The direct effect of cortisol treatment on carp neutrophil viability was examined in vitro. Cortisol treatment caused an inhibition of neutrophil apoptosis. The effect was blocked by glucocorticoid receptor blocker RU486, showing that rescue from apoptosis was receptor mediated. Using binding studies with radioactive cortisol, a single class of glucocorticoid receptors was detected with high affinity (Kd = 2.6 nM) and low capacity (497 receptors/cell) for cortisol binding. Both in vitro and in vivo cortisol treatment did not affect neutrophil respiratory burst activity. These data indicate that cortisol can augment the supply of functional neutrophilic granulocytes in conditions of acute stress, which may be essential for survival, since phagocytes form the first line of defence against micro-organisms.

In humans, alternative splicing of androgen receptor (AR) is usually involved in some diseases. However, our knowledge about the presence of AR variants in other species and its importance for immunity is scant. Here, we report the identification of a constitutively active AR variant lacking the ligand-binding domain (LBD), ARΔLBD, in the fish gilthead seabream. ARΔLBD is expressed in the testis and the head-kidney (HK), and its expression varies with the reproductive stage and is correlated with plasma testosterone (T). In addition, ARΔLBD is expressed in acidophilic granulocytes (AGs), which are the functional equivalent of mammalian neutrophils, but not in macrophages, and its expression is modulated by both T and immune stimuli. Notably, AR and ARΔLBD were able to interact, being the activity of AR dominant at all concentrations tested of the ligand. These results reveal a new mechanism for the regulation of neutrophil biology in vertebrates and explain the conflicting results that suggest that androgens are less important than AR in human and mouse neutrophil homeostasis.

DNA-binding protein Ikaros is a major determinant of haematopoietic lineage, especially in the development, differentiation and proliferation of lymphocytes. In the present study, a Ikaros homologue (designed as CgIkaros-like) was identified and characterized as a vital determinant in the proliferation of haemocytes during haematopoiesis of Pacific oyster Crassostrea gigas. The complete coding sequence of CgIkaros-like was of 1329 bp encoding a predicted polypeptide of 442 amino acids with four ZnF regions, locating at the C-terminus and N-terminus respectively. The highest expression level of CgIkaros-like mRNA was found in gills, followed by haemocytes and gonad. The mRNA transcripts of CgIkaros-like could be detected in all the haemocytes with higher abundance in semi-granulocytes and agranulocytes. CgIkaros-like protein was localized in both of cytoplasm and nucleus with higher abundance in nucleus of oyster haemocytes. The mRNA and protein expression levels of agranulocyte marker CgCD9, granulocyte marker CgAATase, cell cycle related gene CgCDK2, Notch receptor CgNotch and Notch target gene CgHes1 all increased significantly (p < 0.05) after CgIkaros-like was interfered by siRNAs, which were about 27.33-, 2.63-, 24.34-, 4.45- and 6.08-fold of that in the siRNA-NC control group, respectively. While the transcripts of CgGATA3 and CgRunx did not change significantly after CgIkaros-like was interfered. These results demonstrated that CgIkaros-like functioned as a transcription factor combined with Notch pathway to mediate CgCDK2 and regulate the proliferation of oyster haemocytes.

Inflammatory cytokine interleukin-17 (IL-17) binds its receptors (IL-17Rs) to activate the downstream immune signals and plays an important role in host defense. In the present study, an IL-17 receptor (designated as CgIL-17R1) was identified from oyster Crassostrea gigas with an open reading frame of 3141 bp encoding 1047 amino acids. The amino acid sequence of CgIL-17R1 with two conserved FN3 domains shared higher similarity with other known IL-17Rs from mollusc species. The recombinant CgIL-17R1 protein (rCgIL-17R1) displayed high binding affinity to the recombinant CgIL-17 protein (rCgIL-17) in vitro. The mRNA transcripts of CgIL-17R1 were significantly higher expressed in haemocytes, especially in granunolyctes, compared with that in other tissues. After the stimulation with Vibrio splendidus or rCgIL17-1 in vivo, the expressions of CgIL-17R1 and cell proliferation related genes (CgRunx-1, CgCDC-6, CgCDC-45, and CgCDK-2) were significantly up-regulated in haemocytes (p < 0.01). When the CgIL-17R1 expression was interfered by specific CgIL-17R1-dsRNA, the expressions of these cell proliferation related genes reduced significantly, and the proliferation rate of haemocytes declined dramatically at 6 h post V. splendidus stimulation (p < 0.01), compared to that of blank group. These results collectively indicated that CgIL-17R1 expressed in granulocytes mediated the CgIL-17 induced haemocytes proliferation during immune response in oyster C. gigas, which provided novel information about the regulation of haemocyte proliferation in invertebrates.

