Gene expression analyses of GH/IGF axis in triploid crucian carp with growth heterosis.

Hybridization and polyploid breeding are the main approaches used to obtain new aquaculture varieties. Allotriploid crucian carp (3n) with rapid growth performance was generated by mating red crucian carp (RCC) with allotetraploids (4n). Fish growth is controlled by the growth hormone (GH)/insulin-like growth factor (IGF) axis. In the present study, we examined the expression characteristics of GH/IGF axis genes in hybrids F1, 4n, 3n, RCC and common carp (CC). The results showed that GHRa, GHRb, IGF1, IGF2, and IGF-1Ra were highly expressed in 3n compared with RCC and CC, whereas IGF3 was undetectable in the liver in RCC, CC and 3n. GHRa and GHRb had low expression in the 4n group. In hybrid F1, GHRa expression was low, whereas GHRb was highly expressed compared to the levels in RCC and CC. Moreover, in hybrid F1, the expression of IGF3 was higher, and the expression of IGF1 and IGF2 was lower than that in the RCC and CC, whereas the expression of IGF-1Ra was similar to that in RCC and CC. For the IGFBP genes, IGFBP1 had higher expression in 3n compared than that in RCC and CC, while other IGFBP genes were not high expressed in 3n. Among the genes detected in this study, 11 genes were nonadditively expressed in 3n, with 5 genes in the transgressive upregulation model. We proposed that the 11 nonadditive expression of GH/IGF axis genes is related to growth heterosis in 3n. This evidence provides new insights into hybridization and polyploid breeding from the perspective of hormone regulation.

BDE-47-induced damage prevented by melatonin in grass carp hepatocytes (L8824).

Polybrominated diphenyl ethers (PBDEs) are toxic to organisms with melatonin (MT) providing protection for tissues and cells against these. This study investigates the mechanism of damage of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and the cellular protection of MT on grass carp hepatocytes. Grass carp hepatocytes were exposed to 25 µmol/L BDE-47 and/or 40 µmol/L MT for 24 h before testing. Acridine orange/ethidium bromide (AO/EB) double fluorescence staining results showed that BDE-47 could induce cell apoptosis. The expression levels of the endoplasmic reticulum (ER) stress-related genes ire1, atf4, grp78, perk, and chop were also significantly up-regulated (P < 0.01). The levels of the apoptosis-related genes caspase3, bax, and caspase9 were significantly up-regulated (P < 0.0001), while the level of bcl-2 was significantly down-regulated (P < 0.01). Compared with the BDE-47 group, the BDE-47 + MT group showed reduced levels of ER and apoptosis of hepatocytes, while the expression of the ER stress-related genes ire1, atf4, grp78, perk, and chop and the apoptosis-related genes caspase3, bax, and caspase9 were down-regulated (P < 0.05), and the level of bcl-2 was up-regulated (P < 0.01). In conclusion, BDE-47 can activate ER and apoptosis in grass carp hepatocytes, while MT can reduce these responses.

Multi-omics analysis of miRNA-mediated intestinal microflora changes in crucian carp Carassius auratus infected with Rahnella aquatilis.

