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Grass carp reovirus (GCRV) belongs to the genus Aquareovirus and is responsible for causing serious hemorrhagic disease in grass carp (Ctenopharyngodon idella), characterized by high mortality rates. Numerous animal viruses have been shown to activate endoplasmic reticulum stress (ERS). However, the potential for GCRV infection to induce ERS and its implications for viral infection remain unclear. In this study, we demonstrated that GCRV infection induces ERS, activates the protein kinase R-like ER kinase (PERK) pathway, and inhibits both the inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) pathways within the unfolded protein response (UPR). Additionally, we modulated the levels of ERS and UPR pathways in CIK cells through drug treatment and small interfering RNAs (siRNAs). Our findings revealed that the onset of ERS accelerated GCRV infection, while the ATF6 and IRE1 pathways within the UPR negatively regulated GCRV infection. Conversely, the PERK pathway facilitated GCRV infection. Furthermore, we showed that GCRV infection induced oxidative stress, with the production of reactive oxygen species (ROS) being positively regulated by the PERK pathway and the downstream gene endoplasmic reticulum oxidoreductase-1α (ERO1α). Notably, ROS promoted GCRV infection. Collectively, our findings indicate that GCRV infection activates ERS, which in turn promotes viral infection through the PERK-ERO1α-ROS signaling pathway. Thus, the PERK pathway may serve as a novel antiviral target for the prevention of GCRV infection. • GCRV triggers ERS to facilitate viral replication. • GCRV triggers the PERK axis to boost viral infection and inhibits IRE1 and ATF6 antiviral pathways. • The PERK-ERO1α axis regulates GCRV-induced ROS production, and ROS promotes viral replication. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila infection is a severe, acute, and life-threatening disease affecting grass carp (Ctenopharyngodon idella) in aquaculture. Ferroptosis is a novel form of cell death characterized by the accumulation of free iron and harmful lipid peroxides within cells. While selenomethionine (Se-Met) is known to effectively inhibit ferroptosis and alleviate cell damage, its ability to counteract oxidative stress and ferroptosis induced by A. hydrophila remains unclear. The objective of this study is to reveal the possible mechanism behind the ferroptosis phenomenon during A. hydrophila infection. We established a macrophage model of A. hydrophila invasion to monitor the dynamic changes in iron metabolism markers to evaluate the correlation between ferroptotic stress and A. hydrophila infection. A. hydrophila infection induces cytotoxicity and mitochondrial membrane damage via ferroptosis. This damage is attributed to the accumulation of lipid peroxides due to intracellular ferrous ion overload and glutathione depletion. Supplementation of Se-Met reduced mitochondrial damage, enhanced antioxidant enzyme activity and reduced ferroptosis by activating the Nrf2/HO1/GPX4 axis. These findings provide new insights into the regulatory mechanisms of A. hydrophila -induced ferroptosis in teleosts and suggest that targeted inhibition of ferroptosis may offer a novel therapeutic strategy for managing A. hydrophila infections. • A. hydrophila induces macrophage ferroptosis in teleost. • Se-Met activates Nrf2/HO1/GPX4 axis to inhibit A. hydrophila -induced ferroptosis. • Ferroptosis emerges as a promising therapeutic target for A. hydrophila infection. [ABSTRACT FROM AUTHOR]

Interferon-inducible double-stranded RNA-dependent protein kinase (PKR) is one of the key antiviral arms in the innate immune system. The activated PKR performs its antiviral function by inhibiting protein translation and inducing apoptosis. In our previous study, we identified grass carp TARBP2 as an inhibitor of PKR activity, thereby suppressing cell apoptosis. This study aimed to explore the effects of grass carp TARBP2 on PKR activity and cell apoptosis. Grass carp TARBP2 comprises two N-terminal dsRBDs and a C-terminal C4 domain. Subcellular localization analysis conducted in CIK cells revealed that TARBP2-FL (full-length TARBP2), TARBP2-Δ1 (lack of the first dsRBD), and TARBP2-Δ2 (lack of the second dsRBD) are predominantly located in the cytoplasm, while TARBP2-Δ3 (lack of the two dsRBDs) is distributed both in the nucleus and cytoplasm. Colocalization and immunoprecipitation assays confirmed the interaction of TARBP2-FL, TARBP2-Δ1, and TARBP2-Δ2 with PKR, while TARBP2-Δ3 showed no binding. Furthermore, our findings suggested that the inhibitory effect of TARBP2-Δ1 or TARBP2-Δ2 on the PKR-eIF2α pathway is depressed compared to TARBP2-FL. In cell apoptosis assays, it was observed that TARBP2-FL inhibits PKR-mediated cell apoptosis. TARBP2-Δ1 or TARBP2-Δ2 exhibits decreased inhibition to PKR-mediated cell apoptosis, whereas TARBP2-Δ3 nearly completely loses this inhibitory effect. These findings highlight the critical importance of two dsRBDs of TARBP2 in interaction with PKR, as well as in the inhibition of PKR activity, resulting in the suppression of cell apoptosis triggered by prolonged PKR activation. • Ci TARBP2 has two N-terminal dsRBDs and one C-terminal C4 domain. • Ci TARBP2-Δ1 and Ci TARBP2-Δ2 interact with Ci PKR, whereas Ci TARBP2-Δ3 does not. • Ci TARBP2-Δ1 and Ci TARBP2-Δ2 relieve Ci PKR-mediated cell apoptosis. [ABSTRACT FROM AUTHOR]

Krüppel-like factor 2a (KLF2A), a transcription factor of the krüppel-like family, is involved in regulating the immune molecules and is associated with viral infection. However, the function of KLF2A during viral infections in fish remains unclear. In this study, grass carp (Ctenopharyngodon idellus) was used to predict the target genes regulated by KLF2A. The results showed that the candidate target genes included four members of the serpin gene family (serpinb1l2 , serpinc1 , serpinh1a , and serpinh1b). Dual-luciferase experiments showed that klf2a positively regulates serpinc1 expression. Dose-dependent klf2a overexpression in C. idellus kidney (CIK) cells significantly upregulated the expression of serpinc1. Overexpressing klf2a or serpinc1 in CIK cells activated interferon responses and suppressed grass carp reovirus (GCRV) replication. Klf2a and serpinc1 co-expression inhibited GCRV replication. These results show that klf2a upregulates serpinc1 mRNA expression, promotes type 1 interferon responses, and suppresses GCRV infection. This study provides insights into the regulatory role and biological functions of KLF2A in host-virus interactions in fish. • Grass carp KLF2A targets the activation of serpinc1. • KLF2A and serpinc1 are involved in regulating virus-induced interferon responses. • KLF2A upregulates serpinc1 , and suppresses GCRV replication. [ABSTRACT FROM AUTHOR]

Macrophage migration inhibitory factor (MIF) is a pleiotropic protein implicated in a broad spectrum of inflammatory and proliferative disorders. The gene sequence of grass carp (Ctenopharyngodon idella) was identified and the expression level of it was regulated by cadmium exposure in our previous study. To further clarify the immune-regulatory activity of grass carp MIF, MIF was over-expressed and interfered in grass carp peripheral blood leukocytes via transfection of plasmids pcDNA3.1-MIF-EGFP and pLKO.1-shRNA-EGFP-puro, respectively. Subsequently, survival, phagocytic capacity, mitochondrial function and cytokine production of the transfected leukocytes were assayed. The results shown that grass carp MIF was necessary for leukocyte survival, because it enhanced leukocyte viability and inhibited cell apoptosis, while MIF interference disrupted the cell viability and induced leukocyte apoptosis. The effect might benefit from improved mitochondrial function as evidenced by increased ATP production, which was due to maintained mitochondrial trans-membrane potential. In addition, MIF is essential for neutral red uptake into leukocyte, and it provoked chemokine monocyte chemotactic protein-1 (MCP-1), pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα), interleukin 1β (IL1β), interleukin 6 (IL6), interleukin 8 (IL8), and suppressed anti-inflammatory cytokine interleukin 10 (IL10) production. These results indicated that grass carp MIF played a vital role in regulating inflammatory properties and survival of peripheral blood leukocytes. • MIF expression was amplified and diminished in grass carp leukocytes. • MIF was vital for leukocyte survival, enhancing cell viability, inhibiting apoptosis. • MIF increased ATP production and maintained mitochondrial trans -membrane potential. • MIF was essential for phagocytosis of leukocyte. • MIF regulated cytokine network. [ABSTRACT FROM AUTHOR]

Exosomes are important for intercellular "cross talk", but the role of exosomes in communication between hepatocytes and C. idella kidney (CIK) cells remains unknown. In this study, we detected the changes in factors related to immune and oxidative stress to investigate the molecular mechanism by which fatty hepatocyte-derived exosomes (OA-Exos) reduced immunity and induced oxidative stress in CIK cells. After incubation of CIK cells by OA-Exos for 24 h, tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB) and interleukin-1β (IL-1β) were significantly upregulated in the OA-Exos group (P < 0.05), and Mn superoxide dismutase (Mn-SOD) and heme oxygenase-1 (HO-1) were significantly downregulated (P < 0.05). Surprisingly, miR-122 expression was also significantly elevated after OA-Exos incubation. We further identified the expression of miR-122 and found that it was notably increased in OA-Exos compared to hepatocyte-derived exosomes (Exos). Then we transfected CIK cells with miR-122 mimic, consistently, the expression of inflammatory cytokines was also significantly elevated (P < 0.05), and the expression of glutathione peroxidase (GPx), HO-1, and Mn-SOD were dramatically decreased (P < 0.05). Furthermore, HO-1 was improved to be a direct target of miR-122, and transfection with HO-1 siRNA indicated that changes in inflammatory cytokines and genes related to oxidative stress were consistent with the above results of CIK cells incubated with OA-Exos and miR-122 mimic. We concluded that OA-Exos may, through the miR-122/HO-1 pathway, reduce immune function and antioxidant defence in CIK cells. •Fatty hepatocytes-derived exosomes of grass carp (Ctenopharyngodon idella) can specifically recruit miR-122. •The exosomes derived from fatty hepatocytes carry miR-122 into CIK cells and induce oxidative stress and inflammatory response in CIK cells. •HO-1 is the target of miR-122 and interference with HO-1 can reduce the immunity and antioxidant capacity of CIK cells. [ABSTRACT FROM AUTHOR]

