CRISPR/Cas9-mediated knockout of tnf-α1 in zebrafish reduces disease resistance after Edwardsiella piscicida bacterial infection.

Tumor necrosis factor (TNF) is an important cytokine involved in immune responses to bacterial infections in vertebrates, including fish. Although Tnf-α is a well-studied cytokine, there are contradictory findings about Tnf-α function following bacterial infection. In this study, we analyzed the expression and function of the Tnf-α-type I isoform (Tnf-α1) in zebrafish by knockout experiments using the CRISPR/Cas9 gene-editing tool. The open reading frame of tnf-α1 encodes a 25.82 kDa protein with 234 amino acids (aa). The expression of tnf-α1 in the early stages of zebrafish was observed from the 2-cell stage. Adult zebrafish spleens showed the highest expression of tnf-α1. To evaluate the function of Tnf-α1, an 8 bp deletion in the target region, resulting in a short truncated protein of 55 aa, was used to create the tnf-α1 knockout mutant. The pattern of downstream gene expression in 7-day larvae in wild-type (WT) and tnf-α1 knockout fish was examined. We also verified the fish mortality rate after Edwardsiella piscicida challenge and found that it was much higher in tnf-α1 knockout fish than in WT fish. Additionally, downstream gene expression analyses after E. piscicida exposure revealed a distinct expression pattern in tnf-α1 knockout fish compared to that in WT fish. Overall, our study using tnf-α1 deletion in zebrafish confirmed that Tnf-α1 is critical for immune regulation during bacterial infection.

Amphiprion clarkii DDX41 modulates fish immune responses: Characterization by expression profiling, antiviral assay, and macrophage polarization analysis.

DDX41, a member of the DEAD-box helicase family, serves as a vital cytosolic DNA sensor and plays a pivotal role in controlling the activation of type I interferon responses in mammals. However, the functional aspects of fish DDX41 remain relatively unexplored. In this study, we identified and characterized the DDX41 gene in Amphiprion clarkii transcriptomes and designated the gene as AcDDX41. The complete open reading frame of AcDDX41 encoded a putative protein comprising 617 amino acids. Notably, the predicted AcDDX41 protein shared several structural features that are conserved in DDX41, including DEXDc, HELICc, and zinc finger domains, as well as conserved sequence "Asp-Glu-Ala-Asp (D-E-A-D)." AcDDX41 exhibited the highest sequence homology (99.68 % similarity) with DDX41 from Acanthochromis polyacanthus. Phylogenetic analysis revealed that DDX41s from fish formed a branch distinct from that in other animals. All investigated tissues were shown to express AcDDX41 constitutively, with blood showing the highest expression levels, followed by the brain. Furthermore, AcDDX41 expression was significantly induced upon stimulation with poly I:C, lipopolysaccharide, and Vibrio harveyi, indicating its responsiveness to immune stimuli. We confirmed the antiviral function of AcDDX41 by analyzing gene expression and viral replication during viral hemorrhagic septicemia virus infection. Additionally, using a luciferase reporter assay, we validated the ability of AcDDX41 to activate the NF-κB signaling pathway upon stimulation with poly I:C. Finally, AcDDX41 influenced cytokine gene expression and played a regulatory role in macrophage M1 polarization in RAW 264.7 cells. Collectively, these results highlight the significance of AcDDX41 as an immune-related gene that contributes substantially to antiviral defense and regulation of NF-κB activity.

Involvement of tumor necrosis factor receptor-associated factor 6 (TRAF6) in NF-κB activation and antiviral immunity: Molecular and functional characterization of TRAF6 in red-spotted grouper (Epinephelus akaara).

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a member of the TRAF family of adaptor proteins involved in the signal transduction pathways of both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. In this study, red-spotted grouper (Epinephelus akaara) TRAF6 (EaTraf6) was identified and characterized. The open reading frame of EaTraf6, 1713 bp in length, encodes a putative protein of 570 amino acids and has a predicted molecular weight and theoretical isoelectric point of 64.11 kDa and 6.07, respectively. EaTraf6 protein contains an N-terminal RING-type zinc finger domain, two TRAF-type zinc finger domains, a coiled-coil region (zf-TRAF), and a conserved C-terminal meprin and TRAF homology (MATH) domain. EaTraf6 shared the highest amino acid sequence identity with its ortholog from Epinephelus coioides, and phylogenetic analysis showed all fish TRAF6s clustered together and apart from other species. qRT-PCR results revealed that EaTraf6 was ubiquitously expressed in all examined tissues, with the highest level detected in the blood. In the immune challenge, EaTraf6 exhibited modulated mRNA expression levels in the blood and spleen. The subcellular localization analysis revealed that the EaTraf6 protein was predominantly present in the cytoplasm; however, it could translocate into the nucleus following poly (I:C) stimulation. The antiviral function of EaTraf6 was confirmed by analyzing the expression of host antiviral genes and viral genomic RNA during viral hemorrhagic septicemia virus infection. Additionally, luciferase reporter assay results indicated that EaTraf6 is involved in the activation of the NF-κB signaling pathway upon poly (I:C) stimulation. Finally, the effect of EaTraf6 on cytokine gene expression and its role in regulating macrophage M1 polarization were demonstrated. Collectively, these findings suggest that EaTraf6 is a crucial immune-related gene that significantly contributes to antiviral functions and regulation of NF-κB activity in the red-spotted grouper.

