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Ferredoxin (FDX) is a highly conserved iron-sulfur protein that participates in redox reactions and plays an important role as an electron transport protein in biological processes. However, its function in marine fish remains unclear. We identified two ferrodoxin proteins, FDX1 and FDX2, from black scraper (Thamnaconus modestus) to confirm their genetic structures and expression profiles and to investigate their antimicrobial activity properties by fabricating them with antimicrobial peptides based on sequences. The two TmFDXs mRNAs were most abundant in peripheral blood leukocytes of healthy T. modestus. After artificial infection with Vibrio anguillarum, a major pathogen of T. modestus, TmFDX1 mRNA was significantly upregulated in the gills, heart, intestines, kidneys, liver, and spleen, but was consistently downregulated in the brain. The expression levels of TmFDX2 mRNA were significantly upregulated in the heart, intestines, kidneys, liver, and spleen; however, no significant changes in expression were observed in the brain or gills. Based on the 2Fe-2S ferredoxin-type iron-sulfur-binding domain sequence, two peptides (pFDX1 and pFDX2) were synthesized. The bactericidal effect, biofilm formation inhibition, and gDNA-binding activity of these peptides were investigated. These findings highlight the potential as a natural peptide candidate for TmFDXs., 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.)

dsRNA-dependent protein kinase R (PKR) is a key factor of innate immunity. It is involved in translation inhibition, apoptosis, and enhancement of the proinflammatory and IFN responses. However, how these antiviral functions are conserved during evolution remains largely unknown. Overexpression and knockout studies in a Chinook salmon (Oncorhynchus tshawytscha) cell line were conducted to assess the role of salmonid PKR in the antiviral response. Three distinct mRNA isoforms from a unique pkr gene, named pkr-fl (full length), pkr-ml (medium length) and pkr-sl (short length), were cloned and a pkr-/- clonal fish cell line was developed using CRISPR/Cas9 genome editing. PKR-FL includes an N-terminal dsRNA-binding domain and a C-terminal kinase domain, whereas PKR-ML and PKR-SL display a truncated or absent kinase domain, respectively. PKR-FL is induced during IFNA2 stimulation but not during viral hemorrhagic septicemia virus (VHSV) infection. Overexpression experiments showed that only PKR-FL possesses antiviral functions, including activation of apoptosis and inhibition of de novo protein synthesis. Knockout experiments confirmed that PKR is involved in apoptosis activation during the late stage of VHSV infection. Endogenous PKR also plays a critical role in translation inhibition upon poly(I:C) transfection after IFNA2 treatment. It is, however, not involved in translational arrest during VHSV infection. Extra- and intracellular titrations showed that endogenous PKR does not directly inhibit viral replication but apparently favors virion release into the supernatant, likely by triggering late apoptosis. Altogether, our data confirm that salmonid PKR has conserved molecular functions that VHSV appears to bypass with subversion strategies., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Exocyst, a protein complex, plays a crucial role in various cellular functions, including cell polarization, migration, invasion, cytokinesis, and autophagy. Sec3, known as Exoc1, is a key subunit of the Exocyst complex and can be involved in cell survival and apoptosis. In this study, two subtypes of Sec3 were isolated from Epinephelus coioides, an important marine fish in China. The role of E. coioides Sec3 was explored during Singapore grouper iridovirus (SGIV) infection, an important pathogen of marine fish which could induce 90 % mortality. E. coioides Sec3 sequences showed a high similarity with that from other species, indicating the presence of a conserved Sec3 superfamily domain. E. coioides Sec3 mRNA could be detected in all examined tissues, albeit at varying expression levels. SGIV infection could upregulate E. coioides Sec3 mRNA. Upregulated Sec3 significantly promoted SGIV-induced CPE, and the expressions of viral key genes. E. coioides Sec3 could inhibit the activation of NF-κB and AP-1, as well as SGIV-induced cell apoptosis. The results illustrated that E. coioides Sec3 promotes SGIV infection by regulating the innate immune response., (Copyright © 2024. Published by Elsevier Ltd.)

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

The COMMD (Copper Metabolism gene MURR1 Domain) gene family consists of 10 members, which are involved in various biological processes such as copper and sodium transport, NF-κB activity and cell cycle progression. However, the study of COMMD gene family in large yellow croaker (Larimichthys crocea) is largely unknown. In this study, 10 COMMD gene family members (named LcCOMMDs) were successfully identified from large yellow croaker. The results showed that there were differences in the number of LcCOMMDs exons at the level of gene structure, which reflected that they had adjusted and changed accordingly in the process of evolution to adapt to the environment and achieved functional diversification. Through phylogenetic analysis, we found that the LcCOMMDs was highly conserved, indicating their important functions in organisms. It was worth noting that the expression levels of LcCOMMD1, LcCOMMD2, LcCOMMD3, LcCOMMD5 and LcCOMMD10 in the spleen changed significantly after bacterial stress, which suggested that these genes might be involved in the regulation of innate immune response. In addition, the expression levels of LcCOMMD1, LcCOMMD2, LcCOMMD3, LcCOMMD5, LcCOMMD7, LcCOMMD8, LcCOMMD9 and LcCOMMD10 changed significantly after hypoxia exposure, which further proved the role of LcCOMMDs in immune function. In summary, this study not only revealed the important role of COMMD genes in the innate immune response of large yellow croaker, but also provided valuable information for further understanding the regulatory mechanism of COMMD gene family under different conditions., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

IkappaB kinase beta (IKKβ) is a key member of IκB kinases and functions importantly in interferon (IFN) signaling. Phosphorylation and ubiquitination are involved in the activation of IKKβ. A20 is a de-ubiquitin enzyme and functions as a suppressor in inflammation signaling, which has been reported to be phosphorylated and activated by IKKβ. However, the role and relationship of IKKβ and A20 in teleost remains unclear. In this study, IKKβ (bcIKKβ) and A20 (bcA20) of black carp (Mylopharyngodon piceus) have been cloned and characterized. Overexpressed bcIKKβ in EPC cells showed strong anti-viral ability by activating both NF-κB and IFN signaling. EPC cells stable expressing bcIKKβ presented improved anti-viral activity as well. The interaction between bcA20 and bcIKKβ was identified, and overexpression of bcA20 was able to suppress bcIKKβ-mediated activation of NF-κB and IFN signaling. Meanwhile, knock-down of A20 increased host the antiviral ability of host cells. Importantly, it has been identified that bcA20 was able to remove K27-linked ubiquitination and decrease the phosphorylation of bcIKKβ. Thus, our data conclude that bcA20 suppresses the anti-viral activity of bcIKKβ and removes its K27-linked ubiquitination, which presents a new mechanism of IKKβ regulation., 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 a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Prohibitin 1 (PHB1) is ubiquitously expressed in multiple compartments within cells and is involved in the cell cycle, cell signaling, apoptosis, transcriptional regulation, and mitochondrial biogenesis at the cellular level and in the inflammation-associated and immunological functions of B and T lymphocytes. PHB1 is an important protein that performs antioxidant regulation and immune functions inside and outside cells but has not been sufficiently studied in teleost fish. Our study aimed to elucidate the functional properties and gain new insights into the biological processes and immune system of red seabream (Pagrus major), a commercially important fish cultured in South Korea and East Asia. PHB1 mRNA was most abundantly expressed in the head kidney of healthy red seabream, and significant changes in its expression were observed after artificial infection with bacteria and viruses. On analysis, reporter gene was also significantly upregulated by polyinosinic-polycytidylic acid, lipopolysaccharides, and hydrogen peroxide. Consequent to the functional characterization of PHB1 in cells via recombinant protein preparation, the activity of leukocytes was enhanced and the reactive oxygen species-induced stress in red blood cells was reduced. The results reveal the functional characteristics of PHB1 and provide new insights into the biological processes and immune system of P. major, with beneficial implications in the study of stress responses., 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.)