In mammals, DYRK2 increases p53 phosphorylation level by interacting with it and then promotes cell apoptosis. However, the function of fish DYRK2 has not yet been elucidated. In this paper, we cloned and identified the coding sequence (CDS) of a grass carp DYRK2 (CiDYRK2) which is 1773 bp in length and encodes 590 amino acids. SMART predictive analysis showed that CiDYRK2 possesses a serine/threonine kinase domain. Subsequently, we used the dsRNA analog polyinosinic-polycytidylic acid (poly (I:C) and Grass carp reovirus (GCRV) to stimulate grass carp and CIK cells for different times and found that CiDYRK2 mRNA was significantly up-regulated both in fish tissues and cells. To explore the function of CiDYRK2, we carried out overexpression and knockdown experiments of CiDYRK2 in CIK cells. Real-time quantitative PCR (Q-PCR), TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry were used to detect the ratio of BAX/BCL-2 mRNA, the number of TUNEL positive cells, the proportion of Annexin V-positive cells respectively. The results showed that CiDYRK2 significantly up-regulated BAX/Bcl-2 mRNA ratio and increased the number of TUNEL-positive cells, as well as the proportion of Annexin V-positive cells. On the contrary, knock-down of CiDYRK2 significantly down-regulated BAX/Bcl-2 mRNA ratio in the cells. Therefore, CiDYRK2 promoted cell apoptosis. To study the molecular mechanism by which CiDYRK2 promoting cell apoptosis, subcellular localization and immunoprecipitation experiments were used to study the relationship between grass carp DYRK2 and the pro-apoptotic protein p53. The results showed that CiDYRK2 and Cip53 were located and co-localized in the nucleus. Co-immunoprecipitation experiment also showed that CiDYRK2 and Cip53 can bind with each other. We further found that DYRK2 can increase the phosphorylation level of p53. In a word, our results showed that grass carp DYRK2 induces cell apoptosis by increasing the phosphorylation level of p53. • The expression of CiDYRK2 was up-regulated by GCRV and poly (I:C) stimulation. • CiDYRK2 can physically bind to Cip53. • CiDYRK2 promoted apoptosis by increasing the phosphorylation level of p53. [ABSTRACT FROM AUTHOR]

Allograft inflammatory factor-1 (AIF-1) is a 17 kDa calcium-binding protein associated with numerous inflammatory diseases. The full-length cDNA of AIF-1 has been identified in grass carp, Ctenopharyngodon idellus in our previous study, and it was assumed to be a novel molecule involved in immune responses. To clarify this aspect, the level of AIF-1 expression was amplified and reduced in grass carp peripheral blood leukocytes via transfection of vector pcDNA3.1-AIF1-EGFP and pLKO.1-shRNA-EGFP-puro, respectively. Thereafter, AIF-1 stimulated cell proliferation, inhibited cell apoptosis, which might benefit from improved mitochondrial function as evidenced by increased mitochondrial membrane potential, subsequently promoted ATP production. In addition, AIF-1 induced leukocyte migration via up-regulated monocyte chemotactic protein-1(MCP-1) secretion, enhanced neutral red uptake into leukocyte, provoked pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α), interleukin 1β (IL1β), interleukin 6 (IL6), interleukin 8 (IL8) and suppressed anti-inflammatory cytokine interleukin 10 (IL10) production. These results indicated AIF-1 played a critical role in grass carp innate immune system. • AIF-1 expression was amplied and diminished in peripheral blood lymphocytes. • AIF-1 increases cell viability, inhibited cell apoptosis via mitochondrial pathway. • AIF-1 stimulated lymphocyte migration with up-regulated MCP-1. • AIF-1 enhanced cell phagocytosis and TNFα, IL1β, IL6, IL8 production with less IL10. [ABSTRACT FROM AUTHOR]

Grass carp is an important commercial fish in China that is plagued by various diseases, especially the hemorrhagic disease induced by grass carp reovirus (GCRV). Autophagy, a highly conserved biological process among eukaryotes, is pivotal in maintaining cellular homeostasis and managing various stressors, including viral infections. Uncoordinated (Unc) 51-like kinase 2 (ULK2) is considered an initiator of the autophagic process. In this study, we successfully cloned and isolated the ULK2 gene from grass carp. We observed that its expression levels were markedly altered following exposure to GCRV or pathogen-associated molecular patterns (PAMPs). Overexpression of CiULK2 in grass carp ovary cells (GCO) promoted GCRV replication. Conversely, CiULK2 knockdown resulted in inhibited viral loads compared to the control group. Moreover, we also reveal that 2-aminoadipic acid (2-AAA), a representative autophagy related metabolite, can inhibit autophagy and viral replication. Notably, these roles of CiULK2 in autophagy and GCRV replication were reversed upon treatment with the 2-AAA. Collectively, our findings demonstrate the 2-AAA regulates CiULK2 to inhibit GCRV replication. • Grass carp ULK2 gene were cloned and analyzed from grass carp. • CiULK2 co-localizes with the ER and LC3B. • Identification of 2-AAA as a representative autophagy related metabolite. • 2-AAA regulates CiULK2 to inhibit GCRV replication. [ABSTRACT FROM AUTHOR]

Microplastics (MPs) are pollutants widely distributed in the aquatic environments and causing various degrees of aquatic toxicity to aquatic organisms, which has attracted global attention in recent years. Nano-selenium (NSe) has been shown to have the potential to mitigate the harmful impacts of toxic substances. However, there is currently no reported evidence regarding the protective influence of NSe against the adverse effects of MPs. The aim of this study is to determine whether NSe could ameliorate the polystyrene (PS)-MPs-induced injury in grass carp (Ctenopharyngodon idella). The individuals of grass carp were assigned into three groups: (1) the control group fed with basal diet, (2) the PS group fed with basal diet and exposed to PS-MPs, and (3) the NSe group fed with diet supplemented with NSe and exposed to PS-MPs. Our results indicated that NSe administration significantly alleviated the histological damage caused by the PS-MPs in the liver and intestine with lower goblet cell count and larger villus height in the intestine, and significantly lower damage score in the liver. Moreover, NSe mitigated PS-MPs-induced oxidative stress through restoring the activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA)) except the intestinal CAT activity. Furthermore, NSe supplementation could help fish maintain lower transcriptional level of the immune-related genes (Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88)), inflammation-related genes (major histocompatibility complex class II (MHC-II) and interleukin 8 (IL-8)) and antioxidant enzyme-related genes (nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) and kelch-like ECH-associated protein 1 (Keap-1)) after PS-MPs exposure. Besides, NSe supplementation dramatically helped maintain the intestinal microbial composition, for example, the proportion of Proteobacteria in the grass carp intestine of the NSe group (41 %) was similar to that of the control group (34 %) while 85 % of the PS group. NSe also played a significant protective role in intestinal microbial diversity, effectively resisting the damage on intestinal microbial diversity due to PS-MPs exposure. PS-MPs reduced the beneficial bacteria and increased the pathogenic microorganism like Aeromonas , which was undeniable signs of intestinal dysbiosis. Functional analysis indicated that PS-MPs affected intestinal microbiota functions like inhibition of metabolism, while NSe could significantly alleviate the damage. Our findings suggested that NSe could ameliorate PS-MPs-induced injury, which could contribute to the better understanding of the ecotoxicological effects of MPs on fish and help develop relevant mitigation strategies. • Nano-selenium (NSe) could ameliorate the polystyrene (PS)-MPs-induced injury. • NSe administration could significantly alleviate the histological damage. • NSe mitigated PS-MPs-induced oxidative stress. • NSe supplementation could help fish maintain gene expression level. • NSe supplementation could help fish maintain the intestinal microbiome. [ABSTRACT FROM AUTHOR]

Bacterial septicemia represents a significant disease affecting cultured grass carp culture, with the primary etiological agent identified as the Gram-negative bacterium Aeromonas veronii. In response to an outbreak of septicemia in Guangzhou, we developed a formaldehyde-inactivated vaccine against an A. veronii strain designated AV-GZ21-2. This strain exhibited high pathogenicity in experimental infections across at all developmental stages of grass carp. Mortality rates for grass carp weighing 15 ± 5 g ranged from 16 % to 92 % at exposure temperatures of 19 °C–34 °C, respectively. The median lethal dose (LD 50) for grass carp groups weighing 15 ± 5 g, 60 ± 10 g, 150 ± 30 g and 500 ± 50 g were determined to be 1.43, 2.52, 4.65 and 7.12 × 107(CFU/mL), respectively. We investigated the inactivated vaccine in conbination with aluminum hydroxide gel (AV-AHG), Montanide ISA201VG (AV-201VG), and white oil (AV-WO) adjuvants. This study aimed to optimize inactivation conditions and identify the adjuvant that elicits the most robust immune response. The AV-GZ21-2 inactivated bacterial solution (AV),when combined with various adjuvants, was capable of inducing a strong specific immune response in grass carp. The relative percent survival (RPS) following a lethal challenge with AV-GZ21-2 were 94 % for AV-AHG, 88 % for AV-201VG, 84 % for AV-WO and 78 % for AV alone. The minimum immunization dose of the AV-AHG vaccine was determined to be 6.0 × 107 CFU per fish, providing immunity for a duration of six months with an immune protection level exceeding 75 %. Furthermore, the AV-AHG vaccine demonstrated significant protective efficacy against various epidemic isolates of A. veronii. Consequently, we developed an inactivated vaccine targeting a highly pathogenic strain of A. veronii , incorporating an aluminum hydroxide gel adjuvant, which resulted in high immune protection and a duration of immunity exceeding six months. These findings suggest that the AV-AHG vaccine holds substantial potential for industrial application. • Aeronomas veronii strain GZ21-2 was high level virulence and high immunogenicity. • A.V-AHG vaccine was developed with optimal adjvant and produced good immune protection against A.V. • AV-AHG vaccine still had high immune protection rate 6 months after one dose. • AV-AHG vaccine had high immune protection rate against A.V epidemic strains of different regions. [ABSTRACT FROM AUTHOR]

