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Hepcidin belongs to a class of small cationic antimicrobial peptides rich in cysteine. It is synthesized by liver and is widely involved in host antimicrobial, antiviral and other immune responses. We identified and characterized three hepcidin genes (OpHep1, OpHep2 and OpHep3) in spotted knifejaw. All the OpHeps shared high identities with hepcidins in other teleost, containing alpha helix and β-sheets. Three OpHeps were all detected in healthy tissues, with the abundant expression in liver. They were significantly increased after Vibrio harveyi infection in the six immune-relevant tissues (liver, kidney, spleen, gill, skin and intestine). OpHeps knockdown in spotted knifejaw liver cells affected the mRNA levels of inflammation-related genes, including il1β, il6, il8, and nfκb. Further, the recombinant hepcidin proteins were effective in suppressing the growth of both Gram-negative and Gram-positive bacteria. To identify the function of OpHeps in vivo, we performed the overexpression of three OpHeps in zebrafish, and found OpHeps could significantly induce immune-related genes expression in transgenic zebrafish, including myd88, il10, il21, il16, tlr1, tlr3 and lysozyme. When infected with V. harveyi, OpHeps transgenic zebrafishes had a higher survival rate than wild-type zebrafishes. The expression of myd88, il10, il8, il1β, nfκb and lysozyme were all significantly up-regulated in transgenic fishes during bacterial infection. In summary, these results indicated that hepcidin could protect fish fight against pathogen through TLR/NFκB signaling cascade and Lysozyme. Three OpHeps would be potential targets for prevention of bacterial infections in aquaculture industry of spotted knifejaw, which provided a new idea for the molecular breeding of fish disease resistance., Competing Interests: Declaration of competing interest The authors read and approved this version of the final manuscript, confirmed the integrity of this work and declared no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

PHD1 is a member of the prolyl hydroxylase domain protein (PHD1-4) family, which plays a prominent role in the post-translational modification of its target proteins by hydroxylating proline residues. The best-characterized targets of PHD1 are hypoxia-inducible factor α (HIF-1α and HIF-2α), two master regulators of the hypoxia signaling pathway. In this study, we show that zebrafish phd1 positively regulates mavs-mediated antiviral innate immunity. Overexpression of phd1 enhances the cellular antiviral response. Consistently, zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. Further assays indicate that phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, zebrafish phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. However, the enzymatic activity of phd1 is not required for phd1 to activate mavs. In conclusion, this study reveals a novel function of phd1 in the regulation of antiviral innate immunity.IMPORTANCEPHD1 is a key regulator of the hypoxia signaling pathway, but its role in antiviral innate immunity is largely unknown. In this study, we found that zebrafish phd1 enhances cellular antiviral responses in a hydroxylation-independent manner. Phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. Zebrafish lacking phd1 are more susceptible to spring viremia of carp virus infection. These findings reveal a novel role for phd1 in the regulation of mavs-mediated antiviral innate immunity., Competing Interests: The authors declare no conflict of interest.

Zebrafish is a widely used model organism in genetics, developmental biology, pathology, and immunology research. Due to their fast reproduction, large numbers, transparent early embryos, and high genetic conservation with the human genome, zebrafish have been used as a model for studying human and fish viral diseases. In particular, the ability to easily perform forward and reverse genetics and lacking a functional adaptive immune response during the early period of development establish the zebrafish as a favored option to assess the functional implication of specific genes in the antiviral innate immune response and the pathogenesis of viral diseases. In this chapter, we detail protocols for the antiviral innate immunity analysis using the zebrafish model, including the generation of gene-overexpression zebrafish, generation of gene-knockout zebrafish by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, methods of viral infection in zebrafish larvae, analyzing the expression of antiviral genes in zebrafish larvae using qRT-PCR, Western blotting and transcriptome sequencing, and in vivo antiviral assays. These experimental protocols provide effective references for studying the antiviral immune response in the zebrafish model., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

IL-26 is a cytokine that is crucial for the maintenance and function of the gut mucosal barrier. IL-26 signaling pathway relies on a heterodimeric receptor complex, which is composed of two distinct subunits, IL-10R2 and IL-20R1. However, there are no reports on the antibacterial immunity of IL-26 and its receptors in fish. For this purpose, in this study we identified IL-26 and its receptors IL-10R2 and IL-20R1 in Carassius cuvieri × Carassius auratus red var. (named WR-IL-26, WR-IL10R2 and WR-IL20R1, respectively). Phylogenetic analysis confirmed the conservation of these genes, with shared structural motifs similar to those found in higher vertebrates. Upon exposure to Aeromonas hydrophila, a common fish pathogen, there was a significant upregulation of WR-IL-26, WR-IL10R2 and WR-IL20R1 in the gut, indicating a potential role in the immune response to infection. A co-immunoprecipitation assay revealed that WR-IL-26 formed complexes with WR-IL10R2 and WR-IL20R1. In vivo experiments demonstrated that administration of WR-IL-26 activated the JAK1-STAT3 signaling pathway and protected the gut mucosa barrier from A. hydrophila infection. Conversely, silencing WR-IL10R2 and WR-IL20R1 via RNA interference significantly attenuated the activation of WR-IL-26-mediated JAK1-STAT3 pathway. These results provided new insights into the role of IL-26 and its receptors in the gut mucosa barrier and could offer novel therapeutic strategies for managing bacterial infections in aquaculture., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Stimulator of IFN genes (STING) is a critical component of the innate immune system, playing an essential role in defending against DNA virus infections. However, the mechanisms governing basal STING regulation remain poorly understood. In this study, we demonstrate that the basal level of STING is critically maintained by hypoxia-inducible factor 1 (HIF-1)α through transcription. Under normal conditions, HIF-1α binds constitutively to the promoter region of STING, actively promoting its transcription. Knocking down HIF-1α results in a decrease in STING expression in multiple cell lines and zebrafish, which in turn reduces cellular responses to synthetic dsDNAs, including cell signaling and IFN production. Moreover, this decrease in STING levels leads to an increase in cellular susceptibility to DNA viruses HSV-1 and pseudorabies virus. These findings unveil a (to our knowledge) novel role of HIF-1α in maintaining basal STING levels and provide valuable insights into STING-mediated antiviral activities and associated diseases., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Innate immunity is vital for animal homeostasis and survival. First-line immuno-defense for fish larvae involves mucus enriched with leukolectin (LL) secreted by dermal lectocytes. Later during the critical transition from yolk-nutrition to feeding, additional larval immuno-protection in zebrafish (zF) is provided by macrophages containing LL (lectophages). This work investigated new LL-expression in embryos and in blood, structures of fish leukocytic LL and LL-genes, and LL-presence in chicken leukocytes. In zF-embryos, lectophages appear ∼10 hpf, while later, cells co-expressing myeloperoxidase- and LL-mRNA were detected (∼19 hpf). Furthermore, protein-extracts of Atlantic salmon (Ssal) leukocytes contained LL-proteins, compartmentalized in the cytosol. Cloning and sequencing revealed 94 % nt-sequence identity between variants of Ssal-leukolectins. Highly conserved LLs allowed production of epitope-specific anti-LL IgGs. Immuno-fluorescence-analysis demonstrated that most Ssal-bloodcells were LL-negative, but both some large cells with protrusions and some small, rounded cells did express LL. Immunoperoxidase-staining method confirmed LL-expression in some Ssal-leukocytes, identified as macrophages, PMN-leukocytes, thrombocytes and dendritic cells. However, closer examination revealed a dichotomy of these cell-categories into either LL-positive, or LL-negative variants. In situ hybridization demonstrated profuse LL-expression in Ssal head kidney interstitial tissue, while LL-transcripts were absent in large kidney tubules. Both hematopoietic (non-pigmented) marrow cells and melano-macrophages expressed LL-mRNA, implying that leukolectins provide lifelong innate immuno-protection. PCR-amplification using Ssal-leukocytic DNA as template, and direct sequencing yielded a leukocytic ll-gene. Some cells in salmon, cod, halibut, oikopleura and zebrafish embryos express LL-proteins and/or LL-mRNA, and LL-mRNA is detected in salmon, cod and chicken leukocytes. However, current genomes for these species lack recognizable LL-loci except the Ssal_v3.1 Genome-assembly. The data demonstrate an unexpected dichotomy of some leukocyte lineages into LL-positive or LL-negative cell-variants. Such dichotomies suggest exploring differential impacts from the duplicated leukocyte-lineages in health and disease., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Zebrafishes represent a proven model for human diseases and systems biology, exhibiting physiological and genetic similarities and having innate and adaptive immune systems. However, they are underexplored for human vaccinology, vaccine development, and testing. Here we summarize gaps and challenges., Areas Covered: Zebrafish models have four potential applications: 1) Vaccine safety: The past successes in using zebrafishes to test xenobiotics could extend to vaccine and adjuvant formulations for general safety or target organs due to the zebrafish embryos' optical transparency. 2) Innate immunity: The zebrafish offers refined ways to examine vaccine effects through signaling via Toll-like or NOD-like receptors in zebrafish myeloid cells. 3) Adaptive immunity: Zebrafishes produce IgM, IgD,and two IgZ immunoglobulins, but these are understudied, due to a lack of immunological reagents for challenge studies. 4) Systems vaccinology: Due to the availability of a well-referenced zebrafish genome, transcriptome, proteome, and epigenome, this model offers potential here., Expert Opinion: It remains unproven whether zebrafishes can be employed for testing and developing human vaccines. We are still at the hypothesis-generating stage, although it is possible to begin outlining experiments for this purpose. Through transgenic manipulation, zebrafish models could offer new paths for shaping animal models and systems vaccinology.