Singapore grouper iridovirus (SGIV) belongs to the family Iridoviridae and the genus Ranavirus, which is a large cytoplasmic DNA virus. Infection of grouper with SGIV can cause hemorrhage and swelling of the spleen of the fish. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. In the present study, the protein encoded by SGIV ORF128 (VP128) was identified. VP128 is predominantly localized within the endoplasmic reticulum (ER). Overexpression of VP128 significantly promoted SGIV replication. VP128 inhibited the interferon (IFN)-3 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), and TANK-binding kinase 1 (EcTBK1). Moreover, VP128 interacted with EcSTING and EcTBK1. The interaction between VP128 and EcSTING was independent of any specific structural domain of EcSTING. Together, our results demonstrated that SGIV VP128 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion., 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 reports in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aquaculture is a prosperous economic sector threatened by viral infections. Among the viruses threatening fish culture, Betanodavirus (NNV) is extremely important in the Mediterranean Sea affecting to highly traded species as European sea bass. In this context, application of antimicrobial peptides (AMPs) has arisen as a potential biotechnological tool. The aim of this work was to evaluate the therapeutic application of two European sea bass-derived AMPs, NK-lysin (Nkl) and dicentracin (Dic), against NNV infections. Synthetic Dic peptide was able to significantly reduce NNV-induced mortalities while Nkl failed to do so. Although neither Dic nor Nkl peptides were able to alter the transcriptional levels of NNV and the number of infected cells, Nkl seemed to increase the viral load per cell. Interestingly, both Nkl and Dic peptides showed immunomodulatory roles. For instance, our data revealed an interplay among different AMPs, at both gene and protein levels. Otherwise, Nkl and Dic peptides provoked an anti-inflammatory balance upon NNV infection, as well as the recruitment of macrophages and B cells to the target site of the infection, the brain. In conclusion, Dic can be proposed as a therapeutic candidate to combat NNV., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Type I IFNs are a subset of cytokines exerting their antiviral effects mainly through the JAK-STAT signalling. Immunogenetic studies have shown that fish possess key components of IFN-JAK-STAT cascade, but the information about the distinct responses of STAT1 and STAT2 to different IFNs is rather limited in fish. Here, we identified and cloned STAT1 and STAT2 genes (named as On-STAT1 and On-STAT2) from tilapia, Oreochromis niloticus. On-STAT1 and On-STAT2 genes were detected in all orangs/tissues examined, and were rapidly induced in spleen, head kidney, and liver following the stimulation of poly(I:C). In addition, the stimulation of poly(I:C), poly(A:T), and different subgroups of recombinant IFNs could induce the expression of On-STAT1 and On-STAT2 in TA-02 cells with distinct induction levels. Importantly, On-STAT2 was rapidly phosphorylated by all three subgroups of IFNs, but the phosphorylation of On-STAT1 was only observed in IFNc- and IFNh-treated TA-02 cells, reflecting the distinct activation of STAT by different subgroups of fish IFNs. The present results thus contribute to better understanding of the JAK-STAT signalling mediated by different subgroups of IFNs in fish., Competing Interests: Declaration of competing interest The authors have no financial conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chinese seabass (Lateolabrax maculatus) stands out as one of the most sought-after and economically significant species in aquaculture within China. Diseases of L. maculatus occur frequently due to the degradation of the germplasm, the aggravation of environmental pollution of water, and the reproduction of pathogenic microorganisms, inflicting considerable economic losses on the Chinese seabass industry. The Myxovirus resistance (Mx) gene plays pivotal roles in the antiviral immune response ranging from mammals to fish. However, the function of the Mx gene in L. maculatus is still unknown. Firstly, the origin and evolutionary history of Mx proteins was elucidated in this study. Subsequently, an Mx gene from L. maculatus (designed as LmMxA gene) was identified, and its functions in combating antiviral and antibacterial threats were investigated. Remarkably, our findings suggested that while Mx group genes were present in chordates, DYN group genes were present in everything from single-celled animals to humans. Furthermore, our investigation revealed that the LmMxA mRNA level increased in the kidney, spleen and liver subsequent to Vibrio anguillarum and poly(I:C) challenged. Immunofluorescence analysis indicated that LmMxA is predominantly localization in the nucleus and the cytoplasm. Notably, the expression of MAVS, IFN1 and Mx1 increased when LmMxA was overexpression within the EPC cells. Moreover, through assessment via cytopathic effect (CPE), virus titer, and antibacterial activity, it becomes evident that LmMxA exerts a dual role in bolstering both antiviral and antibacterial immune responses. These compelling findings laid the foundation for further exploring the mechanism of LmMxA in response to innate immunity of L. maculatus., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Fish skin plays an important role in defending against pathogens in water, primarily through the secretion of skin mucus containing various immune-related factors. Local immune responses in the skin activate systemic immune responses by inflammatory cytokines. However, it remains unclear whether immune responses in the skin occur after systemic immune responses caused by pathogen invasion into the fish body. This study aimed to clarify the relationship between systemic immune responses and skin responses after intraperitoneal injection of formalin-killed cells (FKC) of Vibrio anguillarum. Although systemic inflammatory responses were observed in the spleen after injection, expression changes in the skin did not show significant differences. In contrast, expression of hemoglobin subunit genes significantly increased in the skin after FKC injection, suggesting that erythrocytes infiltrate extravascularly., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Complement factor H-related protein (CFHR) plays an important role in regulating complement activation and defensive responses. The function of CFHR2 (complement factor H related 2), a member of the CFHR family, in fish remains unclear. Here, we report the genetic relationship, expression characteristics and regulatory mechanism of cfhl5 (complement factor H like 5) gene, which encodes CFHR2 in Chinese tongue sole. We observed that the cfhl5 gene was widely expressed in several tissues, such as brain, heart and immune organs, and was most abundantly expressed in liver. After injection with Vibrio harveyi, the expression of cfhl5 was up-regulated significantly in liver, spleen and kidney at 12 or 24 hours post infection (hpi), suggesting an involvement of this gene in the acute immune response. Knockdown of cfhl5 in liver cells significantly up-regulated the expression of the pro-inflammatory cytokines tnf-α (tumor necrosis factor-alpha) and il1β (interleukin-1beta), the immunomodulatory factor il10 (interleukin-10) and the lectin complement pathway gene masp1 (MBL-associated serine protease 1), and down-regulated the expression of complement components c3 (complement 3) and cfi (complement factor I). In our previous work, we found that cfhl5 gene was significantly higher methylated and lower expressed in the resistant family compared with the susceptible family. Therefore, we used dual-luciferase reporter system to determine the effect of DNA methylation on this gene and found that DNA methylation could inhibit the promoter activity to reduce its expression. These results demonstrated that the expression of cfhl5 is regulated by DNA methylation, and this gene might play an important role in the immune response by regulating the expression of cytokines and complement components genes in Chinese tongue sole., Competing Interests: Declaration of competing interest All the authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Interleukin (IL) 20 is a multifunctional cytokine and plays a vital role in regulating autoimmune diseases, inflammation, and immune responses. IL-20 homologs have been described in fish. However, due to the lack of antibodies, cellular sources and immunological functions of fish IL-20 in response to infections have not been fully characterized. In this study, a monoclonal antibody (mAb) was generated against the recombinant grass carp (Ctenopharyngodon idella) IL-20 protein and characterized by immunoblotting, immunofluorescent microscopy and flow cytometry. It was shown that the IL-20 mAb specifically recognized recombinant IL-20 proteins expressed in the E. coli cells and HEK293 cells. Using confocal microscopy, the IL-20 + cells were identified in the head kidney, gills and intestine of grass carp, and induced after infection with Aeromonas hydrophila. Moreover, the IL-20 protein was found to be secreted mainly by CD3γδ T cells which were located predominantly in the gill filaments and intestinal mucosa. Taken together, our results suggest that IL-20 producing T cells are required for the mucosal immunity against bacterial infection in fish., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mammalian interleukin-22 (IL-22) attenuates organismal injury by inhibiting reactive oxygen species (ROS) and impeding the NLRP3 inflammasome activation. However, the role of fish IL-22 in this process remains unclear. We characterized MaIL-22, an IL-22 homolog in blunt snout bream ( Megalobrama amblycephala ). Despite its low sequence identity, it shares conserved structures and close evolutionary relationships with other teleost IL-22s. Furthermore, Aeromonas hydrophila ( A. hydrophila ) infection leads to tissue injury in M. amblycephala immune organs and concomitantly altered Mail-22 mRNA expression, suggesting that MaIL-22 was involved in the antimicrobial immune response. To explore MaIL-22's biological functions, we produced recombinant MaIL-22 (rMaIL-22) protein and demonstrated it significantly enhanced the survival of M. amblycephala post- A. hydrophila infection. To unravel its protective mechanisms, we explored the ROS/NLRP3 inflammasome axis and its downstream signaling responses. The results showed that rMaIL-22 treatment significantly elevated antioxidant enzyme (T-SOD, CAT and GSH-PX) activities to inhibit MDA activity and scavenge ROS in visceral tissues. Meanwhile, rMaIL-22 impeded the activation of NLRP3 inflammasome by suppressing NLRP3 protein and mRNA expression. This indicated that rMaIL-22 contributed to inhibit A. hydrophila -induced activation of the ROS/NLRP3 inflammasome axis. Consistent with these findings, rMaIL-22 treatment attenuated the expression of proinflammatory cytokines ( il-1β , tnf-α and il-6 ) and proapoptotic genes ( caspase-3 and caspase-8 ) while promoting antiapoptotic genes ( bcl-2b and mcl-1a ) expression, ultimately mitigating tissue injury in visceral tissues. In conclusion, our research underscores MaIL-22's key role in microbial immune regulation, offering insights for developing IL-22-targeted therapies and breeding programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Zhai and Liu.)

