New insight into the biological activity of Salmo salar NK-lysin antimicrobial peptides.

NK-lysin is a potent antimicrobial peptide (AMP) with antimicrobial activity against bacteria, fungi, viruses, and parasites. NK-lysin is a type of granulysin, a member of the saposin-like proteins family first isolated from a pig's small intestine. In previous work, for the first time, we identified four variants of nk-lysin from Atlantic salmon (Salmo salar) using EST sequences. In the present study, we reported and characterized two additional transcripts of NK-lysin from S. salar. Besides, we evaluated the tissue distribution of three NK-lysins from S. salar and assessed the antimicrobial, hemolytic, and immunomodulatory activities and signaling pathways of three NK-lysin-derived peptides. The synthetic peptides displayed antimicrobial activity against Piscirickettsia salmonis (LF-89) and Flavobacterium psychrophilum. These peptides induced the expression of immune genes related to innate and adaptive immune responses in vitro and in vivo. The immunomodulatory activity of the peptides involves the mitogen-activated protein kinases-mediated signaling pathway, including p38, extracellular signal-regulated kinase 1/2, and/or c-Jun N-terminal kinases. Besides, the peptides modulated the immune response induced by pathogen-associated molecular patterns (PAMPs). Our findings show that NK-lysin could be a highly effective immunostimulant or vaccine adjuvant for use in fish aquaculture.

Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus).

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1ß, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila, the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection.

Participation of Hepcidins in the Inflammatory Response Triggered by λ-Carrageenin in Gilthead Seabream (Sparus aurata).

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.

Transcriptome Analysis of Host Anti-Vibrio harveyi Infection Revealed the Pathogenicity of V. harveyi to American Eel (Anguilla rostrata).

Vibrio harveyi, a recently discovered pathogenic bacterium isolated from American eels (Anguilla rostrata), poses uncertainties regarding its pathogenesis in American eel and the molecular mechanisms underlying host defense against V. harveyi infection. This study aimed to determine the LD50 of V. harveyi in American eel and assess the bacterial load in the liver, spleen, and kidney post-infection with the LD50 dose. The results showed that the LD50 of V. harveyi via intraperitoneal injection in American eels over a 14d period was determined to be 1.24 × 103 cfu/g body weight (6.2 × 104 cfu/fish). The peak bacterial load occurred at 36 h post-infection (hpi) in all three organs examined. Histopathology analysis revealed hepatic vein congestion and thrombi, tubular vacuolar degeneration, and splenic bleeding. Moreover, quantitative reverse transcription polymerase chain reaction (qRT-PCR) results indicated significant up or downregulation of 18 host immune- or anti-infection-related genes post 12 to 60 hpi following the infection. Additionally, RNA sequencing (RNA-seq) unveiled 7 hub differentially expressed genes (DEGs) and 11 encoded proteins play crucial roles in the anti-V. harveyi response in American eels. This study firstly represents the comprehensive report on the pathogenicity of V. harveyi to American eels and RNA-seq of host's response to V. harveyi infection. These findings provide valuable insights into V. harveyi pathogenesis and the strategies employed by the host's immune system at the transcriptomic level to combat V. harveyi infection.

A novel protein encoded by circVPS13D attenuates antiviral innate immunity by targeting MAVS in teleost fish.

IMPORTANCE: The expression of circVPS13D was upregulated with SCRV invasion, which proved that circVPS13D was involved in the regulation of the antiviral immune response. Our study revealed that the existence of circVPS13D promoted the replication of SCRV. Functionally, circVPS13D negatively regulates the antiviral responses of fish. Mechanistically, we confirmed that circVPS13D inhibited RLRs antiviral signaling pathway via the encoded protein VPS13D-170aa by targeting MAVS. Our study provided novel insights into the roles of protein-coding circRNAs and supported VPS13D-170aa as a negative regulator in the antiviral immune responses of teleost fish.