The sp1A/ryanodine receptor (SPRY) family members have been reported to involve in important biological pathways, including innate immune signaling, cytokine signaling suppression, development, cell growth, and retroviral restriction. In the present study, a SPRY domain-containing SOCS box protein (named as CgSPSB3) was identified and characterized from oyster Crassostrea gigas. The open reading frame of CgSPSB3 gene was of 699 bp, encoding a polypeptide of 232 amino acid residues with a central SPRY domain and a C-terminal SOCS box motif. CgSPSB3 mRNA transcripts could be detected in all the examined tissues with the highest level in hemocytes, which was about 82.72-fold (p < 0.05) of that in gonad. Furthermore, the expression level of CgSPSB3 mRNA in granulocytes was significantly higher than that in semi-granulocytes and agranulocytes, which was about 2.04-fold (p < 0.05) of the average level of hemocytes. Immunofluorescence assay further revealed that CgSPSB3 protein was mainly distributed in the cytoplasm of granulocytes. The mRNA expression of CgSPSB3 in hemocytes was up-regulated after lipopolysaccharide (LPS) and Vibrio splendidus stimulations. The mRNA expression of CgIFNLP, CgIL17-5 and CgTNF-1 decreased significantly (p < 0.05) at 24 h after the CgSPSB3 mRNA was knocked down by RNAi. These results collectively indicated that CgSPSB3 might play an important role in regulating cytokines production in granulocytes of C. gigas.

Prostaglandins (PGs) are highly reactive small lipophilic molecules derived from polyunsaturated fatty acids of the cell membrane and play a key role in the resolution of inflammation processes. 15-deoxy-Δ12,14-PGJ2 (15dPGJ2) is a cyclopentenone PG (CyPG) of the J series with anti-inflammatory, anti-proliferative and pro-apoptotic effects. This CyPG can signal through: (i) the PGD2 receptor (DP2) and peroxisome proliferator-activated receptor γ (PPARγ) or (ii) by covalent binding to protein nucleophiles, such as, thiols groups of cysteine, lysine or histidine via a Michael addition reaction, modifying its structure and function. In this work we show that acidophilic granulocytes (AGs) of gilthead seabream (Sparus aurata L.), the functional equivalent to mammalian neutrophils, constitutively expressed ppara, pparb and pparg genes, the latter showing the highest expression and up-regulation when stimulated by bacterial DNA. In addition, we tested the ability of 15dPGJ2, and its biotinylated analog, as well as several PPARγ ligands, to modulate reactive oxygen species (ROS) and/or cytokines production during a Toll like receptor (TLR)-mediated granulocyte response. Thus, 15dPGJ2 was able to significantly decrease bacterial DNA-induced ROS production and transcript levels of pparg, interleukin-1β (il1b) and prostaglandin-endoperoxide synthase 2 (ptgs2). In contrast, its biotinylated analog was less potent and a higher dose was required to elicit the same effects on ROS production and cytokine expression. In addition, different PPARγ agonists were able to mimic the effects of 15dPGJ2. Conversely, the PPARγ antagonist T007097 abolished the effect of 15dPGJ2 on DNA bacterial-induced ROS production. Surprisingly, transactivation assays revealed that both 15dPGJ2 and its biotinylated analog signaled via Pparα and Pparβ, but not by Pparγ. These results were further confirmed by HPLC/MS analysis, where Pparβ was identified as an interactor of biotin-15dPGJ2 in naïve and DNA-stimulated leukocytes. Taken together, our data show that 15dPGJ2 acts both through Ppar activation and covalent binding to proteins in fish granulocytes and identify for the first time in vertebrates a role for Pparα and Pparβ in the resolution of inflammation mediated by 15dPGJ2.