The economic importance of lumpfish ( Cyclopterus lumpus ) is increasing, but several aspects of its immune responses are not well understood. To discover genes and mechanisms involved in the lumpfish antiviral response, fish were intraperitoneally injected with either the viral mimic polyinosinic:polycytidylic acid [poly(I:C)] or phosphate-buffered saline (PBS; vehicle control), and head kidneys were sampled 24 hours post-injection (hpi) for transcriptomic analyses. RNA sequencing (RNA-Seq) (adjusted p-value <0.05) identified 4,499 upregulated and 3,952 downregulated transcripts in the poly(I:C)-injected fish compared to the PBS-injected fish. Eighteen genes identified as differentially expressed by RNA-Seq were included in a qPCR study that confirmed the upregulation of genes encoding proteins with antiviral immune response functions (e.g., rsad2 ) and the downregulation of genes (e.g., jarid2b ) with potential cellular process functions. In addition, transcript expression levels of 12 members of the interferon regulatory factor (IRF) family [seven of which were identified as poly(I:C)-responsive in this RNA-Seq study] were analyzed using qPCR. Levels of irf1a , irf1b , irf2 , irf3 , irf4b , irf7 , irf8 , irf9 , and irf10 were significantly higher and levels of irf4a and irf5 were significantly lower in the poly(I:C)-injected fish compared to the PBS-injected fish. This research and associated new genomic resources enhance our understanding of the genes and molecular mechanisms underlying the lumpfish response to viral mimic stimulation and help identify possible therapeutic targets and biomarkers for viral infections in this species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Emam, Kumar, Eslamloo, Caballero-Solares, Hall, Xue, Paradis, Gendron, Santander and Rise.)

Antimicrobial peptides (AMPs) are an alternative to antibiotics for treatment and prevention of infections with a lower risk of bacterial resistance. Pituitary adenylate cyclase activating polypeptide (PACAP) is an outstanding AMP with versatile effects including antimicrobial activity and modulation of immune responses. The objective of this research was to study PACAP immunomodulatory effect on rainbow trout cell lines infected with Aeromonas salmonicida. PACAP from Clarias gariepinus (PACAP1) and a modified PACAP (PACAP5) were tested. RT-qPCR results showed that il1b and il8 expression in RTgutGC was significantly downregulated while tgfb expression was upregulated after PACAP treatment. Importantly, the concentration of IL-1β and IFN-γ increased in the conditioned media of RTS11 cells incubated with PACAP1 and exposed to A. salmonicida. There was a poor correlation between gene expression and protein concentration, suggesting a stimulation of the translation of IL-1β protein from previously accumulated transcripts or the cleavage of accumulated IL-1β precursor. In-silico studies of PACAP-receptor interactions showed a turn of the peptide characteristic of PACAP-PAC1 interaction, correlated with the higher number of interactions observed with this specific receptor, which is also in agreement with the higher PACAP specificity described for PAC1 compared to VPAC1 and VPACA2. Finally, the in silico analysis revealed nine amino acids related to the PACAP receptor-associated functionality., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The transforming growth factor beta-activated kinase 1 (TAK1)/c-Jun N-terminal kinase (JNK) axis is an essential MAPK upstream mediator and regulates immune signaling pathways. However, whether the TAK1/JNK axis harnesses the strength in regulation of signal transduction in early vertebrate adaptive immunity is unclear. In this study, by modeling on Nile tilapia (Oreochromis niloticus), we investigated the potential regulatory function of TAK1/JNK axis on lymphocyte-mediated adaptive immune response. Both OnTAK1 and OnJNK exhibited highly conserved sequences and structures relative to their counterparts in other vertebrates. Their mRNA was widely expressed in the immune-associated tissues, while phosphorylation levels in splenic lymphocytes were significantly enhanced on the 4th day post-infection by Edwardsiella piscicida. In addition, OnTAK1 and OnJNK were significantly up-regulated in transcriptional level after activation of lymphocytes in vitro by phorbol 12-myristate 13-acetate plus ionomycin (P + I) or PHA, accompanied by a predominant increase in phosphorylation level. More importantly, inhibition of OnTAK1 activity by specific inhibitor NG25 led to a significant decrease in the phosphorylation level of OnJNK. Furthermore, blocking the activity of OnJNK with specific inhibitor SP600125 resulted in a marked reduction in the expression of T-cell activation markers including IFN-γ, CD122, IL-2, and CD44 during PHA-induced T-cell activation. In summary, these findings indicated that the conserved TAK1/JNK axis in Nile tilapia was involved in adaptive immune responses by regulating the activation of lymphocytes. This study enriched the current knowledge of adaptive immunity in teleost and provided a new perspective for understanding the regulatory mechanism of fish immunity., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Decay-accelerating factor (DAF) is an essential member of the complement regulatory protein family that plays an important role in immune response and host homeostasis in mammals. However, the immune function of DAF has not been well characterized in bony fish. In this study, a complement regulatory protein named CiDAF was firstly characterized from Ctenopharyngodon idella and its potential roles were investigated in intestine following bacterial infection. Similar to mammalian DAFs, CiDAF has multiple complement control protein (CCP) functional domains, suggesting the evolutionary conservation of DAFs. CiDAF was broadly expressed in all tested tissues, with a relatively high expression level detected in the spleen and kidney. In vivo immune challenge experiments revealed that CiDAF strongly responded to bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and PAMPs (lipopolysaccharide (LPS) or muramyl dipeptide (MDP)) challenges. In vitro RNAi experiments indicated that knockdown of CiDAF could upregulate the expression of complement genes (C4b, C5 and C7) and inflammatory cytokines (TNF-α, IL-1β and IL-8). Moreover, 2000 ng/mL of CiDAF agonist progesterone effectively alleviated LPS- or MDP-induced intestinal inflammation by regulating expression of complement factors, TLR/PepT1 pathway genes and inflammatory cytokines. Overall, these findings revealed that CiDAF may act as a negative regulator of intestinal complement pathway and immune response to bacterial challenge in grass carp., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) have been demonstrated to act as crucial modulators with considerable impacts on the immune system. Cottonseed meal is often used as a protein source in aqua feed, cottonseed meal contains gossypol, which is harmful to animals. However, there is a lack of research on the role of miRNAs in fish exposed to gossypol stress. To determine the regulatory effects of miRNAs on gossypol toxicity, Cyprinus carpio were given to oral administration of 20 mg/kg gossypol for 7 days, and the gossypol concentration in the tissues was tested. Then, we detected spleen index, histology, immune enzyme activities of fish induced by gossypol. The results of miRNA sequencing revealed 8 differentially expressed miRNAs in gossypol group, and miR-214&#95;L-1R+4 was found involved in immune response induced by gossypol. The potential targets of miR-214&#95;L-1R+4 were predicted, and found a putative miR-214&#95;L-1R+4 binding site in the 3'UTR of MyD88a. Furthermore, dual-luciferase reporter assays displayed miR-214&#95;L-1R+4 decreased MyD88a expression through binding to the 3'UTR of MyD88a. Moreover, miR-214&#95;L-1R+4 antagomir were intraperitoneally administered to C. carpio, down-regulated miR-214&#95;L-1R+4 could increase MyD88a expression, as well as inflammatory cytokines and anti-inflammatory cytokines expression. These findings revealed that miR-214&#95;L-1R+4 via the MyD88-dependent signaling pathway modulate the immune response to gossypol in C. carpio spleen., (Copyright © 2024. Published by Elsevier Ltd.)