The transcriptome sequencing analysis reveals immune mechanisms of soybean fermented powder on the loach (Misgurnus anguillicaudatus) in response to Lipopolysaccharide (LPS) infection.

The loach (Misgurnus anguillicaudatus), a small commercial fish that is widely cultivated for its high-quality protein, vitamins, minerals, and essential amino acid, is a member of the genus Misgurnus and the family Cyprinidae. In this study, we gave the LPS-injected loach fermented soybean meal and used transcriptome sequencing to investigate the impact of the fermented soybean powder on the loach's immune system. 3384 up-regulated genes and 12116 down-regulated genes were found among the 15500 differentially expressed genes, according to the results. The differentially expressed genes were shown to be involved in cellular processes, metabolic processes, cellular anatomical entities, and binding, according to the Go functional annotation. Meanwhile, the KEGG enrichment analysis indicated that the soybean fermented powder treated groups showed significant differences in DNA replication, Nucleotide excision repair, Fanconi anemia pathway, and Base excision repair pathways, suggesting that these pathways are closely related to the enhancement of the immune function of loach by soybean fermented powder. The particular conclusions not exclusively can provide a new conception for the rational utilization of soybean fermented powder but also can provide theoretical guidance for the subsequent healthy breeding of loach.

A C-type lectin-like receptor CD302 in yellow drum (Nibea albiflora) functioning in antibacterial activity and innate immune signaling.

Molecular dissection of disease resistance against Vibrio harveyi infection in yellow drum at the genome-wide level uncovered a C-type lectin-like receptor cluster of differentiation CD302 (named as YdCD302) in our previous study. Here, the gene expression pattern of YdCD302 and its function in mediating the defense response to V. harveyi attack were investigated. Gene expression analysis demonstrated that YdCD302 was ubiquitously distributed in various tissues with the highest transcript abundance in liver. The YdCD302 protein exhibited agglutination and antibacterial activity against V. harveyi cells. Binding assay indicated that YdCD302 can physically interact with V. harveyi cells in a Ca2+-independent manner, and the interaction can activate reactive oxygen species (ROS) production in the bacterial cells to induce RecA/LexA-mediated cell death. After infection with V. harveyi, the expression of YdCD302 can be up-regulated significantly in the main immune organs of yellow drum and potentially further trigger the cytokines involved innate immunity. These findings provide insight into the genetic basis of the disease resistance trait in yellow drum and shed light on the functioning of the CD302 C-type lectin-like receptor in host-pathogen interactions. The molecular and functional characterization of YdCD302 is a significant step towards a better understanding of disease resistance mechanisms and the development of new strategies for disease control.

Zebrafish MAP2K7 Simultaneously Enhances Host IRF7 Stability and Degrades Spring Viremia of Carp Virus P Protein via Ubiquitination Pathway.

During viral infection, host defensive proteins either enhance the host immune response or antagonize viral components directly. In this study, we report on the following two mechanisms employed by zebrafish mitogen-activated protein kinase kinase 7 (MAP2K7) to protect the host during spring viremia of carp virus (SVCV) infection: stabilization of host IRF7 and degradation of SVCV P protein. In vivo, map2k7+/- (map2k7-/- is a lethal mutation) zebrafish showed a higher lethality, more pronounced tissue damage, and more viral proteins in major immune organs than the controls. At the cellular level, overexpression of map2k7 significantly enhanced host cell antiviral capacity, and viral replication and proliferation were significantly suppressed. Additionally, MAP2K7 interacted with the C terminus of IRF7 and stabilized IRF7 by increasing K63-linked polyubiquitination. On the other hand, during MAP2K7 overexpression, SVCV P proteins were significantly decreased. Further analysis demonstrated that SVCV P protein was degraded by the ubiquitin-proteasome pathway, as the attenuation of K63-linked polyubiquitination was mediated by MAP2K7. Furthermore, the deubiquitinase USP7 was indispensable in P protein degradation. These results confirm the dual functions of MAP2K7 during viral infection. IMPORTANCE Normally, during viral infection, host antiviral factors individually modulate the host immune response or antagonize viral components to defense infection. In the present study, we report that zebrafish MAP2K7 plays a crucial positive role in the host antiviral process. According to the weaker antiviral capacity of map2k7+/- zebrafish than that of the control, we find that MAP2K7 reduces host lethality through two pathways, as follows: enhancing K63-linked polyubiquitination to promote host IRF7 stability and attenuating K63-mediated polyubiquitination to degrade the SVCV P protein. These two mechanisms of MAP2K7 reveal a special antiviral response in lower vertebrates.

