 and ERK proteins, a significant increase in the expression of IL-6 and TNF-α, and ultimately induction of an inflammatory response of grass carp infected with GCRV. These results provide a theoretical basis for relieving grass carp hemorrhagic diseases by regulating the interaction of C5a and C5aR.

Developing CHCA/PPD as a novel matrix for enhanced matrix-assisted laser desorption/ionization-mass spectrometry imaging for analysis of antibiotics in grass carp tissues.

Developing CHCA/PPD as a novel matrix for enhanced matrix-assisted laser desorption/ionization-mass spectrometry imaging for analysis of antibiotics in grass carp tissues.

DNA methylation profiling and transcriptome sequencing reveal the molecular mechanism of the high-carbohydrate diet on muscle growth of grass carp (Ctenopharyngodon idella)

To investigate the effects of a high-carbohydrate (HC) diet on the growth, serum biochemistry, and DNA methylation and gene transcription profiles of muscle tissue of grass carp (Ctenopharyngodon idella), fish with initial body weight (27.87 ± 0.40) g were fed with a control diet (CON, containing 330 g kg−1 carbohydrate) and an HC diet (containing 490 g kg−1 carbohydrate) for 8 weeks. The results showed that the HC diet had side-effects on muscle growth and health, and caused excessive fat accumulation in the tissues of grass carp. A total of 3135 differentially methylated regions (DMRs) and 530 differentially expressed genes (DEGs) in muscle were screened by bisulfite sequencing and transcriptome sequencing analysis. Twenty-seven DMRs were negatively correlated with DEGs, and eleven DMRs were located in the promoters of DEGs. The methylation level of promoter regions of seven target genes decreased and their transcription levels increased, including FOX protein, diacylglycerol kinase (DGKZ), proto-myosin regulatory protein (tmod1), Notch2, DnaJ homolog (dnajc1), von Willebrand factor D and EGF domains (vwde), and zgc:77838. The methylation level of promoter regions of four target genes increased and their transcription levels decreased, including epidermal growth factor-like domain 6 (EGFL6), estrogen receptor alpha (Erα), cohesin subunit SA-1-like, and LOC795573. These eleven essential genes were mainly involved in GO terms containing cell differentiation, muscle contraction, lipid phosphorylation, PI3K/Akt signaling pathway, and Notch signaling pathway. This study provides a new research direction for the molecular mechanism of the HC diet on muscle growth of grass carp.

Developing CHCA/PPD as a novel matrix for enhanced matrix-assisted laser desorption/ionization-mass spectrometry imaging for analysis of antibiotics in grass carp tissues.

We have used MALDI-TOF-MSI in positive ion mode for the analysis of quinolones and sulfonamides in grass carps. A novel CHCA/PPD matrix was prepared and applied using a simple method to improve the analysis. Compared with the traditional matrix, CHCA/PPD significantly improved the detection intensity of quinolones and sulfonamides with better sensitivity (17.20%-94.30%) and reproducibility. For demonstration, this novel matrix was successfully applied to visualize enrofloxacin (ENR) in grass carp tissues, with the entire abundance differences clearly observed based on MALDI-MSI. The concentration levels in different tissues were determined, with a calibration curve of 10-2000 μg/ml (R2 > 0.993). This study was the first to introduce CHCA/PPD as a novel matrix, and the classical acid-base mixing was used to improve the ionization effect of the traditional matrix CHCA in MALDI. Based on CHCA/PPD, MALDI-MSI detected ENR in different grass carp tissues for the first time and realized the spatial distribution and concentration detection.

Do ectoparasites on fish gills “talk” with gut microbiota far away?

The ectoparasites Dactylogyrus lamellatus usually infest fish gills and cause severe damage to the gill tissues of the host. However, whether the ectoparasites influence gut microbiota is still unknown. In this study, gut microbiota combined with histopathological characteristics, biochemical parameters, and relative expression of immune-related genes were analyzed in grass carp severely infected (SI), mildly infected (MI) and uninfected (UI) with D. lamellatus. Deep amplicon sequencing of the 16S rRNA gene of the gut microbiota revealed that the diversity of the SI group was significantly reduced than UI group (p < 0.05). In parallel with the increased loads of D. lamellatus, the relative abundance of Cetobacterium increased, and that of Bacteroides decreased. Antioxidant indices of the glutathione peroxidase (GPX) in the infected groups were significantly higher than those in the UI group (p < 0.05). The expression levels of several immune-related genes encoding toll-like receptor 3 (TLR3), myeloid differentiation factor 88 (MyD88), major histocompatibility complex II (MHCII), and immunoglobulin M (IgM) were relatively increased the degree of infection in grass carp tissues. Meanwhile, the histopathological characteristics show a large number of immune cells infiltrated in the mucosal and submucosal layers in intestine. It indicates that D. lamellatus infection stimulated both the innate and adaptive immune responses. Our data demonstrated that ectoparasites D. lamellatus infection on the gills resulted in a gut microbiota shift, histopathological changes, and immune responses in the grass carp. Furthermore, ectoparasite- microbiota- host interactions indicate that fish ectoparasites probably have remote “talk” mechanisms with host intestinal microbes. This is the first study of ectoparasites on fish gills affecting the gut microbiota in grass carp from far distance away, which is beneficial for understanding the relationships between parasites, gut microbiota, and host and for the control of parasitic diseases in fish.

