Manipulated C5aR1 over/down-expression associates with IL-6 expression during bacterial inflammation in half-smooth tongue sole (Cynoglossus semilaevis).

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.

The survival, gene expression, and DNA methylation of Paralichthys olivaceus impacted by the decay of green tide and bacterial infection in both laboratory and field simulation experiments.

The recurring appearance of Ulva prolifera green tides has become a pressing environmental issue, especially for marine transportation, tourism, and aquaculture in the stage of decomposition. An abundance of decaying U. prolifera leads to water acidification, hypoxia and pathogenic microorganism proliferation, threatening marine germplasm resources, particularly benthic organisms with weak escape ability. Epigenetic modification is considered to be one of the molecular mechanisms involved in the plastic adaptive response to environmental changes. However, few studies concerning the specific impact of decaying green tide on benthic animals at the epigenetic level. In this study, decomposing algal effluents of U. prolifera, sediments containing uncorrupted U. prolifera, pathogenic microorganism were considered as impact factors, to reveal the effect of decaying U. prolifera on marine economic benthic species, Paralichthys olivaceus, using both field and laboratory simulation experiments. Field simulation experiment showed higher mortality rates and serious histopathological damage than the laboratory simulation experiment. And both the decaying U. prolifera and the sediment containing U. prolifera were harmful to P. olivaceus. Genome-wide DNA methylation and transcription correlation analyses showed that the response of P. olivaceus to green tide stress and bacterial infection was mainly mediated by immune signaling pathways such as PI3K-Akt signaling pathway. DNA methylation regulates the expression of immune-related genes involved in the PI3K-Akt signaling pathway, which enables P. olivaceus to adapt to the adverse environmental stresses by resisting apoptosis. In summary, this research analyzed the potential role of P. olivaceus in decaying U. prolifera, which is of great significance for understanding the impact of decaying green tide on marine commercial fish and also provides some theoretical guidance for the proliferation and release of fish seedlings.

Inflammatory reaction and immune response of half-smooth tongue sole (Cynoglossus semilaevis) after infection with Vibrio anguillarum.

Frequently occurred bacterial diseases have seriously affected the aquaculture industry of half-smooth tongue sole (Cynoglossus semilaevis). Notably, vibriosis, with Vibrio anguillarum as one of the causative pathogens, is the most severe bacterial disease with severe inflammatory response of the host, leading to high mortality rates. In the present study, we explored the relationship between bacterial concentrations and host mortality, inflammatory reaction, and immune response in half-smooth tongue sole after infection with V. anguillarum at different concentrations (Treatment 1, 6.4 × 105 CFU/mL; Treatment 2, 6.4 × 106 CFU/mL). The mortality of Treatment 2 (77.5%) was significantly higher than that of Treatment 1 (10%), corresponding with bacterial concentrations. Although the number of deaths varies, intensive deaths were observed within 24 h post infection (hpi) in both bacterial concentration groups. Histopathological analyses revealed that fish tissues were most severely damaged at 24 or 48 hpi, and Treatment 2 was more severe than Treatment 1. A qRT-PCR-based detection method with virulence factor gene empA was established to quantify the bacterial loads in various tissues, and the bacterial loads were the highest at 24 hpi in Treatment 2, and at 48 hpi in Treatment 1. Additionally, the expression levels of complement genes (C5a, C3, C5, and C6), inflammatory factors (IL-1ß, TNF-α, and IL-10), and other immune-related genes (jak2, NF-κB1, stat3, and tlr3) were increased in various tissues after infection in both treatment groups, with most genes being most expressed at 24 or 48 hpi, and expression levels of inflammatory factors in Treatment 2 were higher than those in Treatment 1. Moreover, the expression of C5a was positively correlated with that of proinflammatory cytokines in both bacterial concentration groups. According to the results of this study, 24-48 hpi was a key node for early vibriosis detection and intervention. Compared with the low mortality of Treatment 1, the mass death of fish in Treatment 2 was suggested to be caused by uncontrolled excessive inflammatory reaction induced by the overactivation of complement system, especially C5a. We believe these results could provide theoretical basis for prevention, evaluation, and treatment of vibrio disease in tongue sole aquaculture, and lay a solid foundation for future functional analyses.