Selenium (Se), a trace element, is vital for the maintenance of cellular redox balance, thyroid hormone metabolism, inflammation, and immunity. Aeromonas hydrophila (A. hydrophila) is a common Gram-negative conditional pathogenic bacterium in fish culture, posing a serious threat to intensive aquaculture. Our study investigated the influence of dietary Se on the intestinal immune function of grass carp (Ctenopharyngodon idella) and the related regulatory mechanisms. The 2160 healthy juvenile grass carp (9.76 ± 0.005 g) were randomly assigned to 6 test groups of 6 replicates each, and fed graded selenomethionine (0.05, 0.20, 0.40, 0.61, 0.77, 0.98 mg Se/kg diet) for 70 days and then injected with A. hydrophila for a 6-day attack test. The results indicated that appropriate Se levels (0.40 mg/kg diet) alleviated intestinal damage caused by A. hydrophila and increased intestinal immune substances C3 and C4 levels as well as the activity of acid phosphatase (ACP) and lysozyme (LZ) (P > 0.05). Appropriate levels of Se (0.40 mg/kg-0.61 mg/kg diet) decreased intestinal pro-inflammatory cytokines (IFN-γ2 , IL-6 , IL-12p35 , IL- 17 A F and IL-17D) mRNA levels (P > 0.05) and increased intestinal anti-inflammatory factors (TGF-β1 , IL-4/13A , IL-4/13B , IL-10 and IL-22) mRNA levels (P > 0.05) in juvenile grass carp. Further studies revealed that Se (0.40 mg/kg-0.61 mg/kg diet) inhibited intestinal endoplasmic reticulum stress (ERS)-related signaling pathway. Furthermore, we found that appropriate levels of Se (0.40 mg/kg-0.61 mg/kg diet) inhibited intestinal autophagy in juvenile grass carp, which may be related to ULK1 , Beclin 1 , ATG5 , ATG12 , LC3 , and P62. In conclusion, appropriate levels of Se can alleviate intestinal inflammation and inhibit ERS and autophagy in juvenile grass carp. A quadratic regression analysis of intestinal ACP and LZ also indicated that the Se requirements of juvenile grass carp were 0.59 and 0.51 mg/kg, respectively. • Se reduced Aeromonas hydrophila infections in fish. • Se enhanced the immune function and improved the enteritis resistance of fish. • Se inhibited intestinal endoplasmic reticulum stress -related signaling pathway. • Se inhibited intestinal autophagy in fish. [ABSTRACT FROM AUTHOR]

Grass carp (Ctenopharyngodon idella) is an intensively cultured and economically important herbivorous fish species in China, but its culture is often impacted by Aeromonas pathogens such as Aeromonas hydrophila and Aeromonas veronii. In this study, healthy grass carp were separately infected with A. hydrophila or A. veronii for 12, 24, 48 or 72 h. The results showed that the mRNA expression levels of intestinal inflammatory factors (tnf-α , il-1β and il-8), complement factors (c3 and c4), antimicrobial peptides (hepcidin , nk-lysin and β-defensin-1), immunoglobulins (igm and igt), and immune pathway-related signaling molecules (tlr1 , tlr2 , tlr4 , myd88 , irak4 , irak1 , traf6 , nf-κb p65 and ap-1) were differentially upregulated in response to A. hydrophila and A. veronii challenge. Additionally, the expression levels of the intestinal pro-apoptotic genes tnfr1 , tnfr2 , tradd , caspase-8 , caspase-3 and bax were significantly increased, whereas the expression of the inhibitory factor bcl-2 was significantly downregulated, indicating that Aeromonas infection significantly induced apoptosis in the intestine of grass carp. Moreover, the expression of intestinal tight junction proteins (occludin , zo-1 , claudin b and claudin c) was significantly decreased after infection with Aeromonas. Histopathological analysis indicated the Aeromonas challenge caused severe damage to the intestinal villi with adhesions and detachment of intestinal villi accompanied by severe inflammatory cell infiltration at 12 h and 72 h. The 16S rRNA sequencing results showed that Aeromonas infection significantly altered the structure of the intestinal microflora of the grass carp at the phylum (Proteobacteria , Fusobacteria , Bacteroidetes and Firmicutes) and genus (Proteus , Cetobacterium , Bacteroides , and Aeromonas) levels. Take together, the findings of this study revealed that Aeromonas infection induces an intestinal immune response, triggers cell apoptosis, destroys physical barriers and alters microflora structure in the intestine of juvenile grass carp; the results will help to reveal the pathogenesis of intestinal bacterial diseases in grass carp. • A. hydrophila and A. veronii infection caused inflammatory response in the intestine of grass carp. • A. hydrophila and A. veronii infection upregulated expression of immune-related genes in the intestine of grass carp. • A. hydrophila and A. veronii infection induced cell apoptosis in the intestine of grass carp. • A. hydrophila and A. veronii infection destroyed intestinal mucosal barrier of grass carp. • A. hydrophila and A. veronii infection altered intestinal microflora structure of grass carp. [ABSTRACT FROM AUTHOR]

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. • A large number of DEGs were screened by RNA-Seq of GCRV resistant and susceptible grass carp. • Forty immunity genes and 28 ISGs related to the IFN pathway were identified. • The DDX and CEBP family play important roles in grass carp's resistance to GCRV. [ABSTRACT FROM AUTHOR]

This study investigates the effects of five thermal processing methods on fish nutritional components, oxidation, thermal degradation and flavor characteristics of grass carp meat, including steam (SM), boil (BO), fry (FY), roast (RO) and sous-vide (SV). The results found that heat treatments reduced the moisture content, total sulfhydryl content, redness, hardness and chewiness of fish meat, increased carbonyl content, TBA value, proteins cross-linking and covalent modification, brightness and yellowness, and altered fatty acid composition and flavor. A total of 136 volatile compounds and 28 fatty acids were identified from the grass carp meat. Correlation analysis suggested that 2,4-Di-tert-butylphenol and tetracosane are formed mainly due to the oxidation of fish meat, which in turn caused changes in C18:2n6c, C20:0, C18:3n3, C24:0 and b*. Heat treatments caused oxidation and thermal degradation of lipids in grass carp meat to produce free fatty acids, which further produced volatile compounds. SV and RO had a more suitable fatty acid composition for human body, while FY had better sensory quality but excessive intake unfavorable to health. This work is expected to provide guidance to consumers in selecting thermal processing methods, as well as offering insights for the development of fish pre-prepared dishes. • Effects of five thermal processing methods on grass carp meat quality was revealed. • Different heat treatments impart a completely different flavor to grass carp meat. • SM and SV groups are more tender, but had a stronger fishy taste. • SV and RO groups had a more suitable fatty acid composition for human body. • FY group with a high oxidation of proteins and lipids and unfavorable to health. [ABSTRACT FROM AUTHOR]

Granzyme (Gzm) B and perforin, both as cytotoxic proteins, can collaborate to induce the death of target cells as well as the microbes. They were originally discovered in cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and confer the cytotoxic activities of these cells. In the present study, the coding sequences of a granzyme b-like (gc gzmbl) and a perforin-like (gc prfl) genes were cloned from grass carp (Ctenopharyngodon idellus) and their specific antibodies were subsequently prepared and validated. The mRNA and protein expression of these two cytotoxic proteins in grass carp peripheral blood neutrophils was demonstrated by quantitative PCR (qPCR) and immunofluorescence staining, respectively. In the same cell model, expression of gcGzmbl and gcPrfl was stimulated by grass carp interleukin (Il)-2 in a dose- and time-dependent manners and Erk, NF-κB and Stat5 pathways were found to be involved in the regulation of Il-2 on the genes' expression. Additionally, glycolysis was proved to play a role in the stimulation of Il-2 on gcGzmbl and gcPrfl expression in peripheral blood neutrophils. As combating the invading microorganisms is one of the main functions of neutrophils, the roles of gcGzmbl and gcPrfl in the anti-bacterial activities of grass carp peripheral blood neutrophils were explored. Results showed that immunoneutralization of gcGzmbl or gcPrfl significantly attenuated the antimicrobial abilities of the neutrophils enhanced by Il-2. These findings shed a light on the expression, regulation and functions of granzyme B- and perforin-like proteins in fish peripheral blood neutrophils and enrich the understanding of Il-2 function in fish innate immunity. • Granzyme B- and perforin-like molecules are expressed in grass carp neutrophils. • gcIl-2 stimulates Granzyme B- and perforin-like genes expression in the neutrophils. • Erk, NF-κB and Stat5 pathways are involved in the stimulatory effects of Il-2. • Glycolysis is required by the cells to express granzyme B- and perforin-like proteins. • Antibodies for these two proteins attenuate the neutrophils' antimicrobial activities. [ABSTRACT FROM AUTHOR]

Vibrio mimicus (V. mimicus) is a pathogen causing serious vibriosis in aquatic animals. Hepcidin and β-Defensin1 are two important antibacterial peptides (AMPs) with broad-spectrum antibacterial activity in fish. In mammals, some evidences demonstrated that interleukin-1β (IL-1β) primarily promote AMPs expression via activating classical NF-κB pathway, but it still remains unclear in fish. Here, the temporal and spatial expression patterns of grass carp IL-1β (gcIL-1β) gene and two AMPs genes (gc hepcidin and gc β-defensin1) in tissues post- V. mimicus infection and anti- V. mimicus activity of these two AMPs in vitro were detected, showing that V. mimicus infection significantly elevated the mRNA levels of these three genes in the immune-related tissues although their expression patterns were not entirely consistent, and both gcHepcidin and gcβ-Defensin1 possessed anti- V. mimicus activity in vitro. Subsequently, the recombinant gcIL-1β (rgcIL-1β) was expressed prokaryotically in an inclusion body, which could promote proliferation of grass carp head kidney leukocytes (gcHKLs) and enhance respiratory burst activity and phagocytic activity of head kidney macrophages. Stimulation with rgcIL-1β was able to significantly regulate the mRNA expression of key regulatory genes (il-1RI , traf6 , tak1 , ikkβ , iκBα and p65) involved in the activation of classical NF-κB pathway, and then induce gcTAK1 phosphorylation, promote gcp65 nuclear translocation and enhance endogenous gcIL-1β expression at both mRNA and protein levels, implying NF-κB pathway was activated. More importantly, exogenous rgcIL-1β stimulation also significantly up-regulated both gcHepcidin and gcβ-Defensin1 mRNA levels against V. mimicus , and the regulatory effect was blocked or inhibited by NF-κB inhibitor PDTC. Taken together, our results demonstrated for the first time that grass carp IL-1β stimulation could significantly enhance the expression of these two anti- V.mimicus AMPs via activating classical NF-κB pathway. • Antimicrobial peptides gcHepcidin and gcβ-Defens possessed anti- V. mimicus activity in vitro. • V. mimicus infection elevated the mRNA expression of gc il-1β , gc hepcidin and gc β-defensin1 in vivo. • rgcIL-1β stimulation could activate classical NF-κB pathway in fish. • rgcIL-1β stimulation up-regulated these two AMPs mRNA levels via activating NF-κB pathway. [ABSTRACT FROM AUTHOR]