A Safe and Efficient Double-Gene-Deleted Live Attenuated Immersion Vaccine to Prevent the Disease Caused by the Infectious Spleen and Kidney Necrosis Virus.

Infectious diseases seriously threaten sustainable aquaculture development, resulting in more than $10 billion in economic losses annually. Immersion vaccines are emerging as the key technology for aquatic disease prevention and control. Here, a safe and efficacious candidate immersion vaccine strain (Δorf103r/tk) of infectious spleen and kidney necrosis virus (ISKNV), in which the orf103r and tk genes were knocked out by homologous recombination, is described. Δorf103r/tk was severely attenuated in mandarin fish (Siniperca chuatsi), inducing mild histological lesions, a mortality rate of only 3%, and eliminated within 21 days. A single Δorf103r/tk immersion-administered dose provided long-lasting protection rates over 95% against lethal ISKNV challenge. Δorf103r/tk also robustly stimulated the innate and adaptive immune responses. For example, interferon expression was significantly upregulated, and the production of specific neutralizing antibodies against ISKNV was markedly induced postimmunization. This work provides proof-of-principle evidence for orf103r- and tk-deficient ISKNV for immersion vaccine development to prevent ISKNV disease in aquaculture production. IMPORTANCE Global aquaculture production reached a record of 122.6 million tons in 2020, with a total value of 281.5 billion U.S. dollars (USD). However, approximately 10% of farmed aquatic animal production is lost due to various infectious diseases, resulting in more than 10 billion USD of economic waste every year. Therefore, the development of vaccines to prevent and control aquatic infectious diseases is of great significance. Infectious spleen and kidney necrosis virus (ISKNV) infection occurs in more than 50 species of freshwater and marine fish and has caused great economic losses to the mandarin fish farming industry in China during the past few decades. Thus, it is listed as a certifiable disease by the World Organization for Animal Health (OIE). Herein, a safe and efficient double-gene-deleted live attenuated immersion vaccine against ISKNV was developed, providing an example for the development of aquatic gene-deleted live attenuated immersion vaccine.

DDX23 of black carp negatively regulates MAVS-mediated antiviral signaling in innate immune activation.

Mammalian DDX23 is involved in multiple biological processes, such as RNA processing and antiviral responses. However, the function of teleost DDX23 still remains unclear. In this paper, we have cloned the DDX23 homologue of black carp (Mylopharyngodon piceus) (bcDDX23) and elucidated its role in the antiviral innate immunity. The coding region of bcDDX23 comprises 2427 nucleotides and encodes 809 amino acids. The transcription of bcDDX23 was promoted by the stimulation of LPS, poly(I:C), and SVCV; and immunoblotting (IB) assay showed that bcDDX23 migrated aground 94.5 kDa. Immunofluorescence (IF) assay revealed that bcDDX23 was mainly distributed in the nucleus, and the amount of cytosolic bcDDX23 was significantly increased after SVCV infection. The reporter assay showed that bcDDX23 inhibited bcMAVS-mediated transcription of the IFN promoter. And the co-immunoprecipitation (co-IP) assays identified the interaction between bcDDX23 and bcMAVS. Furthermore, co-expressed bcDDX23 significantly inhibited bcMAVS-mediated antiviral ability against SVCV in EPC cells, and knockdown of bcDDX23 enhanced the resistance of host cells against SVCV. Overall, our results conclude that bcDDX23 targets bcMAVS and suppresses MAVS-mediated IFN signaling, which sheds light on the regulation of IFN signaling in teleost fish.