Infection by an emerging bacterial pathogen Rahnella aquatilis caused enteritis and septicemia in fish. However, the molecular pathogenesis of enteritis induced by R. aquatilis infection and its interacting mechanism of the intestinal microflora associated with microRNA (miRNA) immune regulation in crucian carp Carassius auratus are still unclear. In this study, C. auratus intraperitoneally injected with R. aquatilis KCL-5 was used as an experimental animal model, and the intestinal pathological changes, microflora, and differentially expressed miRNAs (DEMs) were investigated by multi-omics analysis. The significant changes in histopathological features, apoptotic cells, and enzyme activities (e.g., lysozyme (LYS), alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate transaminase (AST), and glutathione peroxidase (GSH-Px)) in the intestine were examined after infection. Diversity and composition analysis of the intestinal microflora clearly demonstrated four dominant bacteria: Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes. A total of 87 DEMs were significantly screened, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes were mainly involved in the regulation of lipid, glutathione, cytosine, and purine metabolism, which participated in the local immune response through the intestinal immune network for IgA production, lysosome, and Toll-like receptor (TLR) pathways. Moreover, the expression levels of 11 target genes (e.g., TLR3, MyD88, NF-κB, TGF-ß, TNF-α, MHC II, IL-22, LysC, F2, F5, and C3) related to inflammation and immunity were verified by qRT-PCR detection. The correlation analysis indicated that the abundance of intestinal Firmicutes and Proteobacteria was significantly associated with the high local expression of miR-203/NF-κB, miR-129/TNF-α, and miR-205/TGF-ß. These findings will help to elucidate the molecular regulation mechanism of the intestinal microflora, inflammation, and immune response-mediated miRNA-target gene axis in cyprinid fish.

Expression analysis and antiviral activity of koi carp (Cyprinus carpio) viperin against carp edema virus (CEV).

Viperin, also known as radical S-Adenosyl methionine domain containing 2 (RSAD2), is an IFN stimulated protein that plays crucial roles in innate immunity. Here, we identified a viperin gene from the koi carp (Cyprinus carpio) (kVip). The ORF of kVip is 1047 bp in length, encoding a polypeptide of 348 amino acids with neither signal peptide nor transmembrane protein. The predicted molecular weight is 40.37 kDa and the isoelectric point is 7.7. Multiple sequence alignment indicated that putative kVip contains a radical SAM superfamily domain and a conserved C-terminal region. kVip was highly expressed in the skin and spleen of healthy koi carps, and significantly stimulated in both natural and artificial CEV-infected koi carps. In vitro immune stimulation analysis showed that both extracellular and intracellular poly (I: C) or poly (dA: dT) caused a significant increase in kVip expression of spleen cells. Furthermore, intraperitoneal injection of recombinant kVip (rkVip) not only reduced the CEV load in the gills, but also improved the survival of koi carps following CEV challenge. Additionally, rkVip administration effectively regulated inflammatory and anti-inflammatory cytokines (IL-6, IL-1ß, TNF-α, IL-10) and interferon-related molecules (cGAS, STING, MyD88, IFN-γ, IFN-α, IRF3 and IRF9). Collectively, kVip effectively responded to CEV infection and exerted antiviral function against CEV partially by regulation of inflammatory and interferon responses.

Evolutionarily conserved mechanisms regulating stress-induced neutrophil redistribution in fish.

Introduction: Stress may pose a serious challenge to immune homeostasis. Stress however also may prepare the immune system for challenges such as wounding or infection, which are likely to happen during a fight or flight stress response. Methods: In common carp (Cyprinus carpio L.) we studied the stress-induced redistribution of neutrophils into circulation, and the expression of genes encoding CXC chemokines known to be involved in the regulation of neutrophil retention (CXCL12) and redistribution (CXCL8), and their receptors (CXCR4 and CXCR1-2, respectively) in blood leukocytes and in the fish hematopoietic organ - the head kidney. The potential involvement of CXC receptors and stress hormone receptors in stress-induced neutrophil redistribution was determined by an in vivo study with selective CXCR inhibitors and antagonists of the receptors involved in stress regulation: glucocorticoid/mineralocorticoid receptors (GRs/MRs), adrenergic receptors (ADRs) and the melanocortin 2 receptor (MC2R). Results: The stress-induced increase of blood neutrophils was accompanied by a neutrophil decrease in the hematopoietic organs. This increase was cortisol-induced and GR-dependent. Moreover, stress upregulated the expression of genes encoding CXCL12 and CXCL8 chemokines, their receptors, and the receptor for granulocytes colony-stimulation factor (GCSFR) and matrix metalloproteinase 9 (MMP9). Blocking of the CXCR4 and CXCR1 and 2 receptors with selective inhibitors inhibited the stress-induced neutrophil redistribution and affected the expression of genes encoding CXC chemokines and CXCRs as well as GCSFR and MMP9. Discussion: Our data demonstrate that acute stress leads to the mobilization of the immune system, characterized by neutrophilia. CXC chemokines and CXC receptors are involved in this stress-induced redistribution of neutrophils from the hematopoietic tissue into the peripheral blood. This phenomenon is directly regulated by interactions between cortisol and the GR/MR. Considering the pivotal importance of neutrophilic granulocytes in the first line of defense, this knowledge is important for aquaculture, but will also contribute to the mechanisms involved in the stress-induced perturbation in neutrophil redistribution as often observed in clinical practice.