All drugs that can penetrate the blood-brain barrier (BBB) may lead to mental state changes, including the widely used anti-infective drug sulfamethoxazole (SMZ). Herein, we investigated whether lycopene (LYC) could ameliorate SMZ-induced brain injury and the postulated mechanisms involved. A total of 120 grass carps were exposed under SMZ (0.3 μg/L, waterborne) or LYC (10 mg/kg fish weight, diet) or their combination for 30 days. Firstly, brain injury induced by SMZ exposure was suggested by the damage of BBB (decreases of Claudins, Occludin and Zonula Occludens), and the decrease of neurotransmitter activity (AChE). Through inducing oxidative stress (elevations of malondialdehyde and 8-hydroxy-2 deoxyguanosine, inhibition of glutathione), SMZ increased the intra-nuclear level of NF-κB and its target genes (TNF-α and interleukins), creating an inflammatory microenvironment. As a positive feed-back mechanism, apoptosis begins with activation of pro-death proteins (Bax/Bcl-2) and activation of caspases (caspase-9 and caspase-3). Meanwhile, a compensatory upregulation of constitutive Nrf2 and its downstream antioxidative gene expression (NAD(P)H Quinone Dehydrogenase 1 and Heme oxygenase 1) and accelerated autophagy (increases of autophagy-related genes and p62 inhibition) were activated as a defense mechanism. Intriguingly, under SMZ stress, LYC co-administration decreased NF-κB/apoptosis cascades and restored Nrf2/autophagy levels. The neuroprotective roles of LYC make this natural compound a valuable agent for prevention SMZ stress in environment. This study suggests that LYC might be developed as a potential candidate for alleviating environmental SMZ stress in aquaculture. • Sulfamethoxazole exposure leads to oxidative stress, inflammation and apoptosis in the brain. • Lycopene supplementation ameliorates brain damage induced by Sulfamethoxazole. • Lycopene exerts neuroprotection by rebalance of NF-κB/Nrf2 pathway. [ABSTRACT FROM AUTHOR]

Sorting nexin 8 (SNX8), a member of sorting nexin protein family, plays important roles in endocytosis, endosomal sorting, and innate immune response. To date, a few homologs of SNX8 have been found in fish except in mammals. In this study, a teleost SNX8 cDNA was identified from grass carp (Ctenopharyngodon idellus). CiSNX8 was up-regulated significantly after infection with poly I:C or GCRV. We found that SNX8 was mainly distributed in the endoplasmic reticulum (ER) in CIK cells. Further analysis indicated that Ci SNX8 might negatively regulate RLR signaling pathway that is quite distinct from mammalian SNX8. In addition, Ci SNX8 could interact with MAVS, STING, TBK1, IRF3 and IRF7. Either wild type Ci SNX8 or mutants of N-terminal PX domain (aa 1–245) and C-terminal BAR domain (aa 256–519) could associate with STING. These results suggested that fish SNX8 participated in innate immune response through different molecular mechanisms. • An SNX8 ortholog from grass carp (Ctenopharyngodon idella) was cloned. • CiSNX8 was significantly up-regulated after treatment with poly I:C or GCRV. • Ci SNX8 was located in the endoplasmic reticulum (ER) in CIK cells. • Ci SNX8 negatively regulate the key components of RLR pathway such as RIG-I, STING, and TBK1-mediated IFN activation. • Either wild type CiSNX8 or mutants of N-terminal PX domain and C-terminal BAR domain could associate with STING. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) is a multifunctional mediator of the conserved MAPK signaling pathway that plays essential roles in the regulation of immune responses in mammals. However, the function of teleost MAP3K4s in innate immunity, especially in the intestinal immune system, is still poorly understood. In the current study, we identified a fish MAP3K4 homolog (Ci MAP3K4) in Ctenopharyngodon idella as well as its immune function in intestine following bacterial infection in vivo and in vitro. The open reading frame (ORF) of Ci MAP3K4 encodes putative peptide of 1544 amino acids containing a predicted serine/threonine protein kinase (S_TKc) domain with high identity with other fish MAP3K4s. Phylogenetic analysis revealed the Ci MAP3K4 belonged to the fish cluster and showed the closest relationship to Pimephales promelas. Quantitative real-time PCR (qRT-PCR) analysis revealed that Ci MAP3K4 transcripts were widely distributed in all tested tissues, especially with high expression in the muscle and intestine of healthy grass carp. In vitro , Ci MAP3K4 gene expression was upregulated by bacterial PAMPs (lipolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) and muramyl dipeptide (MDP)) and pathogens (Aeromonas hydrophila and Aeromonas veronii) in primary intestinal cells. In vivo, the mRNA expression levels of Ci MAP3K4 in the intestine were significantly induced by bacterial MDP challenge in a time-dependent manner; however, this effect could be inhibited by the bioactive dipeptides β-alanyl- l -histidine (carnosine) and alanyl-glutamine (Ala-Gln). Moreover, Ci MAP3K4 was located primarily in the cytoplasm, and its overexpression increased the transcriptional activity of AP-1 in HEK293T cells. Collectively, these results suggested that Ci MAP3K4 might play an important role in the intestinal immune response to bacterial infections, which paves the way for a better understanding of the intestinal immune system of grass carp. • The first experimental evidence for presence of a functional Ci MAP3K4 gene in grass carp. • Transcript levels of Ci MAP3K4 in intestinal cells could be significantly induced by bacterial PAMPs and pathogens. • The stimulating effects of MDP on intestinal Ci MAP3K4 expression were inhibited by dipeptides carnosine and Ala-Gln. • Ci MAP3K4 is localized mainly in cytoplasm and could enhance transcriptional activities of AP-1-Luc in HEK293T cells. [ABSTRACT FROM AUTHOR]

Grass carp (Ctenopharyngodon idella) is the most productive freshwater aquaculture fish in worldwide. However, the molecular mechanism of its growth traits has not been fully elucidated. Whole transcriptome analysis of the brain and hepatopancreas of 29 six-month-old grass carp with different growth rates was performed. Weighted gene co-expression network analysis (WGCNA) was used to construct a weighted gene co-expression network of mRNA, miRNA and lncRNA separately. A total of 35 hub mRNAs, 47 hub lncRNAs and 33 hub miRNAs were identified from the brain, 37 hub mRNAs, 110 hub lncRNAs and 36 hub miRNAs were identified from the hepatopancreas. The ceRNA networks in the brain and hepatopancreas were involved in brain development and nutrient metabolism, respectively. Overall, this is the first investigation of the growth-related transcriptomic characteristics in the brain and hepatopancreas of grass carp, thus will help us to further explore the molecular mechanism of grass carp growth rate. • The first investigation of the growth-related transcriptomic characteristics in the brain and hepatopancreas of grass carp. • A series of hub mRNAs, lncRNAs and miRNAs highly associated with the growth of grass carp were identified. • The growth-related ceRNA networks of grass carp in the brain and hepatopancreas were constructed. [ABSTRACT FROM AUTHOR]

The heavy use of prophylactic antibiotics in aquaculture leads to elevated antibiotic residues, posing a huge hidden danger in aquaculture products and other natural aquatic environments. Therefore, this study aims to isolate probiotics that can replace antibiotics from the gut of grass carp for disease control. Bacillus velezensis B8 was isolated from the gut of grass carp and showed broad-spectrum antimicrobial activity against several fish pathogenic bacteria, including Aeromonas hydrophilis , Aeromonas veronii , Vibrio parahaemolyticus , Escherichia coli , Edwardsiella tarda and Vibrio mimicus. The safety evaluation showed that the strain B8 was non-toxic to grass carp, had no hemolytic activity, and was sensitive to most antibiotics. In vitro study indicated that strain B8 was viable at pH 2–7, had weak tolerance to 0.1% (w/v) bile salt, and could grow at 10°C–40 °C. The grass carps were fed with diets containing 0 (control), 107, and 109 cfu/g of strain B8 for 4 weeks. Various immune parameters were measured at 1, 2, 3, and 4 weeks of post-feeding. The results of non-specific immunoassay showed that diets supplemented with B8 significantly increased alkaline phosphatase (AKP) and superoxide dismutase (SOD) activity in serum samples (p < 0.05). The expression levels of immune-related genes in the kidney and spleen of grass carp were measured. Among them, the expression levels of IgM and TNF-α both in spleen and kidney were significantly increased after 3 and 4 weeks of post-feeding (p < 0.05). The expression of IgD and MHCI in kidney was significantly upregulated in high-dose groups after 2 and 3 weeks of feeding, respectively (p < 0.05). In addition, after 7 days of challenging with A. veronii , the high-dose group and low-dose group had 48% and 53% survival compared to 25% survival for the control group. These results suggest that B. velezensis B8 has the potential to be developed into a microecological preparation for the alternatives of antibiotics in aquaculture. • Bacillus velezensis B8 isolated from the gut of grass carp possesses prominent probiotic properties. • Bacillus velezensis B8 exerted broad-spectrum antimicrobial activity against fish pathogenic bacteria. • Dietary supplementation of Bacillus velezensis B8 enhanced multiple immune responses in fish. • Dietary supplementation of Bacillus velezensis B8 increased disease resistance against Aeromonas veronii. [ABSTRACT FROM AUTHOR]

In mammals, CD4 is found to be expressed on T cells and innate immune cells, however, teleost cells bearing CD4 have not been well identified and characterized. In this study, we identified two different CD4-1+ cell subsets in grass carp (Ctenopharyngodon idella): CD4-1+ lymphocytes (Lym) and CD4-1+ myeloid cells (Mye), both of which had the highest proportions in the head kidney. The mRNA expression analysis showed that CD4-1 , CD4-2 , TCRβ , CD3γ/δ , and LCK1 are highly expressed in CD4-1+ Lym and also expressed in CD4-1+ Mye. Furthermore, we found that CD4-1+ Lym have a Lym morphology and highly express T-cell cytokines, suggesting that they are CD4+ T cells equivalent to mammalian Th cells. On the other hand, CD4-1+ Mye were found to have a morphology of macrophage and highly express macrophage marker gene MCSFR , indicating that they are macrophages. In addition, functional analysis revealed that CD4-1+ Mye possess phagocytic ability and great antigen-processing ability. Taken together, our study sheds further light on the composition and function of CD4+ cells in teleost fish. • CD4-1+ lymphocytes and CD4-1+ myeloid cells were identified in grass carp. • CD4-1+ lymphocytes highly expressed T-cell-related genes and Th-cell cytokines. • CD4-1+ myeloid cells were found to highly express macrophage-related genes. • CD4-1+ myeloid cells exhibited phagocytic and antigen-processing abilities. [ABSTRACT FROM AUTHOR]

