Two homologous genes encoding interleukin (IL)-34 in the common carp (Cyprinus carpio L.): roles in inflammatory modulation and anti-bacterial defense.

In mammals, interleukin 34 (IL-34) is a ligand for macrophage colony-stimulating factor receptor (M-CSFR), promoting inflammatory responses and inducing the synthesis and secretion of various cytokines. However, studies on its function in lower vertebrates is limited, and its evolutionary relationship with homologous molecules in mammals remains unclear. In this study, two IL-34-encoding genes were cloned and identified in common carp (Cyprinus carpio L.), designated as CcIL-34A and CcIL-34B, with an amino acid sequence similarity of 77.7%. Gene synteny analysis revealed that the IL-34 gene loci are relatively conserved, and both are located downstream of SF3B3. The expression patterns of CcIL-34s were analyzed using qRT-PCR, and this showed that they are expressed across all tested tissues, with higher levels in the liver, spleen, and head kidney and lower levels in the gills and intestines. Following infection with Aeromonas hydrophila, the mRNA expression levels of CcIL-34s in the gills, head kidney, intestines, and spleen were significantly upregulated. Immunofluorescence was also employed to assess changes in CcIL-34 protein expression, showing a significant increase in carp spleens 24 hours after A. hydrophila infection, suggesting that CcIL-34s contribute to host defense against this bacterium. To investigate the immunological function of IL-34 in vivo, pc-CcIL-34A and pc-CcIL-34B eukaryotic expression plasmids were constructed and injected intramuscularly into fish. Five days after injection, the expression levels of inflammation-related cytokines in the head kidney and spleen were significantly altered. Furthermore, 24 h post-A. hydrophila infection, the bacterial loads in the liver, spleen, and kidneys were significantly reduced. Ten days post-infection, the survival rates in the groups with CcIL-34A and CcIL-34B overexpression were 40% and 36.7%, respectively, compared to 16.7% in the control group. These findings suggest that CcIL-34s are involved in modulating inflammatory responses, enhancing the immune response, and improving survival rates in fish following bacterial infection, thus supporting the potential use of IL-34 molecules in aquaculture.

Mucosal immune responses of gut IgM in common carp (Cyprinus carpio) following infection with spring viremia of carp virus (SVCV).

Immunoglobulin M (IgM) specifically recognizes various antigens and can activate complement, mediate cytotoxicity, opsonize and agglutinate pathogens to induce phagocytosis, all of which play an important role in immunity. However, the IgM response of common carp (Cyprinus carpio) in the intestinal mucosa after viral infection has not been thoroughly. Therefore, we successfully produced an anti-carp IgM monoclonal antibody and developed a model of viral infection to study the kinetics of immune responses after viral infection. Our results showed that the expression of IL1-ß and Igs were dramatically increased, implying that common carp exhibited a significant innate and adaptive immune response to viral infection. Furthermore, we found that the IgM responses varied between the two infection strategies. At 14 days post-infection (DPI), a significant population of IgM+ B cells were observed in the gut, accompanied by a sharp rise in IgM levels. The immune response to secondary infection started at 7 DPI, suggesting that the IgM response is faster in the gut after re-infection. Importantly, we also explored the variability of different gut compartments to viral infection, and result revealed a stronger immune response in the hindgut than in the foregut and midgut. Overall, our findings indicate that IgM plays an important role in the intestinal immune response following primary and secondary viral infection, in which the hindgut plays a major immune function.

Interleukin-17B in common carp (Cyprinus carpio L.): Molecular cloning and immune effects as immune adjuvant of Aeromonas veronii formalin-killed vaccine.