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are related cytokines that signal through receptors possessing the β common (βc) chain. As a family, these cytokines combine rather non-specific hematopoietic growth factor properties with a special importance for eosinophils, basophils, and mast cells. In fish the cytokines of this family are called IL-5fam, and the present study, using carp, constitutes their first functional analysis. Carp il-5fam expression was enhanced by stimulation with phytohemagglutinin and killed bacteria. Reminiscent of mammalian IL-3/IL-5/GM-CSF family members, recombinant carp IL-5fam (rcIL-5fam) induced activation of transcription factor STAT5 and efficiently promoted proliferation and colony-formation of eosinophil/basophil/mast-cell type (EBM) granulocytes. Upon addition of recombinant carp βc the growth effect of rcIL-5fam was reduced, suggesting βc participation in the signaling route. In summary, despite differences in individual cytokines and cell populations, fish and mammalian IL-3/IL-5/GM-CSF family members share growth factor functions for non-neutrophil granulocytes.

In our previous study, the differentially expressed proteins have been identified by proteomic analysis in total haemocytes of shrimp (Fenneropenaeus chinensis) after white spot syndrome virus (WSSV) infection. To further investigate the differential response of haemocyte subpopulations to WSSV infection, granulocytes and hyalinocytes were separated from healthy and WSSV-infected shrimp by immunomagnetic bead (IMB) method, respectively. Then two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to analyze the differentially expressed proteins in haemocyte subpopulations between healthy and WSSV-infected shrimp. The results of flow cytometry (FCM) showed that about 98% of granulocytes and about 96% of hyalinocytes in purity were obtained. Quantitative intensity analysis revealed that 26 protein spots in granulocytes and 24 spots in hyalinocytes were significantly changed post WSSV infection. Among them, 24 proteins in granulocytes and 23 proteins in hyalinocytes were identified by MS analysis, which could be divided into eight categories according to Gene Ontology. The identification of prophenoloxidase (proPO), proPO 2 and peroxiredoxin in WSSV-infected granulocytes was consistent with the facts that the proPO-activating system and peroxiredoxin were mainly existed in granulocytes. The phagocytosis of hyalinocytes seemed to be enhanced during the infection, because several proteins that involved in phagocytosis, including clathrin heavy chain, ADP ribosylation factor 4 and Alpha2 macroglobulin were up-regulated in hyalinocytes upon WSSV infection. Our results also reflected the vital biological significance of calcium ion binding proteins in granulocytes and ATPase/GTPase in hyalinocytes during WSSV infection. The data in this study verified the roles of granulocytes and hyalinocytes involved in WSSV infection, and differentially expressed proteins identified in granulocytes and hyalinocytes had a close correlation with their function characteristics.

Carp head kidney (HK) phagocytes can be stimulated by lipopolysaccharide (LPS) to produce nitric oxide (NO). High production of NO can suppress the carp immune system. Carp peripheral blood leukocytes (PBL) are highly susceptible but HK phagocytes are relatively resistant to the immunosuppressive effects of NO. This study demonstrates that the antioxidant glutathione plays an important role in the protection against nitrosative stress. Carp HK phagocytes, especially the neutrophilic granulocytes, contain higher levels of glutathione than PBL. Moreover, freshly isolated carp neutrophilic granulocytes have higher mRNA levels than PBL of glucose-6-phosphate dehydrogenase (G6PD), manganese superoxide dismutase (MnSOD) and gamma-glutamylcysteine synthetase (gamma-GCS). Since these molecules are part of the glutathione redox cycle, neutrophilic granulocytes have a higher capacity than PBL to maintain glutathione in a reduced state following nitrosative stress. When stimulated with LPS, neutrophilic granulocytes upregulate the expression of G6PD, MnSOD and gamma-GCS.

Hemocytes in shrimp play important roles in innate immune responses against pathogens. Although three types of hemocytes including hyalinocytes, semi-granulocytes and granulocytes were identified based on their morphological characters in penaeid shrimp, knowledge about the molecular basis of their functions in the immunity is still very limited. In the present study, three subpopulations of hemocytes were firstly separated by Percoll gradient centrifugation, and their transcriptomes were analyzed. The data showed that significantly differential gene expression patterns existed in different types of hemocytes. The genes encoding phagocytic receptors, lectins and actin cytoskeleton involved in phagocytosis were highly expressed in hyalinocytes, while genes involved in the humoral immunity signaling pathways were highly expressed in semi-granulocytes, and genes encoding prophenoloxidase (proPO)-activating enzyme and serine proteases involved in proPO system activation were highly expressed in granulocytes. Further flow cytometry analysis indicated that hyalinocytes were the main hemocytes subpopulation responsible for ingesting foreign fluorescent beads, and this ingestion process mainly depends on the endocytic way of macropinocytosis. These data provide valuable information for understanding the molecular basis of distinct shrimp hemocytes subpopulations of shrimp in cellular and humoral immunity.