A metallothionein from disk abalone (Haliotis discus discus): Insights into its functional roles in immune response, metal tolerance, and oxidative stress.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential biological markers for assessing metal pollution in various biomonitoring programs. However, the functional properties of these proteins are yet to be comprehensively characterized in most marine invertebrates. In this study, we identified and characterized an MT homolog from the disk abalone (Haliotis discus discus), referred to as disk abalone MT (AbMT). AbMT exhibited the same primary structural features as MTs from other mollusks containing two ß-domains (ß2ß1-form). AbMT protein demonstrated metal-binding and detoxification abilities against Zn, Cu, and Cd, as evidenced by Escherichia coli growth kinetics, metal tolerance analysis, and UV absorption spectrum. Transcriptional analysis revealed that AbMT was ubiquitously expressed in all analyzed tissues and upregulated in gill tissue following challenge with Vibrio parahaemolyticus, Listeria monocytogenes, and viral hemorrhagic septicemia virus (VHSV). Additionally, overexpression of AbMT suppressed LPS-induced NO production in RAW264.7 macrophages, protected cells against H2O2-induced oxidative stress, and promoted macrophage polarization toward the M1 phase. Conclusively, these findings suggest an important role for AbMT in environmental stress protection and immune regulation in disk abalone.

A comprehensive study on the multifunctional properties of galectin-4 in red-lip mullet (Planiliza haematocheilus): Insights into molecular interactions, antimicrobial defense, and cell proliferation.

Galectin-4 belongs to the galactoside-binding protein family and is a type of tandem repeat galectin. Despite previous studies indicating its importance in fish immunology, a comprehensive investigation is necessary to fully understand its role in immunomodulatory functions and cellular dynamics. Therefore, this study aimed to explore the immunomodulatory functions of galectin-4 with a particular focus on its antimicrobial and cellular proliferative properties. The open reading frame of PhGal4 spans 1092 base pairs and encodes a soluble protein of 363 amino acids with a theoretical isoelectric point (IEP) of 6.39 and a molecular weight of 39.411 kDa. Spatial expression analysis under normal physiological conditions revealed ubiquitous expression of PhGal4 across all examined tissues, with the highest level observed in intestinal tissue. Upon stimulation with poly I:C, LPS, and L. garvieae, a significant increase (p < 0.05) in PhGal4 expression was observed in both blood and spleen tissues. Subsequent subcellular localization assay demonstrated that PhGal4 was predominantly localized in the cytoplasm. The recombinant PhGal4 (rPhGal4) exhibited specific binding capabilities to pathogen-associated molecular patterns (PAMPs), including LPS and peptidoglycan, but not poly I:C. The rPhGal4 negatively affected the bacterial growth kinetics. Additionally, rPhGal4 demonstrated complete hemagglutination of fish erythrocytes, which could be inhibited by the presence of D-galactose and α-lactose. The overexpression of PhGal4 in FHM epithelial cells demonstrated a significant suppression of viral replication during VHSV infection. Furthermore, the in vitro scratch assay and WST-1 assay demonstrated a wound healing effect of PhGal4 overexpression in FHM cells, potentially achieved through the promotion of cell proliferation by activating genes involved in cell cycle regulation. In conclusion, the responsive expression to immune stimuli, antimicrobial properties, and cell proliferation promotion of PhGal4 suggest that it plays a crucial role in immunomodulation and cellular dynamics of red-lip mullet. The findings in this study shed light on the multifunctional nature of galectin-4 in teleost fish.

Insights into the functional properties of thioredoxin domain-containing protein 12 (TXNDC12): Antioxidant activity, immunological expression, and wound-healing effect in yellowtail clownfish (Amphiprion clarkii).