Importance: Mitochondrial antiviral signaling protein (MAVS) and stimulator of interferon (IFN) genes (STING) are key adaptor proteins required for innate immune responses to RNA and DNA virus infection. Here, we show that zebrafish transmembrane protein 47 (TMEM47) plays a critical role in regulating MAVS- and STING-triggered IFN production in a negative feedback manner. TMEM47 interacted with MAVS and STING for autophagic degradation, and ATG5 was essential for this process. These findings suggest the inhibitory function of TMEM47 on MAVS- and STING-mediated signaling responses during RNA and DNA virus infection., Competing Interests: The authors declare no conflict of interest.

CD248, also known as endosialin or tumor endothelial marker 1, is a type I single transmembrane glycoprotein. CD248 has been demonstrated to be upregulated in cancers, tumors and many fibrotic diseases in human and mice, such as liver damage, pulmonary fibrosis, renal fibrosis, arthritis and tumor neovascularization. However, no definite CD248 orthologs in fish have been documented so far. In this study, we report the identification of cd248a and cd248b in the zebrafish. Both the phylogenetic analysis and the conserved synteny strongly suggested that zebrafish cd248a and cd248b are orthologs of the human CD248 . Both cd248a and cd248b exhibited similar and dynamic expression pattern in early development, both genes had weak maternal expression, the zygotic transcripts were first seen in anterior somites and head mesenchyme, then shifted to eyes and head mesenchyme, later expanded to branchial arches, and gradually declined with development. The expression profiles of cd248a and cd248b were upregulated upon LPS (Lipopolysaccharide) challenge. Both Cd248a protein and Cd248b protein were localized on the cell membrane and cytoplasm, and overexpression of cd248a and cd248b induced the expression of pro-inflammatory cytokines, in vitro and in vivo. Moreover, deficiency of cd248a or cd248b both downregulated the expression of pro-inflammatory cytokines and upregulated anti-inflammatory cytokine. Additionally, loss of cd248a or cd248b both downregulated the expression of pro-inflammatory cytokines after LPS treatment. Taken together, these results indicated that cd248a and cd248b in zebrafish were involved in immune response and would provide further information to understand functions of Cd248 protein in innate immunity of fish., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Guo, Yang, Zhang, Yin, Teng, Zhang, Ji and Li.)

Visual information is transmitted from the eye to the brain along the optic nerve, a structure composed of retinal ganglion cell (RGC) axons. The optic nerve is highly vulnerable to damage in neurodegenerative diseases, such as glaucoma, and there are currently no FDA-approved drugs or therapies to protect RGCs from death. Zebrafish possess remarkable neuroprotective and regenerative abilities. Here, utilizing an optic nerve transection (ONT) injury and an RNA-seq-based approach, we identify genes and pathways active in RGCs that may modulate their survival. Through pharmacological perturbation, we demonstrate that Jak/Stat pathway activity is required for RGC survival after ONT. Furthermore, we show that immune responses directly contribute to RGC death after ONT; macrophages/microglia are recruited to the retina and blocking neuroinflammation or depleting these cells after ONT rescues survival of RGCs. Taken together, these data support a model in which crosstalk between macrophages/microglia and RGCs, mediated by Jak/Stat pathway activity, regulates RGC survival after optic nerve injury., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

This study investigated the effects of dietary Flos populi extract (FPE) on the growth, antioxidation capability, innate immune response, and disease resistance in gibel carp. A total of 480 fish were fed with five different diets containing 0, 0.5, 1.0, 1.5, or 2.0 g kg -1 FPE (designated as control, D0.5, D1.0, D1.5, or D2.0 groups) for 45 days. The fish were challenged with A. hydrophila after the feeding trial. Compared with the control, the feed efficiency (FE), weight gain (WG), final body weight (FBW), and specific growth rate (SGR) were significantly improved in groups D1.0 and D1.5. Dietary FPE significantly increased serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, as well as glutathione (GSH) content. The contents of protein carbonyl (PCC) and malondialdehyde (MDA) in serum decreased significantly. Additionally, FPE supplementation in diets resulted in significant improvement in serum lysozyme (LZM) and myeloperoxidase (MPO) activities, as well as immunoglobulin M (IgM) and complement 3 (C3) concentrations. The hepatic antioxidant enzymes (CAT and SOD) activities increased, whereas content of MDA decreased in fish treated with dietary FPE than those of control both pre- and post-challenged. After 12 h-challenge, an obvious downregulation of hepatic Kelch-like-ECH-associated protein 1 (Keap1), splenic tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1β, and toll-like receptor 2 (TLR2) mRNA levels was observed in fish treated with dietary FPE, whereas hepatic Nrf2 transcription level was upregulated compared to the control. Furthermore, compared to group D0.5, higher relative percent survival (RPS) was observed in gibel carp fed dietary 1.0-2.0 g/kg FPE. Our results reveal that FPE supplemented diet has a stimulatory effect on antioxidant capacity and nonspecific immune response, along with improved growth performance and enhanced resistance against A. hydrophila infection in juvenile gibel carp., (© 2021. The Author(s).)

Over 99% of ray-finned fishes (Actinopterygii) are teleosts, a clade that comprises half of all living vertebrate species that have diversified across virtually all fresh and saltwater ecosystems. This ecological breadth raises the question of how the immunogenetic diversity required to persist under heterogeneous pathogen pressures evolved. The teleost genome duplication (TGD) has been hypothesized as the evolutionary event that provided the substrate for rapid genomic evolution and innovation. However, studies of putative teleost-specific innate immune receptors have been largely limited to comparisons either among teleosts or between teleosts and distantly related vertebrate clades such as tetrapods. Here we describe and characterize the receptor diversity of two clustered innate immune gene families in the teleost sister lineage: Holostei (bowfin and gars). Using genomic and transcriptomic data, we provide a detailed investigation of the phylogenetic history and conserved synteny of gene clusters encoding diverse immunoglobulin domain-containing proteins (DICPs) and novel immune-type receptors (NITRs). These data demonstrate an ancient linkage of DICPs to the major histocompatibility complex (MHC) and reveal an evolutionary origin of NITR variable-joining (VJ) exons that predate the TGD by at least 50 million years. Further characterizing the receptor diversity of Holostean DICPs and NITRs illuminates a sequence diversity that rivals the diversity of these innate immune receptor families in many teleosts. Taken together, our findings provide important historical context for the evolution of these gene families that challenge prevailing expectations concerning the consequences of the TGD during actinopterygiian evolution., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wu, Fang, Zhang, Bi and Chang.)