Spexin acts as a novel glucose-lowering factor in grass carp (Ctenopharyngodon idella).

At present, the physiological roles of various hormones in fish glucose metabolism have been elucidated. Spexin, a 14-amino acids polypeptide, is highly conserved in many species and has functions such as reducing body weight and improving insulin resistance. In this paper, the open reading frame (ORF) of spx21 in grass carp (Ctenopharyngodon idella) was cloned, and the tissue distribution of spx1 and spx2, their direct and indirect regulatory effects on glucose metabolism of grass carp were investigated. The ORF of spx2 gene in grass carp was 279 bp in length. Moreover, spx1 was highly expressed in the adipose tissue, while spx2 was highly expressed in the brain. In vitro, SPX1 and SPX2 showed opposite effects on the glycolytic pathway in the primary hepatocytes. In vivo, intraperitoneal injection of SPX1 and SPX2 significantly reduced serum glucose levels and increased hepatopancreas glycogen contents. Meanwhile, SPX1 and SPX2 promoted the expression of key genes of glycolysis (pk) and glycogen synthesis (gys) in the hepatopancreas at 3 h post injection. As for indirect effects, 1000 nM SPX1 and SPX2 significantly increased insulin-mediated liver type phosphofructokinase (pfkla) mRNA expression and enhanced the inhibitory effects of insulin on glucose-6-phosphatase (g6pase), phosphoenolpyruvate carboxykinase (pepck), glycogen phosphorylase L (pygl) mRNA expression. Our results show that SPX1 and SPX2 have similar indirect effects on the regulation of glucose metabolism that enhance insulin activity, but they exhibit opposite roles in terms of direct effects.

Dietary exposure to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) induces oxidative damage promoting cell apoptosis primarily via mitochondrial pathway in the hepatopancreas of carp, Cyprinus carpio.

To investigate the mechanisms of BDE-47 on hepatotoxicity in fish, this study examined the effects of dietary exposure to BDE-47 (40 and 4000 ng/g) on carp for 42 days. The results showed that BDE-47 significantly increased carp's condition factor and hepatosomatic index. Pathological results revealed unclear hepatic cord structure, hepatocytes swelling, cellular vacuolization, and inflammatory cell infiltration in the hepatopancreas of carp. Further investigation showed that ROS levels significantly increased on days 7, 14, and 42. Moreover, the activities of antioxidant enzymes SOD, GSH, CAT, and GST increased significantly from 1 to 7 days, and the transcription levels of antioxidant enzymes CAT, Cu-Zn SOD, Mn-SOD, GST, and GPX, and antioxidant pathway genes Keap1, Nrf2, and HO-1 changed significantly at multiple time-points during the 42 days. The results of apoptosis pathway genes showed that the mitochondrial pathway genes Bax, Casp3, and Casp9 were significantly upregulated and Bcl2 was significantly downregulated, while the transcription levels of FADD and PERK were significantly enhanced. These results indicate that BDE-47 induced oxidative damage in hepatopancreas, then it promoted cell apoptosis mainly through the mitochondrial pathway. This study provides a foundation for analyzing the mechanism of hepatotoxicity induced by BDE-47 on fish.