Ring Finger Protein 34 Facilitates Nervous Necrosis Virus Evasion of Antiviral Innate Immunity by Targeting TBK1 and IRF3 for Ubiquitination and Degradation in Teleost Fish.

Ubiquitination, as one of the most prevalent posttranslational modifications of proteins, enables a tight control of host immune responses. Many viruses hijack the host ubiquitin system to regulate host antiviral responses for their survival. Here, we found that the fish pathogen nervous necrosis virus (NNV) recruited Lateolabrax japonicus E3 ubiquitin ligase ring finger protein 34 (LjRNF34) to inhibit the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via ubiquitinating Lateolabrax japonicus TANK-binding kinase 1 (LjTBK1) and interferon regulatory factor 3 (LjIRF3). Ectopic expression of LjRNF34 greatly enhanced NNV replication and prevented IFN production, while deficiency of LjRNF34 led to the opposite effect. Furthermore, LjRNF34 targeted LjTBK1 and LjIRF3 via its RING domain. Of note, the interactions between LjRNF34 and LjTBK1 or LjIRF3 were conserved in different cellular models derived from fish. Mechanically, LjRNF34 promoted K27- and K48-linked ubiquitination and degradation of LjTBK1 and LjIRF3, which in turn diminished LjTBK1-induced translocation of LjIRF3 from the cytoplasm to the nucleus. Ultimately, NNV capsid protein (CP) was found to bind with LjRNF34, CP induced LjTBK1 and LjIRF3 degradation, and IFN suppression depended on LjRNF34. Our finding demonstrates a novel mechanism by which NNV CP evaded host innate immunity via LjRNF34 and provides a potential drug target for the control of NNV infection. IMPORTANCE Ubiquitination plays an essential role in the regulation of innate immune responses to pathogens. NNV, a type of RNA virus, is the causal agent of a highly destructive disease in a variety of marine and freshwater fish. A previous study reported NNV could hijack the ubiquitin system to manipulate the host's immune responses; however, how NNV utilizes ubiquitination to facilitate its own replication is not well understood. Here, we identified a novel distinct role of E3 ubiquitin ligase LjRNF34 as an IFN antagonist to promote NNV infection. NNV capsid protein utilized LjRNF34 to target LjTBK1 and LjIRF3 for K27- and K48-linked ubiquitination and degradation. Importantly, the interactions between LjRNF34 and CP, LjTBK1, or LjIRF3 are conserved in different cellular models derived from fish, suggesting it is a general immune evasion strategy exploited by NNV to target the IFN response via RNF34.

Expression characteristics of non-virion protein of Hirame novirhabdovirus and its transfection induced response in hirame natural embryo cells.

The non-virion (NV) protein is the signature of genus Novirhabdovirus, which has been of considerable concern due to its potential role in viral pathogenicity. However, its expression characteristics and induced immune response remain limited. In the present work, it was demonstrated that Hirame novirhabdovirus (HIRRV) NV protein was only detected in the viral infected hirame natural embryo (HINAE) cells, but absent in the purified virions. Results showed that the transcription of NV gene could be stably detected in HIRRV-infected HINAE cells at 12 h post infection (hpi) and then reached the peak at 72 hpi. A similar expression trend of NV gene was also found in HIRRV-infected flounders. Subcellular localization analysis further exhibited that HIRRV-NV protein was predominantly localized in the cytoplasm. To elucidate the biological function of HIRRV-NV protein, NV eukaryotic plasmid was transfected into HINAE cells for RNA-seq. Compared to empty plasmid group, some key genes in RLR signaling pathway were significantly downregulated in NV-overexpressed HINAE cells, indicating that RLR signaling pathway was inhibited by HIRRV-NV protein. The interferon-associated genes were also significantly suppressed upon transfection of NV gene. This research would improve our understanding of expression characteristics and biological function of NV protein during HIRRV infection process.

