Novel insights into immune stress markers associated with myxosporeans gill infection in Nile tilapia (molecular and immunohistochemical studies).

Nile tilapia (Oreochromis niloticus) is valued in aquaculture because of its quick development and ability to thrive in various environments. Myxosporeans are among the fish parasites that affect fish productivity, as they impact fish growth and reproduction, resulting in large fish deaths in farms and hatcheries. This study has been focused on morpho-molecular identification for the myxosporean parasites infecting Nile tilapia from three governorates in Egypt and assessment of gene expression of different cytokines (Interleukin-1ßeta (IL-1ß), major histocompatibility complex class II (MHC-II), and clusters of differentiation 4 (CD-4) and 8 (CD-8)) in tissues. Additionally, this work aimed to correlate the developed histopathological alterations and inflammatory reactions in gills with immunohistochemical expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α). Finally, the infected fish's cortisol levels and blood glucose were assessed. Results of BLAST sequence analysis of the 18S rRNA for the collected protozoans confirmed Myxobolus agolus, M. brachysporus, M. tilapiae, and Henneguya species. The molecular characterization of the immunological status of gills revealed marked upregulation of different inflammatory cytokines in the gills of infected fish. There was a significantly increased serum cortisol and glucose level in infected fish compared with control, non-infected ones. Severe histopathological alterations were observed in the infected fish gills, associated with increased expression of iNOS and TNF-α and related to myxosporean infection. The present study provides new insights into oxidative stress biomarkers in Nile tilapia infected with Myxosporeans and elucidates the gill's immune status changes as a portal of entry for protozoa that contribute to tissue damage.

Mucosal barrier status in Atlantic salmon fed rapeseed oil and Schizochytrium oil partly or fully replacing fish oil through winter depression.

The study was designed to investigate the effects of replacing fish oil by algal oil and rapeseed oil on histomorphology indices of the intestine, skin and gill, mucosal barrier status and immune-related genes of mucin and antimicrobial peptide (AMP) genes in Atlantic salmon (Salmo salar). For these purposes, Atlantic salmon smolts were fed three different diets. The first was a control diet containing fish oil but no Schizochytrium oil. In the second diet, almost 50 % of the fish oil was replaced with algal oil, and in the third diet, fish oil was replaced entirely with algal oil. The algal oil contained mostly docosahexaenoic acid (DHA) and some eicosapentaenoic acid (EPA). The study lasted for 49 days in freshwater (FW), after which some fish from each diet group were transferred to seawater (SW) for a 48-h challenge test at 33 ppt to test their ability to tolerate high salinity. Samples of skin, gills, and mid intestine [both distal (DI) and anterior (AI) portions of the mid intestine] were collected after the feeding trial in FW and after the SW-challenge test to assess the effects of the diets on the structure and immune functions of the mucosal surfaces. The results showed that the 50 % VMO (Veramaris® algal oil) dietary group had improved intestinal, skin, and gill structures. Principal component analysis (PCA) of the histomorphological parameters demonstrated a significant effect of the algal oil on the intestine, skin, and gills. In particular, the mucosal barrier function of the intestine, skin, and gills was enhanced in the VMO 50 % dietary group after the SW challenge, as evidenced by increased mucous cell density. Immunolabelling of heat shock protein 70 (HSP70) in the intestine (both DI and AI) revealed downregulation of the protein expression in the 50 % VMO group and a corresponding upregulation in the 100 % VMO group compared to 0 % VMO. The reactivity of HSP70 in the epithelial cells was higher after the SW challenge compared to the FW phase. Immune-related genes related to mucosal defense, such as mucin genes [muc2, muc5ac1 (DI), muc5ac1 (AI), muc5ac2, muc5b (skin), and muc5ac1 (gills)], and antimicrobial peptide genes [def3 (DI), def3 (AI), and cath1 (skin)] were significantly upregulated in the 50 % VMO group. PCA of gene expression demonstrated the positive influences on gene regulation in the 50 % VMO dietary group. In conclusion, this study demonstrated the positive effect of substituting 50 % of fish oil with algal oil in the diets of Atlantic salmon. The findings of histomorphometry, mucosal mapping, immunohistochemistry, and immune-related genes connected to mucosal responses all support this conclusion.