Environmental cadmium exposure perturbs systemic iron homeostasis via hemolysis and inflammation, leading to hepatic ferroptosis in common carp (Cyprinus carpio L.).

Cadmium (Cd) pollution is considered a pressing challenge to eco-environment and public health worldwide. Although it has been well-documented that Cd exhibits various adverse effects on aquatic animals, it is still largely unknown whether and how Cd at environmentally relevant concentrations affects iron metabolism. Here, we studied the effects of environmental Cd exposure (5 and 50 µg/L) on iron homeostasis and possible mechanisms in common carp. The data revealed that Cd elevated serum iron, transferrin saturation and iron deposition in livers and spleens, leading to the disruption of systemic iron homeostasis. Mechanistic investigations substantiated that Cd drove hemolysis by compromising the osmotic fragility and inducing defective morphology of erythrocytes. Cd concurrently exacerbated hepatic inflammatory responses, resulting in the activation of IL6-Stat3 signaling and subsequent hepcidin transcription. Notably, Cd elicited ferroptosis through increased iron burden and oxidative stress in livers. Taken together, our findings provide evidence and mechanistic insight that environmental Cd exposure could undermine iron homeostasis via erythrotoxicity and hepatotoxicity. Further investigation and ecological risk assessment of Cd and other pollutants on metabolism-related effects is warranted, especially under the realistic exposure scenarios.

Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus).

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1ß, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila, the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection.

Transcriptomic Responses to Koi Herpesvirus in Isolated Blood Leukocytes from Infected Common Carp.

Koi herpesvirus (KHV, CyHV-3) causes severe economic losses in carp farms. Its eradication is challenging due to the establishment of latency in blood leukocytes and other tissues. To understand the molecular mechanisms leading to KHV infection in leukocytes, common carp were bath-exposed to KHV at 17 °C. After confirming the presence of viral transcripts in blood leukocytes at ten days post infection, RNA-Seq was performed on peripheral blood leukocytes on the Illumina NovaSeq. KHV infection triggered a robust immune response mediated by pattern recognition receptors, mainly toll-like receptors (tlr2, tlr5, tlr7, and tlr13), urokinase plasminogen activator surface receptor-like, galectin proteins, and lipid mediators such as leukotriene B4 receptor 1. Enriched pathways showed increased mitochondria oxidative phosphorylation and the activation of signalling pathways such as mitogen-activated protein kinases (MAPKs) and vascular endothelial growth factor (VEGF). KHV-infected leukocytes showed low production of reactive oxygen species (ROS) and glutathione metabolism, high iron export and phagocytosis activity, and low autophagy. Macrophage polarization was deduced from the up-regulation of genes such as arginase non-hepatic 1-like, macrophage mannose receptor-1, crem, il-10, and il-13 receptors, while markers for cytotoxic T cells were observed to be down-regulated. Further work is required to characterise these leukocyte subsets and the molecular events leading to KHV latency in blood leukocytes.

Hypoxia leads to gill endoplasmic reticulum stress and disruption of mitochondrial homeostasis in grass carp (Ctenopharyngodon idella): Mitigation effect of thiamine.