Thioredoxin domain-containing protein 12 (TXNDC12) is a member of the thioredoxin-like superfamily that contributes to various thiol-dependent metabolic activities in all living organisms. In this research, the TXNDC12 gene from yellowtail clownfish (Amphiprion clarkii) was structurally characterized using in silico tools, assessed for immunological expression, and evaluated for biological activity using recombinant protein and cellular overexpression. The deduced coding sequence of AcTXNDC12 comprised a 522-bp nucleotide, encoding 173 amino acids with a predicted molecular mass of 19.198 kDa. The AcTXNDC12 protein consists of a66WCGAC70 active motif and a170GDEL173 signature. The highest tissue-specific expression of AcTXNDC12 was observed in the brain tissue, with significant modulation observed in the blood and gill tissues following stimulation of polyinosinic: polycytidylic acid, lipopolysaccharides (LPS), and Vibrio harveyi. In functional assays, recombinant AcTXNDC12 protein (rAcTXNDC12) showed insulin disulfide reduction activity, 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) decolorization antioxidant capacity, and ferric (Fe3+) reducing antioxidant potential. Additionally, a significant reduction in nitric oxide production was observed in AcTXNDC12-overexpressed RAW 264.7 cells upon LPS stimulation. Furthermore, genes associated with the regulation of oxidative stress, including nuclear factor erythroid 2-related factor 2 (Nrf2), catalase (Cat), peroxiredoxin 1 (Prx1), and ribonucleotide reductase catalytic subunit M1 (Rrm1) were significantly upregulated in fathead minnow cells overexpressing AcTXNDC12 in response to H2O2 treatment. The scratch wound healing assay demonstrated tissue regeneration and cell proliferation ability upon AcTXNDC12 overexpression. Altogether, the current study elucidated the antioxidant activity, immunological importance, and wound-healing effect of the AcTXNDC12 gene in yellowtail clownfish, providing valuable insights for advancing the aquaculture of A. clarkii fish.

Molecular features, antiviral activity, and immunological expression assessment of interferon-related developmental regulator 1 (IFRD1) in red-spotted grouper (Epinephelus akaara).

Interferon-related developmental regulator 1 (IFRD1) is a viral responsive gene associated with interferon-gamma. Herein, we identified the IFRD1 gene (EaIFRD1) from red-spotted grouper (Epinephelus akaara), evaluated its transcriptional responses, and investigated its functional features using various biological assays. EaIFRD1 encodes a protein comprising 428 amino acids with a molecular mass of 48.22 kDa. It features a substantial domain belonging to the interferon-related developmental regulator superfamily. Spatial mRNA expression of EaIFRD1 demonstrated the highest expression levels in the brain and the lowest in the skin. Furthermore, EaIFRD1 mRNA expression in grouper tissues exhibited significant modulation in response to immune stimulants, including poly (I:C), LPS, and nervous necrosis virus (NNV) infection. We analyzed downstream gene regulation by examining type Ⅰ interferon pathway genes following EaIFRD1 overexpression. The results demonstrated a significant upregulation in cells overexpressing EaIFRD1 compared to the control after infection with viral hemorrhagic septicemia virus (VHSV). A subcellular localization assay confirmed the nuclear location of the EaIFRD1 protein, consistent with its role as a transcriptional coactivator. Cells overexpressing EaIFRD1 exhibited increased migratory activity, enhancing wound-healing capabilities compared to the control. Additionally, under H2O2 exposure, EaIFRD1 overexpression protected cells against oxidative stress. Overexpression of EaIFRD1 also reduced poly (I:C)-mediated NO production in RAW267.4 macrophage cells. In FHM cells, EaIFRD1 overexpression significantly reduced VHSV virion replication. Collectively, these findings suggest that EaIFRD1 plays a crucial role in the antiviral immune response and immunological regulation in E. akaara.

Comparison of specificity and sensitivity of diagnostic methods for Enteromyxum leei and Enteromyxum fugu detected from cultured tiger puffer, Takifugu rubripes in Korea.

Enteromyxum leei and Enteromyxum fugu, which are myxosporean parasites, were first found in cultured tiger puffer Takifugu rubripes in Korea. We collected four tiger puffers that showed severe emaciation signs for our experiments. DNA sequencing was confirmed that the tiger puffers were coinfected with E. leei and E. fugu. Furthermore, similar amounts of E. leei and E. fugu were confirmed using real-time PCR in the intestine. To the best of our knowledge, there have been no reports of E. fugu infection in the olive flounder Paralichthys olivaceus. However, the diagnosis of inflowing water, discharged water and olive flounder samples using highly sensitive diagnostic methods confirmed the presence of E. fugu in water and fish samples from olive flounder farms near the tiger puffer farm. Therefore, the present study aimed to develop highly sensitive diagnostic methods such as real-time and two-step PCR for early diagnosis and follow-up of the emaciation disease and multiplex PCR for rapid diagnosis. The multiplex PCR method exhibited the same sensitivity as the one-step PCR method developed in this study, demonstrating its efficacy for rapid diagnosis. Therefore, the suggested methods can be utilized for the early diagnosis and rapid diagnosis of emaciation diseases and reduction of economic losses through rapid disease control.