Animals acquire nutrients and energy through feeding to achieve a balance between growth and organismal health. When there is a change in nutrient acquisition, the state of growth changes and may also cause changes in the intrinsic immune system. Compensatory growth (CG), a specific growth phenomenon, involves the question of whether changes in growth can be accompanied by changes in innate immunity. The zebrafish ( Danio rerio ), a well-known fish model organism, can serve as a suitable model. In this study, the zebrafish underwent 3 weeks of fasting and refeeding for 3 to 7 day periods. It was found that CG could be achieved in zebrafish. Zebrafish susceptibility to Streptococcus agalactiae increased after starvation. In addition, the amount of melano-macrophage centers increased after fasting and the proportion of injured tubules increased after refeeding for 3 and 5 days, respectively. Furthermore, the kidneys of zebrafish suffering from starvation were under oxidative stress, and the activity of several antioxidant enzymes increased after starvation, including catalase, glutathione peroxidases and superoxide dismutase. Innate immune parameters were influenced by starvation. Additionally, the activity of alkaline phosphatase and lysozyme increased after starvation. The mRNA expression of immune-related genes like il-1β was elevated to a different extent after fasting with or without lipopolysaccharides (LPS) challenge. This study showed that the function of the innate immune system in zebrafish could be influenced by nutrition status.

Inflammation is a crucial step in the development of chronic diseases in humans. Understanding the inflammation environment and its intrinsic mechanisms when it is produced by harmful stimuli may be a key element in the development of human disease diagnosis. In recent decades, zebrafish (Danio rerio) have been widely used in research, due to their exceptional characteristics, as a model of various human diseases. Interestingly, the mediators released during the inflammatory response of both the immune system and nervous system, after its integration in the hypothalamus, could also facilitate the detection of injury through the register of behavioural changes in the fish. Although there are many studies that give well-defined information separately on such elements as the recruitment of cells, the release of pro- and anti-inflammatory mediators or the type of neurotransmitters released against different triggers, to the best of our knowledge there are no reviews that put all this knowledge together. In the present review, the main available information on inflammation in zebrafish is presented in order to facilitate knowledge about this important process of innate immunity, as well as the stress responses and behavioural changes derived from it., (© 2020 John Wiley & Sons Ltd.)

Chasteberry is highly recommended as an herbal medicine across the globe for treating of many gynaecological disorders. In this study, chasteberry extract (CBE) was supplemented in goldfish diet to evaluate the immunity responses at the cellular and molecular levels. Moreover, after the feeding trial, the fish were challenged with Aeromonas hydrophila. The fish (300 individuals, 2.4 ± 0.12 g initial weight) were randomly distributed in 12 tanks and were fed with 0 (control), 5, 10, and 15 g CBE per kg of feed for 8 weeks. Based on the results, lysozyme activity, alkaline phosphatase, and total immunoglobulin (Ig) in the skin mucus samples were significantly enhanced in the fish fed with 15 g/kg CBE (P < 0.05). Moreover, dietary CBE positively affected lysozyme activity, complement components, and IgM content of the serum samples compared to the control group. Also, the number of monocytes and lymphocytes were increased significantly with increasing CBE in the diet (P < 0.05). The highest mRNA levels of tumor necrosis factors (TNF-α, TNF-2α) and Lysozyme were observed in 15 g/kg CBE treatment. After the challenge test, the highest relative percentage survival value (60%) was observed in the fish fed with 15 g/kg CBE. We concluded that dietary CBE especially at 15 g/kg has an immunomodulatory effect in goldfish by stimulating the innate immunity and some inflammatory cytokines as well as disease resistance against A. hydrophila., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Leukocyte immune-type receptors (LITRs) are a multigene family of teleost immunoregulatory proteins that share structural, phylogenetic, and likely functional relationships with several innate immune receptor proteins in other vertebrates, including mammals. Originally discovered in channel catfish (Ictalurus punctatus), representative IpLITR-types have been shown to regulate diverse innate immune cell effector responses including phagocytosis, degranulation, and cytokine secretion. To date, IpLITRs have been primarily characterized using mammalian cell line expression systems, therefore many unanswered questions remain regarding their actual regulatory roles in fish immunity. In the present study, we report on the preliminary molecular characterization of five goldfish (Carassius auratus) CaLITR-types and the identification of several putative splice variants of these receptors cloned from various goldfish tissues and primary myeloid cell cultures. In general, CaLITR mRNA transcripts were detected in all goldfish tissues tested, and also in primary kidney macrophage and neutrophil cultures. Specifically, CaLITR1 is a functionally ambiguous receptor with no charged amino acids in its transmembrane (TM) segment and is devoid of tyrosine-based signaling motifs in its short cytoplasmic tail (CYT) region. CaLITR2 is a putative activating receptor-type that contains immunotyrosine-based activation motifs (ITAMs) within its long CYT region, and CaLITR3 has a positively charged TM segment, suggesting that it may recruit intracellular stimulatory adaptor signaling molecules. CaLITR4 and CaLITR5 appear to have diverse signaling capabilities since they contain various immunoregulatory signaling motifs within their CYT regions including putative Nck and STAT recruitment motifs as well as ITAM-like and ITIM sequences. We also identified putative CaLITR splice variants with altered extracellular Ig-like domain compositions and variable CYT regions. Interestingly, this suggests that alternative splicing-mediated diversification of CaLITRs can generate receptor forms with possible variable binding and/or intracellular signaling abilities. Overall, these findings reveal new information about the teleost LITRs and sets the stage for exploring how alternative splicing leads to the functional diversification of this complex multigene immunoregulatory receptor family., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tripartite motif (TRIM) proteins have attracted particular research interest because of their multiple functions in the antiviral innate immune response. TRIM proteins perform different functions during virus infection, some play a role in inhibiting while others play a role in promoting. In this study, we described a species-specific TRIM gene named ftr67. Analysis of tissue distribution showed that ftr67 was mainly expressed in the gill and liver in five examined tissues of zebrafish. The phylogenic analysis showed that ftr67 was closest to the grass carp TRIM67. Overexpression of ftr67 resulted in a significantly decreased SVCV entry and impaired SVCV replication in FHM cells. Furthermore, overexpression of ftr67 could significantly induce the upregulation of molecular sensor RIG-I, IRF3/7, IFN and ISGs. In addition, RING domain of ftr67 was a required part essential for the antiviral effect. In summary, our results demonstrated that the important role of ftr67 in regulating SVCV infection, which offers a potential target for development of anti-SVCV therapies., Competing Interests: Declaration of competing interest The authors declare to have no conflict of interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tissue damage triggers a rapid and robust inflammatory response in order to clear and repair a wound. Remarkably, many of the cell biology features that underlie the ability of leukocytes to home in to sites of injury and to fight infection-most of which are topics of intensive current research-were originally observed in various weird and wonderful translucent organisms over a century ago by Elie Metchnikoff, the "father of innate immunity," who is credited with discovering phagocytes in 1882. In this review, we use Metchnikoff's seminal lectures as a starting point to discuss the tremendous variety of cell biology features that underpin the function of these multitasking immune cells. Some of these are shared by other cell types (including aspects of motility, membrane trafficking, cell division, and death), but others are more unique features of innate immune cells, enabling them to fulfill their specialized functions, such as encapsulation of invading pathogens, cell-cell fusion in response to foreign bodies, and their self-sacrifice as occurs during NETosis., (© 2020 Weavers and Martin.)