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

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

Upregulation of polymeric immunoglobulin receptor expression in flounder (Paralichthys olivaceus) gill cells by cytokine tumor necrosis factor-α via activating PI3K and NF-κB signaling pathways.

The polymeric immunoglobulin receptor (pIgR) transports secretory immunoglobulins across mucosal epithelial cells into external secretions, playing critical roles in mucosal surface defenses, but the regulation mechanism of pIgR expression is not clarified in teleost fish. In this study, the dynamic changes of flounder (Paralichthys olivaceus) pIgR (fpIgR) and pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) mRNA expression in mucosal tissues were first analyzed post inactivated Vibrio anguillarum immunization, and increased production of TNF-α was found to correlate with increased expression of fpIgR. To determine that cytokine TNF-α influenced fpIgR expression, following confirming that natural fpIgR expressed on flounder gill (FG) cells, FG cells were incubated with various concentrations of recombinant TNF-α for different time, the results showed that the expressions of fpIgR were significantly upregulated at gene and protein levels in a dose-dependent and time-dependent manner, and similar change trend was observed for free secretory component (SC) secreted by fpIgR into the culture supernatant. After FG cells were treated with TNF-α, specific phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, nuclear factor kappa-B (NF-κB) inhibitor Bay11-7082, and the mixtures of TNF-α and wortmannin / Bay11-7082 respectively, the fpIgR protein and mRNA levels, together with SC secretion, obviously decreased in wortmannin- and Bay11-7082-treated cells compared with the untreated control, and cotreatment with wortmannin / Bay11-7082 plus TNF-α resulted in lower expression compared with that upon treatment with TNF-α alone, indicating that the inhibition of PI3K and NF-κB both blocked the ability of TNF-α to increase cellular fpIgR and SC levels. Furthermore, the gene expressions of PI3K and NF-κB were upregulated and present a tendency to increase first and then decrease after TNF-α treatment of FG cells; However, the expression of PI3K mRNA was inhibited significantly by wortmannin but not by Bay11-7082, and the expression of NF-κB mRNA was suppressed obviously by Bay11-7082 but not by wortmannin, suggesting that inhibition of PI3K or NF-κB had no influence on each other. All these results collectively revealed that TNF-α could transcriptionally upregulate fpIgR expression and SC production, and this TNF-α-induced pIgR expression was regulated by complex mechanisms that involved PI3K and NF-κB signaling pathways, which provided evidences for pro-inflammatory cytokine TNF-α acting as a regulator in pIgR expression and better understanding of regulation mechanism of pIgR expression in teleost fish.

Cadmium-induced Oxidative Stress and Immunosuppression Mediated Mitochondrial Apoptosis via JNK-FoxO3a-PUMA pathway in Common Carp (Cyprinus carpio L.) Gills.

Cadmium (Cd)-caused water environment pollution has become a matter of concern. Gill is an organ with respiratory and mucosal immune functions, and is also one of the organs directly attacked by pollutants. It was found that excess Cd could cause Cd accumulation and gill injury in carp. However, the mechanism of Cd-caused damage in common carp gills is still unclear. Oxidative stress, immunosuppression, and apoptosis took part in the mechanism of poisoning caused by some harmful substances. The aim of the study was to investigate complex molecular mechanism of apoptotic injury caused by Cd in common carp gills. Hence, in this study, we established a Cd poisoning model to explore whether excess Cd can induce apoptosis through observing histomorphology and apoptotic cells; and determining mineral elements, oxidative stress-related factors, immune-related, and apoptosis-related genes in common carp gills. Fifty-four fish were randomly separated into the control group and the Cd group and were cultured for 45 days. The water of the control group was drinking water and the water of the Cd group was CdCl2-added drinking water (0.26 mg/L Cd). In our results, we found that excess Cd increased Cd level, decreased the levels of essential mineral elements (Cu, Fe, Zn, and Mn), damaged mitochondria, and increased apoptotic cells in common carp gills, meaning that excess Cd caused Cd accumulation and apoptotic injury via mitochondrion in common carp gills. Furthermore, we found that Cd inhibited anti-apoptosis-related gene Bcl-2 and stimulated pro-apoptosis-related genes (JNK, FoxO3a, PUMA, Bax, Apaf-1, Caspase-9, and Caspase-3) on 15th, 30th, and 45th days. Above data meant that Cd exposure caused apoptosis via mitochondrion and JNK-FoxO3a-PUMA pathway in common carp gills. In addition, in our experiment, Cd treatment increased oxidants (H2O2 and MDA) and decreased antioxidants (CAT, GPx, GST, SOD, T-AOC, and GSH), indicating that Cd caused oxidative stress via oxidation/antioxidation imbalance. Meanwhile, compared to the control group, T-help 17 (Th17) cell-related factors (IL-17, TNF-α, and RORγ) were up-regulated, regulatory T (Treg) cell-related factors (IL-10 and Foxp3) were down-regulated, and IL-17/IL-10, TNF-α/IL-10, and RORγ/Foxp3 were increased in Cd-exposed group; meaning that excess Cd induced immunosuppression via the imbalance of Th17/Treg cells. Taken together, our findings indicated that JNK-FoxO3a-PUMA pathway and mitochondrion participated in oxidative stress and immunosuppression-mediated apoptosis caused by Cd in common carp (Cyprinus carpio L.) gills. Our data provided new perspectives on the negative effects of heavy metal pollutants on fish.

