Lumpfish (Cyclopterus lumpus) Is Susceptible to Renibacterium salmoninarum Infection and Induces Cell-Mediated Immunity in the Chronic Stage.

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum, and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1ß, il8a, il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa, mxb, mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a, cd4b, ly6g6f, cd8a, cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response.

Secretome Profiling of Atlantic Salmon Head Kidney Leukocytes Highlights the Role of Phagocytes in the Immune Response to Soluble ß-Glucan.

ß-Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including macrophages and neutrophils through receptors such as dectin-1 and Complement receptor 3 (CR3), the BG are perceived by the innate immune system of vertebrates as foreign substances known as Pathogen Associated Molecular Patterns (PAMPs). The yeast-derived BG has been recognized for its potent biological activity and it is used as an immunomodulator in human and veterinary medicine. The goal of the current study was to characterize the immunostimulatory activity of soluble yeast BG in primary cultures of Atlantic salmon (Salmo salar) head kidney leukocytes (HKLs) in which phagocytic cell types including neutrophils and mononuclear phagocytes predominate. The effect of BG on the secretome of HKL cultures, including secretion of extracellular vesicles (EVs) and soluble protein55s was characterized through western blotting and mass spectrometry. The results demonstrate that, along with upregulation of proinflammatory genes, BG induces secretion of ubiquitinated proteins (UbP), MHCII-containing EVs from professional antigen presenting cells as well as proteins derived from granules of polymorphonuclear granulocytes (PMN). Among the most abundant proteins identified in BG-induced EVs were beta-2 integrin subunits, including CD18 and CD11 homologs, which highlights the role of salmon granulocytes and mononuclear phagocytes in the response to soluble BG. Overall, the current work advances the knowledge about the immunostimulatory activity of yeast BG on the salmon immune system by shedding light on the effect of this PAMP on the secretome of salmon leukocytes.

A Bioactive Extract Rich in Triterpenic Acid and Polyphenols from Olea europaea Promotes Systemic Immunity and Protects Atlantic Salmon Smolts Against Furunculosis.

In the present study, the modulation of the transcriptional immune response (microarray analysis) in the head kidney (HK) of the anadromous fish Atlantic salmon (Salmo salar) fed a diet supplemented with an olive fruit extract (AQUOLIVE®) was evaluated. At the end of the trial (133 days), in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed. A total number of 1,027 differentially expressed genes (DEGs) (805 up- and 222 downregulated) were found when comparing the transcriptomic profiling of the HK from fish fed the control and AQUOLIVE® diets. The HK transcripteractome revealed an expression profile that mainly favored biological processes related to immunity. Particularly, the signaling of i-kappa B kinase/NF-kappa and the activation of leukocytes, such as granulocytes and neutrophils degranulation, were suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the HK. Moreover, the bacterial challenge with A. salmonicida that lasted 12 days showed that the cumulative survival was higher in fish fed the AQUOLIVE® diet (96.9 ± 6.4%) than the control group (60.7 ± 13.5%). These results indicate that the dietary supplementation of AQUOLIVE® at the level of 0.15% enhanced the systemic immune response and reduced the A. salmonicida cumulative mortality in Atlantic salmon smolts.

The Expression of CD28 and Its Synergism on the Immune Response of Flounder (Paralichthys olivaceus) to Thymus-Dependent Antigen.