Hypoxia in water environment is one of the important problems faced by intensive aquaculture. Under hypoxia stress, the effects of dietary thiamine were investigated on grass carp gill tissue damage and their mechanisms. Six thiamine diets with different thiamine levels (0.22, 0.43, 0.73, 1.03, 1.33 and 1.63 mg/kg) were fed grass carp (Ctenopharyngodon idella) for 63 days. Then, 96-hour hypoxia stress test was conducted. This study described that thiamine enhanced the growth performance of adult grass carp and ameliorated nutritional status of thiamine (pyruvic acid, glucose, lactic acid and transketolase). Additionally, thiamine alleviated the deterioration of blood parameters [glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), glucose, cortisol, lactic dehydrogenase (LDH), erythrocyte fragility, and red blood cell count (RBC count)] caused by hypoxia stress, and reduced reactive oxygen species (ROS) content and oxidative damage to the gills. In addition, thiamine alleviated endoplasmic reticulum stress in the gills, which may be related to its inhibition of RNA-dependent protein kinase-like ER kinase (PERK)/eukaryotic translation initiation factor-2α (eIF2α)/activating transcription factor4 (ATF4), inositol-requiring enzyme 1 (IRE1)/X-Box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6) pathways. Furthermore, thiamine maintaining mitochondrial dynamics balance was probably related to promoting mitochondrial fusion and inhibiting mitochondrial fission, and inhibiting mitophagy may involve PTEN induced putative kinase 1 (PINK1)/Parkin-dependent pathway and hypoxia-inducible factor (HIF)-Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3) pathway. In summary, thiamine alleviated hypoxia stress in fish gills, which may be related to reducing endoplasmic reticulum stress, regulating mitochondrial dynamics balance and reducing mitophagy. The thiamine requirement for optimum growth [percent weight gain (PWG)] of adult grass carp was estimated to be 0.81 mg/kg diet. Based on the index of anti-hypoxia stress (ROS content in gill), the thiamine requirement for adult grass carp was estimated to be 1.32 mg/kg diet.

Impacts of pyraclostrobin on intestinal health and the intestinal microbiota in common carp (Cyprinus carpio L.).

Pyraclostrobin (PYR) is a strobilurin fungicide that is commonly used in agriculture, and its use in agriculture may lead to an increase in its residue in the aquatic environment and may have a deleterious influence on the intestinal health of aquatic creatures. Here, common carp were chronically exposed to PYR (0, 0.5, or 5.0 µg/L) for 30 d to determine its effect on the physical and immunological barrier and intestinal microbiota in the intestine. PYR exposure caused significant histological changes; altered the mRNA expression levels of occludin, claudin-2, and zonula occludens-1 (ZO-1); induced oxidative stress in the common carp intestine; and increased the serum D-lactate and diamine oxidase (DAO) levels. Moreover, PYR significantly increased the protein expression levels of tumour necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), and IL-6 while decreasing the level of transforming growth factor beta (TGF-ß). Further studies revealed that PYR significantly reduced lysozyme (LZM) and acid phosphatase (ACP) activities as well as complement 3 (C3) and immunoglobulin M (IgM) levels. Furthermore, PYR decreased gut microbial diversity while increasing the abundance of pathogenic bacteria such as Aeromonas and Shewanella, causing an intestinal microbial disturbances in common carp. These results imply that PYR has a negative impact on fish intestinal health and may pose serious health risks to fish by disrupting the intestinal microbiota, physical barrier, and immunological barrier in common carp.

Expression and localization of HPG axis-related genes in Carassius auratus with different ploidy.

Introduction: In the Dongting water system, the Carassius auratus (Crucian carp) complex is characterized by the coexistence of diploid forms (2n=100, 2nCC) and polyploidy forms. The diploid (2nCC) and triploid C.auratus (3n=150, 3nCC) had the same fertility levels, reaching sexual maturity at one year. Methods: The nucleotide sequence, gene expression, methylation, and immunofluorescence of the gonadotropin releasing hormone 2(Gnrh2), Gonadotropin hormone beta(Gthß), and Gonadotropin-releasing hormone receptor(Gthr) genes pivotal genes of the hypothalamic-pituitary-gonadal (HPG) axis were analyzed. Results: The analysis results indicated that Gnrh2, follicle-stimulating hormone receptor(Fshr), and Lethal hybrid rescue(Lhr) genes increased the copy number and distinct structural differentiation in 3nCC compared to that in 2nCC. The transcript levels of HPG axis genes in 3nCC were higher than 2nCC (P<0.05), which could promote the production and secretion of sex steroid hormones conducive to the gonadal development of 3nCC. Meanwhile, the DNA methylation levels in the promoter regions of the HPG axis genes were lower in 3nCC than in 2nCC. These results suggested that methylation of the promoter region had a potential regulatory effect on gene expression after triploidization. Immunofluorescence showed that the localization of the Fshß, Lhß, and Fshr genes between 3nCC and 2nCC remained unchanged, ensuring the normal expression of these genes at the corresponding sites after triploidization. Discussion: Relevant research results provide cell and molecular biology evidence for normal reproductive activities such as gonad development and gamete maturation in triploid C. auratus, and contribute to further understanding of the genetic basis for fertility restoration in triploid C. auratus.