The interleukin-17 (IL-17) family of cytokines is critical for host defense responses and mediates different pro- or anti-inflammatory mediators through different signaling pathways. However, the function of the related family member, IL-17B, in teleosts is poorly understood. In the present study, an IL-17B homolog (CcIL-17B) in common carp (Cyprinus carpio) was identified, and sequence analysis showed that CcIL-17B had eight conserved cysteine residues, four of which could form two pairs of disulfide bonds, which in turn formed a ring structure composed of nine amino acids (aa). The deduced aa sequences of CcIL-17B shared 35.79-92.93 % identify with known homologs. The expression patterns were characterized in healthy and bacteria-infected carp. In healthy carp, IL-17B mRNA was highly expressed in the spleen, whereas Aeromonas veronii effectively induced CcIL-17B expression in the liver, head, kidney, gills, and intestine. The recombinant protein rCcIL-17B could regulate the expression levels of inflammatory cytokines (such as IL-1ß, IL-6, TNF-α, and IFN-γ) in primary cultured head kidney leukocytes in vitro. As an adjuvant for the formalin-killed A. veronii (FKA) vaccine, rCcIL-17B induced the production of specific antibodies more rapidly and effectively than Freund's complete adjuvant (FCA). The results of the challenge experiments showed that the relative percent survival (RPS) after vaccination with rCcIL-17B was 78.13 %. This percentage was significantly elevated compared to that observed in the alternative experimental groups (62.5 % and 37.5 %, respectively). Additionally, the bacterial loads in the spleen of the rCcIL-17B + FKA group were significantly lower than those in the control group from 12 h to 48 h after bacterial infection. Furthermore, histological analysis showed that the epithelial cells were largely intact, and the striated border structure was complete in the intestine of rCcIL-17B + FKA group. Collectively, our results demonstrate that CcIL-17B plays a crucial role in eliciting immune responses and evokes a higher RPS against A. veronii challenge compared to the traditional adjuvant FCA, indicating that rCcIL-17B is a promising vaccine adjuvant for controlling A. veronii infection.

Chlorogenic acid improves common carp (Cyprinus carpio) liver and intestinal health through Keap-1/Nrf2 and NF-κB signaling pathways: Growth performance, immune response and antioxidant capacity.

In this experiment, we investigated the effects of adding chlorogenic acid (CGA) to the diet on growth performance, immune function, inflammation response, antioxidant capacity and its related mechanisms of common carp (Cyprinus carpio). A total of 600 fish were selected and randomly divided into five treatment groups and fed with CGA containing 0 mg/kg (CK), 100 mg/kg (L100), 200 mg/kg (L200), 400 mg/kg (L400) and 800 mg/kg (L800) for 56 days. The results of the experiment were as follows: addition of CGA significantly increased the WGR, SGR, FER, and PER of common carp (P < 0.05). The addition of 400-800 mg/kg of CGA significantly increased the serum levels of LZM, AKP activity, C3 and C4 concentration, and increased immune function of common carp (P < 0.05). Regarding antioxidant enzyme activities, adding CGA significantly increased SOD, CAT, and GsH-Px activities, while decreasing MDA content (P < 0.05). Compared with the CK group, the mRNA expression levels of NF-κB, TNF-α, and IL-1ß were decreased. The IL-10 and TGF-ß were increased in the liver and intestines of the CGA supplemented group. Meanwhile, the addition of CGA also significantly up-regulated the mRNA expression levels of Nrf2, HO-1, SOD, CAT, and GPX (P < 0.05). CGA also positively contributed to the development of the carp intestinal tract, as demonstrated by decreased serum levels of DAO, D-LA, and ET-1. And the mucosal fold height was increased significantly with increasing levels of CGA. In conclusion, the addition of CGA in the feed can enhance the growth performance, immune function and antioxidant capacity of common carp, and improve the health of the intestine and liver. According to the results of this experiment, the optimal addition amount in common carp diets was 400 mg/kg.

The function of NLRP3 in anti-infection immunity and inflammasome assembly of common carp (Cyprinus carpio L.).