The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 μm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 μm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes.

We studied a predictive model of gene expression induced by mechanical injury of fish skin, to resolve the confounding effects on the immune system induced by injury and skin parasite-specific molecules. We applied real time quantitative PCR (RQ-PCR) to measure the expression of the pro-inflammatory cytokines CXCa, CXCb, interleukin (IL)1-beta, tumor necrosis factor alpha (TNFalpha), and the receptors IL1R1, CXCR1 and CXCR2 in skin of Cyprinus carpio after mechanical injury. We also studied the expression of the anti-inflammatory cytokine IL-10. Most obvious, specific up-regulation of the chemokine CXCa, the chemokine receptor CXCR1 and the pro-inflammatory cytokine IL-beta was detected at 2-3h after injury. In order to correlate gene expression patterns after injury with cell migration, we studied chemotaxis of head kidney leukocytes towards lysates of epithelioma papulosum cyprini (EPC) cells. Neutrophilic granulocytes were shown to migrate towards epithelial lysates. Using immunohistochemistry we observed that the early inflammatory response after injury involved an influx of cells, most probably neutrophilic granulocytes, into the injured area. This suggests that the increased expression of pro-inflammatory genes is related to a rapid influx of neutrophilic granulocytes.

Cathepsin L1 (CTSL1) is a lysosomal cysteine protease with a papain-like structure. It is known to be implicated in multiple processes of immune response against pathogen infection based on the proteolytic activity. In the present study, a CTSL1 homologue (designated as CgCTSL1) was identified from Crassostrea gigas. It contained a typically single Pept_C1 domain with three conserved catalytically essential residues (Gln25, His135 and Asn178). The mRNA of CgCTSL1 was ubiquitously expressed in oyster tissues with the highest expression level in important immune tissues such as gill and hemocytes. CgCTSL1 proteins were mainly detected in gill and hepatopancreas by immunohistochemistry. Recombinant CgCTSL1 (rCgCTSL1) exhibited proteolytic activity to cleave the substrate Ac-FR-amino-4-trifluoromethyl coumarin (AFC) in a dose-dependent manner, and the inhibitor could reduce its proteolytic activity. After the interference of CgCTSL1 mRNA, the proteolytic activity of oyster hemocytes was significantly down-regulated with the released AFC fluorescence value decreasing from 375.84 to 179.21 (p < 0.05). Flow cytometry analysis revealed that the expression of CgCTSL1 protein was higher in phagocytes with the mean fluorescence intensity (MFI) value of 21,187 (4.13-fold, p < 0.01) compared to the MFI value of 5,130 in non-phagocytic hemocytes. The further confocal analysis demonstrated that the actively phagocytic hemocytes with green bead signals were co-localized with stronger CgCTSL1 positive signals. The mRNA expression levels of CgCTSL1 in phagocyte-like sub-populations of granulocytes and semi-granulocytes were 298.12-fold (p < 0.01) and 2.75-fold (p < 0.01) of that in agranulocytes, respectively. Western blotting analysis of the hemocyte proteins revealed that CgCTSL1 was relatively abundant in granulocytes and semi-granulocytes compared to that in agranulocytes. These results collectively suggested that CgCTSL1, a CTSL1 homologue highly expressed in phagocyte-like hemocytes, was possibly involved in cellular immune response dependent on its conserved proteolytic activity, which might provide clues for the divergence between phagocytes and non-phagocytic hemocytes as well as the identification of promising molecular markers for phagocytes in oyster C. gigas.