Identification of B-Cell Epitopes on Capsid Protein Reveals Two Potential Neutralization Mechanisms in Red-Spotted Grouper Nervous Necrosis Virus.

Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP, 122GYVAGFL128 and 227SLYNDSL233. Site-directed mutational analysis confirmed residues Y123, G126, and L128 and residues L228, Y229, N230, D231, and L233 as the critical residues responsible for the direct interaction with ligand, respectively. According to homologous modeling and bioinformatic evaluation, 122GYVAGFL128 is harbored at the groove of the CP junction with strict conservation among all NNV isolates, while 227SLYNDSL233 is localized in proximity to the tip of a viral protrusion having relatively high evolutionary dynamics in different genotypes. Additionally, 227SLYNDSL233 was shown to be a receptor-binding site, since the corresponding polypeptide could moderately suppress RGNNV multiplication by impeding virion entry. In contrast, 122GYVAGFL128 seemed dedicated only to stabilizing viral native conformation and not to assisting initial virus attachment. Altogether, these findings contribute to a novel understanding of the antigenic distribution pattern of NNV and the molecular basis for neutralization, thus advancing the development of biomedical products, especially epitope-based vaccines, against NNV. IMPORTANCE NNV is a common etiological agent associated with neurological virosis in multiple aquatic organisms, causing significant hazards to the host. However, licensed drugs or vaccines to combat NNV infection are very limited to date. Toward the advancement of broad-spectrum prophylaxis and therapeutics against NNV, elucidating the diversity of immunodominant regions within it is undoubtedly essential. Here, we identified two independent B-cell epitopes on NNV CP, followed by the confirmation of critical amino acid residues participating in direct interaction. These two sites were distributed on the shell and protrusion domains of the virion, respectively, and mediated the neutralization exerted by MAbs via drastically distinct mechanisms. Our work promotes new insights into NNV antigenicity as well as neutralization and, more importantly, offers promising targets for the development of antiviral countermeasures.

Grass carp IL-20 binds to IL-20R2 but induces STAT3 phosphorylation via IL-20R1.

IL-20 is a pleiotropic cytokine that belongs to the IL-10 family and has a variety of biological functions in tissue homeostasis and regulation of host immune defenses. It signals through a heterodimeric receptor composed of a subunit with a long intracellular domain (R1 type receptor) and a subunit with a short intracellular domain (R2 type receptor). In this study, the R1 type receptor (CiIL-20R1/CRFB8) and the R2 type receptor (CiIL-20R2/CRFB16) were identified in grass carp Ctenopharyngodon idella. Expression analysis revealed that IL-20R2 was highly expressed in the gills and skin in healthy fish. Infection with Flavobacterium columnare resulted in the downregulation of both receptors in the gill at 48 and 72 h, whilst infection with grass carp reovirus induced their expression in the head kidney and spleen at 72 h. In the primary head kidney leucocytes, the expression levels of IL-20R1 and IL-20R2 were decreased after stimulation with 250 ng/mL IL-1ß but not affected by IFN-γ. Co-immunoprecipitation analysis showed that CiIL-20R2/CRFB16 but not CiIL-20R1/CRFB8 bound to CiIL-20L. Furthermore, it was shown that CiIL-20R1/CRFB8 was responsible for activating the phosphorylation of STAT3, whilst CiIL-20R2/CRFB16 was not involved. Structural modeling analysis showed that key residues involved in the interaction between IL-20 and receptors were highly conserved between grass carp and humans, suggesting that the signal transduction and functions of IL-20/IL-20R axis are evolutionarily conserved.

Expression and functional characterization of three ß-defensins in grass carp (Ctenopharyngodon idella).