An overview of the structural and functional aspects of immune cells in teleosts.

The immune system of fish consists of two main components, innate and adaptive immunities. Innate immunity is non-specific and acts as the primary line of protection against pathogen invasion, while adaptive immunity is more specific to a certain pathogen/following adaptation. The adaptive immune system consists of the humoral and cellular components. Cytotoxic T-lymphocyte cells are the major component of the cellular immunity that frequently kills viral-, bacterial- or parasitic-infected cells. According to the anatomical location, the mucosal-associated lymphoid tissue (MALT) in teleost fish subdivides into gut-associated lymphoid tissue (GALT), gill-associated lymphoid tissue (GIALT), and skin-associated lymphoid tissue (SALT). The MALTs contain various leukocytes; including, but not limited to, lymphocytes (T and B cells), plasma cells, macrophages, and granulocytes. Macrophages are multifunctional cells that are mainly involved in the immune response, including; phagocytosis and degradation of foreign antigens, tissue remodeling, and production of cytokines, chemokines and growth factors. An interesting feature of teleost macrophages is their ability to form melanomacrophage centers (MMC) in the hemopoietic tissues. Dendritic cells, rodlet cells, mast cells, eosinophilic granular cells (ECGs), telocytes, osteoclasts, club cells, as well as, barrier cells have been recorded in many fish species and have many immunological roles. This paper aims to summarize the current knowledge of the immune cells present in fish tissues serving as anatomical and physiological barriers against external hazards. Increased knowledge of fish immune systems will facilitate the development of novel vaccination strategies in fish.

Polarized Trout Epithelial Cells Regulate Transepithelial Electrical Resistance, Gene Expression, and the Phosphoproteome in Response to Viral Infection.

The burden of disease is a major challenge in aquaculture production. The fish gill characterized with a large surface area and short route to the bloodstream is a major environmental interface and a significant portal of entry for pathogens. To investigate gill responses to viral infection the salmonid gill cell line RTgill-W1 was stimulated with synthetic dsRNA and the salmonid alphavirus subtype 2 (SAV-2). Epithelial integrity in polarized cells can be measured as transepithelial electrical resistance (TEER) which is defined as the electrical resistance across a cell monolayer. TEER is a widely accepted quantitative measure of cellular integrity of a cell monolayer. TEER increased immediately after stimulation with the synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I:C)). In parallel, tight junction and gene expression of innate immune activation markers was modulated in response to poly(I:C). The SAV-2 virus was found to replicate at a low level in RTgill-W1 cells where TEER was disturbed at an early stage of infection, however, gene expression related to tight junction regulation was not modulated. A strong poly(I:C)-driven antiviral response was observed including increases of Rig-like receptors (RLRs) and interferon stimulating genes (ISGs) mRNAs. At the level of signal transduction, poly(I:C) stimulation was accompanied by the phosphorylation of 671 proteins, of which 390 were activated solely in response to the presence of poly(I:C). According to motif analysis, kinases in this group included MAPKs, Ca2+/calmodulin-dependent kinase (CaMK) and cAMP-dependent protein kinase (PKA), all reported to be activated in response to viral infection in mammals. Results also highlighted an activation of the cytoskeletal organization that could be mediated by members of the integrin family. While further work is needed to validate these results, our data indicate that salmonid gill epithelia has the ability to mount a significant response to viral infection which might be important in disease progression. In vitro cell culture can facilitate both a deeper understanding of the anti-viral response in fish and open novel therapeutic avenues for fish health management in aquaculture.