Recent research has highlighted complex and close interaction between miRNAs, autophagy, and viral infection. In this study, we observed the autophagy status in CIK cells infected with GCRV at various time points. We found that GCRV consistently induced cellar autophagy from 0 h to 12 h post infection. Subsequently, we performed deep sequencing on CIK cells infected with GCRV at 0 h and 12 h respectively, identifying 38 DEMs and predicting 9581 target genes. With the functional enrichment analyses of GO and KEGG, we identified 35 autophagy-related target genes of these DEMs, among which akt3 was pinpointed as the most central hub gene using module assay of the PPI network. Then employing the miRanda and Targetscan programs for prediction, and verification through a double fluorescent enzyme system and qPCR method, we confirmed that miR-193 b-3p could target the 3′-UTR of grass carp akt3 , reducing its gene expression. Ultimately, we illustrated that grass carp miR-193 b-3p could promote autophagy in CIK cells. Above results collectively indicated that miRNAs might play a critical role in autophagy of grass carp during GCRV infection and contributed significantly to antiviral immunity by targeting autophagy-related genes. This study may provide new insights into the intricate mechanisms involved in virus, autophagy, and miRNAs. [Display omitted] • GCRV could continuously induce autophagy levels from 0 to 12 hpi in CIK cells. • 38 DEMs were identified after GCRV infection with 35 predicted target genes linked to autophagy enrichment terms. • MiR-193b-3p could promote grass carp autophagy via targeting akt3. [ABSTRACT FROM AUTHOR]

This research elucidates the potential of Lycium barbarum residue (LBR), a by-product rich in bioactive substances, as a dietary supplement in aquaculture, especially for herbivorous fish like grass carp. In a detailed 120-day feeding trial, the impacts of varying LBR levels on juvenile grass carp were assessed, focusing on growth performance, survival rate, biochemical markers, and liver health. The study identified a 6% inclusion rate of LBR as optimal for enhancing survival and growth while mitigating hepatic lipid accumulation. Composition analysis of this diet revealed high concentrations of polysaccharides and flavonoids. Notably, the intake of LBR was found to enhance the antioxidant and immune-related enzymatic activities in the liver. Furthermore, it contributed to a reduction in hepatic fat deposition by decreasing the levels of triglycerides (TG) and total cholesterol (T-CHO) both in the liver and serum. Transcriptomic analysis of the liver highlighted LBR's substantial influence on lipid metabolism pathways, including the PPAR signaling pathway, primary bile acid biosynthesis, cholesterol metabolism, bile secretion, fat digestion and absorption, fatty acid degradation and fatty acid biosynthesis. Further, the expression level of genes pinpointed significant downregulation of fasn and dgat2 , alongside upregulation of genes like pparda , cpt1b , cpt1ab and abca1b , in response to LBR supplementation. Overall, the findings present LBR as a promising enhancer of growth and survival in grass carp, with significant benefits in promoting fat metabolism and liver health, offering valuable insights for aquacultural nutrition strategies. • Dietary 6% LBR significantly maximized the growth performance and survival rate of juvenile grass carp. • Dietary 6% LBR could enhance antioxidant capacity and decrease hepatic fat deposits. • Dietary 6% LBR could regulate the expression of liver genes related to PPAR signaling pathway. [ABSTRACT FROM AUTHOR]

Proteins from the C1q domain-containing (C1qDC) family recognize self-, non-self-, and altered-self ligands and serves as an initiator molecule for the classical complement pathway as well as recognizing immune complexes. In this study, C1qDC gene family members were identified and analyzed in grass carp (Ctenopharyngodon idellus). Members of the C1q subfamily were cloned, and their response to infection with the grass carp virus was investigated. In the grass carp genome, 54 C1qDC genes and 67 isoforms have been identified. Most were located on chromosome 3, with 52 shared zebrafish homologies. Seven substantially differentially expressed C1qDC family genes were identified in the transcriptomes of cytokine-induced killer (CIK) cells infected with grass carp reovirus (GCRV), all of which exhibited sustained upregulation. The opening reading frames of grass carp C1qA, C1qB , and C1qC , belonging to the C1q subfamily, were determined to be 738, 732, and 735 base pairs, encoding 245, 243, and 244 amino acids with molecular weights of 25.81 kDa, 25.63 kDa and 26.16 kDa, respectively. Three genes were detected in the nine collected tissues, and their expression patterns were similar, with the highest expression levels observed in the spleen. In vivo after GCRV infection showed expression trends of C1qA , C1qB , and C1qC in the liver, spleen, and kidney. An N-type pattern in the liver and kidney was characterized by an initial increase followed by a decrease, with the highest expression occurring during the recovering period, and a V-type pattern in the spleen with the lowest expression levels during the death period. In vitro, after GCRV infection showed expression trends of C1qA, C1qB , and C1qC, and this gradually increased within the first 24 h, with a notable increase observed at the 24 h time point. After CIK cells incubation with purified recombinant proteins, rC1qA, rC1qB, and rC1qC for 3 h, followed by GCRV inoculation, the GCRV replication indicated that rC1qC exerted a substantial inhibitory effect on viral replication in CIK cells after 24 h of GCRV inoculation. These findings offer valuable insights into the structure, evolution, and function of the C1qDC family genes and provide a foundational understanding of the immune function of C1q in grass carp. • 54C1qDC genes with 67 isoforms were identified from the genome of grass carp. • 7 upregulated C1qDC family genes were identified in the transcriptome of CIK infected with GCRV. • Genes from the C1q subfamily, namely C1qA, C1qB, and C1qC, were cloned. • The mRNA expression patterns of C1qA, C1qB, and C1qC were elucidated both in vivo and in vitro. • C1qC may play a crucial role of inhibiting the replication of GCRV. [ABSTRACT FROM AUTHOR]

According to the International Agency for Research on Cancer (IARC), aflatoxin B1 (AFB1) has been recognized as a major contaminant in food and animal feed and which is a common mycotoxin with high toxicity. Previous research has found that AFB1 inhibited zebrafish muscle development. However, the potential mechanism of AFB1 on fish muscle development is unknown, so it is necessary to conduct further investigation. In the present research, the primary myoblast of grass carp was used as a model, we treated myoblasts with AFB1 for 24 h. Our results found that 5 μM AFB1 significantly inhibited cell proliferation and migration (P < 0.05), and 10 μM AFB1 promoted lactate dehydrogenase (LDH) release (P < 0.05). Reactive oxygen species (ROS), protein carbonyl (PC) and malondialdehyde (MDA) levels were increased in 15, 5 and 10 μM AFB1 (P < 0.05), respectively. Catalase (CAT), glutathione peroxidase (GPx) and total superoxide dismutase (T-SOD) activities were decreased in 10, 10 and 15 μM AFB1 (P < 0.05), respectively. Furthermore, 15 μM AFB1 induced oxidative damage by Nrf2 pathway, also induced apoptosis in primary myoblast of grass carp. Meanwhile, 15 μM AFB1 decreased MyoD gene and protein expression (P < 0.05). Importantly, 15 μM AFB1 decreased the protein expression of collagen Ⅰ and fibronectin (P < 0.05), and increased the protein levels of urokinase plasminogen activator (uPA), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 2 (MMP-2), and p38 mitogen-activated protein kinase (p38MAPK) (P < 0.05). As a result, our findings suggested that AFB1 damaged the cell morphology, induced oxidative damage and apoptosis, degraded ECM components, in turn inhibiting myoblast development by activating the p38MAPK/urokinase-type plasminogen activator (uPA)/matrix metalloproteinase (MMPs)/extracellular matrix (ECM) signaling pathway. • AFB1 down-regulated the expression of MyoD in primary myoblast of grass carp. • For the first time, we found that AFB1 activated uPA, MMP-2, MMP-9 in primary myoblast of grass carp. • AFB1 inhibited myoblast development by activating the p38MAPK/uPA/MMPs/ECM signaling pathway. [ABSTRACT FROM AUTHOR]

The red blood cells (RBCs) of fish make up around 95% of the total peripheral blood cells, and the long-held paradigm is that RBCs are mainly responsible for transporting oxygen. Previous studies have showed that the RBCs can be involved in the immune response against bacterial infection; however, this mechanism remains enigmatic. Here, we explored the structure of grass carp RBCs (Gc RBCs). The results showed that the Gc RBCs released a pseudopodia-like structure when grown in a 24-well plate, and the transmission electron microscopy (TEM) result showed that Gc RBCs contained some organelle-like structures. To further verify the organelle-like structures might be the mitochondria and lysosome which similar to other immune cells, a fluorescent labeling assay was used to verify it. To decipher the antibacterial immunity of GcRBCs, transcriptomic profiling of grass carp RBCs after the incubation with E. coli was analyzed. The results showed that there were 4099 differently expressed genes (DEGs) of GcRBCs upon E. coli incubation, including 2041 up-regulated and 2058 down-regulated genes. In addition, to validate our transcriptomic data, we checked the expression of several cytokines, such as CCL4, CCL20, IL4, IL12 and IFN-α, and the results showed that all the selected gens were significantly up-regulated after E. coli incubation. Furthermore, E. coli incubation induced hemoglobin oxidation and increased the heme in GcRBCs, which further activated the expression of heme oxygenase 1 (HO-1), autophagy related genes 5 (ATG5), and ferritin. In contrast, E. coli incubation inhibited the expression of Ferroportin-1 (FPN1), which increased intracellular iron levels, induced Fenton reaction to release reactive oxygen species (ROS), and activated the ferroptosis signaling pathway in GcRBCs. Herein, we demonstrate that E. coli can induce teleost RBCs cell death through an iron-mediated ferroptosis pathway, which sheds new light on the interaction between bacteria and teleost RBCs. • E. coli incubation induced 4099 DEGs in grass carp RBCs. • The mRNA expression of some cytokines were significantly increased after E. coli incubation. • E. coli can induce teleost RBCs cell death through an iron-mediated ferroptosis pathway. [ABSTRACT FROM AUTHOR]

In this study, RNA sequencing was used to identify the hepatic gene expression profile in grass carp associated with luteinizing hormone-releasing hormone agonist (LHRH-A) treatment. A total of 93,912,172 reads were generated by HiSeq 4000 sequencing platform. After filtering, 83,450,860 clean reads were mapped to the reference genome. By calculating the FPKM of genes, 1475 differentially expressed genes were identified. PPAR signaling pathway was enriched with upregulated genes in LHRH-A injection group showing the regulation of the lipid metabolism by LHRH-A. The expression of eight key genes in PPAR signaling pathway was confirmed by qPCR and the results suggested that ACSL4A, ACSL4B, ANGPTL4, LPL, RXRBA and SLC27A1B were significantly stimulated by LHRH-A injection. This investigation provides the evidence that LHRH-A could play a role in lipid metabolism. • Profile of liver transcriptomes from grass carp were subjected to treat with saline or LHRH-A. • LHRH-A stimulated PPAR signaling pathway in liver of grass carp. • Several lipid metabolism associated genes were up-regulated by LHRH-A injection. [ABSTRACT FROM AUTHOR]

