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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., Competing Interests: Declaration of competing interest None., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2024. Published by Elsevier Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest regarding the publication of this article., (Copyright © 2024. Published by Elsevier Ltd.)

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aeromonas hydrophila (A. hydrophila) is one of major pathogenic bacteria in aquaculture and potentially virulent to grass carp (Ctenopharyngodon idella). As an essential nutrient for fish, vitamin D3 (VD3) has been reported to play a role against oxidative stress, but the exact mechanism remains to be elusive. In this study, we found that A. hydrophila induced ferrugination and macrophage aggregation in the spleen of grass carp. Along this line, using the splenic macrophages as the model, the effects of VD3 on A. hydrophila-caused iron deposition and subsequent injuries were determined. In the context, 1,25D3 (the active form of VD3) significantly reduced cellular free Fe 2+ , lipid peroxidation and lactic dehydrogenase (LDH) release induced by A. hydrophila in the splenic macrophages, indicating the protective effects of VD3 on A. hydrophila-led to ferroptosis-related injuries. In support of this notion, 1,25D3 was effective in hindering ferroptosis inducers-stimulated LDH release in the same cells. Mechanically, 1,25D3 enhanced iron export protein (ferroportin1) and glutathione peroxidase 4 (GPX4) protein levels, and glutathione (GSH) contents via vitamin D receptor (VDR). Moreover, NF-E2-related factor 2 (Nrf2) pathway mediated the regulation of 1,25D3 on GPX4 protein expression and GSH synthesis. Meanwhile, 1,25D3 maintained the stability of Nrf2 proteins possibly by attenuating its ubiquitination degradation. Furthermore, in vivo experiments showed that 1,25D3 injection could not only improve the survival of fish infected by A. hydrophila, but also enhance GSH amounts and decrease malonaldehyde (MDA) contents and iron deposition in the spleen. In summary, our data for the first time suggest that VD3 is a potential antioxidant in fish to fight against A. hydrophila induced-ferroptotic damages., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are integral to many biological functions, including autophagy, a process recently proven to be closely linked to innate immunity. In this study, we present findings on miR-22a, a teleost homolog of mammalian miR-22, illustrating its capacity to target the autophagy adaptor p62, subsequently inducing downregulation at both mRNA and protein levels. Utilizing Western blot analyses, we demonstrated that miR-22a inhibits the autophagy flux of CIK cells, correlated with an elevated presence of LC3 II. Additionally, the overexpression of miR-22a resulted in the suppression of NF-κB signaling, leading to reduced cellar antimicrobial abilities and increased apoptosis. These findings provide novel insights into the role of miR-22a as an autophagy-related miRNA and its immune mechanisms against pathogens via p62 in teleost, enriching our understanding of the interplay between autophagy and innate immunity., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Grass carp is one of the most economically important fish species. Hemorrhagic diseases caused by grass carp reovirus (GCRV) can seriously damage the economic yield of grass carp. Therefore, antiviral research on grass carp is urgently needed. Membrane-associated RING-CH2 (MARCH2) negatively regulates the innate immune response in mice. However, little is known about the role of march2 in the antiviral innate immune response in teleost fish. Our present study showed that march2 has high homology in grass carp, its orthologs, and mammals, and has the same amino acid sequence in grass carp and crucian carp. Overexpression of Cimarch2 (Ctenopharyngodon idella march2) significantly inhibited interferon (IFN) activation induced by Polyinosinic-polycytidylic acid (poly I: C), spring viremia of carp virus (SVCV), and GCRV. However, knocking down Cimarch2 enhanced the activation of IFN induced by poly I: C, SVCV, and GCRV. Overexpression of Cimarch2 can promotes viral replication. Mechanistically, Cimarch2 tightly bound to TANK-binding kinase 1 (tbk1) and downregulated tbk1 through the proteasome pathway. Our results demonstrated the potential role of Cimarch2 in the antiviral breeding of grass carp., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

miRNAs play a key role in the autophagy process. In recent years, the emerging role of autophagy in regulating immune response has attracted increasing attention. Since then, specific miRNAs have also been found to play an immune function indirectly by modulating autophagy as well. This study proved that miR-23a could downregulate grass carp autophagy simultaneously by targeting ATG3 and ATG12. Besides, both ATG3 and ATG12 mRNA levels were increased in kidney and intestine after being infected by Aeromonas hydrophila; yet almost at the same time, miR-23a was decreased. Besides, we illustrated that grass carp miR-23a could affect antimicrobial capacity, proliferation, migration, and antiapoptotic abilities of CIK cells. These results indicate that miR-23a was related to grass carp autophagy and plays an important role in antimicrobial immunity through targeting ATG3 and ATG12, which provides important information on autophagy-related miRNAs about the defense and immune mechanisms against pathogens in teleost., (Copyright © 2023. Published by Elsevier Ltd.)