NLRP3 inflammasome can be activated by a variety of stimuli and plays an important role in protecting host from pathogen invasion and maintaining homeostasis. However, the activation mechanism of NLRP3 inflammasome in fish is still unclear. In the present study, the NLRP3 gene (CcNLRP3) was identified from common carp, which was 3069 bp in length and encoded a protein with five domains. Sequence analysis showed that NLRP3 was evolutionarily conserved, and CcNLRP3 was closely related to that in grass carp and zebrafish. Real-time PCR showed that CcNLRP3 was widely expressed in various immune-related tissues of healthy common carp, and significantly increased after stimulation with E. tarda, A. hydrophila and Cyprinus spring viremia virus (SVCV), suggesting that CcNLRP3 might be involved in the immune defense of common carp. The results of co-IP, spot formation, oligomerization and fluorescence localization showed that CcNLRP3 could interact with CcASC and assemble into inflammasome. The cytotoxicity assays showed that CcNLRP3 inflammasome was involved in the pyroptosis induced by CcGSDME. At the same time, CcNLRP3 could directly interact with CcCaspase-A/B and result in increased Caspase-B enzyme activity and LDH release, indicating that CcNLRP3 could also form inflammasome through ASC-independent pathway. Taken together, the results provide targets and theoretical basis for the prevention and control of infectious diseases in aquaculture.

CcPTGS2a-like gene-mediated NF-κB/ERK signaling regulation in the common carp (Cyprinus carpio).

Prostaglandin-endoperoxide synthase 2 (PTGS2), a common biological macromolecule, is pivotal for innate immunity and pathogen recognition. In this study, we identified and characterized a CcPTGS2a-like gene in the common carp (Cyprinus carpio) with an open reading frame (ORF) of 1821 bp and epidermal growth factor and peroxidase domains. Our multiple sequence analysis revealed high homology between the amino acid sequence of CcPTGS2a-like and those of its homologs in other fish. CcPTGS2a-like mRNA and protein expressions were significantly upregulated in the spleen, head kidney, liver, and gill tissues upon exposure to Aeromonas hydrophila stimulation. CcPTGS2a-like protein recognized the conserved bacterial surface components and exhibited detectable bacterial binding activity. CcPTGS2a-like overexpression before exposure to A. hydrophila notably enhanced the survival rate of common carp, concomitant with decreased bacterial burden. The NF-κB/ERK signaling pathway initiated the immune response in common carp upon infection with A. hydrophila. CcPTGS2a-like overexpression or interference in the head kidney and Epithelioma papulosum cyprinid cells could modulate the p-NF-κB (p-p-65), p-IκBα, and p-ERK1/2 levels as well as the IL-1ß and IL-6 mRNA expression. These results indicated potential CcPTGS2a-like involvement in the immune response of the common carp to bacterial infections through the NF-κB/ERK signaling pathway.

Selective autophagy receptor p62 promotes antibacterial and antiviral immunity in common carp (Cyprinus carpio).

Sequestosome 1 (SQSTM1/p62) is a selective autophagy adapter protein that participates in antiviral and bacterial immune responses and plays an important regulatory role in clearing the proteins to be degraded and maintaining intracellular protein homeostasis. In this study, two p62 genes were cloned from common carp (Cyprinus carpio), namely Ccp62-1 and Ccp62-2, and conducted bioinformatics analysis on them. The results showed that Ccp62s had the same structural domain (Phox and Bem1 domain, ZZ-type zinc finger domain, and ubiquitin-associated domain) as p62 from other species. Ccp62s were widely expressed in various tissues of fish, and highly expressed in immune organs such as gills, spleen, head kidney, etc. Subcellular localization study showed that they were mainly distributed in punctate aggregates in the cytoplasm. After stimulation with Aeromonas hydrophila and spring viraemia of carp virus (SVCV), the expression level of Ccp62s was generally up-regulated. Overexpression of Ccp62s in EPC cells could inhibit SVCV replication. Upon A. hydrophila challenge, the bacterial load in Ccp62s-overexpressing group was significantly reduced, the expression levels of pro-inflammatory cytokines and interferon factors were increased, and the survival rate of the fish was improved. These results indicated that Ccp62s were involved in the immune response of common carp to bacterial and viral infections.