CRISPR/Cas9-Mediated fech Knockout Zebrafish: Unraveling the Pathogenesis of Erythropoietic Protoporphyria and Facilitating Drug Screening.

Erythropoietic protoporphyria (EPP1) results in painful photosensitivity and severe liver damage in humans due to the accumulation of fluorescent protoporphyrin IX (PPIX). While zebrafish (Danio rerio) models for porphyria exist, the utility of ferrochelatase (fech) knockout zebrafish, which exhibit EPP, for therapeutic screening and biological studies remains unexplored. This study investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated fech-knockout zebrafish larvae as a model of EPP1 for drug screening. CRISPR/Cas9 was employed to generate fech-knockout zebrafish larvae exhibiting morphological defects without lethality prior to 9 days post-fertilization (dpf). To assess the suitability of this model for drug screening, ursodeoxycholic acid (UDCA), a common treatment for cholestatic liver disease, was employed. This treatment significantly reduced PPIX fluorescence and enhanced bile-secretion-related gene expression (abcb11a and abcc2), indicating the release of PPIX. Acridine orange staining and quantitative reverse transcription polymerase chain reaction analysis of the bax/bcl2 ratio revealed apoptosis in fech-/- larvae, and this was reduced by UDCA treatment, indicating suppression of the intrinsic apoptosis pathway. Neutral red and Sudan black staining revealed increased macrophage and neutrophil production, potentially in response to PPIX-induced cell damage. UDCA treatment effectively reduced macrophage and neutrophil production, suggesting its potential to alleviate cell damage and liver injury in EPP1. In conclusion, CRISPR/Cas9-mediated fech-/- zebrafish larvae represent a promising model for screening drugs against EPP1.

Formulation of chitosan microsphere-based oral vaccine against the scuticociliate parasite Miamiensis avidus: Evaluation of its protective immune potency in olive flounder (Paralichthys olivaceus).

Miamiensis avidus is a parasitic pathogen that causes scuticociliatosis, a severe and often lethal marine infection that affects marine fishes worldwide, including olive flounder (Paralichthys olivaceus) in Korea. This parasite infects all size groups of flounder year-round, causing recurring mortalities and huge economic losses to the Korean flounder industry each year. However, few efforts have been made to implement effective remedial measures to control this parasite. Therefore, our study sought to develop a chitosan microsphere (MS)-encapsulated inactivated vaccine (IMa + chitosan) for oral delivery (adsorbed in feed) to flounder fingerlings and assess its protective efficacy at different modalities via three in vivo experimental trials. Immunisation trial-1 was conducted to determine the effective concentration of chitosan. Our findings indicated that an IMa + chitosan 0.05 % vaccine formulation was safe and effective in providing moderate protection [46.67%-53.3 % relative percent survival (RPS)] against M. avidus intraperitoneal (IP) injection challenge at two weeks post-vaccination (wpv) compared to the IMa + chitosan 0.01 % and IMa + chitosan 0.005 % vaccines (0%-13.3 % RPS) irrespective of the antigen doses. In trial-2, the IMa + chitosan 0.05 % vaccine elicited similar protective immunity (30.8%-57.1 % RPS) in olive flounder against M. avidus at varying antigen doses (high: 2.38 × 106 cells/fish; low: 1.5 × 105 cells/fish), immunisation periods (2 and 5 wpv), and challenge modes (IP injection and immersion). Furthermore, experimental trial-3 validated the use of chitosan MS as an IMa antigen carrier to improve survivability (41.7 % RPS) in the host by significantly (p < 0.05) upregulating specific anti-M. avidus antibody titres in the fish sera and mucus of the group immunised with IMa-containing chitosan MS. In contrast, non-specific immunomodulatory effects (16.7 % RPS and enhanced mucosal antibody titres) were observed in the group treated with chitosan MS without IMa. Therefore, our findings suggested that oral administration of chitosan MS (0.05 %)-encapsulated IMa vaccine is a promising immunisation strategy against M. avidus that can protect the IMa antigen from digestive degradation, facilitates its targeted delivery to the host immune organs, and helps in orchestrating protective immune induction in olive flounder, thus controlling parasite infection.

Molecular characterization, immune responses, and functional aspects of atypical prototype galectin from redlip mullet (Liza haematocheila) as a pattern recognition receptor in host immune defense system.