ß-defensins (BDs) are a group of cysteine-rich cationic antimicrobial peptides and play important roles in the first line of defense against infection. In this study, the expression and antibacterial activities of three grass carp (Ctenopharyngodon idella) (Ci) ß-defensin (BD) peptides were comparatively investigated. Expression analysis reveals that CiBD1-3 were constitutively expressed in tissues, with the highest expression detected in the skin. The CiBD-1 transcripts were more abundant than CiBD-2 and CiBD-3. In the primary head kidney leukocytes, CiBDs were induced by PHA, LPS, poly(I:C) and cytokines such as IL-1ß and IFN-γ. In vivo challenge of fish with Aeromonas hydrophila resulted in the up-regulation of CiBDs in the head kidney and hindgut. To determine the biological activities, recombinant CiBD proteins were produced in the HEK293-F cells and purified for the minimum inhibitory concentration assay. It was found that all three recombinant CiBD proteins were effective to inhibit the growth of Gram-negative fish bacterial pathogens including Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare and Klebsiella pneumoniae and Gram-positive Staphylococcus aureus. CiBD-2 and CiBD-3 were more effective than CiBD-1. Our results demonstrate that all the three CiBDs have broad antibacterial activity against fish bacterial pathogens.

α-MSH is partially involved in the immunomodulation of Nile tilapia (Oreochromis niloticus) antibacterial immunity.

α-Melanocyte-stimulating hormone (α-MSH) is a well-studied neuropeptide controlling skin and hair color. Besides, numerous immunomodulation roles of α-MSH were recorded in humans and mice. However, the regulatory effects of α-MSH in teleost immunity haven't been well elucidated. In this study, several precursor molecules of α-MSH (POMCs) and its receptors (MCRs) in Nile tilapia (Oreochromis niloticus) were characterized, and their expression characteristics and specific functions on antibacterial immunity were determined. Overall, POMCs and MCRs were principally detected in the brain, skin, and liver, and were remarkably promoted post Streptococcus agalactiae infection. However, tiny POMCs and MCRs were observed in tilapia immune organs (head kidney and spleen) or lymphocytes, and no evident immunomodulation effect was detected in vitro. Moreover, the in vivo challenge experiments revealed that α-MSH protects tilapia from bacterial infection by regulating responses in the brain and intestine. This study lays theoretical data for a deeper comprehension of the immunomodulation mechanisms of teleost α-MSH and the evolutional process of the vertebrate melanocortin system.

Activated T cells are the cellular source of IL-22 that enhances proliferation and survival of lymphocytes in Nile tilapia.

As a pleiotropic cytokine mainly secreted by CD4+ T cells, interleukin (IL)-22 plays an important role in immune regulation and infection elimination. Despite IL-22 homologues have been identified in non-mammal, whether and how IL-22 participates in the adaptive immune response of early vertebrates have not been fully addressed. In this study, we identified an evolutionarily conserved IL-22 from Nile tilapia Oreochromis niloticus (defined as OnIL-22), proved by its properties regarding sequence, gene structure, functional domain, tertiary structure and phylogeny. IL-22 was broadly expressed in lymphoid-related tissues of tilapia, and with relatively higher levels in skin, gill, intestine and liver. The expression of OnIL-22 in spleen lymphocytes was markedly induced at the adaptive immune stage after Streptococcus agalactiae infection. Moreover, once lymphocytes were activated by PMA plus ionomycin or T-cell specific mitogen PHA in vitro, OnIL-22 expression was obviously up-regulated at both mRNA and protein levels. These results thus suggest that activated T cells produce IL-22 to take part in the adaptive immune response of tilapia. Furthermore, treatment of lymphocytes with recombinant OnIL-22 increased the expression of genes related to proliferation and survival, and further promoted the proliferation and reduced the apoptosis of lymphocytes during bacterial infection or T-cell activation. These cellular effects of IL-22 seem to be associated with JAK1/STAT3 axis downstream of IL-22, because IL-22 application not only elevated the mRNA expression of JAK1 and STAT3, but also enhanced their phosphorylation in lymphocytes. Altogether, we suggest that activated T cells produce IL-22 to promote lymphocyte proliferation and survival probability via JAK1/STAT3 signaling pathway, thus participating in adaptive immune response of Nile tilapia. Our study therefore provides helpful perspective for understanding the function and mechanism of adaptive immune system in teleost.