Distribution of Mpeg1+ cells in healthy grouper (Epinephelus coioides) and after Cryptocaryon irritans infection.

Cryptocaryon irritans is an extremely harmful ciliated obligate parasite that is responsible for large economic losses in aquaculture. C. irritans infection can cause an insect-resistant immune response in fish, and many immune cells can be observed in the local infection site. However, it is unclear whether macrophages are involved in the host defense against C. irritans infection. The Mpeg1 protein can form pores and destroy the cell membrane of invading pathogens, and is also used as a macrophage-specific marker in mammals. Therefore, a polyclonal antibody against grouper recombinant Mpeg1a was produced to mark macrophages in this study, which could recognize both isoforms of Mpeg1 (Mpeg1a/b). Immunofluorescence revealed that EcMpeg1 positive cells were mostly distributed in the head kidney and spleen in healthy grouper. Immunofluorescence and immunohistochemistry showed that the number of EcMpeg1 positive cells increased in the gills after infection with C. irritans, implying that EcMpeg1 positive cells may be involved in the process of grouper resistance against C. irritans infection.

Involvement of a novel Ca2+-independent C-type lectin from Sinonovacula constricta in food recognition and innate immunity.

Bivalve lectins perform a crucial function in recognition of foreign particles, such as microalgae and pathogenic bacteria. In this study, a novel C-type lectin form Sinonovacula constricta (ScCL) was characterized. The full-length cDNA of ScCL was 1645 bp, encoding a predicted polypeptide of 273 amino acids with one typical carbohydrate-recognition domain. ScCL has the highest similarity and closest phylogenetic relationship with the C-type lectin from Solen grandis. Real-time PCR analysis showed that ScCL was expressed in all tested tissues, with the highest expression in the foot and the lowest expression in hemocytes. Agglutination activity of ScCL was Ca2+-independent. ScCL showed the strongest agglutination on Chlorella vulgaris, the modest agglutination on Platymonas subcordiformis, Nannochloropsis sp., and Thalassiosira pseudonana, the weakest agglutination on Chaetoceros sp., and no agglutination on Isochrysis zhanjiangensis. Meanwhile, agglutination tests and western blot analysis revealed that the recombinant ScCL protein could agglutinate Staphylococcus aureus and Vibrio harveyi, but could not agglutinate Vibrio anguillarum, Bacillus cereus, or Vibrio parahaemolyticus. Furthermore, ScCL had a high binding activity with LPS and mannose, a low binding activity with LTA, and no binding activity with PGN. The expression of ScCL in the gill of S. constricta fed with C. vulgaris and T. pseudonana was significantly increased at 1 and/or 3 h. After injection with S. aureus, the expression of ScCL in the gill was significantly increased at 3, 6, and 24 h. These results indicated that ScCL was involved in food particle recognition and immunity of S. constricta.

Osmoregulatory performance and immunolocalization of Na+/K+-ATPase in the branchiopod Artemia salina from the Sebkha of Sidi El Hani (Tunisia).

Artemia salina is an extremophile species that tolerates a wide range of salinity, especially hypertonic media considered lethal for the majority of other aquatic species. In this study, A. salina cysts were hatched in the laboratory and nauplii were acclimated at three different salinities (60, 139 and 212 ppt). Once in the adult phase, their hemolymph osmolality was measured. The animals were strong hypo-osmoregulators in the entire range of tested salinities, with up to 10 fold lower hemolymph osmolalities than their surrounding environment. Immunostaining of Na+/K+-ATPase was done on sections and on whole body mounts of adults in order to localize the ionocytes in different organs. An intense Na+/K+-ATPase immunostaining throughout the cells was observed in the epithelium of the ten pairs of metepipodites. A positive immunoreactivity for Na+/K+-ATPase was also detected in the maxillary glands, in the epithelium of the efferent tubule and of the excretory canal, as well as in the anterior digestive tract. This study confirms the strong hypo-osmotic capacity of this species and affords an overview of the different organs involved in osmoregulation in A. salina adults.