Respiratory burst is a process involving rapid production of reactive oxygen species (ROS) for eliminating invading pathogens. However, excessive ROS production can be fatal to the host organism. The Keap1-Nrf2-ARE (Kelch-like ECH-associated protein 1 [Keap1]; Nuclear factor erythroid-derived 2-like 2 [Nrf2]; Antioxidant responsive element [ARE]) signaling pathway plays an important role in alleviating oxidative stress and preserving cellular homeostasis. However, the role of Keap1 during bacterial infection in fish remains unclear. In this study, we cloned and characterized the Keap1 gene of grass carp (CiKeap1) for the first time. CiKeap1 encodes a 593-amino acid protein of the Keap1b type. The tissue distribution analysis data revealed that the brain contains the highest transcription level of Keap1, followed by the heart and liver. The infection of Aeromonas hydrophila and Staphylococcus aureus obviously modulated the gene transcription and protein expression levels of Keap1, which suggested that the CiKeap1 participates in antibacterial immune responses. Furthermore, in vitro overexpression assays clarified the defensive and regular roles of CiKeap1 in maintaining host redox homeostasis in response to bacterial infection through the Keap1-Nrf2-ARE signaling pathway. In conclusion, the present results provide an expanded perspective on the role of Keap1 in teleost immunology that can guide healthy farming cultivation of grass carp., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

TBK1 is an atypical IκB kinase family member with a set of functions. It is involved in congenital immunization and autophagy in mammals. In this study, we reported that grass carp TBK1 gene expression could be upregulated by bacterial infection. Overexpression of TBK1 could decrease the number of adhesive bacteria in CIK cells. TBK1 could promote cellular migration, proliferation, vitality, and anti-apoptosis ability. Furthermore, the expression of TBK1 could activate the NF-κB signaling pathway by inducing inflammatory cytokines. In addition, we found that the grass carp TBK1 could cause the autophagy level of CIK cells within the decreasing level of p62 protein. Our finding indicated that TBK1 participated in grass carp innate immune progress and autophagy. This study provides evidence of the positive regulation of TBK1 in teleost innate immunity with its multiple functions. It thus may provide important information about the defense and immune mechanisms used by teleost against pathogens., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Toll-like receptor (TLR) is an important pattern recognition receptor, which specifically recognizes microbial components, and TLR5 recognizes bacterial flagellin in vertebrates and invertebrates. In this study, two forms of TLR5 (TLR5a and TLR5b) were identified in grass carp (Ctenopharyngodon idella). Aeromonas hydrophila and Staphylococcus aureus were used to investigate the role of grass carp TLR5a and TLR5b against bacteria (flagellate and non-flagellate) in innate immunity, and the expression of TLR5a and TLR5b genes and proteins were detected in immune-related tissues. Quantitative real-time polymerase chain reaction results showed that TLR5a and TLR5b genes of grass carp were highly expressed in the liver, spleen, and head kidney, and their expression patterns were similar in tissues. Meanwhile, the TLR5b gene expression was higher than TLR5a in most tissues. Following exposure to A. hydrophila and S. aureus, the expression levels of TLR5a and TLR5b genes in the liver, spleen, and head kidney were up-regulated significantly. Moreover, the downstream gene, NF-κB, was up-regulated significantly. After A. hydrophila infection, the expression of TLR5a gene was up-regulated in the liver and spleen at 24 h, while TLR5b was up-regulated at 6 h. In the head kidney, TLR5a was up-regulated at 6 h, while TLR5b was up-regulated at 6 h and 12 h. After S. aureus infection, TLR5a and TLR5b were up-regulated at 6 h in the liver and 12 h in the spleen. However, in the head kidney, TLR5a was down-regulated, while TLR5b was up-regulated. Compared with TLR5a, TLR5b had a higher expression level and stronger response to pathogen stimulation. The immunofluorescence results showed that