Gibel carp (Carassius auratus gibelio) is an important commercial fish that has become one of the most cultured fishes in the region of Yangtze River in China. However, the fish faces increasing hazard due to cyprinid herpesvirus 2 (CyHV-2) infection, which has caused great economic losses. In this study, healthy gibel carp were intraperitoneally injected with different doses of poly I:C at 24 h before CyHV-2 challenge. Results showed that the mortality decreased and peak death time appeared later in the fish injected with poly I:C at a dose of 10 μg/g body weight. To explore what gene plays an important role after poly I:C treatment, the transcriptome analysis of the gibel carp spleen was further performed. Compared with the PBS group, 1286 differentially expressed genes (DEGs) were obtained in the poly I:C-treated fish, including 1006 up-regulated and 280 down-regulated DEGs. GO analysis revealed that the most enriched DEGs responded to "biological regulation", "regulation of cellular process" and "regulation of biological process". Meanwhile, KEGG enrichment analysis showed that the DEGs were mainly mapped on the immune pathways like "TNF signal pathway", "p53 signal pathway" and "JAK-STAT signal pathway", suggesting that these signal pathways may be responsible for the delayed peak of CyHV-2 infection in gibel carp after poly I:C treatment. Taken together, this study provides insights into the immune protection effect of poly I:C against CyHV-2 infection, as well as providing useful information for antiviral defense in gibel carp., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study was aimed to evaluate the effects of partial replacement of fish meal by fermented moringa leaves (FMLs) on growth performance, serum biochemistry, antioxidant status, nonspecific immunity, and resistance against Aeromonas hydrophila in juvenile gibel carp (Carassius auratus gibelio var. CAS III). Four isonitrogenous and isoenergetic balanced diets, including three FML diets (substituting 20%, 40%, 60% of the fish meal in basal diet, F20, F40 and F60, respectively) and a basal diet (a diet containing 10% fish meal) were used. Each diet was randomly allocated to four fish groups (F20, F40, F60 and control) reared in a recirculating system. After 50 days of the feeding trial, fish were challenged by A. hydrophila. The result revealed that final mean body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), feed efficiency (FE) and survival rate (SR) were significantly increased (P < 0.05) in F20 and F40 groups compared with the control group. Decreased hepatosomatic index (HSI), body crude lipid, serum aspartate transaminase (AST) and serum alanine aminotransferase (ALT) activities, and increased serum alkaline phosphatase (AKP) and serum glutathione peroxidase (GPx) activities were observed in F40 and F60 groups compared with the control and F20 groups. All FMLs-supplemented groups increased (P < 0.05) serum superoxide dismutase (SOD), catalase (CAT) and lysozyme activities, complement component 3 (C3) and serum immunoglobulin M (IgM) concentration, or decreased serum malondialdehyde (MDA) and protein carbonyl (PCC) contents (P < 0.05). After the challenge test, the significant downregulation of toll-like receptors2 (TLR2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-8 mRNA transcription levels was observed in spleens of FMLs supplemented groups. Dietary F40 and F60 showed higher (P < 0.05) relative percent survival (RPS) (48.72% and 43.59%, respectively) against A. hydrophila infection than control. These results indicate that, as a dietary fish meal substitute, FMLs enhance the growth, and antioxidant and immune response, and regulate the expression of immune-related genes and increase disease resistance against A. hydrophila via TLR2 pathway in gibel carp, with greatest effects of 40% fish meal substitution., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Early-life exposure to different stressors can lead to various consequences on fish health status in later life development. To evaluate the effects of Aeromonas salmonicida achromogenes infection in the early-life on immunity in adulthood, zebrafish were either early-infected at 18 days post-fertilization (dpf), chronically infected from 18 to 35 dpf, or late infected at 35 dpf and then grown up to 61 dpf to be re-infected with the pathogen. The age of first infection was shown to influence both, level and timing of the immune gene expressions, especially for inflammation-related genes. In addition, evidence for an innate immune memory in zebrafish primarily infected with the pathogen at 35 dpf and re-infected at 61dpf provide new insights to consolidate the concept of a "trained" innate immunity in fish., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Graphene oxide (GO) is an increasingly important nanomaterial that exhibits great promise in the area of bionanotechnology and nanobiomedicine. However, the toxic effects of GO on the vertebrate developmental system are still poorly understood. Here, we aimed to investigate the toxic effects and molecular mechanisms of GO exposure in larval and adult zebrafish. The results showed that the major hepatotoxic phenotype induced by GO in zebrafish embryos was a significant decrease in liver area and a dose-dependent decrease in the hepatocytes. Moreover, the number of macrophages and neutrophils in zebrafish embryos were reduced but the expressions of pro-inflammatory cytokines were increased after GO treatment. High through-put RNA-Seq identified 314 differentially expressed genes (DEGs) in GO-induced zebrafish embryos including 192 up-regulated and 122 down-regulated. KEGG and GO functional analysis revealed that steroid hormone biosynthesis, lipoprotein metabolic process, and PPAR signaling pathway were significantly enriched. Most of the lipid metabolism genes were down-regulated while majority of the immune genes were up-regulated after GO treatment. Moreover, GO induced NF-κB p65 into the nucleus and increased the protein levels of NF-κB p65, JAK2, STAT3, and Bcl2 in adult zebrafish liver. In addition, pharmacological experiments showed that inhibition of ROS and blocking the MAPK signaling could rescue the hepatotoxic phenotypes induced by GO exposure. On the contrary, pharmacological activation of PPAR-α expression have increased the hepatotoxic effects in GO-induced larval and adult zebrafish. Taken together, these informations demonstrated that GO induced hepatic dysfunction mainly through the ROS and PPAR-α mediated innate immune signaling in zebrafish.

Cyclic GMP-AMP synthase (cGAS) is one of the most-characterized cytoplasmic DNA sensors in humans and other mammals. However, knowledge about cGAS homologs in nonmammalian species remains limited. In this study, we report the molecular and functional identification of two cGAS homologs, namely, DrcGASa and DrcGASb, from a zebrafish (Danio rerio) model. DrcGASa and DrcGASb share the same overall conservative structural architectures and functional domains/residues to mammalian cGASs. Both homologs synthesized a 2'3'-cGAMP isomer but not a 3'3'-cGAMP isomer via oligomerization in response to DNA stimulation. Overexpression of DrcGASa/b in HEK293T cells and zebrafish embryos significantly activated NF-κB and IFN-I signaling pathways in a STING-dependent manner. Knockdown of DrcGASa or DrSTING impaired such activations, thereby reducing the host innate immunity against bacterial and viral infections. DrcGASa, but not DrcGASb, was involved in immunoglobulin Z-mediated mucosal immunity in gill-associated lymphoid tissue, suggesting differential functions between the two DrcGASs. This reaction was associated with the DrcGAS-DrSTING-IFNφ1 signaling axis in GALT's γδ T cells. Our findings provide experimental evidence that a modern cGAS-STING pathway that mainly participates in IFN-mediated immunity originated from teleost fish based on the functional constraint of cGAS and STING proteins during vertebrate evolution., (© 2020 Federation of American Societies for Experimental Biology.)

ASGPR (asialoglycoprotein receptor, also known as hepatic lectin) was the first identified animal lectin, which participated in a variety of physiological processes. Yet its detailed immune functions are not well studied in lower vertebrates. After reporting a zebrafish hepatic lectin (Zhl), we identified a novel hepatic lectin (zebrafish hepatic lectin-like, Zhl-l) in zebrafish. The zhl-l was mainly expressed in liver in a tissue specific manner. And challenge with LPS/LTA induced a significant change of zhl-l expression. What's more, recombinant C-type lectin domain (rCTLD) of Zhl-l had the activity of agglutinating and binding to both Gram-negative and Gram-positive bacteria. It promoted the phagocytosis of bacteria by carp macrophages. Moreover, rCTLD could bind to insoluble lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN) independent of Ca 2+ , which was inhibited by galactose. Interestingly, Zhl-l was located in the membrane, and its overexpression could upregulate the production of pre-inflammatory cytokines. Taken together, these results indicated that Zhl-l played a role in immune defense, and would provide further information to understand functions of C-type lectin family and the innate immunity in vertebrates., Competing Interests: Declaration of competing interest None., (Copyright © 2019. Published by Elsevier Ltd.)