Screening of new Paenibacillus polymyxa S3 and its disease resistance of grass carp (Ctenopharyngodon idellus).

A new strain of Paenibacillus polymyxa S3 with antagonistic effects on 11 major fish pathogens (especially Aeromonas hydrophila), but had no toxicity to grass carp, was screened from the sediment of fishponds. In vivo colonization studies showed that strain S3 could be colonized and distributed in the gill and abdomen of the grass carp. Bioassay results showed that the weight growth rate of grass carp in the strain S3 oral group (16.01%) and strain S3 immersion group (16.44%) was significantly higher than those of the control group (8.61%). At the same time, the activities of ACP, AKP, CAT and GSH-Px in the serum of grass carp in oral and immersion groups were significantly higher than those of the control group. In addition, the treatment with strain S3 could significantly upregulate the expression of the antioxidant-related genes and immune-related genes Keap1, Nrf2, C3, LZM, IgM, TLR-4 and MyD-88 in grass carp tissues. The challenge test showed that strain S3 treatment significantly increased the survival rate of grass carp infected with Aeromonas hydrophila. Whole genome sequencing analysis showed that strain S3 had 16 active metabolite gene clusters, indicating that it had abundant gene resources, which provided important support for its development for fish microecological preparations. In summary, a new strain of Paenibacillus polymyxa S3 with antibacterial activity against a variety of fish pathogens was screened in this study and its probiotic function was evaluated, proving its potential value in fisheries.

Functional and expression analysis reveals the involvement of integrin αvβ3 in antiviral immunity of grass carp (Ctenopharyngodon idella)

Integrins are α-β heterodimeric cell receptors that can bind the protein components of pathogens, and play crucial roles in mammalian immune responses, but the immune functions mediated by integrins remains largely unknown in teleost fish. In this study, an integrin αvβ3 (GCαvβ3) originally assembled by αv (GCαv) and β3 (GCβ3) subunits, was identified from a teleost fish grass carp Ctenopharyngodon idella. The pairwise alignment analyses showed that the amino acid sequences of GCαv and GCβ3 shared high similarity (75.2–95.1%) and identity (58.6–90.7%) with their homologs from other vertebrates. Both GCαv and GCβ3 harbored the conserved protein domains and motifs, and were clustered in fish branch of the phylogenetic tree containing the counterparts from various vertebrates. Co-immunoprecipitation displayed that GCβ3 could interact with the grass carp reovirus (GCRV) outer capsid protein VP5. Two incubation experiments revealed that the interaction of GCRV or VP5 proteins with GCβ3 could induce the expressions of type I interferons (IFNs) including IFN2 and IFN3 in grass carp ovary cell line. The functional analysis demonstrated that GCαvβ3 served as a receptor of viral protein components to be involved in antiviral immunity as human integrin αvβ3 did. In addition, both GCαv and GCβ3 were significantly upregulated in various tissues of grass carp after GCRV infection. This study might provide fundamental basis for understanding the molecular characteristics and immune functions of GCαvβ3, and offer a new insight into the antiviral immune mechanism specific to the integrins in grass carp.

Myonectin in grass carp (Ctenopharyngodon idella): Molecular cloning and effect of nutrition state, insulin and glucagon on myonectin expression

As an endocrine organ, skeletal muscle secretes multiple myokines. Myonectin, a myokine, participates in lipid and glucose metabolism. To explore the functions of myonectin in teleosts, the myonectin gene was cloned from the brain tissue of grass carp. The open reading frame (ORF) of isolated myonectin was 813 bp, encoding 270 amino acids. Grass carp myonectin has four predicted domains: signal peptide, N-terminal domain (NTD), collagen protein domain, and C1q/TNF-like domain. In addition, four predicted glycosylation sites and four conserved cysteine residues were located in grass carp myonectin. Myonectin mRNA was detected in multiple tissues of grass carp and was highly expressed in the brain region, heart and kidney. In the oral glucose tolerance test (OGTT), myonectin mRNA levels were significantly induced in the kidney, hypothalamus, muscle and brain after treatment with glucose. In the fast and refeeding experiment, the serum glucose level was decreased in the fast group. However, serum glucose levels were increased in the refeeding group. The myonectin mRNA levels in the kidney, heart, brain and liver were significantly reduced in the fast groups. Moreover, the myonectin expression was markedly increased in the refeeding groups. In grass carp primary hepatocytes, myonectin mRNA levels were induced by treatment with glucose and oleic acid for 12 or 24 h. Myonectin expression was dramatically promoted by treatment with insulin. Moreover, myonectin expression was markedly decreased with glucagon treatment in primary hepatocytes. The results of this study will provide a basis for exploring the roles of grass carp myonectin in the future.