Hematopoiesis is the biological process to generate new blood cells in the living body and reactive oxygen species (ROS) contribute significantly to the regulation of haematopoietic cell homeostasis. In the present study, the involvement of ROS in the proliferation of haemocytes was examined in Pacific oyster Crassostrea gigas. The ROS content in haemocytes increased significantly after lipopolysaccharide (LPS) treatment, but decreased after the treatment with antioxidant N-Acetyl-L-cysteine (NAC, a scavenger of ROS). The percentage of 5-ethynyl-2'-deoxyuridine labeled (EdU+) granulocytes in total haemocytes significantly increased at 12 h (4.12-fold, p < 0.001) and 24 h (2.36-fold, p < 0.001) after LPS treatment, while decreased at 12 h (0.26-fold, p < 0.001) and 24 h (0.61-fold, p < 0.05) after NAC treatment, respectively. Meanwhile, the percentage of haemocytes with autophagosome positive signals significantly increased at 12 h (1.17-fold, p < 0.01) and 24 h (1.19-fold, p < 0.05) after LPS treatment, but significantly reduced at 12 h (0.41-fold, p < 0.001) and 24 h (0.28-fold, p < 0.001) after the NAC treatment, respectively. After ammonium chloride (NH4Cl) treatment, the percentage of haemocytes with autophagosome and EdU+ granulocytes significantly increased at 12 h, which was 1.27-fold (p < 0.01) and 1.70-fold (p < 0.01) of control group, respectively. These results collectively suggested that ROS produced after LPS treatment could act as an inducer for autophagy and involved in regulating the proliferation of some granulocytes in C. gigas.

Numerous CXC chemokines have been identified in fish, however, their role in inflammation is not well established. Here, CXC chemokines of the CXCL8-like (CXCa_L1 and CXCL8_L2) and CXCL9/10/11-like (CXCb) subset were investigated in carp. Recombinant CXCa_L1, CXCL8_L2 and CXCb all stimulated chemotaxis of macrophages and granulocytes in vitro. CXCb also attracted lymphocytes. Distinct effects on phagocyte activation were observed: the CXCL8-like chemokines increase respiratory burst activity, but not nitrite production. The three chemokines differentially induced a moderate increase in IL-1β, CXCa_L1 and CXCL8_L2 gene expression. Intracellular calcium mobilization in granulocytes upon CXCa_L1 stimulation implies signal transduction through G-protein coupled CXC receptors. Notably, upon intraperitoneal administration, carp CXCL8-like chemokines strongly induced in vivo leukocyte recruitment, including neutrophils and monocytes/macrophages, in contrast to CXCb, for which the number of recruited leukocytes was low. The results indicate functional homology for carp CXCL8-like and CXCb chemokines with mammalian CXCL8 and CXCL9-11, respectively.

Hemocyte populations from the ascidian Ciona robusta, separated through a Percoll discontinuous density gradient, are further characterized by May-Grünwald-Giemsa staining and a cytochemical reaction for phenoloxidase. Variability in cell density, acidophilic property and phenoloxidase activity suggest multiple hemocyte type populations, cell lineages and morphotypes that may be involved in distinct cellular responses. Therefore, unilocular refractile granulocytes, typical of this ascidian species, enriched in a fraction separated from the hemolymph show in vitro phenoloxidase-dependent cytotoxic activity against mammalian erythrocytes and a tumor cell lineage, in addition the properties listed above indicate relationships with vacuolated signet ring cells. Finally, bromo-deoxyuridine with, diamino-phenylindole fluorescent reaction and May-Grünwald-Giemsa staining show that in the hemolymph there are hyaline amoebocytes and granulocytes with potential proliferating activity. Present findings and reviewed images of previously reported inflammatory hemocytes in the tunic and pharynx allow us to speculate on theoretical outlines of hemocyte differentiation pathways.

Dopamine β-hydroxylase (DBH) is one of key rate-limiting enzymes converting dopamine to norepinephrine. It locates not only in catecholaminergic neuron system, but also in immunocytes and plays roles in the immune response of vertebrates. However, the knowledge about the function of DBH in immune system is still very limited in invertebrates. In the present study, the DBH gene family with seven members was screened from Crassostrea gigas genome, and their mRNA expressions in various tissues were recorded. Among them, one DBH (designated CgDBH-1) with high expression level in oyster hemocytes was further characterized. The deduced amino acid sequence of CgDBH-1 was predicted to contain a transmembrane domain and shared 30.1% and 30.9% similarity with that in Mus musculus and Homo sapiens, respectively. CgDBH-1 was closely clustered with DBH from Aplysia californica in the phylogenetic tree. The recombinant protein of CgDBH-1 (rCgDBH-1) exhibited significant enzymatic activity (0.54 ± 0.019 pmol L-1 min-1) to synthesize norepinephrine. Importantly, the mRNA transcript of CgDBH-1 was highly expressed in oyster hemocytes, and the highest expression level was observed in granulocytes among the three types of hemocytes, which was 8.18-fold (p < 0.01) of that in agranulocytes. Moreover, the expression of CgDBH-1 in hemocytes was significantly increased at the late stage of immune response. The CgDBH-1 protein was mainly co-localized with the granules and endoplasmic reticulum (ER) of granulocytes. These results collectively suggested that CgDBH-1, as a novel molluscan norepinephrine synthesizing enzyme highly expressed in granulocytes, involved in the late-stage immune response of oysters, which provided vital insight to understand the crosstalk between neuroendocrine and immune systems in invertebrates.