TLR5a and TLR5b proteins in the liver of grass carp infected with A. hydrophila and S. aureus were similar but different in the spleen and head kidney. The results indicated that TLR5a and TLR5b play a critical role in resisting bacterial infection, and TLR5a and TLR5b had obvious tissue and pathogen specificity. TLR5b may play a major role in immune tissues, while TLR5a may play an auxiliary regulatory role in early infection. In addition, TLR5a and TLR5b have an irreplaceable regulatory role in response to flagellate and non-flagellate bacteria. This lays a foundation to explore further the role of TLR5 in resisting flagellate and non-flagellate infections in fish and provides a reference for the innate immunity research of grass carp., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Septicemia is a systemic inflammatory response to bacterial infection that results in a hyper-inflammatory state, which could lead to septic shock and death in grass carp (Ctenopharyngodon idella). The aim of this study was to determine the underlying mechanism of microRNA (miR-130a) in bacteria-infected grass carp. Expression levels of miR-130a against Aeromonas hydrophila (A. hydrophila) infection in Ctenopharyngodon idella kidney cells (CIK) were analyzed. Luciferase reporter assay, quantitative reverse transcription-polymerase chain reaction were performed to explore whether Ctenopharyngodon idella growth arrest and DNA damage-inducible 45 (CiGadd45bb) was a target of miR-130a. MiR-130a mimic, inhibitor and miR-control were transfected to CIK respectively. After transfection, the expression levels of proinflammatory genes were determined. Here we show that CiGadd45bb as a target of miR-130a. We also confirmed that miR-130a levels were significantly higher after being stimulated for 4 h and lower after 12 h (P < 0.01). Overexpressing miR-130a strikingly inhibited p38, JNK, ERK and TNF-a genes (P < 0.01) and silencing miR-130a activated p38, JNK, ERK, TNF-a, IFN and IL-8 (P < 0.01). Our results provide a theoretical basis for studying the molecular mechanism underlying the regulation of inflammation by miR-130a in grass carp., (Copyright © 2023. Published by Elsevier Ltd.)

4-tert-butylphenol (4-tBP) is a monomer widely used in the synthesis of industrial chemicals, and posed a high risk to aquatic animals. Our study focused on toxic phenotype and mechanism of detoxification in grass carp hepatocytes (L8824) after 4-tBP-treatment. In this experiment, L8824 displayed hallmark phenotypes of apoptosis and necroptosis after 4-tBP exposure, as evidenced by changes in cell morphology, increased rates of apoptosis and necrosis, the loss of MMP, the accumulation of ROS, and changes in associated factors (PARP1, JNK, Bid, Bcl-2, Bax, AIFM1, CytC, Caspase 9, APAF1, Caspase 3, TNF-α, TNFR1, RIPK1, RIPK3, and MLKL). Furthermore, we found that 4-tBP-induced apoptosis and necroptosis were reversed by pretreating with N-Acetylcysteine (a ROS scavenger) and 3-Aminobenzamide (a PARP1 inhibitor), indicating that 4-tBP induced the onset of mitochondrial apoptosis and necroptosis in L8824 via activating ROS-PARP1 axis. Nano-selenium (Nano-Se) is a novel form of Se with a noteworthy antioxidant capacity. Here, Nano-Se was found to have preventive, therapeutic, and resistance effects on 4-tBP-induced L8824 apoptosis and necroptosis. Nano-Se co-treatment with 4-tBP was an optimal way to alleviate 4-tBP-induced apoptosis and necroptosis. We demonstrated for the first time that Nano-Se protected L8824 against 4-tBP-induced mitochondrial apoptosis and necroptosis through ROS-PARP1 pathway. This study will provide a new theoretical basis for 4-tBP toxicology researches and aquatic animal protection., Competing Interests: Declaration of competing interest The authors declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Long noncoding RNAs (lncRNAs) play important roles in many biological processes including the immune response against virus infection. However, their roles in grass carp reovirus (GCRV) pathogenicity are largely unknown. In this study, the next-generation sequencing (NGS) technology was used to analyze the profiles of lncRNAs in GCRV-infected and mock-infected grass carp kidney (CIK) cells. Our results showed that 37 