Metabolomic Investigation of Ultraviolet Ray-Inactivated White Spot Syndrome Virus-Induced Trained Immunity in Marsupenaeus japonicus.

Trained immunity is driven by metabolism and epigenetics in innate immune cells in mammals. The phenomenon of trained immunity has been identified in invertebrates, including shrimp, but the underlying mechanisms remain unclear. To elucidate mechanisms of trained immunity in shrimp, the metabolomic changes in hemolymph of Marsupenaeus japonicus trained by the UV-inactivated white spot syndrome virus (UV-WSSV) were analyzed using tandem gas chromatography-mass/mass spectrometry. The metabolomic profiles of shrimp trained with UV-WSSV followed WSSV infection showed significant differences comparison with the control groups, PBS injection followed WSSV infection. 16 differential metabolites in total of 154 metabolites were identified, including D-fructose-6-phosphate, D-glucose-6-phosphate, and D-fructose-6-phosphate, and metabolic pathways, glycolysis, pentose phosphate pathway, and AMPK signaling pathway were enriched in the UV-WSSV trained groups. Further study found that histone monomethylation and trimethylation at H3K4 (H3K4me1 and H3K4me3) were involved in the trained immunity. Our data suggest that the UV-WSSV induced trained immunity leads to metabolism reprogramming in the shrimp and provide insights for WSSV control in shrimp aquaculture.

Grouper PKR activation inhibits red-spotted grouper nervous necrosis virus (RGNNV) replication in infected cells.

The double-stranded RNA-activated protein kinase (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, PKR inhibits or promotes viral replication. In the present study, PKR homologues of orange-spotted grouper (Epinephelus coioides) (EcPKR) were cloned and the involvement of EcPKR during Red-spotted grouper nervous necrosis virus (RGNNV) infection was investigated. EcPKR encodes a 621-amino acid polypeptide that is closely related to the equivalent protein in Larimichthys crocea. EcPKR encoded two dsRNA binding domains and a Serine/Threonine protein kinase domain. Quantitative real-time PCR (qRT-PCR) analysis indicated that EcPKR was present in all examined tissues, with higher expression in spleen, intestine and gill. When stimulated with poly(I:C), the expression of EcPKR in the grouper spleen was increased, with highest expression 12 h post stimulation. EcPKR concentration was significantly increased in RGNNV-infected cells, with highest expression at 36 h post stimulation. EcPKR is mainly present in the cytoplasm. Overexpression of EcPKR in grouper spleen (GS) cells inhibits the transcription of the RGNNV genes. Furthermore, our results show that EcPKR overexpression significantly enhances the immune response of interferon and the activation of interferon-beta (IFN-ß), interferon stimulated response element (ISRE) and nuclear factor-kappa B (NF-κB). Taken together, these results are important for better understanding of the function of PKR in fish and reveal its involvement in host response to immune challenges in RGNNV.

HRI of Epinephelus coioides is a critical factor in the grouper immune response to RGNNV infection.

Phosphorylation of eukaryotic initiation factor 2 alpha subunit (eIF2α) occurs under a variety of conditions, including viral infection. Heme-regulated inhibitor (HRI) is an eIF2α kinase that modifies this phosphorylation. In this study, a HRI homologue (EcHRI) from the orange-spotted grouper (Epinephelus coioides) was cloned and its roles during fish viral infection were characterized. EcHRI encodes a 664-amino acid polypeptide that shares a high degree of similarity with HRIs from other species. Quantitative real-time polymerase chain reaction analysis indicated that EcHRI was distributed in all examined tissues. Expression of EcHRI in the spleen of E. coioides was up-regulated when challenged with the synthetic analog of double-stranded RNA (dsRNA) of polyinosine-polycytidylic acid (poly I:C). EcHRI was significantly increased in red-spotted grouper nervous necrosis virus (RGNNV) infected cells. EcHRI was abundantly distributed in the nucleus of grouper spleen (GS) cells. Overexpression of EcHRI inhibited the expression of red-spotted grouper nervous necrosis virus (RGNNV) genes in GS cells. Furthermore, our results showed that EcHRI overexpression significantly increased the expression of interferon (IFN)-related cytokines and enhanced activation of IFN-ß, interferon-sensitive response element (ISRE), and nuclear factor κB (NF-κB). Taken together, these results suggest that EcHRI is involved in the fish immune response to virus challenge.