Myostatin, through type I receptor (kinase 4, 5, ALK4/5), functions to participate in the immune system and negatively regulate muscle growth in mammals. However, the role of myostatin (mstn) in the immune system of teleosts is largely unknown. In a previous study, we cloned the mstn1 cDNA encoding myostatin in Qi river crucian carp (Carassius auratus). In the present study, we have cloned mstn2 cDNA, which was characterized and analyzed together with mstn1. Tissue distribution analysis showed that both mstn genes are expressed in numerous tissues, with mstn1 dominantly expressed in the muscle and brain, whereas mstn2 is mainly expressed in the brain. During embryogenesis, mstn1 and mstn2 exhibit different expression patterns. Both mstn1 and mstn2 expression increased stepwise in the brain at different developmental stages. Furthermore, both genes are differentially regulated during different periods of fasting/re-feeding. Following the exposure of C. auratus to polyI:C, lipopolysaccharide (LPS), and Aeromonas hydrophila, both genes were upregulated in different tissues, which indicated that they might be involved in the immune response against pathogenic invasion. Blocking the Mstn signal pathway with SB-431542 (a chemical inhibitor of ALK4/5) resulted in significantly increased body length and weight. However, the mortality of SB-431542-treated fish was higher after A. hydrophila challenge. Moreover, decreased expression of lysozymes (lyz), complement component 3 (c3), β-defensin 3 (defb3), and interferon γ (ifnγ) were exhibited in treated fish, compared with the controls. Furthermore, the expression of nf-κb1, three pro-inflammatory cytokines (il1β, il6, and tnfα), and inflammatory cytokines (il8 and il10) were significantly increased in both the SB-431542-treated group and the control after A. hydrophila infection, suggesting that the NF-κB pathway was not suppressed in the SB-431542-treated fish. Taken together, our data suggest that both mstn1 and mstn2 play important roles in early body development, muscle growth, and the immune system by acting downstream of the NF-κB signal pathway., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aryl hydrocarbon receptor (AhR), a ligand-dependent transcriptional factor that responds to environmental chemicals, has been recently found to be closely associated with immune response in mammals. Pseudomonas plecoglossicida (P. plecoglossicida) is a temperature-dependent bacterial pathogen of visceral white spot disease in fish. Using dual RNA-seq, we previously evaluated the expression levels of ahr1a, ahr1b, ahr2 and cyp1a in the spleen of Epinephelus coioides at different time points after infection with P. plecoglossicida. In the present study, the expression levels of ahr1a, ahr1b, ahr2 and cyp1a in different organs of E. coioides and Danio rerio showed similar trends after being infected by P. plecoglossicida. It also was noted that liver, intestine, spleen, and heart were the most obviously affected organs, and ahr2 particularly showed a dramatically increase in the spleen. Subsequently, macrophages of E. coioides were isolated, and then infected by P. plecoglossicida, followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay, which revealed that the expression level of ahr1a in macrophages was significantly down-regulated, while expression levels of ahr1b, ahr2 and cyp1a were noticeably up-regulated. Eventually, it was noted that ahr1b and ahr2 were knocked-down in macrophages, and intracellular survival rate and immune escape rate of P. plecoglossicida were markedly improved. Taken together, ahr1a, ahr1b, ahr2 and cyp1a participate in the immune response to P. plecoglossicida in different organs of fish, while ahr1b and ahr2 may play pivotal roles in the immune response of spleen and macrophages., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aquaculture system based on biofloc technology (BFT) showed positive effects on prevention of Cyprinid herpesvirus 2 (CyHV-2) infection in gibel carp (Carassius auratus gibelio), which is detrimental to health and causes seriously economic losses to aquaculture. However, the enhancement mechanism of BFT regarding immunity and disease resistance of cultured species is scarce. Poly-β-hydroxybutyrate (PHB) has been proved as one of bioactive compounds in bioflocs. In this study, two groups (4% PHB supplementation diets and control with basal diets) with 30-day feeding were set to study the effect of PHB supplementation on immune-related gene expression by qRT-PCR, time-course CyHV-2 replication in vivo by qPCR and intestinal microbiota by illumine high-throughput sequencing. PHB supplementation significantly up-regulated transcriptional levels of eight immune-related genes, decreased cumulative mortality of gibel carp and early CyHV-2 replication in spleen in vivo (P < 0.05). Additionally, PHB changed the microbial structure but not diversity, and significantly increased beneficial bacteria such as Bacillus sp. KEGG pathway analysis by PICRUSt demonstrated that oral administration of PHB up-regulated abundances of genes responsible for seven pathways and down-regulated genes in eleven pathways. Histological structures of foregut, mindgut and hindgut were also affected. Our findings suggested that profitable effects of PHB on immunity and disease resistance might be gut microbiota-related, and regulated through pathways of enzymes secretion, replication and repair, and host immune system. This study will provide new insights into understanding the enhancing mechanism of BFT on immunity and disease resistance of cultured animals, and developing prebiotics/probiotics-based immunotherapies to improve animal health and disease resistance., Competing Interests: Declaration of competing interest The research was conducted in the absence of any commercial or financial relationships. We declare no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

One of the most important emerging pathogens in the aquaculture industry is Edwardsiella tarda, and it causes extensive losses in farmed fish globally. The identification of protective immunogens against E. tarda is increasingly valued. We previously investigated 20 recombinant proteins of 38 E. tarda extracellular secretory proteins and identified 10 as protective immunogens in a zebrafish model. Here, we clone 10 of the remaining 18 genes, and the resulting recombinant proteins are used for evaluation of immune protection. ETAE&#95;2147 (FliK), ETAE&#95;0654 (PpdD), and ETAE&#95;3259 (DamX) are identified as protective immunogens. Furthermore, their protection mechanism is explored by the detection of innate immunity genes encoding IL-1b, IL-6, IL-8, C3b, and NF-κB. The three protective immunogens stimulate zebrafish to produce higher and more lasting expression of the five immunity genes than non-protective immunogens during the first 48 h of infection. In addition, these protective immunogens are prone to be regulated by host products, which is helpful for cross-talk between host and pathogen, and thus they become vaccine candidates. These results highlight the way to understand the working mechanisms of protective immunogens., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The specific roles of the two major innate immune cell types - neutrophils and macrophages - in response to infection and sterile inflammation are areas of great interest. The larval zebrafish model of innate immunity, and the imaging capabilities it provides, is a source of new research and discoveries in this field. Multiple methods have been developed in larval zebrafish to specifically deplete functional macrophages or neutrophils. Each of these has pros and cons, as well as caveats, that often make it difficult to directly compare results from different studies. The purpose of this Review is to (1) explore the pros, cons and caveats of each of these immune cell-depleted models; (2) highlight and place into a broader context recent key findings on the specific functions of innate immune cells using these models; and (3) explore future directions in which immune cell depletion methods are being expanded., Competing Interests: Competing interestsThe author declares no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Objectives: Cell-culture studies reported that prokaryotic RNA molecules among the various microbe-associated molecular patterns (MAMPs) were uniquely present in live bacteria and were categorized as viability-associated MAMPs. They also reported that specific nucleotide modifications are instrumental in the discrimination between self and nonself RNAs. The aim of this study was to characterize the in vivo immune induction potential of prokaryotic and eukaryotic ribosomal RNAs (rRNAs) using zebrafish embryos as novel whole animal model system. Additionally, we aimed to test the possible role of rRNA modifications in immune recognition., Results: We used three immune markers to evaluate the induction potential of prokaryotic rRNA derived from Escherichia coli and eukaryotic rRNAs from chicken (nonself) and zebrafish (self). Lipopolysaccharide (LPS) of Pseudomonas aeruginosa served as a positive control. E. coli rRNA had an induction potential equivalent to that of LPS. The zebrafish innate immune system could discriminate between self and nonself rRNAs. Between the nonself rRNAs, E. coli rRNA was more immunogenic than chicken rRNA. The in vitro transcript of zebrafish 18S rRNA gene without the nucleotide modifications was not recognized by its own immune system. Our data suggested that prokaryotic rRNA is immunostimulatory in vivo and could be useful as an adjuvant.