This paper presents data from an investigation of the mode of action of five different crustacean immunostimulants presented to European lobster (Homarus gammarus) granulocytes cultured in vitro. The experiments were designed to test whether or not the immunostimulants could cause the short-term up-regulation of genes coding for immune proteins without causing the cells to degranulate. Quantitative measurements of mRNA transcript abundance were made using real-time PCR and it was first necessary to isolate the complete gene sequences coding for the proteins prophenoloxidase (proPO), beta-1,3-glucan binding protein (betaGBP) and beta-actin (beta-act) in the lobster. These sequences were used to design TaqMan primer and fluorescent probe sets. The presented data indicated that the majority of the tested immunostimulants did not induce the up-regulation of immune-related gene expression in the granulocytes in isolation. Alternative modes of action, including the in vivo up-regulation of gene expression in haemopoetic tissues, are discussed.

Prostaglandin E2 (PGE2) plays an important role in immune activities in teleost fish, including seabream. However, receptors involved in PGE2 signaling, as well as the pathways activated downstream, are largely unknown. In this study, one ortholog of mammalian PTGER1, PTGER3 and PTGER4, and two of PTGER2 (Ptger2a and Ptger2b) were identified and characterized in gilthead seabream. In silico analysis showed that all these receptors possessed the organization domain of G protein-coupled receptors, with the exception of Ptger2b. The corresponding in vivo studies revealed that they were expressed in all the tissues examined, the highest mRNA levels of ptger1 and ptger3 being observed in the spleen and of ptger2a and ptger4 in the blood. Bacterial infection induced higher mRNA levels of ptger2a, ptger3 and ptger4 in peritoneal exudate (the site of bacterial injection). In addition, head kidney acidophilic granulocytes and macrophages displayed different ptger1, ptger2a, ptger3 and ptger4 expression profiles. Furthermore, in macrophages the expression of the receptors was weakly affected by stimulation with bacterial DNA or with PGE2, while in acidophilic granulocytes stimulation resulted in the upregulation of ptger2a and ptger4. Taken together, these results suggest different roles for seabream PGE2 receptors in the regulation of the immune responses.

Adjuvants have emerged as the best tools to enhance the efficacy of vaccination. However, the traditional adjuvants used in aquaculture may cause adverse alterations in fish making necessary the development of new adjuvants able to stimulate the immune system and offer strong protection against infectious pathogens with minimal undesirable effects. In this respect, flagellin seems an attractive candidate due to its ability to strongly stimulate the immune response of fish. In the present study, we have evaluated the ability of recombinant flagellin from Marinobacter algicola (MA) and Vibrio vulnificus (Vvul), a non-pathogenic and a pathogenic bacteria, respectively, to stimulate the innate immune system of gilthead seabream (Sparus aurata L.) and compare the effect with that of the classical flagellin from Salmonella enterica serovar Typhimurium (Salmonella Typhimurium, STF). Intraperitoneal injection of MA and Vvul resulted in a strong inflammatory response characterized by increased reactive oxygen species production and the infiltration of acidophilic granulocytes at the injection site. Interestingly, however, only flagellin from MA consistently induced the expression of the gene encoding pro-inflammatory interleukin-1β. These effects were further confirmed in vitro, where a dose-dependent activation of macrophages and acidophilic granulocytes by MA and Vvul flagellins was observed. In contrast, STF flagellin was found to be less potent in both in vivo and in vitro experiments. Our results suggest the potential use of MA and Vvul flagellins as immunostimulants and adjuvants for fish vaccination.