Galectins are a family of lectins that are widely distributed ß-galactoside-binding proteins identified in diverse organisms. Galectin family have appeared as pattern recognition receptors (PRRs) responsible for initiating and controlling the innate immunity. The present study aimed to study the binding ability and potential role in PRRs of galectin-related protein B-like (LhGal B-like) from redlip mullet (Liza haematocheila) involved in the host immune responses. We constructed a cDNA library of redlip mullet and identified the LhGal B-like sequence. By sequence analysis and multiple sequence alignment, we revealed that LhGal B-like contains a conserved carbohydrate recognition domain (CRD) and consists of 135 amino acids with a predicted molecular weight of 16.07 kDa. In addition, pairwise comparison results showed that LhGal B-like shares higher sequence identity (82.2-95.2%) and similarity (89-95.9%) with fish species than those (34.1-37.8% and 57.2-58.1%, respectively) with other species. The phylogenetic tree showed that LhGal B-like clustered into the fish group and was evolutionally related to Mastacembelus armatus. The tissue distribution results revealed that LhGal B-like was expressed ubiquitously in all the tested tissues, where it was highly expressed in the brain, followed by gills and muscle. The immune modulated expression of LhGal B-like was observed by injecting lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). According to the results, in the gills, the mRNA expression of LhGal B-like was significantly upregulated upon LPS treatment after 48 h and upon poly I:C treatment after 48 and 72 h. In addition, the result showed significant upregulations upon LPS and poly I:C treatment after 24 h. However, significant downregulation was also shown in the earlier phase after injection of poly I:C and L. garvieae in gills. Further, the binding affinity of recombinant LhGal B-like (rLhGal B-like) was evaluated using carbohydrate, pathogen-associated molecular patterns (PAMP) and bacterial binding assays. The rLhGal B-like could bind all the examined carbohydrates but had a higher affinity to α-lactose. PAMPs and bacterial binding experiments verified a wide range of PAMP molecules and bacterial strains that rLhGal B-like could bind to. Moreover, we examined the agglutination activity of rLhGal B-like, and the result showed that it could aggregate all the gram-positive and gram-negative bacteria. Taken together, our findings reveal the functional aspects of LhGal B-like as a PRR and the potential involvement of LhGal B-like in the innate immunity of redlip mullet.

TRAF family member-associated NF-κB activator (TANK) regulates the antiviral function and NF-κB activation in red-spotted grouper (Epinephelus akaara).

The TRAF family member-associated nuclear factor kappa B (NF-κB) activator (TANK) regulates the NF-κB activation through the TRAF-mediated signaling pathway and is involved in the antiviral pathway by inducing the interferon (IFN) production. In the present study, we identified a TANK ortholog from the red-spotted grouper (Epinephelus akaara) and analyzed its immunological functions. The coding sequence of EaTANK consists of 1047 base pairs and encodes a 348 amino acids protein. The predicted molecular weight and theoretical isoelectric point (pI) were 38.92 kDa and 5.39, respectively. According to the phylogenetic analysis, EaTANK was closely clustered with fish TANK orthologs, exhibiting the highest identity (97.1 %) and similarity (97.1 %) to that of Epinephelus lanceolatus. A highly conserved TBK1/IKKi binding domain (TBD) was identified between 110 and 164 residues. Our tissue distribution analysis showed that EaTANK mRNA was ubiquitously expressed in 12 tested tissues, with the highest expression in the spleen and peripheral blood cells (PBCs). According to the immune challenge experiments, EaTANK mRNA expression in PBCs was significantly elevated following stimulation with polyinosinic:polycytidylic acid [poly (I:C)], lipopolysaccharide (LPS), or nervous necrosis virus (NNV). We also observed a significant elevation in the mRNA expression of downstream antiviral pathway-related genes (ISG15, IRF3, and IRF7) in EaTANK-overexpressing fathead minnow (FHM) cells against poly (I:C) stimulation. Moreover, the replication of 6 genes in the VHSV genome was inhibited by the overexpression of EaTANK. Finally, we confirmed that the expression of NFKB1 mRNA and promoter binding activity of NF-κB was significantly increased in poly (I:C)-stimulated EaTANK-overexpressing FHM cells. In conclusion, the results of this study suggest that TANK significantly contributes to the antiviral response and regulation of NF-κB activity in red-spotted grouper.

Expression profile and molecular function of beclin-1 in Epinephelus akaara in response to immune stimuli and oxidative stress.