Histopathology and transcriptome profiling reveal features of immune responses in gills and intestine induced by Spring viremia of carp virus.

The immune system of bony fish closely resembles that of mammals, comprising both specific (adaptive) and non-specific (innate) components. Notably, the mucosa-associated lymphoid tissue (MALT) serves as the first line of defense within the non-specific immune system, playing a critical role in protecting these aquatic organisms against invading pathogens. MALT encompasses a network of immune cells strategically distributed throughout the gills and intestines, forming an integral part of the mucosal barrier that interfaces directly with the surrounding aquatic environment. Spring Viremia of Carp Virus（SVCV）, a highly pathogenic agent causing substantial harm to common carp populations, has been designated as a Class 2 animal disease by the Ministry of Agriculture and Rural Affairs of China. Utilizing a comprehensive array of research techniques, including Hematoxylin and Eosin (HE)、Alcian Blue Periodic Acid-Schiff (AB-PAS)、transcriptome analysis for global gene expression profiling and Reverse Transcription-Polymerase Chain Reaction (RT-qPCR), this study uncovered several key findings: SVCV is capable of compromising the mucosal architecture in the gill and intestinal tissues of carp, and stimulate the proliferation of mucous cells both in gill and intestinal tissues. Critically, the study revealed that SVCV's invasion elicits a robust response from the carp's mucosal immune system, demonstrating the organism's capacity to resist SVCV invasion despite the challenges posed by the pathogen.

Subgenomic T cell receptor alpha and delta (TRA/TRD) loci in common carp.

In jawed vertebrates, the T cell receptor alpha (TRA) and delta (TRD) genes, which encode the TRα and TRδ chains, respectively, are located as a nested structure on a single chromosome. To date, no animal has been reported to harbor multiple TRA/TRD loci on different chromosomes. Therefore, herein, we describe the first full annotation of the TRA/TRD genomic regions of common carp, an allo-tetraploid fish species that experiences cyprinid-specific whole-genome duplication (WGD) in evolution. Fine genomic maps of TRA/TRD genomic regions 1 and 2, on LG30 and LG22, respectively, were constructed using the annotations of complete sets of TRA and TRD genes, including TRA/TRD variable (V), TRA junction (J), and constant (C), TRD diversity (D), and the J and C genes. The structure and synteny of the TRA/TRD genomic regions were highly conserved in zebrafish, indicating that these regions are on individual chromosomes. Furthermore, analysis of the variable regions of the TRA and TRD genes in a monoclonal T cell line revealed that both subgenomic regions 1 and 2 were indeed rearranged. Although carp TRAV and TRDV genes were phylogenetically divided into different lineages, they were mixed and organized into the TRA/TRD V gene clusters on the genome, similar to that in other vertebrates. Notably, 285 potential TRA/TRD V genes were detected in the TRA/TRD genomic regions, which is the most abundant number of genes in vertebrates and approximately two-fold that in zebrafish. The recombination signal sequences (RSSs) at the end of each V gene differed between TRAV and TRDV, suggesting that RSS variations might separate each V gene into a TRα or TRδ chain. This study is the first to describe subgenomic TRA/TRD loci in animals. Our findings provide fundamental insights to elucidate the impact of WGD on the evolution of immune repertoire.

Comparative analysis of the immune responses of CcIgZ3 in mucosal tissues and the co-expression of CcIgZ3 and PCNA in the gills of common carp (Cyprinus carpio L.) in response to TNP-LPS.

