Galectin-8-like isoform X1 mediates antibacterial, antiviral, and antioxidant responses in red-lip mullet (Planiliza haematocheilus) through positive modulation of pro-inflammatory cytokine, chemokine, and enzymatic antioxidant activity.

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 ß-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 H2O2 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.

Molecular characterization, cytoprotective, DNA protective, and immunological assessment of peroxiredoxin-1 (Prdx1) from yellowtail clownfish (Amphiprion clarkii).

Peroxiredoxin-1 (Prdx1) is a thiol-specific antioxidant enzyme that detoxifies reactive oxygen species (ROS) and regulates the redox status of cells. In this study, the Prdx1 cDNA sequence was isolated from the pre-established Amphiprion clarkii (A. clarkii) (AcPrdx1) transcriptome database and characterized structurally and functionally. The AcPrdx1 coding sequence comprises 597 bp and encodes 198 amino acids with a molecular weight of 22.1 kDa and a predicted theoretical isoelectric point of 6.3. AcPrdx1 is localized and functionally available in the cytoplasm and nucleus of cells. The TXN domain of AcPrdx1 comprises two peroxiredoxin signature VCP motifs, which contain catalytic peroxidatic (Cp-C52) and resolving cysteine (CR-C173) residues. The constructed phylogenetic tree and sequence alignment revealed that AcPrdx1 is evolutionarily conserved, and its most closely related counterpart is Amphiprion ocellaris. Under normal physiological conditions, AcPrdx1 was ubiquitously detected in all tissues examined, with the most robust expression in the spleen. Furthermore, AcPrdx1 transcripts were significantly upregulated in the spleen, head kidney, and blood after immune stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and Vibrio harveyi injection. Recombinant AcPrdx1 (rAcPrdx1) demonstrated antioxidant and DNA protective properties in a concentration-dependent manner, as evidenced by insulin disulfide reduction, peroxidase activity, and metal-catalyzed oxidation (MCO) assays, whereas cells transfected with pcDNA3.1(+)/AcPrdx1 showed significant cytoprotective function under oxidative and nitrosative stress. Overexpression of AcPrdx1 in fathead minnow (FHM) cells led to a lower viral copy number following viral hemorrhagic septicemia virus (VHSV) infection, along with upregulation of several antiviral genes. Collectively, this study provides insights into the function of AcPrdx1 in defense against oxidative stressors and its role in the immune response against pathogenic infections in A. clarkii.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Highly stable and durable ZnIn2S4 nanosheets wrapped oxygen deficient blue TiO2(B) catalyst for selective CO2 photoreduction into CO and CH4.

Developing new and highly stable efficient photocatalysts is crucial for achieving high performance and selective photocatalytic CO2 conversion. In this paper, we designed a one-dimensional oxygen-deficient blue TiO2(B) (BT) catalyst for improved electron mobility and visible light accessibility. In addition, hexagonal ZnIn2S4 (ZIS) nanosheets with a low bandgap and great visible light accessibility are employed to produce effective heterostructures with BT. The synthesized materials are tested for photocatalytic conversion of CO2 into solar fuels (H2, CO and CH4). The optimized composite yields 71.6 and 10.3 µmol g-1h-1 of CO and CH4, three and ten times greater than ZIS, respectively. When ZIS nanosheets are combined with a one-dimensional oxygen-deficient BT catalyst, improved electron mobility and visible light accessibility are achieved, charge carriers are effectively segregated, and the transfer process is accelerated, resulting in efficient CO2 reduction. The photocatalytic CO2 conversion activity of the constructed BT/ZIS heterostructures is very stable over a 10-day (240-hour) period, and CO and CH4 production rates increase linearly with time; however, as time goes on, the rates of H2 production decrease. Further, a five-time recycling test confirmed this, revealing essentially equal activity and selectivity throughout the experiment. As a result, CO2 to CO and CH4 conversion has high selectivity and longer durability. The band structure of the BT/ZIS composite is determined using Mott-Schottky measurement, diffuse reflectance spectroscopy, and valence band X-ray photoelectron spectroscopy. This research demonstrates a novel approach to investigating effective, stable, and selective photocatalytic CO2 reduction systems for solar-to-chemical energy conversion.

Characterization and biocontrol efficacy of lytic phage (KPP-1) that infects multidrug resistant Klebsiella variicola.

Klebsiella variicola strain was identified from a natural water stream. Novel phage (KPP-1) infecting K. variicola was isolated and characterized. The biocontrol efficacy of KPP-1 against K. variicola-infected adult zebrafish was also investigated. The host K. variicola strain was resistant to six of the antibiotics tested and comprised the virulence genes kfuBC, fim, ureA, and Wza-Wzb-Wzccps. Morphological analysis by transmission electron microscopy revealed that KPP-1 has icosahedron head and tail structures. The latent period and burst size of KPP-1 were 20 min and 88 PFU per infected cell, respectively, at a multiplicity of infection of 0.1. KPP-1 was stable over a broad pH range (3-11), temperature (4-50 °C), and salinity (0.1-3%). KPP-1 inhibits the growth of K. variicola in vitro and in vivo. In the zebrafish infection model, treatment with KPP-1-infected K. variicola demonstrated 56% of cumulative survival. This suggests the possibility of developing KPP-1 as a potential biocontrol agent against multidrug-resistant K. variicola that belongs to the K. pneumoniae complex.