Beclin-1, the mammalian ortholog of the yeast autophagy-related gene 6 (Atg 6), is a key regulator of autophagy. A variety of health and disease conditions in mammals are intricately related to the broad spectrum of beclin-1 functions. Nevertheless, few studies have investigated the role of beclin-1 in fish. In this study, we identified and cloned the beclin-1 cDNA (EaBECN-1) of Epinephelus akaara (red-spotted grouper) and carried out in silico analysis, tissue-specific expression analysis, immune challenge experiment, and in vitro analysis of its roles against viral infection and oxidative stress. The open reading frame was 1344 bp long and encoded 447 amino acids with a molecular weight of 51.2 kDa. Beclin-1 consisted of a conserved N-terminal BH3 and APG6 domains, and shared more than 88% identity with other vertebrates, according to a pairwise sequence alignment. EaBECN-1 expression profile analysis in E. akaara revealed that it is mostly expressed in the blood. Moreover, transcriptional modulation of EaBECN-1 was observed following stimulation with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly (I:C)), and nervous necrosis virus. During the viral hemorrhagic septicemia virus challenge, increased viral gene expression was observed at 12 h post-infection in FHM cells ectopically expressing EaBECN-1, and decreased thereafter at 24 h post-infection compared to control cells. However, increased antiviral gene expression at 12 and 24 h confirmed the antiviral function of EaBECN-1. Furthermore, EaBECN-1 overexpression protected the cells against H2O2-mediated apoptosis, as evidenced by the MTT assay, analysis of mRNA expression levels of apoptotic genes, and AO-EtBr staining. Overall, our study demonstrated the protective role of EaBECN-1 against viral pathogenesis and oxidative stress through autophagy, increasing our understanding of the role of beclin-1 in fish.

Ablation of myd88 alters the immune gene expression and immune cell recruitment during VHSV infection in zebrafish.

Myeloid differentiation primary response protein-88 (MYD88) is an essential adaptor molecule in pathogen-related pattern recognition signaling pathways. Toll-like and interleukin receptors recognize numerous signals and are funneled through MyD88 to express genes responsible for the innate and adaptive immune systems. In the present study, the relevance of MyD88 in viral hemorrhagic septicemia virus (VHSV) was investigated by generating myd88-/- zebrafish. The model was challenged with VHSV, and viral propagation was quantified by evaluating clinical symptoms, mortality, and VHSV copy number. The infected fish showed abnormal morphologies, such as subcutaneous hemorrhages, abdominal swelling, and bulging eyes, which were comparatively more intense in myd88-/- fish than in the wild-type. An injury infection experiment conducted in zebrafish larvae indicated a substantial spread of VHSV in the wound site. The number of neutrophils and macrophages recruited to the wounded area were markedly reduced in myd88-/- fish. According to gene expression analysis, VHSV NP gene expression was considerably upregulated in myd88-/- fish. Substantial gene expression and immune cell marker modulation were observed in the mutant model compared to that in the wild-type. These results suggest that the lack of a significant adaptor protein for immune signal transduction results in enhanced VHSV replication.

Identification, expression profiling, and functional characterization of cystatin C from big-belly seahorse (Hippocampus abdominalis).

Cystatins are natural inhibitors of lysosomal cysteine proteases, including cathepsins B, L, H, and S. Cystatin C (CSTC) is a member of the type 2 cystatin family and is an essential biomarker in the prognosis of several diseases. Emerging evidence suggests the immune regulatory roles of CSTC in antigen presentation, the release of different inflammatory mediators, and apoptosis in various pathophysiologies. In this study, the 390-bp cystatin C (HaCSTC) cDNA from big-belly seahorse (Hippocampus abdominalis) was cloned and characterized by screening the pre-established cDNA library. Based on similarities in sequence, HaCSTC is a homolog of the teleost type 2 cystatin family with putative catalytic cystatin domains, signal peptides, and disulfide bonds. HaCSTC transcripts were ubiquitously expressed in all tested big-belly seahorse tissues, with the highest expression in ovaries. Immune challenge with lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae caused significant upregulation in HaCSTC transcript levels. Using a pMAL-c5X expression vector, the 14.29-kDa protein of recombinant HaCSTC (rHaCSTC) was expressed in Escherichia coli BL21 (DE3), and its protease inhibitory activity against papain cysteine protease was determined with the aid of a protease substrate. Papain was competitively blocked by rHaCSTC in a dose-dependent manner. In response to viral hemorrhagic septicemia virus (VHSV) infection, HaCSTC overexpression strongly decreased the expression of VHSV transcripts, pro-inflammatory cytokines, and pro-apoptotic genes; while increasing the expression of anti-apoptotic genes in fathead minnow (FHM) cells. Furthermore, HaCSTC overexpression protected VHSV-infected FHM cells against VHSV-induced apoptosis and increased cell viability. Our findings imply the profound role of HaCSTC against pathogen infections by modulating fish immune responses.

Molecular characterization, immune expression, and functional delineation of peroxiredoxin 1 in Epinephelus akaara.