Molecular and immunological characterizations of interleukin-11 in large yellow croaker (Larimichthys crocea).

Interleukin (IL)-11 is a multifunctional cytokine that exerts a series of important immunomodulatory effects and exists in many tissues and cells. A 1106-bp nucleotide sequence representing the complete cDNA of IL-11 was obtained from large yellow croaker (Larimichthys crocea), containing an open reading frame (ORF) of 603 bp encoding for 200 amino acids (aa). The predicted LcIL-11 protein included a 12aa signal peptide and a conserved IL-11 domain. The polypeptide sequence identities between LcIL-11 and its counterparts in mammals and other fish are from 84% to 92% with known fish IL-11a and 22%-27% with fish IL-11b. LcIL-11 mRNA existed in most tissues with the most predominant expression in the gill. After immune challenge, the expression levels of LcIL-11 were induced largely in vivo and in vitro, with the peak-value of 32 times as much as the control in the liver at 24 h after Vibrio parahaemolyticus injection (p < 0.05) and the greatest value of 13.9 times as much as the control in LCK cells at 12 h after poly I:C stimulation (p < 0.05). Furthermore, the overexpression vector pcDNA3.1-LcIL-11 was constructed and transfected to LCK cells. Our results showed that the transcriptional expression levels of tumor necrosis factor (TNF)-α and myxovirus resistant protein (Mx) significantly up-regulated in LCK cells after LcIL-11 overexpression (p < 0.05). However, no significant changes of IL-1ß, janus kinase (JAK)2 and signal transducers and activators of transcription (STAT)5 was detected. Our finding indicated that LcIL-11 might enhance TNF-α and antiviral protein Mx expression in large yellow croaker.

Identification of a Novel Pattern Recognition Receptor DM9 Domain Containing Protein 4 as a Marker for Pro-Hemocyte of Pacific Oyster Crassostrea gigas.

DM9 refers to an uncharacterized protein domain that is originally discovered in Drosophila melanogaster. Two proteins with DM9 repeats have been recently identified from Pacific oyster Crassostrea gigas as mannose-specific binding pattern-recognition receptors (PRRs). In the present study, a novel member of DM9 domain containing protein (designated as CgDM9CP-4) was identified from C. gigas. CgDM9CP-4, about 16 kDa with only two tandem DM9 domains, was highly enriched in hemocytes and gill. The transcripts level of CgDM9CP-4 in circulating hemocytes were decreased after LPS, PGN and Vibrio splendidus stimulations. The recombinant protein of CgDM9CP-4 (rCgDM9CP-4) displayed a broad binding spectrum towards various pathogen-associated molecular patterns (PAMPs) (LPS, PGN, ß-glucan and Mannose) and microorganisms (Staphylococcus aureus, Micrococcus luteus, V. splendidus, V. anguillarum, Escherichia coli, Pichia pastoris and Yarrowia lipolytica). CgDM9CP-4 was mostly expressed in gill and some of the hemocytes. Flow cytometry analysis demonstrated that the CgDM9CP-4-positive hemocytes accounted for 7.3% of the total hemocytes, and they were small in size and less in granularity. CgDM9CP-4 was highly expressed in non-phagocytes (~82% of total hemocytes). The reactive oxygen species (ROS) and the expression levels of cytokines in CgDM9CP-4-positive hemocytes were much lower than that in CgDM9CP-4-negative hemocytes. The mRNA expression level of CgDM9CP-4 in hemocytes was decreased after RNAi of hematopoietic-related factors (CgGATA, CgRunt, CgSCL, and CgNotch). In addition, CgDM9CP-4-positive cells were found to be much more abundant in hemocytes from gill than that from hemolymph, with most of them located in the gill filament. All these results suggested that CgDM9CP-4 was a novel member of PRR that expressed in undifferentiated pro-hemocytes to mediate immune recognition of pathogens.

Interactive effect of thermal and hypoxia on largemouth bass (Micropterus salmoides) gill and liver: Aggravation of oxidative stress, inhibition of immunity and promotion of cell apoptosis.