Norfloxacin nicotinate (NOR-N), an adduct of norfloxacin (NOR) and nicotinic acid, has been widely used for replacing NOR in animal husbandry and fishery industry. Nowadays, increasing evidences showed that NOR could pose toxic effects on fish and other aquatic organisms, but as its adduct, whether NOR-N could cause adverse effects on aquatic organisms is still unclear. To evaluate the toxic effects of NOR-N on the early life stage of zebrafish, we determined the changes in embryonic development (hatching rate, body length, malformation rate and mortality), antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx)) activities, malondialdehyde (MDA) content and gene expression levels related to antioxidant enzymes (Cu/Zn-sod, Mn-sod, CAT and Gpx) and innate immune system (tumor necrosis factor α (TNFα), interferon (IFN), Interleukin-1 beta (IL-1β), IL-8, CXCL-clc, CC-chemokine, lysozyme (Lzy) and complement factors (C3)) after embryonic exposure to NOR-N till 96 hpf. The results showed that NOR-N exposure could decreased the hatching rate and body length, and increased abnormality and mortality as concentration-dependent during embryonic development process. NOR-N induced oxidative stress in zebrafish larvae through increasing the contents of MDA and the activities of SOD, CAT and Gpx, as well as the mRNA levels of genes related to these antioxidant enzymes. Moreover, the expression of TNFα, IFN, IL-1β, IL-8, CXCL-clc, CC-chemokine, Lzy and C3 genes were significantly up-regulated after exposure to high concentration (5 and/or 25 mg/L) of NOR-N till 96 hpf, indicating that the innate immune system in zebrafish larvae was disturbed by NOR-N. Overall, our results suggested that NOR-N caused development toxicity, oxidative stress and immunotoxicity on the early life stage of zebrafish. Thus, widespread application of NOR-N might pose potential ecotoxicological risk on aquatic ecosystems., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Fatty acids (FAs) are key elements that affect not only growth but also different immune functions, and therefore, nutrition is important for growing healthy fish. Zebrafish (Danio rerio) is a good model for assessing the beneficial effects of immunostimulants, including FAs, before applying them in aquaculture. Accordingly, this study evaluated the effects of palmitic acid (PA) treatment on different immune parameters of zebrafish and on the mortality caused by the spring viremia of carp virus (SVCV). The results suggest that PA modulates the infection outcome in vivo, which benefits zebrafish and results in reduced mortality and viral titres. The antiviral protection elicited by this FA seems to be associated with the inhibition of autophagy and is independent of other immune processes, such as neutrophil proliferation or type I interferon (IFN) activity. The use of PA as an immunostimulant at low concentrations showed great potential in the prevention of SVCV infections; therefore, this FA could help to prevent the mortality and morbidity caused by viral agents in aquacultured fish. Nevertheless, the potentially detrimental effects of suppressing autophagy in the organism should be taken into account., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We investigated cellular functional and targeted immune cytokine responses of farmed Chinook salmon (Oncorhynchus tshawytscha) peripheral blood mononuclear cells (PBMCs) in vitro to LPS from Escherichia coli (E. coli) serotypes O111: B4 and O55: B5, and a phorbol ester phorbol 12-myristate 13-acetate (PMA). Bacterial LPS and PMA significantly (p < 0.05) induced reactive oxygen species (ROS) production in O. tshawytscha PBMCs, and enhanced by interferon (IFN)-inducible cytokine production. Cellular phagocytosis was significantly enhanced with PMA and E. coli serotype O111: B4 LPS after 1 and 2 h respectively. At the molecular level, LPS and PMA significantly (p < 0.05) upregulated pro-inflammatory cytokine gene transcripts for IFNγ, TNF-α, and anti-inflammatory IL-10, 24 h post-stimulation. This response is postulated to be mediated via the MyD88 and TRIF pathways in TLR4, or synergistic TLR1 and TLR2 receptors. This is the first report of LPS induced immune related in vitro responses in farmed O. tshawytscha PBMCs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Streptococcus agalactiae is a major aquaculture pathogen infecting various saltwater and freshwater fish. To better understand the mechanism of the immune responses to S. agalactiae in wildtype zebrafish, the transcriptomic profiles of two organs containing mucosal-associated lymphoid tissues from S. agalactiae-infected and non-infected groups were obtained using RNA-seq techniques. In the intestines, 6735 and 12908 differently expressed genes (DEGs) were identified at 24 hpi and 48 hpi, respectively. Among 66 and 116 significantly enriched pathways, 15 and 21 pathways were involved in immune system or signal transduction at 24 hpi and 48 hpi, respectively. A number of genes involved in Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, T cell receptor signaling pathway, B cell receptor signaling pathway, Antigen processing and presentation, NF-kappa B signaling pathway and PI3K-Akt signaling pathway were significantly downregulated. In the skins, 3113 and 4467 DEGs were identified at 24 hpi and 48 hpi, respectively. Among 24 and 56 significantly enriched pathways, 4 and 13 pathways were involved in immune system or signal transduction at 24 hpi and 48 hpi, respectively. More immune-related signaling pathways including Leukocyte transendothelial migration, Cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, IL-17 signaling pathway, MAPK signaling pathway, TNF signaling pathway, Complement and coagulation cascades, Hematopoietic cell lineage and Jak-STAT signaling pathway were differently enriched for upregulated DEGs at 48 hpi, which were completely different from that in the intestines. Furthmore, comparative transcriptome analysis revealed that the downregulated 1618 genes and upregulated 1622 genes existed both at 24 hpi and 48 hpi for the intestine samples. In the skins, the downregulated 672 genes and upregulated 428 genes existed both at 24 hpi and 48 hpi. Three pathways related to immune processes were significantly enriched for downregulated DEGs both in the intestines and skins collected at 24 hpi and 48 hpi, which included Antigen processing and presentation, Intestinal immune network for IgA production and Hematopoietic cell lineage. Interaction network analysis of DEGs identified the main DEGs in the sub-network of complement and coagulation cascades both in the intestines and skins. Twenty of DEGs involved in complement and coagulation cascades were further validated by Real-time quantitative PCR. Altogether, the results obtained in this study will provide insight into the immune response of zebrafish against S. agalactiae XQ-1 infection in fatal conditions, and reveal the discrepant expression pattern of complement and coagulation cascades in the intestines and skins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Skin plays an important role in the innate immune responses of fish, particularly towards bacterial infection. To understand the molecular mechanism of mucosal immunity of fish during bacterial challenge, a de novo transcriptome assembly of crucian carp Carassius auratus skin upon Aeromonas hydrophila infection was performed, the latter with Illumina Hiseq 2000 platform. A total of 118111 unigenes were generated and of these, 9693 and 8580 genes were differentially expressed at 6 and 12 h post-infection, respectively. The validity of the transcriptome results of eleven representative genes was verified by quantitative real-time PCR (qRT-PCR) analysis. A comparison with the transcriptome profiling of zebrafish skin to A. hydrophila with regards to the mucosal immune responses revealed similarities in the complement system, chemokines, heat shock proteins and the acute-phase response. GO and KEGG enrichment pathway analyses displayed the significant immune responses included TLR, MAPK, JAK-STAT, phagosome and three infection-related pathways (ie., Salmonella, Vibrio cholerae and pathogenic Escherichia coli) in skin. To our knowledge, this study is the first to describe the transcriptome analysis of C. auratus skin during A. hydrophila infection. The outcome of this study contributed to the understanding of the mucosal defense mechanisms in cyprinid species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The immune system is composed of two subsystems-the innate immune system and the adaptive immune system. The innate immune system is the first to respond to pathogens and does not retain memory of previous responses. Innate immune responses are evolutionarily older than adaptive responses and elements of innate immunity can be found in all multicellular organisms. If a pathogen persists, the adaptive immune system will engage the pathogen with specificity and memory. Several components of the adaptive system including immunoglobulins (Igs), T cell receptors (TCR), and major histocompatibility complex (MHC), are assumed to have arisen in the first jawed vertebrates-the Gnathostomata. This review will discuss and compare components of both the innate and adaptive immune systems in Gnathostomes, particularly in Chondrichthyes (cartilaginous fish) and in Osteichthyes [bony fish: the Actinopterygii (ray-finned fish) and the Sarcopterygii (lobe-finned fish)]. While many elements of both the innate and adaptive immune systems are conserved within these species and with higher level vertebrates, some elements have marked differences. Components of the innate immune system covered here include physical barriers, such as the skin and gastrointestinal tract, cellular components, such as pattern recognition receptors and immune cells including macrophages and neutrophils, and humoral components, such as the complement system. Components of the adaptive system covered include the fundamental cells and molecules of adaptive immunity: B lymphocytes (B cells), T lymphocytes (T cells), immunoglobulins (Igs), and major histocompatibility complex (MHC). Comparative studies in fish such as those discussed here are essential for developing a comprehensive understanding of the evolution of the immune system., (Copyright © 2019 Smith, Rise and Christian.)