Peroxiredoxin 1 is a member of the typical 2-Cys peroxiredoxin family, which serves diverse functions in gene expression, immune and inflammatory responses, and tumor progression. In this study, we aimed to analyze the structural, functional, and immunomodulatory properties of peroxiredoxin 1 from Epinephelus akaara (EaPrx1). The open reading frame of EaPrx1 is 597 base pairs in length, encoding 198 amino acids, with a molecular weight of approximately 22 kDa. The in silico analysis revealed that EaPrx1 shares a conserved thioredoxin fold and signature motifs that are critical for its catalytic activity and oligomerization. Further, EaPrx1 is closely related to Epinephelus lanceolatus Prx1 and clustered in the Fishes group of the vertebrate clade, revealing that EaPrx1 was conserved throughout evolution. In terms of tissue distribution, a high level of EaPrx1 expression was observed in the spleen, brain, and blood tissues. Likewise, in immune challenge experiments, significant transcriptional modulations of EaPrx1 upon lipopolysaccharide, polyinosinic:polycytidylic acid, and nervous necrosis virus injections were noted at different time points, indicating the immunological role of EaPrx1 against pathogenic infections. In the functional analysis, rEaPrx1 exhibited substantial DNA protection, insulin disulfide reduction, and tissue repair activities, which were concentration-dependent. EaPrx1/pcDNA™ 3.1 (+)-transfected fathead minnow cells revealed high cell viability upon arsenic toxicity, indicating the heavy metal detoxification activity of EaPrx1. Taken together, the transcriptional and functional studies imply critical roles of EaPrx1 in innate immunity, redox regulation, apoptosis, and tissue-repair processes in E. akaara.

Thioredoxin domain-containing protein 12 (TXNDC12) in red spotted grouper (Epinephelus akaara): Molecular characteristics, disulfide reductase activities, and immune responses.

Thioredoxins are small ubiquitous redox proteins that are involved in many biological processes. Proteins with thiol-disulfide bonds are essential regulators of cellular redox homeostasis and diagnostic markers for redox-dependent diseases. Here, we identified and characterized the thioredoxin domain-containing protein 12 (EaTXNDC12) gene in red spotted grouper (Epinephelus akaara), evaluated transcriptional responses, and investigated the activity of the recombinant protein using functional assays. EaTXNDC12 is a 19.22-kDa endoplasmic reticulum (ER)-resident protein with a 522-bp open reading frame and 173 amino acids, including a signal peptide. We identified a conserved active motif (66WCGAC70) and ER retention motif (170GDEL173) in the EaTXNDC12 amino acid sequence. Relative EaTXNDC12 mRNA expression was analyzed using 12 different tissues, with the highest expression seen in brain tissue, while skin tissue showed the lowest expression level. Furthermore, mRNA expression in response to immune challenges was analyzed in the head kidney, blood, and gill tissues. EaTXNDC12 was significantly modulated in response to bacterial endotoxin lipopolysaccharide (LPS), nervous necrosis virus (NNV), and polyinosinic:polycytidylic acid (poly(I:C)) challenges in all of the tested tissues. Recombinant EaTXNDC12 (rEaTXNDC12) displayed antioxidant ability in an insulin reductase assay, and a capacity for free radical inhibition in a 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay. In addition, a DNA nicking assay revealed that purified rEaTXNDC12 exhibited concentration-dependent DNA protection activity, while results from 2-hydroxyethyl disulfide and L-dehydroascorbic assays indicated that rEaTXNDC12a possesses reducing ability. Furthermore, fathead minnow (FHM) cells transfected with EaTXNDC12-pcDNA demonstrated significantly upregulated cell survival against H2O2-induced apoptosis. Collectively, the results of this study strengthen our knowledge of EaTXNDC12 with respect to cellular redox hemostasis and immune regulation in Epinephelus akaara.

Molecular characterization and immune response of suppressor of cytokine signaling 5b from redlip mullet (Planiliza haematocheilus): Disclosing its anti-viral potential and effect on cell proliferation.