In this study, the compound antimicrobial peptides was added to the basic diet to probe its effects on grass carp for 8 weeks. There were 6 groups in our study, including M0 (0 mg/kg) and 5 additional groups: M1 (100 mg/kg), M2 (200 mg/kg), M3 (400 mg/kg), M4 (800 mg/kg) and M5 (1600 mg/kg). The results are as follows: (1) The grass carp's FBW, WGR and SGR in M4 were significantly increased than M0 (p < 0.05). (2) In the hepatopancreas, the amylase activity of M2, M3 and M5 were significant up-regulation than other groups (p < 0.05). (3) The activity of T-AOC in the M3 and M4 was significantly higher compared to the other groups in grass carp hepatopancreas, while MDA was significantly reduced compared to the control group (p < 0.05). The activity of SOD in M5 was significantly increased than the other groups (p < 0.05). (4) The expression of MnSOD (except head kidney), IL8 and TNF-α and IL-1β (except hepatopancreas) were significantly increased (p < 0.05), while TGF-β and were significantly reduced in the hepatopancreas, spleen and head kidney at M3 (p < 0.05), and IL10 were significantly decreased in the hepatopancreas at M3 (p < 0.05). In addition, expression of IL8 and TNF-α were significant down-regulation, whereas TGF-β (expect head kidney) were significant up-regulation in hepatopancreas, spleen and head kidney in M3 after challenge with A. hydrophila. The expression of IL-1β in spleen and head kidney were also down-regulated, whereas IL10 were significantly up-regulated in the hepatopancreas at M3 after challenge with A. hydrophila (p < 0.05). The results of this study showed that grass carp fed a diet supplemented compound antimicrobial peptides was added with 400–800 mg/kg, which could improve the growth performance, antioxidant and digestive capabilities, and could also enhance the expression of immune-related genes and control to A. hydrophila. Image 1 • Study has shown that antimicrobial peptides from non-natural sources also have excellent antibacterial activity. • Antimicrobial peptides has effects on grass carp growth, digestion, anti-oxidation and immunity. • Antimicrobial peptides could improve the growth performance, antioxidant and digestive capabilities. • Antimicrobial peptides can be used as the substitute of antibiotics. [ABSTRACT FROM AUTHOR]

Interleukin (IL) −15 belongs to the common cytokine receptor γ chain (γC) family and has diverse functions in regulating the development, proliferation and activation of NK and T cells. It activates a hetero-trimeric receptor complex consisting of IL-2Rα, IL-2Rβ and a common γ chain (γC). In this study, the full-length cDNA sequences of IL-15 and IL-2Rβ were identified in grass carp (Ctenopharyngodon idella, Ci) and their expression profiles analysed. The Ci IL-15 and Ci IL-2Rβ were shown to be broadly expressed in tissues, with the highest levels detected in the spleen. Moreover, the Ci IL-15 and Ci IL-2Rβ were modulated in primary head kidney leucocytes (HKLs) and splenocytes by immunostimulants and cytokines, and in the head kidney and spleen of fish after infection of Flavobacterium columnare and grass carp reovirus. The bioactivity of bacteria derived recombinant Ci IL-15 protein was evaluated in the primary leucocytes. The Ci IL-15 was shown to induce signature genes of type 1 immune response (IFN-γ and T-bet) and NK cell activation (perforin and Eomesa), whilst exhibiting inhibitory effects on the genes involved in the type 2 immune response (IL-4 / 13, IL-10 and Gata3). Our data suggest that IL-15 is a key regulator in promoting the type 1 immune response and NK cell activation in fish. • Homologues of IL-15 and IL-2Rβ were identified in grass carp. • Ci IL-15 and Ci IL-2Rβ were upregulated in the spleen and head kidney after bacterial and viral infection. • Ci IL-15 upregulated signature genes of type 1 immune response and NK cell activation in primary leucocytes. • Ci IL-15 inhibited genes involved in type 2 immune response. [ABSTRACT FROM AUTHOR]

Functional ingredients such as Bacillus subtilis are used in aquaculture to improve fish condition, modulate microbiota and promote a healthy intestinal system. However, the underlying mechanisms of grass carp treated with B. subtilis are not fully characterized. This study investigated the gut microbes of grass carp after treated with B. subtilis H2 (106 CFU/mL) and Aeromonas hydrophila (106 CFU/mL). The intestinal flora was found that the dominant bacterial phyla identified in all samples were Proteobacteria , Actinobacteria , Fusobacteria , Bacteroidetes and Acidobacteria. Compared with the control group, the relative abundance of Proteobacteria and Bacteroidetes in B. subtilis group were significantly increased. In addition, the relative abundances of Aeromonas and Shewanella in A. hydrophila group were more than the control group. For the intestinal transcriptomic profiling of the grass carp treated with B. subtilis H2, 824 different expressed genes (DEGs) between the B. subtilis H2 treated and non-treated groups were detected, including 365 up-regulated and 459 down-regulated genes. Six DEGs were randomly selected for further validation by quantitative real-time RT-PCR (qRT-PCR) and the results were consistent with the RNA-seq data. Additionally, eight immunomodulatory genes (IL-4 , IL-11 , IFN-α , CSF , FOSB , MAPK12b , IGHV3-11 and IGHV3-21) were significantly up-regulated after treated with B. subtilis H2. Furthermore, almost all the lipid metabolism-associated genes were significantly up-regulated after treated with B. subtilis H2 according to the lipid metabolism pathways. Eleven lipid metabolism-associated genes were selected by qRT-PCR, which showed that the expressions of almost all the selected genes were increased, especially Apob-48 , ABCG8 and DGAT. Taken together, our results support that B. subtilis could modulate the immune response, fat metabolism and bacterial assembly in the gut of grass carp. • B. subtilis H2 enhanced the intestinal bacterial diversity of grass carp. • B. subtilis H2 induced 824 differentially expressed genes (DEGs) from transcriptome analysis. • B. subtilis H2 activated the immune response of the intestines of grass carp. • B. subtilis H2 improved the expression of fat metabolism-related genes. [ABSTRACT FROM AUTHOR]

Complement component 4 (C4) has critical immunological functions in vertebrates. In the current study, a C4 homolog (gcC4) was identified in grass carp (Ctenopharyngodon idella). The full-length 5458 bp gcC4 cDNA contained a 5148 bp open reading frame (ORF) encoding a protein of 1715 amino acids with a signal peptide and eight conservative domains. The gcC4 protein has a high level of identity with other fish C4 counterparts and is phylogenetically clustered with cyprinid fish C4. The gcC4 transcript shows wide tissue distribution and is inducible by Aeromonas hydrophila in vivo and in vitro. Furthermore, its expression also fluctuates upon lipopolysaccharide or flagellin stimulation in vitro. During infection, the gcC4 protein level decreases or increases to varying degrees, and the intrahepatic C4 expression location changes. With gcC4 overexpression, interleukin 1 beta, tumor necrosis factor alpha, and interferon transcripts are all upregulated by A. hydrophila infection. Meanwhile, overexpression of gcC4 reduces bacterial invasion or proliferation. Moreover, gcC4 may activate the NF-κB signaling pathway. These findings demonstrate the vital role of gcC4 in the innate immunity of grass carp. • A C4 homolog (gcC4) is identified in grass carp (Ctenopharyngodon idella). • gcC4 mRNA or protein expression is induced by A. hydrophila or PAMPs. • Overexpression of gcC4 inhibits the invasion and proliferation of A. hydrophila. • Overexpression of gcC4 induces the expression of immune-related genes. • Overexpression of gcC4 activates the NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, plays an important regulatory role in the activation of T cells induced by mitogenic or antigenic stimuli. However, the immunologic property of MIF in freshwater fish is limitedly known by now. In the present study, MIF gene was identified in grass carp. Bioinformatics analysis revealed that the molecular weight of grass carp MIF protein was 12.377 kDa and it could also bind to CD74. MIF gene was predominantly expressed in immune tissues including spleen and head kidney, then liver, skin, gill, intestine and blood, while a relative low level expression in heart, brain, fat and red muscle. The predicted receptor and tissues distribution of MIF implied the immunologic activity of grass carp MIF. Then grass carp MIF antigen and the polyclonal antibodies against it were prepared. Using cadmium as an immunosuppressive agent, MIF expression in spleen and head kidney was depressed in a dose-dependent manner with cadmium consumption. On the same time, white blood cell count decrease displayed a similar pattern with MIF expression, which suggested a possible positive correlation between MIF and white blood cell count. Thereafter, MIF enhanced the viability of grass carp peripheral blood leukocytes and inhibited cell apoptosis with depressed reactive oxygen species production in vitro. In addition, recombinant grass carp MIF promoted tumor necrosis factor-alpha (TNF-α), interleukin 1β (IL1β) and interleukin 6 (IL6) secretion from peripheral blood leukocytes. These results indicated the immunologic property of grass carp MIF. • Grass carp MIF was identified and highly expressed in spleen and head kidney. • MIF antigen and poly-antibodies against it were prepared. • MIF expression pattern was similar with white blood cell count under Cd exposure. • MIF enhanced leukocyte viability and inhibited cell apoptosis with depressed ROS. • MIF induced inflammatory factor TNF-α, IL1β and IL6 secretion from leukocytes. [ABSTRACT FROM AUTHOR]

In vertebrates, IL-10 is an anti-inflammatory factor that serves as a key inhibitory role in a wide range of immune responses. IRAK1 (IL-1 receptor-associated kinase 1), a key molecule in the inflammatory signal of IL-1R/TLR, plays an important pivotal role in regulating the autoimmunity of body. STAT3 (Signal transducer and activator of transcription 3) activated by IRAK1 participates in inflammation, tumorigenesis, metabolic disorders and immune response. Under the stimulation of LPS, IRAK1 enters the nucleus to form a dimer with STAT3 and regulates the expression of IL-10. However, the relationship between fish IRAK1 and STAT3 has not been reported. To explain the anti-inflammation in fish, we amplified and identified the complete open reading frame of grass carp IRAK1 (CiIRAK1) and STAT3 (CiSTAT3) based on the existing sequences. The expression of CiIRAK1 and CiSTAT3 were up-regulated significantly under the stimulation of LPS. This result suggests that both Ci IRAK1 and Ci STAT3 may be involved in LPS-induced TLR4 pathway. The subcellular localization experiment revealed that Ci IRAK1 is distributed in cytoplasm and enters nucleus after LPS stimulation. Ci STAT3 is distributed in both cytoplasm and nucleus with or without LPS stimulation. Immunoprecipitation assay revealed that Ci IRAK1 interacted with Ci STAT3 under LPS stimulation. However in absence of LPS stimulation, Ci IRAK1 and Ci STAT3 cannot interact with each other. Subsequently, immunofluorescence colocalization experiment further proved the interaction of Ci IRAK1 and Ci STAT3 in nucleus under LPS stimulation. The dual luciferase reporter assays indicated that the binding of Ci IRAK1 and Ci STAT3 synergistically enhanced the activity of CiIL-10 promoter. • Grass carp IRAK1 and STAT3 can respond to LPS stimulation. • LPS promotes grass carp IRAK1 nuclear translocation. • Grass carp IRAK1 interacts with STAT3 in nuclear. • Grass carp IRAK1 and STAT3 up-regulates synergistically the promoter activity of IL-10. [ABSTRACT FROM AUTHOR]