CD28 is well known as a critical T-cell costimulatory receptor involved in T cell activation by binding to its ligands. In this study, CD28 was cloned, and its expression profiles were characterized in flounder (Paralichthys olivaceus); variations of CD28+ cells after being stimulated with different types of antigens and the function of the CD28 costimulatory pathway on T-cell activation were investigated in vitro. fCD28 consists of four exons and three introns, and the full-length cDNA of fCD28 was 675-bp encoded 224 amino acids. The conserved motif (121TFPPPF126) binding to the CD80/86 ligand exists in the Ig-superfamily homology domain. The high expression of fCD28 is in gills, PBLs, head kidney, and spleen. CD28+ cells were co-localized with CD4+ T lymphocytes but not on IgM+ B lymphocyte cells. Moreover, the expression of CD28 was significantly varied in flounder after being stimulated by keyhole limpet hemocyanin (KLH) at both the transcriptional and cellular levels, while no significant differences were observed between lipopolysaccharide (LPS) stimulation and the control group. Notably, treatment of PBLs cultured in vitro with CD28 molecule-specific antibody (anti-CD28 Abs) and PHA produced more cell colonies and stimulated the proliferation of cultured leukocytes compared to PHA stimulation alone and the control group, and a higher level of IL-2 was detected in the culture medium. Meanwhile, anti-CD28 Abs increased the percent of CD28+ cells (10.41 ± 1.35%), CD4+ T lymphocytes (18.32 ± 2.15%), and CD28+/CD4+ double-positive cells (6.24 ± 1.52%). This effect also resulted in significant variations in the genes of cell membrane-bound molecules, cytokines, and related signaling pathways in cultured leukocytes, with significant changes in the genes of interleukin-2 (IL-2) and nuclear factor of activated T cells (NFAT) in the early stages of culture, and the expression of other molecules increased over time. These results proved the localization of the CD28 molecule on T lymphocytes in flounder, and anti-CD28 may act as the B7 ligand involved in T cell activation after antigen stimulation. These data provide a basis for a more in-depth study of the mechanism of the CD28 costimulatory pathway in T cell activation.

Pro-Inflammatory and B Cell Regulating Capacities of TWEAK in Rainbow Trout (Oncorhynchus mykiss).

Tumor necrosis factor (TNF)-like weak inducer of apoptosis or TWEAK is a member of the TNF superfamily involved in the regulation of many biological processes. In mammals, TWEAK has been shown to play a role in some autoimmune or inflammatory conditions, but its immune role is not yet clearly defined. In teleost fish, although a few studies have identified homologues to mammalian TWEAK, their biological effects have never been investigated. In the current study, we have studied the transcriptional regulation of two TWEAK homologues (TWEAK 1 and 2) identified in rainbow trout (Oncorhynchus mykiss) throughout different tissues, in response to parasitic or viral infections, or in head kidney (HK) leukocytes stimulated with different stimuli. Although the transcription of both homologues was modulated when HK leukocytes were exposed to several immune stimuli, only TWEAK 1 was significantly modulated upon pathogenic exposure. Thus, we performed a characterization of the functions exerted by this cytokine in HK leukocytes. Recombinant TWEAK 1 strongly up-regulated the transcription of pro-inflammatory genes and antimicrobial peptides in HK leukocytes, with differential transcriptional effects in IgM+ B cells, IgM- lymphocytes and myeloid cells. TWEAK 1 also increased the survival and promoted the differentiation of B cells in HK leukocyte cultures. Our results demonstrate that in teleost fish, TWEAK 1 is involved in the response to different types of pathogens, through the modulation of antimicrobial and pro-inflammatory genes in different leukocytes subsets. Furthermore, a role for TWEAK as a B cell differentiation factor has also been established in rainbow trout.

Efficacy of ulvan on immune response and immuno-antioxidant gene modulation in Labeo rohita against columnaris disease.

This study reports the effect of ulvan enriched diet on the influence of growth, changes in hemato-biochemical indices, improvement of antioxidant system, enhancement of innate-adaptive immunity and modification of immuno-antioxidant genes expression in Labeo rohita against Flavobacterium columnaris. The weight gain (WG) was significantly high (P > 0.05) in unchallenged normal and challenged fish fed with diets enriched with 25 and 50 mg kg-1 ulvan; the FCR was better (P > 0.05) when fed with 50 mg kg-1 enriched diet. In normal fish fed with or without ulvan supplementation was noted 100% survival rate (SR). In both groups, the red blood cell (RBC) and while blood cell (WBC) counts increased significantly (P > 0.05) when fed with 50 mg kg-1 ulvan diet whereas the hemoglobin (Hb) level increased significantly on being fed with 25 and 50 mg kg-1 ulvan diets. The SOD activity was enhanced significantly in both groups fed with any dose of ulvan diets whereas the MDA and GPx activity increased only with 25 and 50 mg kg-1 ulvan diets. The phagocytic (PC) activity significantly increased with any enriched diet and control diet groups while the respiratory burst (RB) activity increased only with 50 mg kg-1 ulvan diet. The alternate complement pathway (ACP), activity of lysozyme (Lyz), and immunoglobuline M (IgM) were better in both groups fed with 50 mg kg-1 ulvan diet. The SOD and GPx antioxidant gene expression were significantly high in both groups fed with any ulvan diet while the Nrf2 gene expression was high with 50 mg kg-1 ulvan diet. The IL-1ß, TNFα, hepcidin, Lyz, and IgM cytokines or proteins mRNA expression were significant in both groups fed with all ulvan supplement diet whereas the ß-2M expression was significant only with 50 mg kg-1 ulvan diet. The present research indicates that both L. rohita groups fed with 50 mg kg-1 ulvan diet significantly improved growth, antioxidant system, immune defense system, and immuno-antioxidant related gene expression against F. columnaris.