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.

CRISPR/Cas9-Induced Knockout of Sting Increases Susceptibility of Zebrafish to Bacterial Infection.

Stimulator of interferon genes (STING) is an adapter protein that is activated when cyclic dinucleotides (CDNs) are present. CDNs originate from the cytosolic DNA of both pathogens and hosts. STING activation promotes efficient immune responses against viral infections; however, its impact in bacterial infections is unclear. In this study, we investigated the role of Sting in bacterial infections by successfully creating a sting-deficient (sting(-/-) with a 4-bp deletion) knockout zebrafish model using CRISPR/Cas9. The transcriptional modulation of genes downstream of cGAS (cyclic GMP-AMP synthase)-Sting pathway-related genes was analyzed in seven-day-old wild-type (WT) and sting(-/-) embryos, as well as in four-day-old LPS-stimulated embryos. The expression of downstream genes was higher in sting(-/-) than in healthy WT fish. The late response was observed in sting(-/-) larvae following LPS treatment, demonstrating the importance of Sting-induced immunity during bacterial infection by activating the cGAS-STING pathway. Furthermore, adult sting(-/-) fish had a high mortality rate and significantly downregulated cGAS-STING pathway-related genes during Edwardsiella piscicida (E. piscicida) infection. In addition, we assessed NF-κB pathway genes following E. piscicida infection. Our results show fluctuating patterns of interleukin-6 (il6) and tumor necrosis factor-α (tnfα) expression, which is likely due to the influence of other NF-κB pathway-related immune genes. In summary, this study demonstrates the important role of Sting against bacterial infection.

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.

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.

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.

Genome Characterization of Bacteriophage KPP-1, a Novel Member in the Subfamily Vequintavirinae, and Use of Its Endolysin for the Lysis of Multidrug-Resistant Klebsiella variicola In Vitro.

Multidrug-resistant members of the Klebsiella pneumoniae complex have become a threat to human lives and animals, including aquatic animals, owing to the limited choice of antimicrobial treatments. Bacteriophages are effective natural tools available to fight against multidrug-resistant bacteria. The bacteriophage KPP-1 was found to be strictly lytic against K. variicola, a multidrug-resistant isolate, producing clear plaques. The genome sequence analysis of KPP-1 revealed that it comprised 143,369 base pairs with 47% overall GC content. A total of 272 genes (forward 161, complementary 111) encode for 17 tRNAs and 255 open reading frames (ORFs). Among them, 32 ORFs could be functionally annotated using the National Center for Biotechnology Information (NCBI) Protein Basic Local Alignment Search Tool (BLASTp) algorithm while 223 were found to code for hypothetical proteins. Comparative genomic analysis revealed that the closest neighbor of KPP-1 can be found in the genus Mydovirus of the subfamily Vequintavirinae. KPP-1 not only markedly suppressed the growth of the host but also worked synergistically with ampicillin. Useful genes for pathogen control such as endolysin (locus tag: KPP_11591) were found to have activity against multidrug-resistant isolate of K. variicola. Further studies are necessary to develop a strategy to control the emerging pathogen K. variicola using bacteriophages such as KPP-1.

Ortholinea nupchi n. sp. (Myxosporea: Ortholineidae) from the urinary bladder of the cultured olive flounder Paralichthys olivaceus, South Korea.

A new myxosporean parasite, Ortholinea nupchi n. sp. (Myxozoa; Bivalvulida), was isolated from the urinary bladder of the olive flounder Paralichthys olivaceus cultured on Jeju Island, Korea. Mature spores were subspherical in the valvular and apical views and ellipsoidal in the sutural view. The spores measured 7.6 ± 0.5 µm in length, 6.7 ± 0.3 µm in thickness, and 7.3 ± 0.5 µm in width. Two pyriform polar capsules measured 3.2 ± 0.1 µm in length and 2.7 ± 0.1 µm in width and were located at the same level at the anterior half of the myxospores. The suture line was straight in the middle of the spores, and the surface ridges ranged between five and seven, forming an intricate pattern. The result of the 18S rDNA comparison showed ≤ 93.0% similarity with other Ortholinea species. The phylogenetic tree demonstrated that O. nupchi n. sp. was closest to O. auratae and clustered with oligochaete-infecting myxosporeans (OIM) having urinary system infection tropism. Based on the comparison of environmental and host factors in the phylogenetic groups of the OIM clade, we propose that the infection of O. nupchi n. sp. originated from marine oligochaetes.