Fish live in an aquatic environment rich in various microorganisms and pathogens. Fish mucosal-associated lymphoid tissue (MALT) plays a very important role in immune defence. This study was conducted to characterize the immune response mediated by CcIgZ3 in common carp (Cyprinus carpio.) and investigate the proliferating CcIgZ3+ B lymphocytes in gill. We determined the expression of CcIgZ3 in many different tissues of common carp following stimulation by intraperitoneal injection of TNP-LPS (2,4,6-Trinitrophenyl hapten conjugated to lipopolysaccharide) or TNP-KLH (2,4,6-Trinitrophenyl hapten conjugated to Keyhole Limpet Hemocyanin). Compared with TNP-KLH, TNP-LPS can induce greater CcIgZ3 expression in the head kidney, gill and hindgut, especially in the gill. The results indicate that the gill is one of the main sites involved in the immune response mediated by CcIgZ3. To examine the distribution of CcIgZ3+ B lymphocytes, immunohistochemistry (IHC) experiments were performed using a polyclonal antibody against CcIgZ3. The results indicated that CcIgZ3 was detected in the head kidney, hindgut and gill. To further examine whether CcIgZ3+ B lymphocytes proliferate in the gills, proliferating CcIgZ3+ B cells were analysed by immunofluorescence staining using an anti-CcIgZ3 polyclonal antibody and an anti-PCNA monoclonal antibody. CcIgZ3 and PCNA (Proliferating Cell Nuclear Antigen) double-labelled cells in the gills were located within the epithelial cells of the gill filaments of common carp stimulated with TNP-LPS at 3 dps and 7 dps, and relatively more proliferating CcIgZ3+ B cells appeared in the gills of common carp at 7 dps. These data imply that CcIgZ3+ B cells in the gills might be produced by local proliferation following TNP-LPS stimulation. In summary, compared with those in TNP-KLH, CcIgZ3 preferentially affects the gills of common carp following challenge with TNP-LPS. CcIgZ3+ B cells proliferate in the gills to quickly produce the CcIgZ3 antibody. In addition, CcIgZ3+ B cells can be activated to induce a strong immune response very early locally in the gill and produce the antibody CcIgZ3, which helps exert an immune-protective effect. These results suggest that an effective vaccine can be designed to promote production of the mucosal antibody CcIgZ3.

Cloning and expression profiling of peptide YY in the brain of common carp, Cyprinus carpio.

Peptide YY (PYY) is an anorectic brain-gut pancreatic peptide that helps in feeding regulation by reducing appetite and is well characterized in mammals. The role of PYY in relation to brain is least studied in mammals as well as in lower vertebrates including fish, however high expression was evident in male reproductive tissue. In this regard, this study attempts to evaluate the significance of PYY in the brain of common carp, Cyprinus carpio. As a first step, the cDNA of PYY from brain of adult male carp was cloned. Following which expression analysis was performed using juvenile and adult fish. The differential distribution pattern in various regions of brain and ontogeny expression analysis indicated that PYY may involve in physiological processes related to brain-pituitary axis. In addition, a significant decrease in neuropeptide Y expression was observed upon PYY- endoribonuclease-prepared small interfering RNA transfection in brain cells, in vitro indicating plausible PYY-NPY interaction in brain-pituitary axis of common carp.

Autophagy and PPARs/NF-κB-associated inflammation are involved in hepatotoxicity induced by the synthetic phenolic antioxidant 2,4-di-tert-butylphenol in common carp (Cyprinus carpio).