Cyprinid herpesvirus 2 (CyHV-2) is the causative pathogen of herpesviral haematopoietic necrosis disease, which has caused huge economic losses to aquaculture industry in China. In this study, nine truncated CyHV-2 membrane glycoproteins (ORF25, ORF25C, ORF25D, ORF30, ORF124, ORF131, ORF136, ORF142A, ORF146) and a GFP reporter protein were respectively expressed using baculovirus surface displaying system. Western blot showed that the proteins were successfully packaged in the recombinant virus particles. In baculovirus transduced gibel carp kidney cells, the target proteins were expressed and displayed on the fish cell surface. Healthy gibel carp were immunized by immersion with the recombinant baculoviruses and the fish treated with phosphate-buffered saline (PBS) were served as mock group. The expression of interleukin-11 (IL-11), interferon α (IFNα) and a complement component gene C3 were significantly up-regulated in most experimental groups, and interferon γ (IFNγ) expression in some groups were also induced after immunization. Subsequently, the immunized gibel carp were challenged by intraperitoneal injection of CyHV-2 virus. All the immunized groups exhibited reduced mortality after CyHV-2 challenge. In the groups immunized with baculoviruses displaying and expressing ORF25, ORF25C and ORF146, the relative percentage survival values reached 83.3%, 87.5% and 70.8%, respectively. Our data suggested that baculovirus-displayed ORF25, ORF25C and ORF146 could be potential vaccine candidates for the prevention of CyHV-2 infection in gibel carp., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The use of probiotics as alternatives to antibiotics for disease control is a relatively eco-friendly approach in aquaculture; hence, studies isolating and assessing the benefit of potential probiotics to fish farming are common. The zebrafish is an excellent model system for validating beneficial functions of potential probiotics before their practical application in aquaculture. Here, a potentially probiotic Chromobacterium aquaticum was isolated from lake water samples and characterized by biochemical analysis and 16S rDNA sequencing. The probiotic produced extracellular enzymes (protease and xylanase) and a bacteriocin-like substance, which exhibited tolerance to extreme pH and high-temperature conditions and broad-spectrum bactericidal activity against diverse pathogens, including aquatic, foodborne, clinical and plant pathogens. The effects of C. aquaticum on zebrafish nutrient metabolism, growth performance and innate immunity were evaluated by measuring the expression of indicator genes after C. aquaticum feeding for 8 weeks. Fish administered the probiotic exhibited significantly increased hepatic mRNA expression of carbohydrate metabolism-related genes, including glucokinase (GK), hexokinase (HK), glucose-6-phosphatase (G6Pase), and pyruvate kinase (PK-L), and growth-related genes, including the growth hormone receptor (GHR) and insulin-like growth factor-1 (IGF-1). Innate immune-related genes (IL-1β, IL-6, TNF-α, IL-10, IL-21, NF-κb, lysozyme and complement C3b) were induced in fish with probiotic supplementation. Probiotic-treated fish exhibited a higher survival rate than control fish after challenge with Aeromonas hydrophila and Streptococcus iniae. Together, these data suggest that C. aquaticum, as a probiotic feed supplement, could enhance nutrient metabolism and growth performance and could modulate innate immunity against A. hydrophila and S. iniae in zebrafish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Although aquaculture is among the fastest growing food production sectors in the world, one of the bottlenecks for the continuity of its expansion is the dependence of animal protein on commercial feed formulations. Vegetable proteins are an alternative due to the low cost and high availability. However, this protein source is accompanied by a series of antinutritional and pro-inflammatory compounds, including phytate. Phytases can be added in feed for phytate degradation and increase nutrient availability. However, the use of purified phytases significantly increases the production costs. An interesting alternative is to use probiotics genetically modified as bioreactors for phytase production. In the present study, a strain of Bacillus subtilis secreting a fungal phytase was used to evaluate the effect of a feed with high content of soybean meal on zebrafish (Danio rerio). We analysed the condition factor (K) of fish, and the expression of genes related to the immune system, inflammatory response and oxidative. stress. The results obtained demonstrate that the transgenic probiotic was efficient in improving the fish condition factor, stimulating the immune system, reducing the inflammatory response and oxidative stress. Thus, probiotics acting as phytase bioreactors can be considered an interesting tool for the adaptation of commercial species to feed of lower cost., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Innate immunity is regulated by phagocytic cells and is critical for host control of bacterial infection. In many bacteria, the type VI secretion system (T6SS) can affect bacterial virulence in certain environments, but little is known about the mechanisms underlying T6SS regulation of innate immune responses during infection in vivo. Here, we developed an infection model by microinjecting bacteria into the tail vein muscle of 3-day-post-fertilized zebrafish larvae, and found that both macrophages and neutrophils are essential for bacterial clearance. Further study revealed that EvpP plays a critical role in promoting the pathogenesis of Edwardsiella piscicida (E. piscicida) via inhibiting the phosphorylation of Jnk signaling to reduce the expression of chemokine (CXC motif) ligand 8 (cxcl8a), matrix metallopeptidase 13 (mmp13) and interleukin-1β (IL-1β) in vivo. Subsequently, by utilizing Tg (mpo:eGFP +/+ ) zebrafish larvae for E. piscicida infection, we found that the EvpP-inhibited Jnk-caspy (caspase-1 homolog) inflammasome signaling axis significantly suppressed the recruitment of neutrophils to infection sites, and the caspy- or IL-1β-morpholino (MO) knockdown larvae were more susceptible to infection and failed to restrict bacterial colonization in vivo. taken together, this interaction improves our understanding about the complex and contextual role of a bacterial T6SS effector in modulating the action of neutrophils during infection, and offers new insights into the warfare between bacterial weapons and host immunological surveillance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The Notch signaling pathway is known to regulate innate immunity by influencing macrophage function and interacting with the Toll-like receptor (TLR) signaling pathway. However, the comprehensive role of the Notch signaling pathway in the innate immune response remains unknown. To assess the function of Notch1a in immunity, we examined the innate immune responses to Vibrio parahaemolyticus strain Vp13 of wild-type (WT) and notch1a -/- zebrafish larvae generated using the clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system. The median lethal dose (LD 50 ) of V. parahaemolyticus was significantly lower in notch1a -/- larvae than in WT larvae 3 days post fertilization (dpf). Transcriptome data analysis revealed 359 significantly differentially expressed genes (DEGs), including 246 significantly down-regulated genes and 113 significantly up-regulated genes, in WT infected groups compared with WT control groups. In contrast, 986 significantly DEGs were found in notch1a -/- infected groups compared with notch1a -/- control groups, of which 82 genes were significantly down-regulated and 904 genes were significantly up-regulated. These DEGs belonged to the tumor necrosis factor (TNF), complement, nuclear factor kappa B (NF-κB), cathepsin, interleukin (IL), chemokine, serpin peptidase inhibitor, matrix metallopeptidase, innate immune cells, pattern recognition receptor (PRR), and other cytokine families. Our results indicate that Notch1a plays roles in inhibiting many immunity-related genes and could comprehensively mediate the innate immune response by regulating TLRs, nucleotide-binding-oligomerization-domain-like receptors (NLRs), lectins, complement, ILs, chemokines, TNF, cathepsin, and serpin. Further studies are required to understand the specific mechanism of Notch1a in innate immunity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Here we present immunostimulant-loaded nanoliposomes (NL c ) as a strategy to protect zebrafish larvae against bacterial infection. The NL c encapsulate crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid (Poly I:C), a synthetic analogue of viral dsRNA. Fluorescently-labeled NL c were ingested by zebrafish larvae 4 days post fertilization, when administrated by bath immersion, and accumulated in the intestine. RT-qPCR analysis showed the expression of innate immune related genes (tnfα, il1β, nos2a, irf1a and ptgs2a) was significantly upregulated at 48 h post NL c treatment. A zebrafish larvae infection model for Aeromonas hydrophila was set up by bath immersion, achieving bacterial-dose-dependent significant differences in survival at day 5 post infection in both injured and non-injured larvae. Using this model, NL c protected non-injured zebrafish larvae against an A. hydrophila lethal infection. In contrast, neither the empty nanoliposomes nor the mixture of immunostimulants could protect larvae against lethal challenges. Our results demonstrate that nanoliposomes could be further developed as an efficient carrier, widening the scope for delivery of other immunostimulants in aquaculture., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Fibroblast growth factor receptor (FGFR) 3 is one of the four distinct membrane-spanning tyrosine kinases required for proper skeletal development. In fish, the role of FGFR3 is still unclear. In this article, we reveal that zebrafish FGFR3 is a negative regulator of interferon (IFN) production in the innate immune response by suppressing the activity of TANK-binding kinase 1 (TBK1) in the process of virus infection. qPCR experiments demonstrate that the transcriptional level of cellular FGFR3 was upregulated by infection with spring viremia of carp virus (SVCV), indicating that FGFR3 might be involved in the process of host cell response to viral infection. Then, overexpression of FGFR3 significantly impeded the IFN promoter activity induced by a stimulator. In addition, the capabilities of a retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) system to activate IFN promoter were decreased during the overexpression of FGFR3. Subsequently, FGFR3 decreased the phosphorylation of interferon regulatory factor 3 (IRF3) and mediator of IRF3 activation (MITA) by TBK1. These findings suggest that zebrafish FGFR3 is a negative regulator of IFN by attenuating the kinase activity of TBK1, leading to the suppression of IFN expression., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cytidine/uridine monophosphate kinase 2 (CMPK2) is known as a nucleoside monophosphate kinase in mitochondria to maintains intracellular UTP/CTP, and could be induced by immunostimulants LPS and Poly (I:C) in mammals, suggesting its potential antiviral and antibacterial role. In this study, CMPK2 was cloned and characterized in Fathead minnow (FHM) cells. In vivo analysis of tissue distribution revealed that CMPK2 transcript was detected in all the tissues of zebrafish (Danio rerio) examined in this study, particularly abundant in liver, spleen and kidney. In addition, indirect immunofluorescence showed that CMPK2 was localized in the cytoplasm of FHM cells. Expression of CMPK2 mRNA was significantly up-regulated following challenge with Spring viraemia of carp virus (SVCV), poly(I:C), or zebrafish IFN1 and IFN3 both in vitro and in vivo. Furthermore, overexpression and RNA interference of CMPK2 in SVCV-infected FHM cells showed significantly antiviral effect. In summary, this study for the first time shows the presence and distribution of CMPK2 in different tissues of zebrafish, but also demonstrates its antiviral potential against SVCV infection in vivo. These new findings could contribute to explain the molecular mechanism of the CMPK2 mediated antiviral function., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In recent years, microRNAs (miRNAs) have been shown to play important roles in immunity. Analyses of the functions of miRNAs and their targets are useful in understanding the regulation of the immune response. To understand the relationships between miRNAs and their targets during infection, we used zebrafish as an infection model in which to characterize the miRNA and mRNA transcriptomes of zebrafish larvae infected with Vibrio parahaemolyticus. We identified the differentially expressed miRNAs and mRNAs. Overall, 37 known zebrafish miRNAs were differentially expressed in the infection group and 107 predicted target genes of 26 miRNAs were differentially expressed in the mRNA transcriptome. These targets with specific Gene Ontology (GO) terms, such as peripheral nervous system neuron axonogenesis, organophosphate metabolic process, heme binding, protein binding, tetrapyrrole binding, protein dimerization activity, and aromatase activity, which regulate nerve conduction, energy metabolism, hematopoiesis, and protein synthesis. They were also associated with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways such as phototransduction, tryptophan metabolism, notch signaling, and purine metabolism. Our findings indicate that miRNAs regulate the innate immune response via complex networks, and zebrafish (Danio rerio, dre)-miR-205-3p, dre-miR-141-5p, dre-miR-200a-5p, dre-miR-92a-2-5p, dre-miR-192, and dre-miR-1788 may play important roles in the innate immune response by regulating target genes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Tripartite motif (TRIM) proteins were shown to play an important role in innate antiviral immunity. FinTRIM (ftr) is a new subset of TRIM genes that do not possess obvious orthologs in higher vertebrates. However, little is known about its function. In this study, we used bioinformatic analysis to examine the phylogenetic relationships and conserved domains of zebrafish (Danio rerio) ftr01, ftr42, and ftr58, as well as qualitative real-time PCR to examine their expression patterns in zebrafish embryonic fibroblast (ZF4) cells and zebrafish tissues. Sequence analysis showed that the three finTRIMs are highly conserved, and all contain a RING domain, B-box domain, and SPRY-PRY domain. In addition, ftr42 and ftr58 had one coiled-coil domain (CCD), whereas ftr01 had two CCDs. Tissue expression analysis revealed that the mRNA level of ftr01 was the highest in the liver, whereas those of ftr42 and ftr58 were the highest in the gill; the expression of these finTRIMs was clearly upregulated not in the eyes, but in the liver, spleen, kidney, gill, and brain of zebrafish following spring viremia of carp virus (SVCV) infection. Similarly, the expression of these three finTRIM genes also increased in ZF4 cells after SVCV infection. Our study revealed that ftr01, ftr42, and ftr58 may play an important role in antiviral immune responses, and these findings validate the need for more in-depth research on the finTRIM family in the future.