ß-glucan mimics tissue damage signaling and generates a trade-off between head kidney and spleen to activate acquired immunity in vaccinated tilapia (Oreochromis niloticus).

The association of vaccines with immunostimulants such as ß-glucan, promote the production of cytokines, competent immune cells and antibodies. However, differences between ß-glucan types and trials make it difficult to understand ß-glucan's mechanism of action. In this study, three trials were carried out with control and fish fed ß-glucan, the first trial occurred at 15 days; the second trial occurred at 30 days when we associated ß-glucan and vaccine; and the third trial occurred at 15 days post-challenge with Streptococcus agalactiae in tilapia (O. niloticus) in order to investigate immune-related gene expression in the head kidney and spleen using real-time qPCR. We found increases in HSP70, IL-6, IL-1ß, TNF-α, IL-10, Lys and C3 predominantly in the head kidney, except for IgM expression, which prevailed in the spleen, under vaccinated + ß-glucan action. This demonstrates the trade-off presented by the head kidney and spleen after immunostimulation in order to produce acquired immunity, as well as an increase in HSP70 expression in vaccinated + ß-glucan fish. The results suggest that ß-glucan stimulates the immune response through damage-associated molecular patterns (DAMPs) recognition. Therefore, these dynamics of the immune response promote a more robust defense against disease.

Characterisation and analysis of IFN-gamma producing cells in rainbow trout Oncorhynchus mykiss.

IFN-γ is one of the key cytokines involved in Th1 immune responses. It is produced mainly by T cells and NK cells, which drive both innate and adaptive responses to promote protection against infections. IFN-γ orthologues have been discovered to be functionally conserved in fish, suggesting that type I immunity is present in early vertebrates. However, few studies have looked at IFN-γ protein expression in fish and its role in cell mediated immunity due to a lack of relevant tools. In this study, four monoclonal antibodies (mAbs) V27, N2, VAB3 and V91 raised against short salmonid IFN-γ peptides were developed and characterised to monitor IFN-γ expression. The results show that the IFN-γ mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-γ protein and rainbow trout IFN-γ produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-γ at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3-5% IFN-γ secreting cells were detected in peripheral blood leucocytes (PBL), which increased significantly when stimulated in vitro with PAMPs (Aeromonas salmonicida bacterin), a mitogen (PHA) and recombinant cytokine (IL-2). Similarly, after injection of live bacteria (Aeromonas salmonicida) or poly I:C the number of IFN-γ+ cells increased in the lymphoid population of PBL, as well as in the myeloid population after infection, with the myeloid cells increasing substantially after both treatments. Immunohistochemistry was used to visualise the IFN-γ+ cells in spleen and head kidney following vaccination, which increased in intensity of staining and number relative to tissue from saline-injected control fish. These results show that several types of cells can produce IFN-γ in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-γ producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish.

Modulatory effects of laurel-leaf cistus (Cistus laurifolius) ethanolic extract on innate immune responses and disease resistance in common carp (Cyprinus carpio).

Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg-1 laurel-leaf cistus extract for 45 days. After 30 days, superoxide anion production (SAP) increased in CL0.1 and CL0.5 fish groups and at the end of the study all experimental fish groups had higher SAP compared to that of the control (P ˂ 0.05). Lysozyme activity (LA) was elevated in CL0.5 and CL1 treated groups on 30th day (P < 0.05), and this increase was only observed in C0.1 fish group at the end of study compared to control (P ˂ 0.05). Myeloperoxidase activity was significantly increased in CL0.5 and CL1 fish groups at the end of study. IL-1ßgene expression was significantly increased in treated fish in a dose-depended manner. Similar results were observed for transcription of IL-6 and IL-8 (P < 0.05). Anti-inflammatory cytokines, IL-10 and TGF-ß were highly up-regulated in the intestine and head kidney of CL treated fish groups compared to control (P < 0.05). At the end of experiment, significantly higher final body weight, weight gain, and specific growth rate were obtained in CL0.1 treated fish group compared to control. However, growth was negatively affected in CL1 fish group (P < 0.05). CL1 fish group had also a significantly higher FCR. Amylase activity was significantly increased in all experimental fish groups compared to control (P ˂ 0.05). Trypsin activity was decreased in CL0.1 and CL1 fish groups (P ˂ 0.05). WBC and RBC were significantly increased (P ˂ 0.05) in CL0.5 and CL1 fish groups, whereas haemoglobin, haematocrit, mean cell, mean cell haemoglobin contents were no significantly changed among control and treatment groups. Result of challenge test with A. hydrophila exhibited that survival rate in all treatment groups was significantly higher than that of control. These findings demonstrated that laurel-leaf cistus at 0.1 g kg-1 can be a suitable candidate for growth promotion, immune system induction and infection control in fish.