Extracellular ATP- and adenosine-mediated purinergic signaling modulates inducible nitric oxide synthase (iNOS) gene expression, enzyme activity and nitric oxide production in common carp (Cyprinus carpio) head kidney macrophages.

Inducible nitric oxide (NO) synthase (iNOS) is a key immune mediator for production of inflammatory mediator NO from l-arginine. Tight regulation of iNOS expression and enzyme activity is critical for proper NO productions under inflammation and infection conditions. However, the regulatory mechanism for iNOS expression and enzyme activity in fish remains largely unknown. Here, we show that extracellular ATP treatment significantly up-regulates iNOS gene expression and enzyme activity, and consequently leads to enhanced NO production in Cyprinus carpio head kidney macrophages (HKMs). We further show that the extracellular ATP-induced iNOS enzyme activity and NO production can be attenuated by pharmacological inhibition of the ATP-gated P2X4 and P2X7 receptors with their respective specific antagonists, but enhanced by overexpression of P2X4 and P2X7 receptors in grass carp ovary cells. In contrast, adenosine administration significantly reduces iNOS gene expression, enzyme activity and NO production in carp HKMs, and these inhibitory effects can be reversed by pharmacological inhibition of adenosine receptors with the antagonist XAC. Furthermore, LPS- and poly(I:C)-induced iNOS gene expression, enzyme activity, and NO production are significantly attenuated by blockade of P2X4 and P2X7 receptors with their respective specific antagonists in carp HKMs, while overexpression of P2X and P2X7 receptors results in enhanced iNOS gene expression, enzyme activity and NO production in LPS- and poly(I:C)-treated grass carp ovary cells. Taken together, we firstly report an opposite role of extracellular ATP/adenosine-mediated purinergic signaling in modulating iNOS-NO system activity in fish.

Ginger polysaccharide alleviates the effects of acute exposure to carbonate in crucian carp (Carassius auratus) by regulating immunity, intestinal microbiota, and intestinal metabolism.

Alkaline stress poses a significant challenge to the healthy growth of fish. Ginger polysaccharide (GP) is one of the main active substances in ginger and has pharmacological effects, such as anti-oxidation and immune regulation. However, the physiological regulatory mechanism of GP addition to diet on alkalinity stress in crucian carp remains unclear. This study aimed to investigate the potential protective effects of dietary GP on antioxidant capacity, gene expression levels, intestinal microbiome, and metabolomics of crucian carp exposed to carbonate (NaHCO3). The CK group (no GP supplementation) and COG group (NaHCO3 stress and no GP supplementation) were set up. The GPCS group (NaHCO3 stress and 0.4% GP supplementation) was stressed for seven days. Based on these data, GP significantly increased the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), acid phosphatase (ACP), and alkaline phosphatase (AKP) in carp under alkalinity stress (p < 0.05) and decreased the activity of malon dialdehyde (MDA) (p < 0.05). GP restored the activity of GSH-PX, ACP, and AKP to CK levels. The expression levels of tumor necrosis factor ß (TGF-ß), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin 8 (IL-8) genes were decreased, and the expression levels of determination factor kappa-B (NF-κB) and interleukin 10 (IL-10) genes were increased (p < 0.05). Based on 16 S rRNA high-throughput sequencing, GP improved the changes in the intestinal microbial diversity and structural composition of crucian carp caused by NaHCO3 exposure. In particular, GP increased the relative abundance of Proteobacteria and Bacteroidetes and decreased the relative abundance of Actinobacteria. The metabolic response of GP to NaHCO3 exposed crucian carp guts was studied using LC/MS. Compared to the COG group, the GPCS group had 64 different metabolites and enriched 10 metabolic pathways, including lipid metabolism, nucleotide metabolism, and carbohydrate metabolism. The addition of GP to feed can promote galactose metabolism and provide an energy supply to crucian carp, thus alleviating the damage induced by alkalinity stress. In conclusion, GP can mitigate the effects of NaHCO3 alkalinity stress by regulating immune function, intestinal flora, and intestinal metabolism in crucian carp. These findings provide a novel idea for studying the mechanism of salt-alkali tolerance in crucian carp by adding GP to feed.