Rainbow trout is an important fish species for Peruvian artisanal aquaculture, comprising over 60 % of the total aquaculture production. However, their industry has been highly affected by several bacterial agents such as Yersinia ruckeri. This pathogen is the causative agent of Enteric Redmouth Disease, and causes high mortality in fingerlings and chronic infection in adult rainbow trout. To date, the immune response of rainbow trout against Y. ruckeri has been well studied in laboratory-controlled infection studies (i.e. intraperitoneal infection, bath immersion), however, the immune response during natural infection has not been explored. To address this, in this study, 35 clinically healthy O. mykiss without evidence of lesions or changes in behavior and 32 rainbow trout naturally infected by Y. ruckeri, were collected from semi-intensive fish farms located in the Central Highlands of Peru. To evaluate the effect on the immune response, RT-qPCR, western blotting, and ELISA were conducted using head kidney, spleen, and skin tissues to evaluate the relative gene expression and protein levels. Our results show a significant increase in the expression of the pro-inflammatory cytokines il1b, tnfa, and il6, as well as ifng in all three tissues, as well as increases in IL-1β and IFN-γ protein levels. The endogenous pathway of antigen presentation showed to play a key role in defense against Y. ruckeri, due to the upregulation of mhc-I, tapasin, and b2m transcripts, and the significant increase of Tapasin protein levels in infected rainbow trout. None of the genes associated with the exogenous pathway of antigen presentation showed a significant increase in infected fish, suggesting that this pathway is not involved in the response against this intracellular pathogen. Finally, the transcripts of immunoglobulins IgM and IgT did not show a modulation, nor were the protein levels evaluated in this study., 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 a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-β (IFN-β) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-β (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) represent a prominent category of pattern recognition receptors that have been extensively investigated for their pivotal role in combating pathogen incursions. Despite this, there has been a notable absence of comprehensive identification and exploration of the immune response associated with the TLR family genes in C. altivelis. This study successfully identified and named fourteen genes as Catlr1-1, Catlr1-2, Catlr2-1, Catlr2-2, Catlr3, Catlr5, Catlr7, Catlr8, Catlr9, Catlr13-1, Catlr13-2, Catlr18, Catlr21, and Catlr22. A series of bioinformatic analysis were performed, encompassing analysis of protein properties, examination of gene structures, evolutionary assessments, and prediction of protein tertiary structures. The expression patterns of Catlr genes were analyzed in five immune tissues: liver, spleen, kidney, gill, and intestine, in both healthy and bacterial stimulated-fish. The results showed that different tissue and different genes showed differed expression patterns after V. harveyi infection, indicating the involvement of all Catlr members in mounting immune responses following infection in various tissues. Additionally, histological evaluations of immune tissues unveiled varying levels of damage. In conclusion, this investigation into the TLR gene family offers novel information that contribute to a more profound comprehension of the immune response mechanisms in C. altivelis., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Takifugu rubripes is a highly valued cultured fish in Asia, while pathogen infections can result in severe diseases and lead to substantial economic losses. Toll-like receptors (TLRs), as pattern recognition receptors, play a crucial role on recognition pathogens and initiation innate immune response. However, the immunological properties of teleost-specific TLR23 remain largely unknown. In this study, we investigated the biological functions of TLR23 (TrTLR23) from T. rubripes, found that TrTLR23 existed in various organs. Following bacterial pathogen challenge, the expression levels of TrTLR23 were significantly increased in immune related organs. TrTLR23 located on the cellular membrane and specifically recognized pathogenic microorganism. Co-immunoprecipitation and antibody blocking analysis revealed that TrTLR23 recruited myeloid differentiation primary response protein (MyD88), thereby mediating the activation of the ERK signaling pathway. Furthermore, in vivo showed that, when TrTLR23 is overexpressed in T. rubripes, bacterial replication in fish tissues is significantly inhibited. Consistently, when TrTLR23 expression in T. rubripes is knocked down, bacterial replication is significantly enhanced. In conclusion, these findings suggested that TrTLR23 played a critical role on mediation TLR23-MyD88-ERK axis against bacterial infection. This study revealed that TLR23 involved in the innate immune mechanism, and provided the foundation for development disease control strategies in teleost., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

RLR helicases RIG-I and MDA5, which are known as pattern recognition receptors to sense cytoplasmic viral RNAs and trigger antiviral immune responses, are DExD/H-box helicases. In teleost, whether and how non-RLR helicases regulate RLR helicases to affect viral infection remains unclear. Here, we report that the non-RLR helicase DHX40 from grass carp (namely gcDHX40) is a negative regulator of grass carp reovirus (GCRV) infection and RLR-mediated type I IFN production. GcDHX40 was a cytoplasmic protein. Ectopic expression of gcDHX40 facilitated GCRV replication and suppressed type I IFN production induced by GCRV infection and by those genes involved the RLR antiviral signaling pathway. Mechanistically, gcDHX40 promoted the generation of viral inclusion bodies (VIBs) by interacting with the NS38 protein of GCRV. Additionally, gcDHX40 interacted with RLR helicase, and impaired the formation of RLR-MAVS functional complexes. Taken together, our results indicate that gcDHX40 is a novel important proviral host factor involving in promoting the generation of GCRV VIBs and inhibiting the production of RLR-mediated type I IFNs., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The non-specific cytotoxic cell receptor protein 1 (NCCRP1) is considered the universal marker for teleost non-specific cytotoxic cells (NCCs). However, the specific distribution characteristics and response patterns of NCCRP1, and the confirmed existence of NCCs in fish species remain debatable. In this study, we investigated the distribution of NCCRP1 in the croaker and observed the most dominant abundance in the head kidney. While most common markers of cytotoxicity were localized in the trunk kidney lymphocytes (TKLs) and spleen lymphocytes (SPLs), NCCRP1-positive cells were predominantly detected in head kidney lymphocytes (HKLs) with a positive rate of approximately 10 %, where present a huge amount of macrophages (Mϕ) as well. Furthermore, the remarkable induction evidence of NCCRP1 in HKLs was determined. Collectively, these findings contribute significantly to comprehending the immunological function of NCCRP1 in fish species and enhancing our understanding of its evolutionary development., 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 a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cytokine-like factor 1 (CYTL1) is a small cytokine and has diverse biological functions in mammals. However, whether CYTL1 exists in lower vertebrates is not clear. In this study, we identified cytl homologs in fish and characterized the immune functions in a teleost species, grass carp (Ctenopharyngodon idella). Fish CYTL1 homologs share conserved molecular features with their mammalian counterparts, including 6 cysteine residues in the mature peptide, genomic organization and synteny. Gene expression analysis revealed that cytl1 was constitutively expressed in tissues of grass carp, with the highest expression detected in the heart. Upon infection with Aeromonas hydrophila (A. hydrophila), cytl1 was downregulated in the hindgut, head kidney, skin, and spleen. In the primary head kidney leukocytes (HKLs), stimulation with inactivated A. hydrophila, LPS, poly(I:C), IL-22, IFN-a or IFN-γrel resulted in downregulation of cytl1 expression. Recombinant grass carp CYTL1 protein produced in the HEK293-F cells was potent to induce il-10 expression, but had little effect on the expression of il-1β and il-6. In vivo experiments revealed that CYTL1 was effective to recruit macrophages to the muscle injected with cytl expression plasmids. Taken together, our results indicate that CYTL1 is a potent chemokine for recruitment of macrophages in fish., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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

The EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is involved in connective tissue development, elastic fiber formation, and tumor growth. In this study, we characterized the cDNA of EFEMP2 (PoEFEMP2), a member of the fibulin family of ECM proteins, in the olive flounder Paralichthys olivaceus. The coding region of PoEFEMP2 encodes a protein that contains six calcium-binding EGF-like (EGF-CA) domains and four complement Clr-like EGF-like (cEGF) domains. PoEFEMP2 shows 67.51-96.77 % similarities to orthologs in a variety of fish species. PoEFEMP2 mRNA was detected in all tissues examined; the highest levels of PoEFEMP2 mRNA expression were observed in the heart, testis, ovary and muscle. The PoEFEMP2 mRNA level increases during early development. In addition, the PoEFEMP2 mRNA level increased at 3 h post-infection (hpi) and decreased from 6 to 48 hpi in flounder Hirame natural embryo (HINAE) cells infected with viral hemorrhagic septicemia virus (VHSV). Disruption of PoEFEMP2 using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system resulted in a significant upregulation of VHSV G mRNA levels and immune-related genes expression in knockout cells. These findings implicate PoEFEMP2 in antiviral responses in P. olivaceus., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

3-phosphoinositide-dependent protein kinase-1 (PDK-1) is a key kinase regulating the activity of the PI3K/AKT pathway and a major regulator of the AGC protein kinase family. It is essential in the physiological activities of cells, embryonic development, individual development and immune response. In this study, we have identified for the first time an analogue of PDK-1 in the most primitive vertebrate, lamprey, and named it PDK-1-like. The protein sequence similarity of lamprey PDK-1-like to human, mouse, chicken, African xenopus and zebrafish PDK-1 were 64.4 %, 64.5 %, 65.0 %, 61.3 % and 63.2 %, respectively. The phylogenetic tree showed that PDK-1-like of lamprey were located at the base of the vertebrate branch, in line with the trend of biological evolution. Meanwhile, homology analysis showed that PDK-1 proteins across species shared a conserved kinase structural domain and a Pleckstrin Homology (PH) domain. Genomic synteny analysis revealed that the large-scale duplication blocks were not found in lamprey genome and neighbor genes of lamprey PDK-1-like presented dramatic differences compared with jawed vertebrates. More importantly, qPCR analysis showed that PDK-1-like was widely expressed in lamprey. Its mRNA expression levels varied in response to different pathogenic stimuli, and its expression was generally up-regulated under Polyinosinic-Polycytidylic acid (Poly(I:C)) stimulation. Pearson's correlation analysis showed that PDK-1-like was involved in co-expressed with MyD88-independent TLR-3 pathway during the immune response of lamprey, instead of MyD88-dependent TLR-3 pathway. In summary, our composite results offer valuable clues to the origin and evolution of PDK-1, and imply that PDK-1 s are among the most ancestral immune regulators in vertebrates., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Heme oxygenase-1 (HO-1), an inducible rate-limiting metabolic enzyme, exerts critical immunomodulatory functions by potential anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Although accumulative studies have focused on the immune functions of HO-1 in mammals, the roles in fish are poorly understood, and the reports on involvement in the defensive and immune response are very limited. In this study, On-HO-1 gene from Oreochromis niloticus was successfully cloned and identified, which contained an open reading frame (ORF) of 816 bp and coded for a protein of 271 amino acids. The On-HO-1 protein phylogenetically shared a high homology with HO-1 in other teleost fish (76.10%-98.89 %) and a lowly homology with HO-1 in mammals (38.98%-41.55 %). The expression levels of On-HO-1 were highest in the liver of healthy tilapias and sharply induced by Streptococcus agalactiae or Aeromonas hydrophila. Besides, On-HO-1 overexpression significantly increased non-specific immunological parameters in serum during bacterial infection, including LZM, SOD, CAT, ACP, and AKP. It also exerted anti-inflammatory and anti-apoptotic effects in response to the immune response of the infection with S. agalactiae or A. hydrophila by upregulating anti-inflammatory factors (IL-10, TGF-β), autophagy factors (ATG6, ATG8) and immune-related pathway factors (P65, P38), and down-regulating pro-inflammatory factors (IL-1β, IL-6, TNF-α), apoptotic factors (Caspase3, Caspase9), pyroptosis factor (Caspase1), and inflammasome (NLRP3). These results suggested that On-HO-1 involved in immunomodulatory functions and host defense in Nile tilapia., 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 a potential conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Type I interferons (IFN-I) play a pivotal role in vertebrate innate immunity against viruses. This study is an analysis of IFN-I genes in an updated version of the Atlantic salmon genome published in 2021 (version Ssal&#95;v3.1), revealing 47 IFN-I genes in the Atlantic salmon genome. The GH1 locus of chromosome (Chr) 3 harbors 9 IFNa genes, 5 IFNb genes, 6 IFNc genes, 11 IFNe genes and 1 IFNf gene. The GH2 locus on Chr6 contains 1 IFNa gene, 12 IFNc genes and 1 IFNf gene while Chr19 carries a single IFNd gene. Intraperitoneal injection of Atlantic salmon presmolts with poly I:C, a mimic of virus double-stranded RNA, significantly up-regulated IFNc genes from both Chr3 and Chr6 in heart, with lower expression in head kidney. IFNe expression increased in the heart, but not in the head kidney while IFNf was strongly up-regulated in both tissues. Antiviral activity of selected IFNs was assessed by transfection of salmon cells with IFN-expressing plasmids followed by infectious pancreatic necrosis virus infection, and by injection of fish with IFN-plasmids followed by measuring expression of the antiviral Mx1 gene. The results demonstrated that IFNc from both Chr3 and Chr6 provided full protection of cells against virus infection, whereas IFNe and IFNf showed lesser protection. IFNc from Chr3 and Chr6 along with IFNe and IFNf, up-regulated the Mx1 gene in the muscle, while only the IFNcs caused induction of Mx1 in liver. Overall, this study reveals that Atlantic salmon possesses an even more potent innate immune defense against viruses than previously understood., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Receptors of type I interferon (IFNR) play a vital role in the antiviral immune response. However, little is known about the negative regulatory role of the IFNR. Nervous necrosis virus (NNV) is one of the most significant viruses in cultured fish, resulting in great economic losses for the aquaculture industry. In this study, two orange-spotted grouper (Epinephelus coioides) cytokine receptor family B (CRFB) members, EcCRFB3 and EcCRFB4 were cloned and characterized from NNV infected grouper brain (GB) cells. The open reading frame (ORF) of EcCRFB3 consists of 852 bp encoding 283 amino acids, while EcCRFB4 has an ORF of 990 bp encoding 329 amino acids. The mRNA levels of EcCRFB3 or EcCRFB4 were significantly upregulated after NNV infection and the stimulation of poly (I:C) or NNV-encoded Protein A. In addition, EcCRFB3 or EcCRFB4 overexpression facilitated NNV replication, whereas EcCRFB3 or EcCRFB4 silencing resisted NNV replication. Overexpressed EcCRFB3 or EcCRFB4 inhibited the expression of IFN-I-induced ISGs. Taken together, our research provides the first evidence in fish demonstrating the role of IFNRs to regulate the IFN signaling pathway negatively. Our findings enrich the understanding of the functions of IFNRs and reveal a novel escape mechanism of NNV., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Innate immunity is vital for animal homeostasis and survival. First-line immuno-defense for fish larvae involves mucus enriched with leukolectin (LL) secreted by dermal lectocytes. Later during the critical transition from yolk-nutrition to feeding, additional larval immuno-protection in zebrafish (zF) is provided by macrophages containing LL (lectophages). This work investigated new LL-expression in embryos and in blood, structures of fish leukocytic LL and LL-genes, and LL-presence in chicken leukocytes. In zF-embryos, lectophages appear ∼10 hpf, while later, cells co-expressing myeloperoxidase- and LL-mRNA were detected (∼19 hpf). Furthermore, protein-extracts of Atlantic salmon (Ssal) leukocytes contained LL-proteins, compartmentalized in the cytosol. Cloning and sequencing revealed 94 % nt-sequence identity between variants of Ssal-leukolectins. Highly conserved LLs allowed production of epitope-specific anti-LL IgGs. Immuno-fluorescence-analysis demonstrated that most Ssal-bloodcells were LL-negative, but both some large cells with protrusions and some small, rounded cells did express LL. Immunoperoxidase-staining method confirmed LL-expression in some Ssal-leukocytes, identified as macrophages, PMN-leukocytes, thrombocytes and dendritic cells. However, closer examination revealed a dichotomy of these cell-categories into either LL-positive, or LL-negative variants. In situ hybridization demonstrated profuse LL-expression in Ssal head kidney interstitial tissue, while LL-transcripts were absent in large kidney tubules. Both hematopoietic (non-pigmented) marrow cells and melano-macrophages expressed LL-mRNA, implying that leukolectins provide lifelong innate immuno-protection. PCR-amplification using Ssal-leukocytic DNA as template, and direct sequencing yielded a leukocytic ll-gene. Some cells in salmon, cod, halibut, oikopleura and zebrafish embryos express LL-proteins and/or LL-mRNA, and LL-mRNA is detected in salmon, cod and chicken leukocytes. However, current genomes for these species lack recognizable LL-loci except the Ssal&#95;v3.1 Genome-assembly. The data demonstrate an unexpected dichotomy of some leukocyte lineages into LL-positive or LL-negative cell-variants. Such dichotomies suggest exploring differential impacts from the duplicated leukocyte-lineages in health and disease., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To achieve insights in antiviral immune defense of the central nervous system (CNS), we investigated T cells and CD45 cells in the marine fish model Dicentrarchus labrax infected with the CNS-tropic virus betanodavirus. By employing markers for pan-T cells (mAb DLT15) and CD45-cells (mAb DLT22) in immunofluorescence (IIF) of leukocytes from brain, we obtained 3,7 ± 2.3 % of T cells and 7.3 ± 3.2 % of CD45 + cells. Both IIF and immunoelectron microscopy confirmed a leukocyte/glial morphology for the immunoreactive cells. Quantitative immunohistochemistry (qIHC) of brain/eye sections showed 1.9 ± 0.8 % of T + cells and 2 ± 0.9 % of CD45 + cells in the brain, and 3.6 ± 1.9 % and 4.1 ± 2.2 % in the eye, respectively. After in vivo RGNNV infection the number of T cells/CD45 + leukocytes in the brain increased to 8.3 ± 2.1 % and 11.6 ± 4.4 % (by IIF), and 26.1 ± 3.4 % and 45.6 ± 5.9 % (by qIHC), respectively. In the eye we counted after infection 8.5 ± 4.4 % of T cells and 10.2 ± 5.8 % of CD45 cells. Gene transcription analysis of brain mRNA revealed a strong increase of gene transcripts coding for: antiviral proteins Mx and ISG-12; T-cell related CD3ε/δ, TcRβ, CD4, CD8α, CD45; and for immuno-modulatory cytokines TNFα, IL-2, IL-10. A RAG-1 gene product was also present and upregulated, suggesting somatic recombination in the fish brain. Similar transcription data were obtained in the eye, albeit with differences. Our findings provide first evidence for a recruitment and involvement of T cells and CD45 + leukocytes in the fish eye-brain axis during antiviral responses and suggest similarities in the CNS immune defense across evolutionary distant vertebrates., Competing Interests: Declaration of competing interest Authors declare there are no competing financial interests in relation to the work described., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The suppressor of cytokine signaling (SOCS) gene family is a group of genes involved in the negative regulation of cytokine signal transduction. The members of this family play a crucial role in regulating immune and inflammatory processes. However, comprehensive investigations of these genes have not yet been conducted in the economically significant fish large yellow croaker (Larimichthys crocea). In this study, a total of 13 SOCS genes (LcSOCS1a, LcSOCS1b, LcSOCS2, LcSOCS3a, LcSOCS3b, LcSOCS4, LcSOCS5a, LcSOCS5b, LcSOCS6, LcSOCS7a, LcSOCS7b, LcCISHa and LcCISHb) were identified and analyzed in L. crocea. The phylogenetic tree revealed a high conservation of SOCS genes in evolution, and the gene structure and motif analysis indicated a high similarity in the structure of LcSOCSs in the same subfamily. In addition, the expression patterns of LcSOCSs showed that LcSOCS1b was significantly down-regulated in all time under acute hypoxia stress, but it was markedly up-regulated throughout the entire process after P. plecoglossicida infection, revealing its different immune effects to two stresses. Besides, LcSOCS2a, LcSOCS6 and LcSOCS7a only participated in acute hypoxic stress, while LcSOCS5a was more sensitive to P. plecoglossicida infection. In summary, these results indicated that SOCS genes were involved in stress responses to both biological and non-biological stimuli, setting the foundation for deeper study on the functions of SOCS genes., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The scavenger receptors (SRs) gene family is considered as the membrane-associated pattern recognition receptors that plays important roles in the immune responses of organisms. However, there is currently limited research on the systematic identification of the SRs gene family in teleost and their role in the innate immunity of S. schegelii. In this study, we identified and annotated 15 SRs genes in S. schegelii. Through phylogenetic analysis, analysis of conserved domains, gene structure, and motif composition, we found that SRs gene family within different classes were relatively conserved. Additionally, we