The synthetic phenolic antioxidant 2,4-di-tert-butylphenol (2,4-DTBP) is an emergent contaminant and can disrupt the delicate balance of aquatic ecosystems. This study aimed to investigate 2,4-DTBP-induced hepatotoxicity in common carp and the underlying mechanisms involved. Sixty common carp were divided into four groups and exposed to 0 mg/L, 0.01 mg/L, 0.1 mg/L or 1 mg/L 2,4-DTBP for 30 days. Here, we first demonstrated that 2,4-DTBP exposure caused liver damage, manifested as hepatocyte nuclear pyknosis, inflammatory cell infiltration and apoptosis. Moreover, 2,4-DTBP exposure induced hepatic reactive oxygen species (ROS) overload and disrupted antioxidant capacity, as indicated by the reduced activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). In addition, transmission electron microscopy revealed that 2,4-DTBP exposure induced autophagosome accumulation in the liver of common carp. Western blot analysis further revealed that 2,4-DTBP exposure significantly decreased the protein levels of mTOR and increased the LC3II/LC3I ratio. Furthermore, 2,4-DTBP exposure inhibited lysozyme (LZM) and alkaline phosphatase (AKP) activity; decreased immunoglobulin M (IgM), complement 3 (C3), and complement 4 (C4) levels in the serum; increased the mRNA levels of proinflammatory cytokines (NF-κB, TNF-α, IL-1ß and IL-6); and increased the mRNA levels of three types of proliferator-activated receptors (PPARs) (α, ß/δ and γ). Molecular docking revealed that 2,4-DTBP directly binds to the internal active pocket of PPARs. Overall, we concluded that 2,4-DTBP exposure in aquatic systems could induce hepatotoxicity in common carp by regulating autophagy and controlling inflammatory responses. The present study provides new insights into the hepatotoxicity mechanism induced by 2,4-DTBP in aquatic organisms and furthers our understanding of the effects of 2,4-DTBP on public health and ecotoxicology.

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.

Symmetric expression of ohnologs encoding conserved antiviral responses in tetraploid common carp suggest absence of subgenome dominance after whole genome duplication.

Allopolyploids often experience subgenome dominance, with one subgenome showing higher levels of gene expression and greater gene retention. Here, we address the functionality of both subgenomes of allotetraploid common carp (Cyprinus carpio) by analysing a functional network of interferon-stimulated genes (ISGs) crucial in anti-viral immune defence. As an indicator of subgenome dominance we investigated retainment of a core set of ohnologous ISGs. To facilitate our functional genomic analysis a high quality genome was assembled (WagV4.0). Transcriptome data from an in vitro experiment mimicking a viral infection was used to infer ISG expression. Transcriptome analysis confirmed induction of 88 ISG ohnologs on both subgenomes. In both control and infected states, average expression of ISG ohnologs was comparable between the two subgenomes. Also, the highest expressing and most inducible gene copies of an ohnolog pair could be derived from either subgenome. We found no strong evidence of subgenome dominance for common carp.

Risk Assessment of Fenpropathrin: Cause Hepatotoxicity and Nephrotoxicity in Common Carp (Cyprinus carpio L.).

The synthetic pyrethroid pesticide fenpropathrin (FEN) is extensively used worldwide and has frequently been detected in biota and the environment, whilst the negative effects and toxicological mechanisms of FEN on non-target organisms are still unknown. In the present study, healthy immature common carp were treated with FEN (0.45 and 1.35 µg/L) for a duration of 14 days, and the negative impacts and possible mechanisms of FEN on fish were investigated. Biochemical analyses results showed that FEN exposure altered the levels of glucose (GLU), total cholesterol (T-CHO), triglyceride (TG), albumin (ALB), alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate transaminase (AST) in carp serum, and caused histological injury of the liver and kidney, indicating that FEN may cause hepatotoxicity and nephrotoxicity in carp. In addition, FEN also altered the activities of superoxide dismutase (SOD) and catalase (CAT) in carp serum, upregulated the levels of reactive oxygen species (ROS), and elevated the levels of malondialdehyde (MDA) in the liver and kidney. Meanwhile, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) levels were also upregulated, indicating that oxidative stress and inflammatory reaction may be involved in the hepatotoxicity and nephrotoxicity caused by FEN. Furthermore, RNA-seq analysis results revealed that FEN treatment induced a diverse array of transcriptional changes in the liver and kidney and downregulated differentially expressed genes (DEGs) were concentrated in multiple pathways, especially cell cycle and DNA replication, suggesting that FEN may induce cell cycle arrest of hepatocytes and renal cells, subsequently inducing hepatotoxicity and nephrotoxicity. Overall, the present study enhances our comprehension of the toxic effects of FEN and provides empirical evidence to support the risk assessment of FEN for non-target organisms.