Functional characterization of MEKK3 in the intestinal immune response to bacterial challenges in grass carp (Ctenopharyngodon idella).

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) is an evolutionarily conserved Ser/Thr protein kinase of the MEKK family that is essential for the host immune response to pathogen challenges in mammals. However, the immune function of MEKK3s in lower vertebrate species, especially in bony fish, remains largely unknown. In this study, a fish MEKK3 (designated CiMEKK3) gene was cloned and identified from grass carp (Ctenopharyngodon idella). The present CiMEKK3 cDNA encoded a 620 amino acid polypeptide containing a conserved S-TKc domain and a typical PB1 domain. Several potential immune-related transcription factor-binding sites, including activating protein 1 (AP-1), nuclear factor kappa B (NF-κB) and signal transducer and activator of downstream transcription 3 (STAT3), were observed in the 5’ upstream DNA sequence of CiMEKK3. A phylogenetic tree showed that CiMEKK3 exhibits a close evolutionary relationship with MEKK3s from Cyprinus carpio and Carassius auratus. Quantitative real-time PCR analysis revealed that CiMEKK3 transcripts were widely distributed in all selected tissues of healthy grass carp, with a relatively high levels observed in the gill, head kidney and intestine. Upon in vitro challenge with bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and pathogen-associated molecular patterns (PAMPs) (lipopolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-mDAP (Tri-DAP) and muramyl dipeptide (MDP)), the expression levels of CiMEKK3 in the intestinal cells of grass carp were shown to be significantly upregulated in a time-dependent manner. In vivo injection experiments revealed that CiMEKK3 transcripts were significantly induced by MDP challenge in the intestine; however, these effects could be inhibited by the nutritional dipeptides carnosine and Ala-Gln. Moreover, subcellular localization analysis and luciferase reporter assays indicated that CiMEKK3 could act as a cytoplasmic signal-transducing activator involved in the regulation of NF-κB and MAPK/AP-1 signaling cascades in HEK293T cells. Taken together, these findings strongly suggest that CiMEKK3 plays vital roles in the intestinal immune response to bacterial challenges, which will aid in understanding the pathogenesis of inflammatory bowel disease in bony fish.

Transcriptomics analysis provides new insights into the fish antiviral mechanism and identification of interferon-stimulated genes in grass carp (Ctenopharyngodon idella)

Grass carp is an economically important freshwater fish in China, and haemorrhagic disease caused by GCRV has seriously restricted its farming scale. To understand the host molecular basis for antiviral defence and explore the effector molecules, a global transcriptional profiling of four major immune tissues (liver, spleen, head kidney, and trunk kidney) of GCRV-infected grass carp was established. A total of 192.65 Gb clean data was obtained with 6.11 Gb per sample and stored in the NCBI Sequence Read Archive (with accession number PRJNA759556). Based on the GO and KEEG analyses, 108 unique GO terms were enriched in the four tissues. Thirty-five enriched pathways were obtained, with 21 metabolism-related pathways mainly gained in the liver and trunk kidney, and 14 immune response pathways were enriched in the spleen and head kidney. Also demonstrated was that GCRV stimulates not only the expression of interferon-stimulated genes (ISGs) but also proinflammatory cytokines. 27 ISGs were screened from the candidate DEGs, and eight ISGs were identified for the first time in grass crap. These ISGs were classified into three categories by their function found in mammals: (i) positively regulates the IFN signalling pathway (RIG-I, MDA5, IRF7, IRF9, STAT2, and TRIM25); (ii) negatively regulates the IFN signalling pathway (usp18 and SOCS1); and (iii) exerts direct antiviral activity such as Mx1, ISG15, ISG56, ISG58, viperin, and PKR. Eight major ISGs and four typical differentially inflammatory cytokines were used for further expression analysis with prominent expression in the liver, spleen and kidney. The onset time of IFN-mediated antiviral response was trunk kidney (12–24 h) > liver (48 h) > spleen (96–120 h), and the intensity was liver > spleen > trunk kidney. Notably, the inflammatory reaction occurs early in the liver and trunk kidney. This result implies that ISGs may act synergistically and that the IFN response is closely related to the inflammatory response against GCRV infection. The transcriptomic profiles obtained and the function of ISGs predicted in this study provide new insights into fish antiviral mechanisms and developing effective therapeutic directions.