used qRT-PCR to analyze the expression patterns of SRs genes in immune-related tissues from healthy and Acinetobacter johnsonii-infected S. schegelii. The results showed that SRs genes exhibited different tissue expression patterns and the expression of SRs genes significantly changed after A. johnsonii infection. These results provided a valuable basis for further understanding of the functions of SRs in the innate immune response of S. schegelii., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Meningitis caused by Gram-negative bacteria is a serious public health problem, causing morbidity and mortality in both children and adults. Here, we propose a novel experimental model using Nile tilapia (Oreochromis niloticus) to study neuroinflammation. The fish were infected with Aeromonas hydrophila, and the course of infection was monitored in the peripheral blood. Septicemia was obvious in the blood, while in the brain tissue, infection of the meninges was present. The histopathological examination showed suppurative meningitis, and the cellular immune response in the brain tissue during infection was mediated by microglia. These cells were morphologically characterized and phenotyped by MHC class II markers and CD68. The increased production of TNF-α, IL-1β and iNOS supported the infiltration of these cells during the neuroinflammatory process. In the proteomic analysis of A. hydrophila isolated from brain tissue, we found chemotactic and transport proteins, proteolytic enzymes and enzymes associated with the dismutation of nitric oxide (NO), as well as motor proteins and those responsible for cell division. After characterizing the most abundant proteins during the course of infection, we investigated the druggability index of these proteins and identified promising peptide sequences as molecular targets that are similar among bacteria. Thus, these findings deepened the understanding of the pathophysiology of meningitis caused by A. hydrophila. Moreover, through the proteomics analysis, important mechanisms and pathways used by the pathogen to subvert the host response were revealed, providing insights for the development of novel antibiotics and vaccines., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that has caused significant economic losses to the grouper aquaculture industry. So far, the structure and function of SGIV proteins have been successively reported. In the present paper, the protein of SGIV VP146 was cloned and identified. VP146 was whole-cell distributed in GS cells. VP146 promoted SGIV replication and inhibited the transcription of interferon-related genes as well as pro-inflammatory cytokines in GS cells. In addition, VP146 was involved in the regulation of the cGAS-STING signaling pathway, and decreased cGAS-STING induced the promoter of ISRE and NF-κB. VP146 interacted with the proteins of cGAS, STING, TBK1, and IRF3 from grouper, but did not affect the binding of grouper STING to grouper TBK1 and grouper IRF3. Interestingly, grouper STING was able to affect the intracellular localization of VP146. Four segment structural domains of grouper STING were constructed, and grouper STING-CTT could affect the intracellular localization of VP146. VP146 had no effect on the self-binding of EcSITNG, nor on the binding of EcSTING to EcTBK1 and EcIRF3. Together, the results demonstrated that SGIV VP146 modulated the cGAS-STING signaling pathway to escape the interferon immune response., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Red-spotted grouper nervous necrosis virus (RGNNV) is a major viral pathogen of grouper and is able to antagonize interferon responses through multiple strategies, particularly evading host immune responses by inhibiting interferon responses. Ovarian tumor (OTU) family proteins are an important class of DUBs and the underlying mechanisms used to inhibit interferon pathway activation are unknown. In the present study, primers were designed based on the transcriptome data, and the ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) and OTUB2 genes of Epinephelus coioides (EcOTUB1 and EcOTUB2) were cloned and characterized. The homology alignment showed that both EcOTUB1 and EcOTUB2 were most closely related to E. lanceolatus with 98 % identity. Both EcOTUB1 and EcOTUB2 were distributed to varying degrees in grouper tissues, and the transcript levels were significantly up-regulated following RGNNV stimulation. Both EcOTUB1 and EcOTUB2 promoted replication of RGNNV in vitro, and inhibited the promoter activities of interferon stimulated response element (ISRE), nuclear transcription factors kappaB (NF-κB) and IFN3, and the expression levels of interferon related genes and proinflammatory factors. Co-immunoprecipitation experiments showed that both EcOTUB1 and EcOTUB2 could interact with TRAF3 and TRAF6, indicating that EcOTUB1 and EcOTUB2 may play important roles in interferon signaling pathway. The results will provide a theoretical reference for the development of novel disease prevention and control techniques., 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 reports in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to β-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 μg/mL or 100 μg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H 2 O 2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Interleukin-10 (IL-10) is an immunosuppressive cytokine, which plays a vital role in regulating inflammation for inhibiting the generation and function of pro-inflammatory cytokines in vivo or in vitro. In the present study, the full length cDNA of IL-10 was characterized from Nibea albiflora (named as NaIL-10) of 1238 base pairs (bp), containing a 5'-UTR (untranslated region) of 350 bp, a 3'-UTR of 333 bp and an open reading frame (ORF) of 555 bp (Fig. 1A) to encode 184 amino acid residues with a signal peptide at the N-terminus. The sequence analysis showed that NaIL-10 possessed the typical IL-10 family symbolic motif and conversed cysteine residues, similar to its teleost orthologues. Real-time PCR indicated that NaIL-10 had wide distribution in different healthy tissues, with a relatively high expression in immune-related tissues (head kidney, spleen, kidney, liver and gill). Significantly, up-regulations of NaIL-10 after infection against Vibrio parahaemolyticus, Vibrio alginolyticus and Poly I:C were also observed. Subcellular localization manifested that NaIL-10 mainly distributed in the cytoplasm unevenly and aggregately, and there was also a small amount on the cell membrane, indicating that NaIL-10 was secreted to the extracellular space as the known IL-10 homologous molecules. It could co-locate with IL-10 Rα on the membrane of HEK293T cells for their potential interaction, and GST pull-down and Co-IP studies certified the specific and direct interaction between NaIL-10 and NaIL-10 Rα, confirming that an IL-10 ligand-receptor system existed in N.albiflora. The expression of pro-inflammatory cytokines, including TNF-α, IL-6, IL-1β, were dramatically inhibited in LPS-stimulated RAW264.7 macrophages pre-incubated with recombinant NaIL-10 protein, demonstrating its anti-inflammatory roles. Taken together, the results demonstrated the existence of IL-10 ligand-receptor system in N.albiflora for the first time, and indicated the suppressive function of NaIL-10 on pro-inflammatory cytokine expression in inflammatory response, which would be conducive to better comprehending the role of IL-10 in the immunomodulatory mechanisms of teleost., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The complement component 5a/complement component 5 receptor 1 (C5a/C5aR1) pathway plays a crucial role in the onset and development of inflammation, but relevant studies in fish are lacking. In this study, we successfully characterized the relationship between half-smooth tongue sole (Cynoglossus semilaevis) C5aR1 (CsC5aR1) and bacterial inflammation. First, we showed that the overexpression of CsC5aR1 significantly increased bacterial pathological damage in the liver and intestine, whereas inhibition attenuated the damage. The in vitro experiments suggested that CsC5aR1 was able to positively regulate the phagocytic activity and respiratory burst of tongue sole macrophages. In terms of both transcriptional and translational levels, overexpression/inhibition of CsC5aR1 was followed by a highly consistent up-regulation/decrease of its downstream canonical inflammatory factor interleukin-6 (CsIL-6). Furthermore, we stimulated macrophages by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and found a broad-spectrum response to bacterial infections by the C5a/C5aR1 complement pathway together with the downstream inflammatory factor CsIL-6. Subsequently, we directly elucidated that CsIL-6 is an indicator of C5a/C5aR1-mediated inflammation at different infection concentrations, different infectious bacteria (Vibrio anguillarum and Mycobacterium marinum), and different detection levels. These results might provide a new inflammation bio-marker for early warning of bacteria-induced hyperinflammation leading to fish mortality and a promising target for the treatment of bacterial inflammation in teleost., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Leukocyte-derived chemotaxin-2 (LECT2) is a multifunctional immunoregulator that plays several pivotal roles in the host's defense against pathogens. This study aimed to elucidate the specific functions and mechanisms of LECT2 (CaLECT2) in the northern snakehead (Channa argus) during infections with pathogens such as Nocardia seriolae (N. seriolae). We identified CaLECT2 in the northern snakehead, demonstrating its participation in the immune response to N. seriolae infection. CaLECT2 contains an open reading frame (ORF) of 459 bp, encoding a peptide of 152 amino acids featuring a conserved peptidase M23 domain. The CaLECT2 protein shares 62%-84 % identities with proteins from various other fish species. Transcriptional expression analysis revealed that CaLECT2 was constitutively expressed in all examined tissues, with the highest expression observed in the liver. Following intraperitoneal infection with N. seriolae, CaLECT2 transcription increased in the spleen, trunk kidney, and liver. In vivo challenge experiments showed that injecting recombinant CaLECT2 (rCaLECT2) could protect the snakehead against N. seriolae infection by reducing bacterial load, enhancing serum antibacterial activity and antioxidant capacity, and minimizing tissue damage. Moreover, in vitro analysis indicated that rCaLECT2 significantly enhanced the migration, respiratory burst, and microbicidal activity of the head kidney-derived phagocytes. These findings provide new insights into the role of LECT2 in the antibacterial immunity of fish., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The grass carp (Ctenopharyngodon idella) constitutes a significant economic resource within the aquaculture sector of our nation, yet it has been chronically afflicted by the Grass Carp Reovirus (GCRV) disease. The complement system, a vital component of fish's innate immunity, plays a crucial role in combating viral infections. This research investigates the potential role of MASP1, a key molecule in the lectin pathway of the complement system, in the GCRV infection in grass carp. An analysis of the molecular characteristics of MASP1 in grass carp revealed that its identity and similarity percentages range from 35.10 to 91.00 % and 35.30-91.00 %, respectively, in comparison to other species. Phylogenetically, MASP1 in C. idella aligns closely with species such as Danio rerio, Cyprinus carpio, and Carassius carassius, exhibiting chromosomal collinearity with the zebrafish. Subsequent tissue analysis in both healthy and GCRV-infected grass carp indicated that MASP1's basal expression was predominantly in the liver. Post-GCRV infection, MASP1 expression in various tissues exhibited temporal variations: peaking in the liver on day 5, spleen on day 7, and kidney on day 14. Furthermore, employing Complement Component 3 (C3) as a benchmark for complement system activation, it was observed that MASP1 could activate and cleave C3 to C3b. MASP1 also demonstrated an inhibitory effect on GCRV replication (compared with the control group, VP2 and VP7 decreased by 6.82-fold and 4.37-fold) and enhanced the expression of antiviral genes, namely IRF3, IRF7 and IFN1 (compared with the control group, increased 2.25-fold, 45.38-fold and 22.37-fold, respectively). In vivo protein injection experiments substantiated MASP1's influence on the relative mRNA expression levels of C3 in various tissues and its protein expression in serum. This study also verified that C3 could modulate the expression of antiviral genes such as IFN1 and IRF3., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The goal of the present study was to establish a rapid, simple method for simultaneous allergy testing of sera from multiple fish-allergic patients. Sera from fish-allergic patients were pooled and used for capturing allergens in fish muscle of crucian carp ( Carassius auratus ), which was studied as a fish model. Sarcoplasmic proteins of crucian carp ( Carassius auratus ) were extracted for the analysis of allergens. Anti-human IgE antibody-functionalized magnetic beads were utilized to collect IgE antibodies from human pooled sera. The isolation of allergenic proteins was immunomagnetically performed in microfluidic channels, and the elution of the captured allergenic proteins was done with 5% (v/v) acetic acid aqueous solution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting were used for the analysis of tryptic digests of eluted proteins. Ten potential allergenic proteins were identified from crucian carp ( Carassius auratus ). The present protocol provides a rapid, efficient, and simple method for simultaneous detection of multiple allergens, based on multitargeted antibodies from pooled sera of allergic patients. The constructed multiple antibody-modified MBs can be applied for the deallergenicity of food matrices. The efficiency of allergen detection can be greatly improved, with promising application in allergen discovery and filtration for other muscle-based foods.