lncRNAs and 1039 mRNA transcripts exhibited differential expression in CIK cells after GCRV infection compared with the mock infection. Functional analysis through the gene ontology and Kyoto Encyclopedia of Genes and Genomes databases (KEGG) indicated that target genes of the differentially expressed lncRNAs were mainly enriched in the biological processes - biological regulation, cellular process, metabolic process and regulation of the biological process, such as MAPK signaling pathway and Notch signaling. Furthermore, we observed that the lncRNA3076 (ON693852) was markedly upregulated after the GCRV infection. In addition, silencing lncRNA3076 decreased the GCRV replication, which indicates that it might play an important role in the replication of GCRV., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A 70-day feeding trial was conducted to study the effects of dietary nucleotide, yeast cell wall (containing 20% β -glucan) and their combination on growth performance, feed utilization and immune response of grass carp (Ctenopharyngodon idella) with 69.97 ± 0.05 g of initial body weight. Four isonitrogenous (about 38% crude protein) and isolipidic (about 5% crude lipid) diets were established. Based on the control diet (CD), the other three experimental diets were prepared by adding 0.01% of nucleotide (NT), 0.1% of yeast cell wall (YCW) and NT (0.01%) +YCW (0.1%), respectively. Results showed that no significant difference was found in survival of grass carp ranging from 94.44% to 97.78% among all the groups (P > 0.05). Compared with the control group, weight gain rate, muscle crude protein content, serum protein, trypsin and chymotrypsin activities in midgut, lysozyme and immunoglobulin M in serum significantly increased in fish fed the YCW diet (P < 0.05). The significantly highest weight gain rate, villus height and digestive enzyme activities in midgut and innate immune parameters in serum were found in fish fed the NT + YCW diet (P < 0.05). The gene expressions of β-defensin, hepcidin, il-10 and tgf-β1 in the midgut, and tor and s6k1 in liver significantly increased in fish fed the NT + YCW diet. Meanwhile, the gene expressions of il-1β and tnf-α in the midgut decreased significantly (P < 0.05). The liver histology showed the better development in dietary NT and/or YCW supplemented groups than those in the control group. In conclusion, combination of dietary NT and YCW had significantly synergetic improvements on the growth, feed utilization, digestive enzymes, innate immunity and histology of midgut and liver of grass carp., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Interleukin (IL) 4 and 13 are signature cytokines orchestrating Th2 immune response. Teleost fish have two homologs, termed IL-4/13A and IL-4/13B, and have been functionally characterized. However, what cells express IL-4/13A and IL-4/13B has not been investigated in fish. In this work, the recombinant IL-4/13A and IL-4/13B proteins of grass carp (Ctenopharyngodon idella) were produced in the Escherichia coli (E. coli) cells and purified. Monoclonal antibodies (mAbs) against the recombinant CiIL-4/13A and CiIL-4/13B proteins were prepared and characterized. Western blotting analysis showed that the CiIL-4/13A and CiIL-4/13B mAbs could specifically recognize the recombinant proteins expressed in the E. coli cells and HEK293T cells and did not cross-react with each other. Confocal microscopy revealed that the CiIL-4/13A + and CiIL-4/13B + cells were present in the gills, intestine and spleen and could be upregulated in fish infected with Flavobacterium columnare (F. columnare). Interestingly, the cells expressing CiIL-4/13A and CiIL-4/13B were mostly CD3γ/δ + cells. The CD3γ/δ + /IL-4/13A + and CD3γ/δ + /IL-4/13B + cells were significantly upregulated in the gill filaments and the intestinal mucosa after F. columnare infection. Our results imply that the CD3γ/δ + /IL-4/13A + and CD3γ/δ + /IL-4/13B + cells are important for homeostasis and the regulation of mucosal immunity., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest All the authors declare that they have no conflict of interest., (Copyright © 2022. Published by Elsevier Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2022. Published by Elsevier Ltd.)