In vivo evidence of sea buckthorn relieving oxidative stress and improving immune performance of common carp (Cyprinus carpio L.).

BACKGROUND: Sea buckthorn has the functions of antioxidation, antitumor, anti-inflammation and regulating energy metabolism. In order to investigate the effects of sea buckthorn powder and sea buckthorn flavonoids on the antioxidant properties, immune function and muscle fatty acid composition of common carp, an oral feeding experiment was carried out. RESULTS: The administration of glucose significantly reduced the levels of glutathione and the activity of total antioxidant capacity enzyme in serum and hepatopancreas, while concurrently upregulating the level of malondialdehyde (MDA)(P < 0.05). Conversely, oral intake of sea buckthorn powder and flavonoids increased antioxidant enzyme activity and decreased MDA levels. In terms of antioxidant molecular indicators, sea buckthorn powder and sea buckthorn flavonoids significantly increased the mRNA levels of nuclear factor NF-E2-related factor (nrf2) in the hepatopancreas and muscle. Meanwhile, mRNA expression levels of downstream antioxidant-related genes (gr, cat, gpx, and sod) regulated by Nrf2 were also upregulated. In the immune aspects, the mRNA expression levels of proinflammatory cytokines, such as interleukin-6 (il-6), interleukin-1ß (il-1ß) and nuclear factor-κB (nf-κb), were reduced but the expressions of anti-inflammatory cytokines, such as growth factor-ß (tgf-ß) and interleukin-10 (il-10), were enhanced in the head kidney and spleen tissues after oral administration with sea buckthorn. In terms of muscle fatty acid composition, the ratio of n-3 polyunsaturated fatty acid (PUFA)/n-6 PUFA was notably higher after administering sea buckthorn flavonoids than that of the glucose group (P < 0.05). CONCLUSION: This study demonstrated that oral administration of sea buckthorn powder and sea buckthorn flavonoids significantly enhanced the antioxidant capacity and immune response and improved the muscle fatty acid compositions in common carp, and also mitigated the adverse effects of glucose treatment to a certain extent. © 2024 Society of Chemical Industry.

Season affects the estrogen system and the immune response of common carp.

The physiology of ectothermic animals, including fish, is strictly regulated by season-related external factors such as temperature or photoperiod. The immune response and the production of hormones, such as estrogens, are therefore also subject to seasonal changes. This study in common carp aimed to determine how the season affects the estrogen system and the immune response, including the antibacterial response during Aeromonas salmonicida infection. We compared the immune reaction in spring and autumn in the head kidney and liver and found that carp have higher levels of blood 17ß-estradiol in autumn, while in the liver of these fish there is a higher constitutive expression of genes encoding vitellogenin, estrogen receptors and Cyp19 aromatase than in spring. Fish sampled in autumn also exhibited higher expression of immune-related genes in the liver. In contrast, in the head kidney from fish sampled in the autumn, the expression of genes encoding estrogen receptors and aromatase was lower than in spring, and a similar profile of expression was also measured in the head kidney for inos, arginases and il-10. In turn, during bacterial infection, we observed higher upregulation of the expression of inos, il-12p35, ifnγ-2, arginase 2 and il-10 in the liver of carp sampled in spring. In the liver of carp infected in spring a higher upregulation of the expression of the genes encoding CRPs was observed compared to fish infected during autumn. The opposite trend occurred in the head kidney, where the upregulation of the expression of the genes involved in the immune response was higher in fish infected in autumn than in those infected in spring. During the infection, also season-dependent changes occurred in the estrogen system. In conclusion, we demonstrated that season differentially affects the estrogenic and immune activity of the head kidney and liver. These results reinforce our previous findings that the endocrine and immune systems cooperate in maintaining homeostasis and fighting infection.