Fish-specific Toll-like receptor 14 (TLR14) from Asian swamp eel (Monopterus albus) is involved in immune response to bacterial infection.

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that play a critical role in innate immune responses against pathogens. In the present study, a fish-specific TLR14 was identified and characterized from Monopterus albus (named MaTLR14), which consisted of a 2658 bp open reading frame encoding a protein of 885 amino acids. Phylogenetic analysis revealed that MaTLR14 belong to the TLR1 subfamily and shared the highest similarity to Paralichthys olivaceus TLR14. Immunohistochemistry assay showed that MaTLR14 mainly located in intestinal epithelial cells of hindgut. Immunofluorescence revealed that MaTLR14 largely localized to the intracellular region and partially co-localized with cell membrane of HeLa cells. The expression levels of MaTLR14 were upregulated in the liver, spleen, foregut and hindgut post infection with Aeromonas hydrophila. When stimulated with LPS and Flagellin, the MaTLR14 expression was elevated in isolated peripheral blood leukocytes. Further studies showed that recombinant MaTLR14-LRR could bind to both the gram-negative and gram-positive bacteria and cause agglutination. Subsequently, the signaling pathway of MaTLR14 was investigated. Confocal microscopy and co-immunoprecipitation assay demonstrated that MaTLR14 recruited MyD88 as adaptor. When overexpressed, MaTLR14 augmented the expression of TRAF6 and phosphorylation of ERK and p65, activated NF-κB and AP-1 and elicited the expression of il-6 and tnf-α. Collectively, MaTLR14 plays an important role in the microorganism recognition and signaling transduction.

Don't Let It Get Under Your Skin! - Vaccination Protects the Skin Barrier of Common Carp From Disruption Caused by Cyprinid Herpesvirus 3.

Vaccination is the best form of protecting fish against viral diseases when the pathogen cannot be contained by biosecurity measures. Vaccines based on live attenuated viruses seem to be most effective for vaccination against challenging pathogens like Cyprinid herpesvirus 3. However, there are still knowledge gaps how these vaccines effectively protect fish from the deadly disease caused by the epitheliotropic CyHV-3, and which aspects of non-direct protection of skin or gill integrity and function are important in the aquatic environment. To elucidate some elements of protection, common carp were vaccinated against CyHV-3 using a double deletion vaccine virus KHV-T ΔDUT/TK in the absence or presence of a mix of common carp beta-defensins 1, 2 and 3 as adjuvants. Vaccination induced marginal clinical signs, low virus load and a minor upregulation of cd4, cd8 and igm gene expression in vaccinated fish, while neutralisation activity of blood serum rose from 14 days post vaccination (dpv). A challenge infection with CyHV-3 induced a severe disease with 80-100% mortality in non-vaccinated carp, while in vaccinated carp, no mortality was recorded and the virus load was >1,000-fold lower in the skin, gill and kidney. Histological analysis showed strongest pathological changes in the skin, with a complete destruction of the epidermis in non-vaccinated carp. In the skin of non-vaccinated fish, T and B cell responses were severely downregulated, inflammation and stress responses were increased upon challenge, whereas vaccinated fish had boosted neutrophil, T and B cell responses. A disruption of skin barrier elements (tight and adherence junction, desmosomes, mucins) led to an uncontrolled increase in skin bacteria load which most likely exacerbated the inflammation and the pathology. Using a live attenuated virus vaccine, we were able to show that increased neutrophil, T and B cell responses provide protection from CyHV-3 infection and lead to preservation of skin integrity, which supports successful protection against additional pathogens in the aquatic environment which foster disease development in non-vaccinated carp.