Dual-Organ Transcriptomic Analysis of Rainbow Trout Infected With Ichthyophthirius multifiliis Through Co-Expression and Machine Learning.

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective.

Expression and Function Analysis of Interleukin-17A/F1, 2, and 3 Genes in Yellow Catfish (Pelteobagrus fulvidraco): Distinct Bioactivity of Recombinant IL-17A/F1, 2, and 3.

In mammals, Interleukin-17 cytokine family plays critical roles in both acute and chronic inflammatory responses. In fish species, three Interleukin-17A/F (IL-17A/F) genes have been identified to be homologous to mammalian IL-17A and IL-17F, but little is known about their functional activity. In this study, Pf_IL-17A/F1, 2 and 3 genes were cloned from yellow catfish (Pelteobagrus fulvidraco) and they differed in protein structure and exon length, implying that they may have divergent bioactivity. Real-time quantitative PCR analyses revealed that three Pf_IL-17A/F genes were highly expressed in blood and mucosal tissues (skin+mucus and gill) from healthy adult fish. The mRNA expressions of Pf_IL-17A/F1, 2 and 3 genes were significantly up-regulated in the gill, skin+mucus, head kidney and spleen after challenge with Edwardsiella ictaluri and in the isolated peripheral blood leucocytes (PBLs) of yellow catfish after stimulation with phytohaemagglutinin (PHA), lipopolysaccharides (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (Poly I:C). These results indicate that Pf_IL-17A/F1, 2 and 3 genes may play a vital role in the regulation of immune against pathogens. Additionally, the recombinant (r) Pf_IL-17A/F1, 2 and 3 proteins significantly induced the mRNA expressions of proinflammatory cytokines, chemokines and antibacterial peptides genes, and the rPf_IL-17A/F 2 and 3 proteins promoted phagocytosis of PBLs more powerfully than the rPf_IL-17A/F1. Furthermore, the rPf_IL-17A/F1, 2 and 3 proteins might activate the NF-κB and MAPK signal pathways by IL-17RA, ACT1, TRAF6, TRAF2, TRAF5 and TAK1, indicating that the three Pf_IL-17A/F proteins may play different roles in promoting inflammatory response.

TLR22-mediated activation of TNF-α-caspase-1/IL-1ß inflammatory axis leads to apoptosis of Aeromonas hydrophila-infected macrophages.

Toll-like receptors (TLRs) represent first line of host defence against microbes. Amongst different TLRs, TLR22 is exclusively expressed in non-mammalian vertebrates, including fish. The precise role of TLR22 in fish-immunity remains abstruse. Herein, we used headkidney macrophages (HKM) from Clarias gariepinus and deciphered its role in fish-immunity. Highest tlr22 expression was observed in the immunocompetent organ - headkidney; nonetheless expression in other tissues suggests its possible involvement in non-immune sites also. Aeromonas hydrophila infection up-regulates tlr22 expression in HKM. Our RNAi based study suggested TLR22 restricts intracellular survival of A. hydrophila. Inhibitor and RNAi studies further implicated TLR22 induces pro-inflammatory cytokines TNF-α and IL-1ß. We observed heightened caspase-1 activity and our results suggest the role of TLR22 in activating TNF-α/caspase-1/IL-1ß cascade leading to caspase-3 mediated apoptosis of A. hydrophila-infected HKM. We conclude, TLR22 plays critical role in immune-surveillance and triggers pro-inflammatory cytokines leading to caspase mediated HKM apoptosis and pathogen clearance.