Molecular cloning and characterization of Aos1 and Uba2 from the orange-spotted grouper (Epinephelus coioides).

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are essential regulators of a wide range of cellular processes. Covalent attachment of SUMO requires an enzymatic cascade consisting of a single E1-activating enzyme (Aos1 and Uba2 heterodimer), a single E2-conjugating enzyme (Ubc9), and one of several E3 ligases that facilitate transfer of SUMO from Ubc9 to the substrate. In the present study, the Aos1 and Uba2 homologues (EcAos1 and EcUba2) from the orange-spotted grouper (Epinephelus coioides) were cloned and their possible roles in fish immunity were analyzed. The open reading frame (ORF) of EcAos1 contains 1050 base pairs (bp) encoding a 350 amino acid protein with a predicted molecular mass of 38.97 kDa EcAos1 has a nuclear localization signal (NLS) at residues 193-203. The ORF of EcUba2 contains 1950 bp encoding a 650 amino acid protein with a predicted molecular mass of 71.3 kDa EcUba2 has a NLS at residues 608-630. Quantitative real-time polymerase chain reaction analysis indicated that both EcAos1 and EcUba2 were distributed in all examined tissues. The expression levels of EcAos1 and EcUba2 in the spleen and head kidney of E. coioides were differentially up-regulated when challenged with polyinosine-polycytidylic acid. Green fluorescence of both pEGFP-C1-EcAos1 and pEGFP-C1-EcUba2 was distributed in the nucleus of GS cells. When the NLSs of EcAos1 and EcUba2 were deleted, the cellular localizations all changed. Over-expression of EcAos1 and EcUba2 inhibited red-spotted grouper nervous necrosis virus infection and replication. These results are important for better understanding of the SUMO pathway in fish and provide insights into the regulatory mechanism of viral infection in E. coioides under farmed conditions.

Involvement of eIF2α of Epinephelus coioides in the fish immune response to virus infection.

The eukaryotic initiation factor 2 alpha subunit (eIF2α) is a key translation regulator that plays an important role in different cellular pressures and stimuli, including virus infection. In the present study, an eIF2α homolog (EceIF2α) from the orange-spotted grouper (Epinephelus coioides) was cloned and its roles during fish viral infection were characterized. EceIF2α encodes a putative protein of 315 amino acid residues, and shares a high degree of similarity with eIF2αs from other species. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that EceIF2α was distributed in all examined tissues. Both of the expression levels of EceIF2α in the spleen and head kidney of E. coioides were up-regulated when challenged with polyinosine-polycytidylic acid (poly[I:C]). EceIF2α was abundantly distributed in both the cytoplasm and nucleus in grouper spleen (GS) cells. Over-expression of EceIF2α improved the expression of red-spotted grouper nervous necrosis virus (RGNNV) genes in GS cells. In addition, EceIF2α depressed the activation of NK-κB and IFN-ß. Furthermore, dephosphorylation inhibitor treatment led to a significant decrease of RGNNV gene transcription. Taken together, these results suggest that EceIF2α might be involved in the fish immune response to virus challenge.

Molecular cloning and characterization of FADD from the orange-spotted grouper (Epinephelus coioides).