In mammals, interleukin 21 (IL-21) is a broad pleiotropic cytokine that plays critical roles in the development of several inflammatory and autoimmune diseases. In fish, functional information of Il-21 is limited, and its role in immune response is largely unknown. In the present study, we cloned a coding sequence of grass carp (Ctenopharyngodon idella) il21 gene (gcil21). To characterize the release patterns and biological activity of gcIl-21, we prepared recombinant gcIl-21 (rgcIl-21) and obtained the polyclonal antibody with gcIl-21 specificity. Western blotting analysis showed that in grass carp head kidney leukocytes (HKLs), gcIl-21 was undetected in culture supernatant of untreated cells but drastically induced by heat-killed Aeromonas hydrophila (A. hydrophila), uncovering the release features of gcIl-21 and its possible involvement in immune response. Subsequent functional experiments revealed that rgcIl-21 did not affect the mRNA expression of grass carp il1b and tgfb , but induced a strong expression of grass carp il10 , and to a lesser extent of grass carp tnfa in HKLs, suggesting a dominant effect of gcIl-21 in modulating Il-10 signaling as seen in rainbow trout and mammals. Furthermore, in vivo studies showed that intraperitoneal injection of rgcIl-21 was able to increase the survival rate of grass carp infected with live A. hydrophila , and reduce the pathological responses caused by the same pathogenic bacteria in head kidney and intestine. Taken together, these results for the first time revealed the close relationship of fish Il-21 production and function with inflammatory responses, and highlighted its anti-bacterial and anti-inflammatory ability, thereby providing a new insight into host defense mechanisms in fish. • Recombinant gcIl-21 (rgcIl-21) is prepared by using prokaryotic expression system. • Bacterial challenge drastically induces gcIl-21 release from head kidney leukocytes. • RgcIl-21 markedly induces il10 expression in head kidney leukocytes. • RgcIl-21 is able to increase the survival rate of bacteria-infected grass carp. • RgcIl-21 can improve bacteria-induced pathological responses in vivo. [ABSTRACT FROM AUTHOR]

Ctenopharyngodon idella growth arrest and DNA damage-inducible 45 ab (CiGadd45ab) is a subtype of the Gadd45a gene of the Gadd45 family in grass carp. There is increasing evidence that microRNAs (miRNAs) are involved in the regulation of inflammatory and apoptotic responses. However, little is known about the regulatory effects of miRNAs on CiGadd45ab expression. In the present study, CiGadd45ab was identified as a target gene of miR-23a-3p and miR-23a-5p, based on miRNA expression profiling and a dual-luciferase reporter assay. In addition, miR-23a-3p and miR-23a-5p were both confirmed to be involved in the inflammatory response following infection with Aeromonas hydrophila by targeting CiGadd45ab. Transfection with miR-23a-3p and miR-23a-5p mimics and inhibitor altered proinflammatory gene expression and apoptosis rate, thereby suggesting that miRNAs regulate immune response and anti-apoptosis by targeting CiGadd45ab in grass carp. Our results provide a theoretical basis for exploring the molecular mechanisms by which miR-23a-3p and miR-23a-5p target CiGadd45ab to regulate inflammation and apoptosis against bacterial infection in grass carp. • miRNAs play a role in regulating immune and apoptotic responses in grass carp. • CiGadd45ab was identified as a target gene of miR-23a-3p and miR-23a-5p. • A negative correlation between CiGadd45ab and miRNA expression was reported. • Overexpression of CiGadd45ab had pro-apoptotic and miRNAs mimics had anti-apoptotic effects in CIK cells. • miRNAs modulate inflammatory response via activation of the MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

Mannose-binding lectin (MBL) is a crucial pattern recognition receptor in the host innate immune system. Previously, we reported the biological function of Ctenopharyngodon idella MBL (CiMBL) in initiating the lectin pathway of the complement system. In the present study, we further explored its biological function including the agglutinating ability, binding capacity and protective role in vitro and in vivo. After Aeromonas hydrophila infection, western blot analysis revealed that the CiMBL were fluctuated and expressed in the serum and major immune-related tissues. The result of quantitative PCR (qPCR) showed that the recombinant CiMBL (rCiMBL) significantly inhibited the mRNA expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in liver, spleen and hepatic cells. Due to rCiMBL bound to d -mannose, d -galactose, d -glucose, N-acetyl- d -glucosamine (GlcNAc), lipopolysaccharide (LPS), peptidoglycan (PGN) and Agar in the presence of Ca2+, herein gram-positive (Staphylococcus aureus and Micrococcus luteus) and gram-negative (A. hydrophila and Vibrio anguillarum) bacteria were agglutinated by rCiMBL in a Ca2+-dependent manner. More importantly, rCiMBL enhanced the survival rate of grass carp following bacterial infection. Overall, the results provide an evidence that CiMBL can protect grass carp against A. hydrophila infection in aquaculture. • The rCiMBL enhanced the survival rate of grass carp following bacterial infection. • The CiMBL agglutination activity against both gram-positive and gram-negative bacteria. • The rCiMBL inhibited CiIL-1β and TNF-α expression in the liver, spleen and hepatic cells. [ABSTRACT FROM AUTHOR]

Grass carp Ctenopharyngodon idella Hsp70 has been identified to play a functional role in viral attachment of type III grass carp reovirus, GCRV-104. However, it remains to be clarified whether Hsc70, sharing 86% identity with Hsp70, plays a similar role during viral infection. In this study, grass carp Hsp70 was shown to be induced by GCRV-104 in different grass carp cell lines, whereas Hsc70 was expressed in a relatively constant level during the infection. The expression patterns of Hsc70 and Hsp70 were similar to their homologs in mammals. Notably, both inhibitor and over-expression assays indicated that Hsp70 was required for efficient viral replication. Thus, our study supported a novel pro-viral property of Hsp70 besides its reported role in the viral attachment. Results herein presented also suggested that the heat shock response of grass carp might be manipulated by aquareovirus to facilitate its replication in fish cells. • Hsp70 is induced by infection of GCRV-104. • Hsc70 is expressed in a constant level independent of viral infection. • Hsp70 is required for efficient viral replication. • GCRV-104 selectively induces heat shock response gene(s). [ABSTRACT FROM AUTHOR]

Interleukin (IL)-12p40, a component of IL-12 and IL-23, can be secreted as monomer and homodimer in mammals. Our previous study has proved the existence of natural three p40 isoforms and their proinflammatory properties in grass carp. In the present study, we unexpectedly found that recombinant grass carp p40a/b/c (rgcp40a, rgcp40b and rgcp40c) were able to enhance the mRNA levels of grass carp il-17a/f1 (gcil-17a/f1) in a dose- and time-dependent manner in head kidney leukocytes (HKLs). In agreement with these findings, the enzyme-linked immunosorbent assay (ELISA) showed that rgcp40a, rgcp40b and rgcp40c markedly stimulated gcIl-17a/f1 secretion from the HKLs. Together with their stimulatory effects on grass carp gcil-22 and gcil-26 expression, our data suggested their potential to mediate Th17-like response in grass carp. To support this notion, we investigated the underlying mechanisms for the regulation of rgcp40 isoforms on gcil-17a/f1 expression, and found that three rgcp40 isoforms significantly induced the activation of Erk, Jnk and Stat3 pathways in a time-dependent oscillation in the same cell model. Moreover, three rgcp40 isoforms-induced gcil-17a/f1 mRNA expression was suppressed by the inhibition on Erk, Jnk and Stat3 pathways, suggesting the signaling pathways in the p40 isoforms-mediating il-17a/f1 transcription. These studies for the first time proved the involvement of three gcp40 isoforms in mediating Th17 signature cytokine expression in fish immune cells, therefore providing new insights into the roles of p40 in teleost immunity. • Three grass carp p40 isoforms induce the transcription of Th17 signature cytokines. • Three p40 isoforms significantly stimulate Il-17a/f1 protein release. • Three p40 isoforms can activate Erk, Jnk and Stat3 pathways. • Erk, Jnk and Stat3 pathways mediate three isoforms-induced il-17a/f1 transcription. [ABSTRACT FROM AUTHOR]

In this study, a Streptomyces strain was isolated from the soil samples of Yanghu Wetland Park in Changsha, Hunan Province. This strain showed excellent antimicrobial activity against 10 fish pathogens, as indicated by the results of the agar-diffusion and oxford cup assays. After 16s rDNA sequencing and physiological & biochemical analyses, it was identified as Streptomyces amritsarensis , namely for S. amritsarensis N1-32. Cytotoxicity test was performed, and the results exhibited that this strain had no toxicity to hepatic L8824 cell line from grass carp liver. The diets supplemented strain N1-32 at concentrations of 1 × 107 cfu/g and 1 × 109 cfu/g was used to feed fish. After 28 days, the expression levels of antioxidant-related genes Nrf2 and Keap1 in the liver and spleen were significantly up-regulated, and the expression of immune-related gene IgM was notably increased in the liver, kidney, head-kidney, and spleen. Toll-like receptor 4 (TLR4) gene expression was up-regulated in the spleen, and TLR4, myeloid differentiation factor 88 (MyD88) gene were up-regulated in the kidney. The survival rate of grass carp was significantly improved after pathogen infection. Whole-genome analysis of N1-32 showed that the strain harbored related genes, capability for producing substances that enhance the immunity of grass carp and inhibit pathogens. A total of 22 gene clusters were identified in the genome, including 5 terpene gene clusters, 4 nonribosomal peptide-synthetase (NRPS) gene clusters and 2 lantipeptide gene clusters. In summary, these results showed that strain N1-32 as a feed additive could regulate grass carp immunity and enhance the resistance of grass carp against fish pathogens. • A new strain, Streptomyces amritsarensis N1-32, was isolated and prospective to be applied to freshwater fish aquaculture. • Streptomyces amritsarensis N1-32 has broad-spectrum antibacterial activity against freshwater fish pathogens. • Streptomyces amritsarensis as probiotics was firstly applicated in aquaculture. • Streptomyces amritsarensis N1-32 could significantly promote the immunity and growth of grass carps. • Genome analysis of S. amritsarensis N1-32 revealed its potential for antimicrobial synthesis. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is a strong gram-negative bacterium that can cause a mass death of grass carp, and result in the huge economic loss. Development of practical vaccines is the best way to control the outbreak of this bacterial disease. In this study, a whole-cell inactivated vaccine was obtained via sonication, and then single-walled carbon nanotubes (SWCNTs) was used to link to the bacterial lysate (BL) for a novel vaccine (SWCNTs-BL). A total of 400 fish were vaccinated with BL and SWCNTs-BL via immersion (5, 10 mg L−1) or injection (5, 10 μg/fish) before challenge with live A. hydrophila at the 28 days post immunization (d.p.i.). The results showed that the antibody titer, enzymatic activity, expression of some immune-related genes (especially IgM and TNF-α) and RPS of fish in the injection groups were significantly increased compared to the control group after 28 d.p.i. For the immersion groups, immunological parameters were increased compared to the control group. Furthermore, the immuno-protective effects of SWCNTs-BL were better than BL. The above results indicated that BL of A. hydrophila can effectively induce specific immune response of grass carp, and BL linked with functionalized SWCNTs could enhance the protective effect of immersion immunization. Our results may provide a practical vaccine, with a simple production, to fight against bacterial diseases in aquaculture industry. • The bacterial lysate of A. hydrophila can effectively induce specific immune response of grass carp. • The bacterial lysate linked with functionalized SWCNTs could enhance the protective effect of immersion immunization. • Related immunity index was significantly enhanced after vaccination with the bacterial lysate of A. hydrophila. [ABSTRACT FROM AUTHOR]