Immunoprotective effect of silybin through blocking p53-driven caspase-9-Apaf-1-Cyt c complex formation and immune dysfunction after difenoconazole exposure in carp spleen.

As a broad-spectrum and efficient triazole fungicide, difenoconazole is widely used, which not only pollutes the environment but also exerts toxic effects on non-target organisms. The spleen plays an important role in immune protection as an important secondary lymphoid organ in carp. In this study, we assessed the protective impact of silybin as a dietary additive on spleen tissues of carp during exposure to difenoconazole. Sixty carp were separated into four groups for this investigation including control group, difenoconazole group, silybin group, and silybin and difenoconazole group. By hematoxylin-eosin staining, dihydroethidium staining, immunohistochemical staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, quantitative real-time PCR assay, Western blot analysis, biochemical assays, and immune function indicator assays, we found that silybin could prevent difenoconazole-induced spleen tissue damage, oxidative stress, and immune dysfunction, and inhibited apoptosis of carp spleen tissue cells by suppressing the formation of p53-driven caspase-9-apoptotic protease activating factor-1-cytochrome C complex. The results suggested that silybin as a dietary additive could improve spleen tissue damage and immune dysfunction induced by difenoconazole in aquaculture carp.

DNA Methylation and Chromatin Accessibility Impact Subgenome Expression Dominance in the Common Carp (Cyprinus carpio).

DNA methylation and chromatin accessibility play important roles in gene expression, but their function in subgenome expression dominance remains largely unknown. We conducted comprehensive analyses of the transcriptome, DNA methylation, and chromatin accessibility in liver and muscle tissues of allotetraploid common carp, aiming to reveal the function of epigenetic modifications in subgenome expression dominance. A noteworthy overlap in differential expressed genes (DEGs) as well as their functions was observed across the two subgenomes. In the promoter and gene body, the DNA methylation level of the B subgenome was significantly different than that of the A subgenome. Nevertheless, differences in DNA methylation did not align with changes in homoeologous biased expression across liver and muscle tissues. Moreover, the B subgenome exhibited a higher prevalence of open chromatin regions and greater chromatin accessibility, in comparison to the A subgenome. The expression levels of genes located proximally to open chromatin regions were significantly higher than others. Genes with higher chromatin accessibility in the B subgenome exhibited significantly elevated expression levels compared to the A subgenome. Contrastingly, genes without accessibility exhibited similar expression levels in both subgenomes. This study contributes to understanding the regulation of subgenome expression dominance in allotetraploid common carp.

Transferrin maintains the motility rate, ATP content, and DNA integrity of common carp spermatozoa during short-term storage.