Nervous necrosis virus (NNV), an aquatic RNA virus belonging to Betanodavirus , infects a variety of marine and freshwater fishes, leading to massive mortality of cultured larvae and juveniles and substantial economic losses. The enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is widely recognized as a central component in the innate immune response to cytosolic DNA derived from different pathogens. However, little is known about the response of cGAS to aquatic RNA viruses. This study found that Epinephelus coioides cGAS (EccGAS) overexpression inhibited NNV replication, whereas EccGAS silencing promoted NNV replication. The anti-NNV activity of EccGAS was involved in interferon (IFN) signaling activation including tumor necrosis factor receptor-associated factor family member-associated NF-kappa-B activator-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor 3 (IRF3) nuclear translocation, and the subsequent induction of IFNc and ISGs. Interestingly, NNV employed its capsid protein (CP) or Protein A (ProA) to negatively or positively modulate EccGAS-mediated IFN signaling by simultaneously targeting EccGAS. CP interacted with EccGAS via the arm-P, S-P, and SD structural domains and promoted its polyubiquitination with K48 and K63 linkages in an EcUBE3C (the ubiquitin ligase)-dependent manner, ultimately leading to EccGAS degradation. Conversely, ProA bound to EccGAS and inhibited its ubiquitination and degradation. In regulating EccGAS protein content, CP's inhibitory action was more pronounced than ProA's protective effect, allowing successful NNV replication. These novel findings suggest that NNV CP and ProA dynamically modulate the EccGAS-mediated IFN signaling pathway to facilitate the immune escape of NNV. Our findings shed light on a novel mechanism of virus-host interaction and provide a theoretical basis for the prevention and control of NNV.IMPORTANCEAs a well-known DNA sensor, cGAS is a pivotal component in innate anti-viral immunity to anti-DNA viruses. Although there is growing evidence regarding the function of cGAS in the resistance to RNA viruses, the mechanisms by which cGAS participates in RNA virus-induced immune responses in fish and how aquatic viruses evade cGAS-mediated immune surveillance remain elusive. Here, we investigated the detailed mechanism by which EccGAS positively regulates the anti-NNV response. Furthermore, NNV CP and ProA interacted with EccGAS, regulating its protein levels through ubiquitin-proteasome pathways, to dynamically modulate the EccGAS-mediated IFN signaling pathway and facilitate viral evasion. Notably, NNV CP was identified to promote the ubiquitination of EccGAS via ubiquitin ligase EcUBE3C. These findings unveil a novel strategy for aquatic RNA viruses to evade cGAS-mediated innate immunity, enhancing our understanding of virus-host interactions., Competing Interests: The authors declare no conflict of interest.