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

To study the effect of dietary supplementation of Bacillus licheniformis FA6 on the growth, survival and intestinal health of grass carp, we assessed the antioxidant capacity, intestinal barrier, expression levels of immune genes, and the resistance to Aeromonas hydrophila AH-1 infection. Experimental setup comprised three groups (90 specimens each; average initial weight = 16.5 g): the control group was fed the basal diet without B. licheniformis , the low-dose (LD) group was supplemented with B. licheniformis at the concentration of 1 × 105 cfu/g, and the high-dose (HD) group with 1 × 106 cfu/g. After 56 days of growth trial, the challenge test with A. hydrophila AH-1 was conducted for 14 days. The results revealed that the grass carp in LD group and HD group had significantly (p < 0.05) improved percent weight gain (PWG) and specific growth rate (SGR) parameters. Additionally, the antioxidant status was improved, which included increased superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) levels in the serum, and upregulated mRNA levels of antioxidant enzymes MnSOD and catalase (CAT) in the intestine. Meanwhile, B. licheniformis FA6 supplementation groups exhibited a decreased mRNA expression of proinflammatory cytokines (such as IL-1β and TNF-α) and increased the expression of anti-inflammatory cytokine IL-10. Histological (villi length was increased) and gene expression (qPCR: upregulated ZO-1 , occludin , and claudin-c) analyses suggested improved functioning of the intestinal barrier. Post-challenge mortality rates in LD and HD groups were significantly lower (56.6% and 70% respectively) than in the control group (100%). Overall, these results indicated that dietary supplementation of B. licheniformis FA6 can improve growth, antioxidant capacity, intestinal barrier functions and disease resistance of grass carp. • Supplementing Bacillus licheniformis FA6 enhanced growth performance of grass carp. • ...increased the antioxidant capacity of grass carp. • ...strengthened the tight junctions and down-regulated pro-inflammatory cytokines in the intestine. • ...and enhanced its resistance against Aeromonas hydrophila. [ABSTRACT FROM AUTHOR]

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

Small sample capacity is a critical limitation for the industrialization of the high voltage electrostatic field assisted freezing (HVEFF) technique. In this study, using grass carp as a sample, the effects of ultrahigh permittivity ceramic enhanced HVEFF system on the quality attributes of grass carp (Ctenopharyngodon idella) fillets during freezing and frozen storage were investigated. The appropriate electrostatic field strength for freezing grass carp fillets was determined first with the HVEFF Peltier cooling platform and then used for the freezing and frozen storage of grass carp fillets in an HVEFF refrigerator at −18 °C. The biochemical qualities, and ice crystal morphology of the samples were measured every 2, 4, 6, and 8 weeks during frozen storage. Compared to the frozen control, this novel HVEFF system could effectively delay the deterioration of texture and water migration, and reduce TVB-N, TBARS and the equivalent ice crystal diameters. This study not only showed the positive effect of this novel HVEEF system on the frozen preservation of grass carp fillets but more importantly provided a solution to the bottleneck of HVEFF industrialization. • A high voltage electrostatic field-assisted freezing (HVEFF) system was built. • The distance between electrodes was increased from common 10–20 mm–50 mm. • Freezing at 5.21 × 104 V/m effectively maintained the qualities of grass carp. • The study provided a solution to the bottleneck of HVEFF industrialization. [ABSTRACT FROM AUTHOR]

miRNAs are increasingly recognized for their crucial role in autophagy processes. Recent research has highlighted the significant function of autophagy in modulating immune responses. Within this context, specific miRNAs have been identified as indirect mediators of immune functions through their modulation of autophagy. In this study, we verified that miR-193b-5p simultaneously targeted the grass carp autophagy-related gene deptor , thereby reducing autophagy levels in CIK cells. Moreover, we found the expression levels of miR-193b-5p and deptor responding to pathogen infections in the GCRV-infected CIK cells. Notably, the overexpression of miR-193b-5p was found to induce the GCRV replication and reduce the irf3 , irf7 and IFN1 expression. These findings also demonstrated that grass carp miR-193b-5p impacted the proliferation, migration, and antiapoptotic abilities of CIK cells. All the above results indicated that miR-193b-5p was linked to grass carp autophagy and played a vital role in antiviral immunity by targeting deptor. Our study may provide important insights into autophagy-related miRNAs and their roles in defense and immune mechanisms against pathogens in teleost. • miR-193b-5p simultaneously targets grass carp deptor and blocks autophagy flux in CIK cells. • miR-193b-5p can be downregulated by GCRV stimulation and its overexpression promotes virus replication. • miR-193b-5p can exacerbate cell apoptosis and weaken the survivability of CIK cells. [ABSTRACT FROM AUTHOR]

Edwardsiella piscicida (E. piscicida) is a gram-negative pathogen that survives in intracellular environment. Currently, the interplay between E. piscicida and host cells has not been completely explored. In this study, we found that E. piscicida disturbed iron homeostasis in grass carp monocytes/macrophages to maintain its own growth. Further investigation revealed the bacteria induced an increase of intracellular iron, which was subjected to the degradation of ferritin. Moreover, the autophagy inhibitor impeded the degradation of ferritin and increase of intracellular iron in E. piscicida -infected monocytes/macrophages, implying possible involvement of autophagy response in the process of E. piscicida -broken iron homeostasis. Along this line, confocal microscopy observed that E. piscicida elicited the colocalization of ferritin with LC3-positive autophagosome in the monocytes/macrophages, indicating that E. piscicida mediated the degradation of ferritin possibly through the autophagic pathway. These results deepened our understanding of the interaction between E. piscicida and fish cells, hinting that the disruption of iron homeostasis was an important factor for pathogenicity of E. piscicida. They also indicated that autophagy was a possible mechanism governing intracellular iron metabolism in response to E. piscicida infection and might offer a new avenue for anti- E. piscicida strategies in the future. • E. piscicida enhances intracellular iron in grass carp monocytes/macrophages. • E. piscicida induces the degradation of ferritin in the monocytes/macrophages. • Autophagy inhibitor impedes E. piscicida -induced disorders of iron homeostasis. • E. piscicida elicits the colocalization of ferritin with autophagosome. [ABSTRACT FROM AUTHOR]

Since disinfectants are used all over the world to treat illnesses in people and other animals, they pose a major risk to human health. The comprehensive effects of disinfectant treatments on fish liver, especially the impacts on oxidative stress, toxicological effects, transcriptome profiles, and apoptosis, have not yet been fully analyzed. In the current investigation, healthy grass carp were exposed to 80 μg/L glutaraldehyde or 50 μg/L povidone-iodine for 30 days. First, the findings of enzyme activity tests demonstrated that the administration of glutaraldehyde could considerably increase oxidative stress by lowering T-SOD, CAT, and GPx and raising MDA. Furthermore, KEGG research revealed that exposure to glutaraldehyde and povidone-iodine stimulated the PPAR signal pathway. To further elucidate the transcriptome results, the relative expressions of related DEGs in the PPAR signal pathway were verified. Glutaraldehyde induced apoptosis in liver tissue of grass carp; however, it activated cytotoxicity and apoptosis in grass carp hepatocytes when exposed to glutaraldehyde or povidone-iodine. According to the current study, disinfectants can cause the impairment of the immune system, oxidative stress, and attenuation of the PPAR signal pathway in the liver of grass carp, making them detrimental as dietary supplements for grass carp, particularly in the aquaculture sector. • Glutaraldehyde or povidone-iodine has effects on the immune system of the liver of grass carp. • Povidone-iodine or glutaraldehyde affects the PPAR signalling pathway. • Glutaraldehyde and povidone-iodine induce apoptosis in grass carp hepatocytes. [ABSTRACT FROM AUTHOR]

Submerged macrophytes are vital in shallow lakes, as they provide critical ecosystem functions and services and can stabilize the clear-water conditions by various mechanisms. Nutrient enrichment reduces the resilience of macrophyte dominance in shallow lakes, thereby making them susceptible to shifts towards phytoplankton dominance following perturbations. Here, we conducted a mesocosm experiment to examine the individual and combined effects of nutrient enrichment and the addition of grass carp (Ctenopharyngodon idella) on the abundance of submerged macrophytes, epiphyton, and phytoplankton. We hypothesized that moderate nutrient enrichment facilitates macrophyte abundance, but also phytoplankton abundance after macrophyte removal by herbivorous fish. Our data showed that herbivory by grass carp could trigger a shift from macrophytes to algal dominance in mesocosms with moderate nutrient concentrations, but not in those with low nutrient concentrations. Moderate nutrient enrichment alone promoted submerged macrophyte growth, whereas the introduction of grass carp induced a collapse of submerged macrophytes regardless of nutrient conditions. Moreover, the introduction of grass carp showed more negative effects on light conditions of the water column in mesocosms with moderate nutrient concentrations compared to those with low nutrient concentrations. A recovery of submerged macrophytes might thus be limited by low light availability in lakes with moderate nutrient conditions suffering grass carp perturbation. Our results suggest that submerged macrophyte-dominated shallow lakes with moderate nutrient conditions are vulnerable to perturbation by herbivorous fish such as grass carp. In turn, managing the abundance of herbivores in these lakes can support the dominance of macrophytes and associated clear water conditions. • Moderate nutrient enrichment alone promoted submerged macrophyte growth. • Nutrient enrichment reduces the resilience of shallow, macrophyte-dominated lakes. • Grass carp introduction led to the collapse of submerged macrophytes. • Submerged macrophyte recovery would be limited by nutrient enrichment and grass carp. [ABSTRACT FROM AUTHOR]