Identification and characterization of Trpm4 gene involved in regulating Japanese flounder (Paralichthys olivaceus) inflammatory response.

The transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed Ca2+ -impermeable cation channel involved in modulating inflammatory and immune responses in mammals. However, the role of TRPM4 channel in fish immunity remains unclear. In this report, from a comparative immunological point of view, we identified and characterized a Trpm4 gene from Japanese flounder (Paralichthys olivaceus) and analysed its potential role in regulating the fish inflammatory response. The Japanese flounder Trpm4 gene is expressed in a wide range of tissues and encodes a 1264-amino acid protein which expresses on the cell surface and shares several conserved domains with its mammalian counterparts. In vitro inflammatory challenge and in vivo bacterial infection experiments revealed that Japanese flounder Trpm4 expression was significantly modulated following different immune challenges, indicating the implication of Trpm4 in the fish immune response. Overexpression of TRPM4 significantly attenuated LPS- and poly(I:C)-induced pro-inflammatory cytokine expression in Japanese flounder FG-9307 cells. In contrast, pharmacological inhibition of the endogenous TRPM4 channel activity in Japanese flounder head kidney macrophages resulted in increased pro-inflammatory cytokine expression following LPS and poly(I:C) stimulations. Taken together, these findings indicate that TRPM4 channels may play a conserved role in regulating inflammatory response(s) in fish.

Molecular cloning and characterization of chemokine C-C motif ligand 34 (CCL34) genes from olive flounder (Paralichthys olivaceus).

Chemokines are a superfamily of chemotactic cytokines that regulate the migration and immune responses of leukocytes. Depending on the arrangement of the first two cysteine residues, chemokines are divided into four groups: CXC (α), CC (ß), C (γ), and CX3C (δ). Chemokine C-C motif ligand 34 (CCL34) is a member of the CC chemokine family and is known as a fish-specific CC chemokine. In this experiment, we analyzed the molecular cloning and characterization of the PoCCL34 gene in olive flounder (Paralichthys olivaceus), including CCL34a.3 (PoCCL34a.3) and CCL34b.3 (PoCCL34b.3). The amino acid sequence of PoCCL34 has four highly conserved cysteine residues and it has a C-C motif. Phylogenetic analysis revealed that PoCCL34 was phylogenetically clustered in the fish CCL34 subcluster. Recombinant PoCCL34 induced chemotaxis of head kidney leukocytes in a dose-dependent manner. Head kidney leukocytes stimulated with PoCCL34 also exhibited significant respiratory burst activity and increased expression of pro-inflammatory cytokines (IL-1ß, IL-6, and CXCL8), but the overall expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15) did not increase. Olive flounder injected with recombinant PoCCL34 demonstrated increased expression of pro-inflammatory cytokines (IL-1ß and IL-6) in the head kidney. However, there was no increase in the expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15). Additionally, recombinant PoCCL34 induced high lysozyme activity in the serum of the flounder. These results indicate that although PoCCL34 is not involved in the antiviral response, it may play a significant role in the overall immune response of the flounder, particularly in mediating the inflammatory response.

Molecular characterization and role in virus infection of Beclin-1 in large yellow croaker (Larimichthys crocea).

Beclin-1, the ortholog of yeast autophagy-related gene 6 (Atg6), has a central role in autophagy, which has been linked to diverse biological processes including immunity, development, tumor suppression, and lifespan extension. However, understanding of function of fish Beclin-1 is limited now. In this study, the complete Beclin-1 cDNA of large yellow croaker Larimichthys crocea (LcBeclin-1) was cloned, whose open reading frame (ORF) is 1344 bp long and encodes a protein of 447 amino acids (aa). The deduced LcBeclin-1 possesses a typical Bcl-2 homology domain 3(BH3) and an APG6 domain that contains a central coiled-coil domain (CCD, residues 174 to 231) and a C-terminal evolutionarily conserved domain (ECD, residues 241 to 334). LcBeclin-1 shared a high amino acid identity of 81.66-98.66% with reported Beclin-1 molecules from other vertebrate species. LcBeclin-1 gene was constitutively expressed in all tissues tested, with the highest levels in heart. LcBeclin-1 transcripts were also detected in primary head kidney granulocytes (PKGs), primary head kidney macrophages (PKMs), primary head kidney leukocytes (PKLs), and large yellow croaker head kidney cell line (LYCK), and were significantly upregulated by poly (I:C) in PKMs and LYCK cells. Subcellular localization showed that LcBeclin-1 was evenly distributed in the cytoplasm and nucleus of LYCK cells. Overexpression of LcBeclin-1 significantly increased the replication of SVCV, as evidenced by increased severity of the cytopathic effects, enhanced viral titre, and upregulated transcriptional levels of viral genes. Further studies showed that LcBeclin-1 induced the occurrence of autophagy in LYCK cells. Additionally, LcBeclin-1 also decreased the expression levels of large yellow croaker interferons (IFNs; IFNc, IFNd, and IFNh), interferon regulatory factor 3 (IRF3) and IRF7, IFN-stimulated genes (ISGs; Mx, PKR, and Viperin) in LYCK cells. All these data suggest that LcBeclin-1 promoted the viral replication possibly by inducing autophagy or negatively modulating IFN response, which will help us to further understand the function of fish Beclin-1.