Fas-associated protein with death domain (FADD) is the key adaptor protein that transmits apoptotic signals mediated by the main death receptors. Besides being an essential instrument in cell death, FADD is also implicated in proliferation, cell cycle progression, tumor development, inflammation, innate immunity, and autophagy. In the present study, a FADD homologue (EcFADD) from the orange-spotted grouper (Epinephelus coioides) was cloned and its possible role in fish immunity was analyzed. The full length cDNA of EcFADD contains 808 base pairs (bp), including a 573 bp open reading frame that encodes a 190 amino acid protein with a predicted molecular mass of 21.81 kDa. Quantitative real-time polymerase chain reaction analysis indicated that EcFADD was distributed in all examined tissues. The expression of EcFADD in the spleen of E. coioides was differentially up-regulated when challenged with Singapore grouper iridovirus (SGIV) or polyinosine-polycytidylic acid(poly[I:C]). EcFADD was abundantly distributed in both the cytoplasm and nucleus in grouper spleen (GS) and fathead minnow (FHM) epithelial cells. Over-expression of EcFADD inhibited SGIV infection and replication and SGIV-induced apoptosis. To achieve antiviral and anti-apoptosis activities, FADD promoted the activation of interferon-stimulated response element (ISRE) and type I interferon (IFN) genes in the antiviral IFN signaling pathway and inhibited activation of apoptosis-related transcription factors p53. Our results not only characterize FADD but also reveal new immune functions and the molecular mechanisms by which FADD responds to virus infection and virus-induced apoptosis.

Expression and functional characterization of TRIF in orange-spotted grouper (Epinephelus coioides).

Antiviral immune responses are triggered by the innate immune recognition of viral infection. Toll/interleukin-1 receptor (TIR) domain containing adapter inducing interferon-ß (TRIF) is an adapter in responding to activation of Toll-like receptors, which provides early clearance of viral pathogens. Our study focuses on the functional characterization of grouper TRIF (EcTRIF) based on the comparison of its sequence and functional evolution from grouper fish to mammals. The results show that the open reading frame of EcTRIF encoded a protein of 580 amino acids. Real-time PCR analysis indicates that EcTRIF was constitutively expressed in all the analyzed tissues in healthy grouper. EcTRIF was significantly induced in spleen post-LPS and poly (I:C) stimulation. Fluorescence microscopy shows that EcTRIF is colocalized with a Golgi apparatus marker, implying its unique subcellular localization in the Golgi apparatus. Luciferase reporter assays confirmed that EcTRIF was able to activate the IFN and NF-κB promoter. Overexpression of EcTRIF in grouper brain cells inhibited the replication of red-spotted grouper nervous necrosis virus (RGNNV). These results indicate that EcTRIF plays an important role in modulating antiviral innate immune responses. Our results have applications in functional studies on TRIF in teleost fish and immune evolution.

TRAF6 is a critical factor in fish immune response to virus infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adaptor molecule in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. In our previous study, the molecular characteristics of EtTRAF6 (TRAF6 from Epinephelus tauvina), the tissue distributions, expression patterns after challenging with bacterial and viral pathogens were investigated. Here we identified EtTRAF6 as an important regulator of virus-triggered signaling pathway. Overexpression of EtTRAF6-ORF and truncated forms of EtTRAF6, including EtTRAF6-C (delete the MATH domain), EtTRAF6-N (delete the RING domain) and EtTRAF6-MATH, inhibited IFN-ß activity strongly in grouper spleen (GS) cells. Overexpression of EtTRAF6 repressed virus-induced production of type I IFNs. When EtTRAF6 cotransfected with EcIRF3 or EcIRF7, EtTRAF6 inhibited IRF-induced activation of IFN-ß. Over-expressed EtTRAF6 inhibited the transcription of SGIV genes significantly in GS cells. Although TRAF6 has a role in apoptosis regulation, it is not known if EtTRAF6 has any role in apoptosis regulation. Strikingly, when over-expressed in fathead minnow (FHM) cells, EtTRAF6 protected them from cell death induced by SGIV. Therefore, these results suggest that TRAF6 may play a critical role in their response to SGIV infection, through regulation of a cell death pathway that is common to fish and humans.