Type I interferons, as a class of multipotent cytokines, play a key role in host antiviral immune responses. In this study, a type I IFN coding gene of gibel carp, Carassius auratus gibelio, CagIFNa was cloned and sequenced. The full-length cDNA sequence of CagIFNa consists of 724 nucleotides that encode a predicted protein of 183 amino acids. CagIFNa has two highly conserved cysteine residues in the deduced protein, which is mostly conserved in the fish group I type I IFNs. CagIFNa was identified as a member of the IFNa subgroup of group I type I IFNs by phylogenetic analysis. CagIFNa transcripts were detected in all investigated tissues with higher levels in the liver, intestine, spleen and head kidney of gibel carp. Following injection with Cyprinid herpesvirus 2 (CyHV-2), CagIFNa gene expression was significantly inhibited in the spleen but delayed and then increased in head kidneys. Similarly, while CagIFNa expression was rapidly induced in gibel carp brain (GiCB) cells by poly I:C stimulation and its high induction level was delayed following CyHV-2 infection. CagIFNa overexpression in GiCB cells drastically reduced virus CPE and titer. Furthermore, several genes associated with type I IFN signaling pathway including IRF3, IRF7, IRF9, STAT1, Mx1 and PKR were induced in GiCB cells overexpressing CagIFNa upon CyHV-2 infection. These results show that CagIFNa plays a role in antiviral immune system in gibel carp., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The zebrafish has unique advantages for understanding the evolution of vertebrate immunity and to model human diseases. In this review, we will firstly give an overview of the current knowledge on vertebrate innate immune receptors with special emphasis on the inflammasome and then summarize the main contribution of the zebrafish model to this field, including to the identification of novel inflammasome components and to the mechanisms involved in its activation, assembly and clearance of intracellular bacteria., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tripartite motif (TRIM) proteins have been demonstrated to exhibit critical functions in multiple cellular processes, including development, carcinogenesis, and programmed cell death, and are also widely recognized to be important antiviral restriction factors or modulators of immune and inflammatory signaling pathways. However, in teleosts, additional TRIM members have been identified and their functions remain largely unknown. Here, a novel finTRIM gene from orange spotted grouper (EcfinTRIM82) was cloned and characterized. Sequence analysis indicated that EcfinTRIM82 encoded a 575 amino acid peptide which shared 94% and 82% identity with Asian sea bass (Lates calcarifer), and zebrafish (Danio rerio) finTRIM82, respectively. EcfinTRIM82 contained three conserved domains, including a RING, B-Box, and SPRY domain. Using fluorescence microscopy, we found that green fluorescence aggregates were observed in the cytoplasm of EcfinTRIM82-EGFP transfected grouper spleen (GS) cells. As the infection proceeded, EcfinTRIM82 transcription was significantly upregulated in Singapore grouper iridovirus (SGIV) or red-spotted grouper nervous necrosis virus (RGNNV) infected GS cells. This suggests that EcfinTRIM82 might be involved in fish virus infection. The in vitro overexpression of EcfinTRIM82 in GS cells significantly enhanced the replication of SGIV and RGNNV, evidenced by increased expression of viral genes, including the SGIV major capsid protein (MCP), VP19, ICP-18, RGNNV coat protein (CP), and RNA-dependent RNA polymerase (RdRp). Furthermore, the ectopic expression of EcfinTRIM82 significantly decreased the expression of interferon (IFN)-related signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon stimulated gene 15 (ISG15), ISG56, IFP35, and myxovirus resistance gene (MXI), suggesting that EcfinTRIM82 regulated viral replication via the negative regulation of the host IFN response. In addition, EcfinTRIM82 overexpression substantially decreased the level of proinflammatory cytokine transcription. Furthermore, the ectopic expression of EcfinTRIM82 significantly weakened the melanoma differentiation-associated protein 5 (MDA5), mediator of IRF3 activation (MITA) and mitochondrial antiviral-signaling (MAVS) protein-induced IFN response by detecting the transcription of interferon related cytokines and the promoter activity of IFN. Together, our results demonstrate that finTRIM82 negatively regulates the innate antiviral immune response against grouper virus infection., (Copyright © 2019. Published by Elsevier Ltd.)