Hot water extract of Pleurotus pulmonarius stalk waste enhances innate immune response and immune-related gene expression in red hybrid tilapia Oreochromis sp. following challenge with pathogen-associated molecular patterns.

In aquaculture, commercial fish such as red hybrid tilapia are usually raised at high density to boost the production within a short period of time. This overcrowded environment, however, may cause stress to the cultured fish and increase susceptibility to infectious diseases. Antibiotics and chemotherapeutics are used by fish farmers to overcome these challenges, but this may increase the production cost. Studies have reported on the potential of mushroom polysaccharides that can act as immunostimulants to enhance the immune response and disease resistance in fish. In the current study, hot water extract (HWE) from mushroom stalk waste (MSW) was used to formulate fish feed and hence administered to red hybrid tilapia to observe the activation of immune system. Upon 30 days of feeding, the fish were challenged with pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (poly (I:C)) to mimic bacterial and viral infection, respectively. HWE supplementation promoted better feed utilisation in red hybrid tilapia although it did not increase the body weight gain and specific growth rate compared to the control diet. The innate immunological parameters such as phagocytic activity and respiratory burst activity were significantly higher in HWE-supplemented group than that of the control group following PAMPs challenges. HWE-supplemented diet also resulted in higher mRNA transcription of il1b and tnfa in midgut, spleen and head kidney at 1-day post PAMPs injection. Tlr3 exhibited the highest upregulation in the HWE fed fish injected with poly (I:C). At 3-days post PAMPs injection, both ighm and tcrb expression were upregulated significantly in the spleen and head kidney. Results showed that HWE supplementation enhances the immune responses of red hybrid tilapia and induced a higher serum bactericidal activity against S. agalactiae.

Functional characterization of four TIR domain-containing adaptors, MyD88, TRIF, MAL, and SARM in mandarin fish Siniperca chuatsi.

Toll/interleukin-1 receptor (TIR) domain-containing adaptors, serve as pivotal signal transduction molecules in Toll-like receptor (TLR) signalling pathway to mediate downstream signalling cascades. In this study, four TIR-domain containing adaptors, MyD88, TRIF, MAL and SARM, were identified in mandarin fish Siniperca chuatsi, and they all contain TIR domains, of which MyD88 and SARM had high sequence homology with their vertebrate homologues. The expression analysis at mRNA level indicated that these genes were ubiquitously distributed in different tissues, being high in immune- and mucosa-related tissues such as head-kidney and intestine. The transcripts of these adaptor genes were up-regulated by poly(I:C) and LPS stimulation in isolated head-kidney lymphocytes (HKLs) of mandarin fish. Fluorescence microscopy revealed that all these molecules were localized in cytoplasm, and further investigations showed that the over-expression of MyD88, TRIF and MAL activated the NF-κB, ISRE or type Ι IFN promoters and inhibited SVCV replication, whereas their antiviral effects were significantly impaired when co-transfected with SARM. It was also confirmed by co-immunoprecipitation (Co-IP) that SARM interacts separately with MyD88, TRIF and MAL, and MAL interacts with MyD88. However, the regulatory mechanisms of these adaptors involved in signalling pathways of different TLRs should be of interest for further research.

Conservation of structural and interactional features of CD226 and Necl5 molecules from Nile tilapia (Oreochromis niloticus).