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 (gcgzmbl) and a perforin-like (gcprfl) 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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 (gchepcidin and gcβ-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., (Copyright © 2021. Published by Elsevier Ltd.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 CiSNX8 might negatively regulate RLR signaling pathway that is quite distinct from mammalian SNX8. In addition, CiSNX8 could interact with MAVS, STING, TBK1, IRF3 and IRF7. Either wild type CiSNX8 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 (CiMAP3K4) 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 CiMAP3K4 encodes putative peptide of 1544 amino acids containing a predicted serine/threonine protein kinase (S&#95;TKc) domain with high identity with other fish MAP3K4s. Phylogenetic analysis revealed the CiMAP3K4 belonged to the fish cluster and showed the closest relationship to Pimephales promelas. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMAP3K4 transcripts were widely distributed in all tested tissues, especially with high expression in the muscle and intestine of healthy grass carp. In vitro, CiMAP3K4 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 CiMAP3K4 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, CiMAP3K4 was located primarily in the cytoplasm, and its overexpression increased the transcriptional activity of AP-1 in HEK293T cells. Collectively, these results suggested that CiMAP3K4 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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), 10 7 , and 10 9  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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 (GcRBCs). The results showed that the GcRBCs released a pseudopodia-like structure when grown in a 24-well plate, and the transmission electron microscopy (TEM) result showed that GcRBCs 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 CiIL-15 and CiIL-2Rβ were shown to be broadly expressed in tissues, with the highest levels detected in the spleen. Moreover, the CiIL-15 and CiIL-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 CiIL-15 protein was evaluated in the primary leucocytes. The CiIL-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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 (10 6  CFU/mL) and Aeromonas hydrophila (10 6  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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 CiIRAK1 and CiSTAT3 may be involved in LPS-induced TLR4 pathway. The subcellular localization experiment revealed that CiIRAK1 is distributed in cytoplasm and enters nucleus after LPS stimulation. CiSTAT3 is distributed in both cytoplasm and nucleus with or without LPS stimulation. Immunoprecipitation assay revealed that CiIRAK1 interacted with CiSTAT3 under LPS stimulation. However in absence of LPS stimulation, CiIRAK1 and CiSTAT3 cannot interact with each other. Subsequently, immunofluorescence colocalization experiment further proved the interaction of CiIRAK1 and CiSTAT3 in nucleus under LPS stimulation. The dual luciferase reporter assays indicated that the binding of CiIRAK1 and CiSTAT3 synergistically enhanced the activity of CiIL-10 promoter., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 × 10 7  cfu/g and 1 × 10 9  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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest All authors must disclose any financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work. Examples of potential competing interests include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. Authors must disclose any interests in two places: 1. a summary declaration of interest statement in the title page file (if double-blind) or the manuscript file (if single-blind). If there are no interests to declare then please state this: ‘Declarations of interest: none'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 Ca 2+ , herein gram-positive (Staphylococcus aureus and Micrococcus luteus) and gram-negative (A. hydrophila and Vibrio anguillarum) bacteria were agglutinated by rCiMBL in a Ca 2+ -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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ctenopharyngodon idellagrowth 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 (gcil2) 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)