Autophagy induced by Cyprinid herpesvirus 3 (CyHV-3) facilitated intracellular viral replication and extracellular viral yields in common carp brain cells.

Autophagy is a conservative and important process that exists in all eukaryotic cells in nature. Cyprinid herpesvirus 3 (CyHV-3), also known as KHV (Koi Herpesvirus), is a pathogen that mainly infecting common carp and koi. In the present study, we identified the CcLC3B gene, with a length of 379 bp and displaying a close evolutionary relationship with other sixteen different species, the tissue distribution and expression pattern of CcLC3 were also identified. We found that CyHV-3 infection could promote autophagy in CCB cells at the early stage but inhibit autophagy at the late stage by using confocal fluorescence microscopy, transmission electron microscopy and western blotting. And we measured the protein levels associated with the Akt/mTOR signalling pathway, intracellular replication of CyHV-3 at the mRNA and protein levels as well as viral titters. Collectively, the results taken together suggested that CyHV-3 infection could promote autophagy in CCB cells at the early stage but inhibit autophagy at the late stage via mTOR and that promoting autophagy could facilitate CyHV-3 intracellular replication and extracellular viral yields in CCB cells. These findings revealed the relationship between CyHV-3 and autophagy and provided a novel treatment strategy targeting the autophagy signalling pathway against CyHV-3 infection.

New reports on iron related proteins: Molecular characterization of two ferroportin genes in common carp (Cyprinus carpio L.) and its expression pattern.

Iron uptake, transport, and storage require the involvement of several proteins, including ferroportin (fpn), the sole known iron efflux transporter. Due to its critical function fpn has been studied, particularly in humans. Here, we characterized the ferroportin gene in common carp (Cyprinus carpio L.) and performed RNA-seq analysis to evaluate its constitutive transcription levels across different tissues. Our results indicate that C. carpio possesses two functional fpns with distinct expression patterns, highlighting the potential for functional divergence and expression differentiation among fpns in this species.

Common carp (Cyprinus carpio) CD81 promoting CyHV-3 virus replication via regulating autophagy and RLRs-interferon signaling pathway.

Cyprinid herpesvirus type 3 (CyHV-3), also called Koi herpesvirus (KHV), which leads to mass cyprinid mortality and enormous economic losses. To establish an infection, CyHV-3 needs to counteract host antiviral responses. CD81 belongs to the evolutionary conserved tetraspanin family of proteins. Several studies have shown that different members of the tetraspanin superfamily modulates different virus infectious processes. Here we aimed at analysing the role of CD81 in CyHV-3 infection. In this study, we cloned and characterized the CD81 of Common Carp, the open reading frame of CcCD81 gene was 702 bp, which encoded 234 amino acids with four transmembrane domains (TM1 to TM4), a small extracellular loop (SEL), and a large extracellular loop (LEL). Tissue distribution analysis showed that CcCD81 was widely expressed in all the tested tissues with the highest expression in head kidney, followed by a high expression in brain. Subsequently, expression levels of CcCD81 were significantly increased in CCB cells within the first 3h after infection, meanwhile, the expression of viral gene VP136 was reduced after CcCD81 knockdown in CCB cells post CyHV-3 infection. Furthermore, CcCD81 knockdown can significantly reduce the autophagy process and increase the promoter activity of ISRE and IFN-1 in the CCB cells after viral infection, as well as other genes involved in the IFN signaling pathway, including RIG-1、MDA5、MAVS、TBK1 and IRF3. Taking the data together, we revealed that CcCD81 mediates autophagy and blocks RIG-1-mediated antiviral signaling and negatively regulates the promoter activity of type I interferon (IFN) promoting virus replication. These results reveal a new link between autophagy and four-transmembrane-domain protein superfamily and contribute to elucidate the mechanism of CyHV-3 infection.