High temperatures and low oxygen in aquatic environments, such as intensive aquaculture or in natural watersheds, inevitably cause stress in fish. Fish are exposed to high temperatures during the summer, which exacerbates hypoxia. Hypoxia (1.2 ± 0.2 mg/L) under 20 °C (20 HG) and 26 °C (26 HG) was simulated to induce stress in largemouth bass (Micropterus salmoides). Related enzymes and genes involved in antioxidant, immune, and apoptotic responses were selected to explore the interactive effects of temperature and hypoxia on largemouth bass. The results showed that malondialdehyde (MDA) levels in plasma, gill, and liver increased in the 26 HG (p < 0.05). Liver superoxide dismutase (SOD) activity increased in the 26 HG. Peak SOD (SOD1, SOD2, SOD3a, and SOD3b), CAT, and GSH-Px mRNA levels in the gill and liver were observed at 12-24 h of stress. The levels of gill and liver total antioxidant capacity, catalase (CAT), glutathione peroxidase (GSH-Px) activities and other enzyme activities and genes in the 26 HG were higher than those in the 20 HG (p < 0.05). The gill and liver acid phosphatase and alkaline phosphatase activities increased with time in the 26 HG (p < 0.05), while gill and liver lysozyme activities in the 26 HG were lower than those in the 20 HG (p < 0.05). Tumor necrosis factor-α mRNA level was upregulated in the gill and downregulated in the liver at 24 h in the 26 HG. Interleukin (IL)-1ß and IL-8 mRNA levels were upregulated in the gill and liver in the 26 HG at 24 h, whereas IL-15 mRNA level was downregulated in the 26 HG at 12 h. Transforming growth factor-ß1 mRNA level was upregulated in the gill in the 20 HG at 24 h, but downregulated in gill and liver in the 26 HG at 24 h. Similarly, IL-10, Hepcidin-1, and Hepcidin-2 showed lower expression levels in the 26 HG. Gill and liver caspase-3 activities were higher in the 26 HG (p < 0.05), and gill caspase-3 activity was higher than that in the liver. The mRNA levels of proapoptotic genes (caspase-3, caspase-8, and caspase-9) were higher in the 26 HG. The present study demonstrates the interactive effects of temperature and hypoxia on stress in largemouth bass gill and liver. These results will be helpful to understand the mechanisms of stress induced by temperature and hypoxia in fish and provide a theoretical basis for aquaculture management.

Molecular characterization and expression analysis of fibrinogen related protein (FREP) genes of Manila clam (Ruditapes philippinarum) after lipopolysaccharides challenge.

Ruditapes philippinarum has high economic value and is distributed all over the world. Fibrinogen associated protein (FREP) is a type of pattern recognition receptor, participates in the innate immune response to eliminate pathogens after the invasion of pathogenic microorganisms. In this study, three FREP genes (FREP-1, FREP-2, and FREP-3) were identified and characterized from R. philippinarum. The protein sequence of FREPs were highly conserved with those homologous in vertebrates, and FBG domain possessed significantly high structural conservation in polypeptide binding site and Ca2+ binding site. The tissues expression analysis of FREPs in three shell color strains and two population of R. philippinarum were examined, with the highest expression level in gill and hepatopancreas. Besides, FREP genes were demonstrated to be induced by lipopolysaccharides injection, the significantly changes were observed after LPS injected. Our results suggest the involvement of FREPs in response to LPS injection, and it might exert a significant function on the innate immune defense of the Manila clam.

Induction of IL-22 protein and IL-22-producing cells in rainbow trout Oncorhynchus mykiss.

IL-22 is a critical cytokine which is involved in modulating tissue responses during inflammation, and is produced mainly by T cells and innate leucocytes. In mammals, IL-22 is a key component in mucosal defences, tissue repair, epithelial cell survival and proliferation. In teleosts, IL-22 has been cloned and studied in several species, and the transcript is highly expressed in mucosal tissues and induced by pathogen associated molecular patterns (PAMPs), suggesting IL-22 also functions as an important component of the innate immune response in fish. To investigate these immune responses further, we have validated and characterised two monoclonal antibodies (mAbs) which were raised against two different peptide immunogens of salmonid IL-22. Our results show that both mAbs specifically react to their own peptide immunogens and recombinant IL-22, and are able to detect the induction of native protein expression after stimulation. In flow cytometry, an increase in IL-22 positive cells was detected after stimulation in vitro with cytokines and PAMPs and in vivo after bacterial challenge. The immunohistochemistry results showed that IL-22 is highly upregulated in the gills after challenge, both in cells within the gill filaments and in the interbranchial lymphoid tissue. Such results suggest IL-22 may have a role in triggering local antimicrobial defences in fish that may facilitate efficient microbial clearance. Hence monitoring IL-22 producing cells/protein secretion may provide an alternative mean to assess the effectiveness of mucosal vaccines.