CD226 interacts with its ligand Necl5 as a costimulatory signal. In this study, we cloned a CD226 from Nile tilapia (Oreochromis niloticus, named OnCD226) and a Necl5 (named OnNecl5). The open reading frame of OnCD226 was 1071 bp, encoding a protein of 356 amino acids. Sequence alignment analysis indicated that OnCD226 contained two Ig-like domains in ectodomain. The open reading frame of OnNecl5 was 1155 bp, encoding a protein of 384 amino acids, and there are three lg-like domains in the extracellular domain. In healthy tilapia, OnCD226 was distributed in all tested tissues and relatively higher in the brain, while OnNecl5 was relatively higher in the skin. After Streptococcus agalactiae infection, OnCD226 has the same up-regulated expression pattern as OnNecl5 in different tissues. After HKLs stimulation with S. agalactiae and Poly I:C, respectively. OnCD226 was significantly up-regulated (0.01 < p < 0.05) at 12 h and extremely significant up-regulation was observed (p < 0.01) at 48 h and 96 h, the peak was observed at 96 h after stimulation by S. agalactiae. After stimulation by Poly I:C, OnCD226 expression was extremely significant (p < 0.01) at 72 h and 96 h, the peak was observed at 96 h. After stimulation by Keyhole limpet hemocyanin (KLH), a classical T cell-dependent antigen, the expression of OnCD226 was significantly up-regulated in blood, head kidney, spleen, and thymus. Moreover, when compared with the first challenge, the gene expression of OnCD226 which response to the second challenge was up-regulated earlier. Subcellular co-localization studies showed that OnCD226 and OnNecl5 were distributed mainly in the cytomembrane. Yeast two-hybrid results, indicated a strong interaction between OnCD226 and OnNecl5. These results suggested that OnCD226 plays an important role during pathogens infection, and the interaction between CD226 and Necl5 is conserved in Nile tilapia.

The evolutionary puzzle solution for the origins of the partial loss of the Cτ2 exon in notothenioid fishes.

Cryonotothenioidea is the main group of fishes that thrive in the extremely cold Antarctic environment, thanks to the acquisition of peculiar morphological, physiological and molecular adaptations. We have previously disclosed that IgM, the main immunoglobulin isotype in teleosts, display typical cold-adapted features. Recently, we have analyzed the gene encoding the heavy chain constant region (CH) of the IgT isotype from the Antarctic teleost Trematomus bernacchii (family Nototheniidae), characterized by the near-complete deletion of the CH2 domain. Here, we aimed to track the loss of the CH2 domain along notothenioid phylogeny and to identify its ancestral origins. To this end, we obtained the IgT gene sequences from several species belonging to the Antarctic families Nototheniidae, Bathydraconidae and Artedidraconidae. All species display a CH2 remnant of variable size, encoded by a short Cτ2 exon, which retains functional splicing sites and therefore is included in the mature transcript. We also considered representative species from the three non-Antarctic families: Eleginopsioidea (Eleginops maclovinus), Pseudaphritioidea (Pseudaphritis urvillii) and Bovichtidae (Bovichtus diacanthus and Cottoperca gobio). Even though only E. maclovinus, the sister taxa of Cryonotothenioidea, shared the partial loss of Cτ2, the other non-Antarctic notothenioid species displayed early molecular signatures of this event. These results shed light on the evolutionary path that underlies the origins of this remarkable gene structural modification.

CCL19 variants mediate chemotactic response via CCR7 in grass carp Ctenopharyngodon idella.

CC chemokine ligand 19 (CCL19) plays a key role in the regulation of immune responses including homeostasis, inflammation, and immune tolerance. In this study, two variants of CCL19 homologues (CCL19a2 and CCL19b) and CCR7 were investigated in grass carp Ctenopharyngodon idella. The three genes were widely expressed in immune tissues and could be modulated by stimulation with LPS, PHA and poly(I:C), and infection with Flavobacterium columnare and grass carp reovirus. In an in vitro chemotaxis assay, the recombinant CCL19a2 and CCL19b were active to promote the migration of HEK293 T cells expressing CCR7 and leucocytes isolated from the gills, head kidney and spleen. Moreover, their chemotactive effects were validated in vivo. We found that the cells recruited by CCL19a2 and CCl19b are mainly monocytes/macrophages expressing high levels of IL-1ß, IFN-γ, colony stimulating factor 1 receptor (CSF1R) and MHC II. Our work suggests that CCL19a2 and CCl19b are involved in recruitment of antigen presenting cells in fish.