Short-term sperm storage is a straightforward and cost-effective method of managing logistics in large scale fish hatchery operations but may result in decline in sperm quality. For effective artificial reproduction of fish, use of an appropriate additive to optimize sperm storage conditions is essential. In this study, it was investigated the effect of purified seminal plasma transferrin (Tf) at 10 µg/ml on relevant parameters in common carp Cyprinus carpio sperm during short-term storage. We compared sperm motility and curvilinear velocity, adenosine triphosphate (ATP) content and DNA fragmentation of fresh spermatozoa to that stored for 24, 48, 72, and 144 h with or without Tf. The percentage of motile cells and the curvilinear velocity of spermatozoa in stored samples for 72 h with transferrin supplementation were greater compared to samples with no added protein. The ATP content in samples without added transferrin was reduced (P < 0.05) after 72 h of storage, in contrast to the levels observed in transferrin-supplemented sperm. A time-dependent increase in DNA fragmentation was observed. Significantly lower DNA damage, expressed as percent tail DNA (10.99 ±â€¯1.28) and olive tail moment (0.54 ±â€¯0.12), was recorded in Tf-supplemented samples stored for 48 h compared to that with no Tf. Hence, it is concluded that the beneficial effects of transferrin on common carp sperm could serve as an additional tool for developing and enhancing short-term sperm preservation procedures commonly used in aquaculture.

IGFBP1a is a nutrient deficient response factor that can inhibit fish reproduction through the hypothalamus-pituitary-ovary axis†.

Reproduction is a high energy consuming process, so long-term malnutrition can significantly inhibit gonadal development. However, little is known about the molecular mechanism by which fasting inhibits reproduction. Our present study found that fasting could dramatically induce insulin-like growth factor binding protein 1 (IGFBP1) expression in the liver, hypothalamus, pituitary and ovaries of grass carp. In addition, IGFBP1a in the hypothalamus-pituitary-gonad axis could inhibit the development of gonads. These results indicated that fasting may participate in the regulation of fish gonadal development through the mediation of IGFBP1a. Further studies found that IGFBP1a could markedly inhibit gonadotropin-releasing hormone 3 expressions in hypothalamus cells. At the pituitary level, IGFBP1a could significantly reduce the gonadotropin hormones (LH and FSH) expression by blocking the action of pituitary insulin-like growth factor 1. Interestingly, IGFBP1a could also directly inhibit the expression of lhr, fshr, and sex steroid hormone synthase genes (cyp11a, cyp17a, and cyp19a1) in the ovary. These results indicated that IGFBP1a should be a nutrient deficient response factor that could inhibit fish reproduction through the hypothalamus-pituitary-ovary axis.

Bio-nanocomposite active packaging films based on carboxymethyl cellulose, myrrh gum, TiO2 nanoparticles and dill essential oil for preserving fresh-fish (Cyprinus carpio) meat quality.

This study developed a composite film for packaging refrigerated common carp fillets using carboxymethyl cellulose (CMC) (1.5 % w/v)/Myrrh gum (MG) (0.25 % w/v) base with the addition of titanium dioxide nanoparticles (TiO2 NPs) (0.25 %, 0.5 %, and 1 %) and Dill essential oil (DEO) (1.5 %, 2.25 %, and 3 %). The film was produced using a casting method and optimized for mechanical and barrier properties. The incorporation of DEO and TiO2 NPs into CMC/MG composite films significantly reduced moisture content (MC) and water vapor permeability (WVP), improved their tensile strength (TS), and increased their antimicrobial and antioxidant properties. Moreover, MG can improve the physicomechanical properties of the CMC/MG composite films. The film components had good compatibility without significant aggregation or cracks. In conclusion, the optimized CMC/MG (1.5 %/0.25 %) film containing TiO2 NPs (0.5 %), and DEO (2.25 %) has the best overall performance and can be a good source for making edible film. Functionally, this bioactive nanocomposite film significantly increased the shelf life of refrigerated fish fillet samples for 12 days by inhibiting microbial growth and reducing the oxidation rate compared to the control sample. The knowledge obtained from this study can guide the development of bio-nanocomposite and biodegradable food packaging films based on CMC/MG to increase the shelf life of food products and environmental protection.