Transcriptome sequencing of the gill and barbel of Southern catfish (Silurus meridionalis) revealed immune responses and novel rhamnose-binding lectins (RBLs).

Southern catfish (Silurus meridionalis) is an economically important species widely cultured in China. It is well known for its fast growth, strong resistance to diseases and euryphage. However, little is known about the mechanisms for its powerful immune systems. Our Fish-T1K project has finished its first phase of 200 fish transcriptomes, with sequencing of gills in most examined fishes. In this study, we performed transcriptome sequencing of the gill and the maxillary barbel of Southern catfish, with the latter as a control. High expression of immune-related transcripts were observed in these two tissues. We observed that genes in the T cell receptor signaling pathway had higher transcription values in the gill than in the barbel. In addition, eight new rhamnose-binding lectins (RBLs) were identified and their carbohydrate recognition domains (CRDs) were classified according to the eight conserved cysteine residues and two conserved motifs (-YGR- and -DPC-). This is the first transcriptome report by high-throughput sequencing of the Southern catfish. Our genomic data and discovery of novel RBLs in this project should be able to promote better understandings of the roles of gills in immune responses and disease prevention for further aquaculture.

A Novel Antigen-Sampling Cell in the Teleost Gill Epithelium With the Potential for Direct Antigen Presentation in Mucosal Tissue.

In mammals, M cells can take up antigens through mucosal surfaces of the gut and the respiratory tract. Since M cells are deficient of lysosomes and phagosomes, the antigens are directly delivered to the mucosa-associated lymphoid tissue (MALT) without degradation. In teleost fish, the entire body surface (gills, skin, and intestinal system) is covered by mucus; however, specific antigen-sampling cells have not yet been identified in their mucosal tissues. Here, we show that two phenotypes of antigen-sampling cells take up antigens through epithelial surfaces of the rainbow trout gill. One phenotype of antigen-sampling cells has features of monocyte/macrophage/dendritic cell-type cells; they have large vacuoles in the cytoplasm and express PTPRC (CD45), CD83, IL-1ß, and IL-12p40b. The second phenotype exhibits similar characteristics to mammalian M cells; the corresponding cells bind the lectin UEA-1 but not WGA and show expression of M cell marker gene Anxa5. In contrast to mammalian M cells, teleost M-type cells were found to exhibit small vacuoles in their cytoplasm and to express almost all genes related to the "phagosome", "lysosome," and "antigen processing and presentation" pathways. Furthermore, MHC class II was constitutively expressed on a fraction of M-type cells, and this expression was significantly increased after antigen uptake, suggesting that the MHC class II is inducible by antigen stimulation. Here, we suggest that teleost M-type cells play a role in the phylogenetically primitive teleost immune system, similar to bona-fide M cells. In addition, the presence of MHC class II expression suggests an additional role in antigen presentation in the gills, which are an organ with high T cell abundance, especially in interbranchial lymphoid tissue. The present results suggest an unconventional antigen presentation mechanism in the primitive mucosal immune system of teleosts, which generally lack highly organized lymphoid tissues. Moreover, the results of this work may be valuable for the development of mucosal vaccines that specifically target M-type cells; mucosal vaccines significantly reduce working costs and the stress that is usually induced by vaccination via injection of individual fish.

Modulation of immune genes mRNA levels in mucosal tissues and DNA damage in red blood cells of Sparus aurata by gold nanoparticles.