Hsp40-Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein-protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida . dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40-Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.

Galectins (Gals) are a type of S-type lectin that are widespread and evolutionarily conserved among metazoans, and can act as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). In this study, 10 Gals ( ToGals ) were identified in the Golden pompano ( Trachinotus ovatus ), and their conserved domains, motifs, and collinearity relationships were analyzed. The expression of ToGals was regulated following infection to Cryptocaryon irritans and Streptococcus agalactiae , indicating that ToGals participate in immune responses against microbial pathogens. Further analysis was conducted on one important member, Galectin-3, subcellular localization showing that ToGal-3like protein is expressed both in the nucleus and cytoplasm. Recombinant protein obtained through prokaryotic expression showed that rToGal-3like can agglutinate red blood cells of rabbit, carp and golden pompano and also agglutinate and kill Staphylococcus aureus, Bacillus subtilis, Vibrio vulnificus, S. agalactiae, Pseudomonas aeruginosa , and Aeromonas hydrophila . This study lays the foundation for further research on the immune roles of Gals in teleosts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pan, Liang, Zhu, Guo, Liu, Zhang, Xian, Zhu and Zhang.)

E3 ubiquitin ligases, key components of the ubiquitin proteasome system, orchestrate protein degradation through ubiquitylation and profoundly impact cellular biology. Small HERC E3 ligases (HERC3-6) have diverse functions in mammals, including roles in spermatogenesis, protein degradation, and immunity. Until now, only mammals' HERC3, HERC5, and HERC6 are known to participate in immune responses, with major involvement in the antiviral response. Interestingly, an exclusive HERC7 has been characterized in fish showing great molecular conservation and antiviral roles. Thus, this study identifies and characterizes the herc7 gene in the European sea bass teleost. The European sea bass herc7 gene and the putative protein show good conservation of the promoter binding sites for interferons and the RCC1 and HECT domains characteristic of HERC proteins, respectively. The phylogenetic analysis shows a unique cluster with the fish-exclusive HERC7 orthologues. During ontogeny, the herc7 gene is expressed from 3 days post-fertilization onwards, being constitutively and widely distributed in adult tissues. In vitro, stimulated leucocytes up-regulate the herc7 gene in response to mitogens and viruses, pointing to a role in the immune response. Furthermore, sea bass herc7 expression is related to the interferon response intensity and viral load in different tissues upon in vivo infection with red-grouper betanodavirus (RGNNV), suggesting the potential involvement of fish HERC7 in ISGylation-based antiviral activity, similarly to mammalian HERC5. This study broadens the understanding of small HERC proteins in fish species and highlights HERC7 as a potential contributor to the immune response in European sea bass, with implications for antiviral defense mechanisms. Future research is needed to unravel the precise actions and functions of HERC7 in teleost fish immunity, providing insights into direct antiviral activity and viral evasion.