The suppressor of cytokine signaling (SOCS) proteins family comprising eight proteins (SOCS1-7 and cytokine-inducible SH2-containing (CIS)) are classical feedback inhibitors of cytokine signaling. Although the biological role of CIS and SOCS1-3 have been extensively studied, the biological functions of SOCS4-7 remain unclear. Here, we elucidated the molecular characteristics, expression profile, immune response, anti-viral potential, and effect on cell proliferation of Phsocs5b, a member of the SOCS protein family from redlip mullet (Planiliza haematocheilus); phsocs5b comprised 1695 nucleotides. It was 564 amino acids long with a molecular weight of 62.3 kDa and a theoretical isoelectric point of 8.95. Like SOCS4-7 proteins, Phsocs5b comprised an SH2 domain, SOCS box domain, and a long N-terminal. SH2 domain is highly identical to its orthologs in other vertebrates. Phsocs5b, highly expressed in the brain tissue, was localized in the cytoplasm. Temporal changes in phsocs5b expression were observed following immune stimulation with polyinosinic: polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. In FHM cells, Phsocs5b overexpression suppressed the viral hemorrhagic septicemia virus (VHSV) infection and epidermal growth factor receptor (egfr) expression but increased the mRNA levels of pi3k, akt, pro-inflammatory cytokines (il1ß and il8), and anti-viral genes (isg15 and ifn). Overall, our findings suggest that Phsocs5b attenuates VHSV infection, either by hindering the cell entry via degradation of Egfr, enhancing pro-inflammatory cytokines and anti-viral factor production, or both. The results also indicated that Phsocs5b could directly activate Pi3k/Akt pathway by itself, thus enhancing the proliferation and migration of cells. Taken together, Phsocs5b may be considered a potential therapeutic target to enhance immune responses while positively regulating the proliferation and migration of cells.

Galectin 9 restricts viral replication in teleost via autophagy-antiviral pathway and polarizes M2 macrophages for anti-inflammatory response: New insights into functional properties of fish Galectin-9 from Planiliza haematocheilus.

Galectin 9 (Gal9) is a tandem repeat type ß-galactoside-binding galectin that mediates various cellular biochemical and immunological functions. Many studies have investigated the functional properties of Gal9 in mammals; however, knowledge of fish Gal9 is limited to antibacterial studies. In this context, our aim was to clone Gal9 from Planiliza haematocheilus (PhGal9) and investigate its structural and functional characteristics. We discovered the PhGal9 open reading frame, which was 969 base pairs long and encoded a 322 amino acid protein. PhGal9 had a projected molecular weight of 35.385 kDa but no signal peptide sequence. PhGal9 mRNA was ubiquitously produced in all investigated tissues but was predominant in the intestine, spleen, and brain. Its mRNA expression was increased in response to stimulation by Poly(I:C), LPS, and L. garvieae. The rPhGal9 exhibited a dose-dependent agglutination potential toward gram-positive and gram-negative bacteria at a minimum concentration of 50 µg/mL. Overexpression of PhGal9 promoted M2-like phenotype changes in mouse macrophages, and RT-qPCR analysis of M1 and M2 marker genes confirmed M2 polarization with upregulation of M2 marker genes. In the antiviral assay, the expression levels of Viral Hemorrhagic Septicemia Virus (VHSV) glycoproteins, phosphoproteins, nucleoproteins, non-virion proteins, matrix proteins, and RNA polymerase were significantly reduced in PhGal9-overexpressed cells. Furthermore, the mRNA expression of autophagic genes (sqstm1, tax1bp1b, rnf13, lc3, and atg5) and antiviral genes (viperin) were upregulated in PhGal9 overexpressed cells. For the first time in teleosts, our study demonstrated that PhGal9 promotes M2 macrophage polarization by upregulating M2-associated genes (egr2 and cmyc) and suppressing M1-associated genes (iNOS and IL-6). Furthermore, our results show that exogenous and endogenous PhGal9 prevented VHSV attachment and replication by neutralizing virion and autophagy, respectively. Gal9 may be a potent modulator of the antimicrobial immune response in teleost fish.

Molecular features, antioxidant potential, and immunological expression assessment of thioredoxin-like protein 1 (TXNL1) in yellowtail clownfish (Amphiprion clarkii).

Thioredoxin-like protein 1 (TXNL1) is a redox-active protein belonging to the thioredoxin family, which mainly controls the redox status of cells. The TXNL1 gene from Amphiprion clarkii (AcTXNL1) was obtained from a pre-established transcriptome database. The AcTXNL1 is encoded with 289 amino acids and is predominantly localized in the cytoplasm and nucleus. The TXN domain of AcTXNL1 comprises a34CGPC37 motif with redox-reactive thiol (SH-) groups. The spatial distribution pattern of AcTXNL1 mRNA was examined in different tissues, and the muscle was identified as the highest expressed tissue. AcTXNL1 mRNA levels in the blood and gills were significantly increased in response to different immunostimulants. In vitro antioxidant capacity of the recombinant AcTXNL1 protein (rACTXNL1) was evaluated using the ABTS free radical-scavenging activity assay, cupric ion reducing antioxidant capacity assay, turbidimetric disulfide reduction assay, and DNA nicking protection assay. The potent antioxidant activity of rAcTXNL1 exhibited a concentration-dependent manner in all assays. Furthermore, in the cellular environment, overexpression of AcTXNL1 increased cell viability under H2O2 stress and reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). Collectively, the experimental results revealed that AcTXNL1 is an antioxidant and immunologically important gene in A. clarkii.