Gold nanoparticles (AuNP) effects on Sparus aurata were evaluated on skin, gills and intestine by assessing the expression of immune genes and in peripheral blood evaluating genetic damage. Fish were exposed to 0.5 and 50 µg/L AuNP for 96 h. Results showed that exposure to 50 µg/L AuNP induced an upregulation in the expression of innate immune genes in gills (c3, lys, il1ß, tnfα, il6, il10 and tgfß) and intestine (il1ß, tnfα and il6). Furthermore, mRNA levels of hsp70 and hsp90 were increased in gills after exposure to 0.5 µg/L AuNP, when compared to 50 µg/L. Present data demonstrated the sensitivity of gills and intestines to AuNP exposure supporting their use in the study of fish responses to other nanoparticles. Genotoxic potential of AuNP was demonstrated by increased DNA strand breaks in red blood cells of fish exposed to AuNP, suggesting that AuNP represent a potential hazard to fish.

Identification of tumor necrosis factor receptor-associated factor 6 in the pearl mussel Hyriopsis cumingii and its involvement in innate immunity and pearl sac formation.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) acts as a central intracellular signal adapter molecule that mediates the tumor necrosis factor receptor superfamily and the interleukin-1 receptor/Toll-like receptor family in vertebrates and invertebrates. In the present study, HcTRAF6, a molluscan homologue of TRAF6 from Hyriopsis cumingii, has been cloned and identified. The entire open reading frame of HcTRAF6 was found to comprise a 1965-bp region that encodes a predicted protein of 654 amino acids, which contains conserved characteristic domains including a RING domain, two TRAF-type zinc finger domains, a typical coiled coil and the MATH domain. Phylogenetic analysis revealed that HcTRAF6 was aggregated closely with CsTRAF6 from Cyclina sinensis in the invertebrate cluster of mollusks. Further, qRT-PCR analysis showed that HcTRAF6 mRNA was extensively distributed in mussel tissues with a high expression in gills. After immune stimulation with Aeromonas hydrophila and lipopolysaccharides, the transcription of HcTRAF6 was obviously induced in the gills and hemocytes. In addition, significant fluctuation in HcTRAF6 expression was observed in the pearl sac, gills and hemocytes after mantle implantation. These findings confirmed its role in the alloimmune response. Dual-luciferase reporter assay showed that over-expression of HcTRAF6 could enhance the activity of the NF-κB reporter in a dose-dependent manner. Further, the RNA interference showed that the up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in HcTRAF6-silenced mussels and that depletion of HcTRAF inhibited the elimination of A. hydrophila. All these findings together prove that HcTRAF6 functions as an efficient regulator in innate immune mechanisms against invading pathogens and the alloimmune mechanism after mantle implantation in H. cumingii.

Kunitz-type serine protease inhibitor is a novel participator in anti-bacterial and anti-inflammatory responses in Japanese flounder (Paralichthys olivaceus).

Kunitz-type serine protease inhibitor (KSPI) interacts with serine protease (SP) to regulate cascade reactions in vivo and plays essential roles in innate immunity. Theoretical considerations support various functions of kspi, but further studies are required for full characterization of these functions. In this study, a KSPI molecule was identified from Japanese flounder (Paralichthys olivaceus), and was named Pokspi. The full-length cDNA sequence of Pokspi was 2810 nt, containing an open reading frame of 1527 nt, which encoded a polypeptide of 509 amino acid residues. PoKspi protein contained five conversed domains, namely, MANEC, PKD, LDLa and two Kunitz domains. Homology analysis revealed that Pokspi shared the highest similarity (83%) with its homolog in Cynoglossus semilaevis. Phylogenetic analysis indicated that Pokspi clustered with the homologs in other fishes. The mRNA transcripts of Pokspi were detected in all tested tissues, with the highest expression level in gill, followed by kidney and intestine. Its elevated expression in response to the application of Edwardsiella tarda (in vivo) and pathogen-associated molecular pattern (in vitro) suggested the involvement of Pokspi in the essential immune defense against various pathogens. Recombinant PoKspi (rPoKspi) purified from Escherichia coli exhibited not only serine protease inhibitor activities but also a broad spectrum of anti-microbial effect in a manner that was independent of any host factors. In addition, the recombinant PoKspi protein could cause the down-regulation of pro-inflammatory factors TNF-α and IL-1ß. In conclusion, Pokspi is a biologically active serine protease inhibitor endowed with anti-bacterial and anti-inflammatory property. This study provides strong evidences for understanding the innate immune defense in Japanese flounder.