RNA-seq analysis of the head-kidney transcriptome response to handling-stress in the red cusk-eel (Genypterus chilensis).

Stress is a primary contributing factor of fish disease and mortality in aquaculture. We have previously reported that the red cusk-eel (Genypterus chilensis), an important farmed marine fish, demonstrates a handling-stress response that results in increased juvenile mortality, which is mainly associated with skeletal muscle atrophy and liver steatosis. To better understand the systemic effects of stress on red cusk-eel immune-related gene expression, the present study assessed the transcriptomic head-kidney response to handling-stress. The RNA sequencing generated a total of 61,655,525 paired-end reads from control and stressed conditions. De novo assembly using the CLC Genomic Workbench produced 86,840 transcripts and created a reference transcriptome with a N50 of 1426bp. Reads mapped onto the assembled reference transcriptome resulted in the identification of 569 up-regulated and 513 down-regulated transcripts. Gene ontology enrichment analysis revealed a significant up-regulation of the biological processes, like response to stress, response to biotic stimulus, and immune response. Conversely, a significant down-regulation of biological processes is associated with metabolic processes. These results were validated by RT-qPCR analysis for nine candidate genes involved in the immune response. The present data demonstrated that short term stress promotes the immune innate response in the marine teleost G. chilensis. This study is an important step towards understanding the immune adaptive response to stress in non-model teleost species.

Characterization, expression, and evolutionary analysis of new TLR3 and TLR5M genes cloned from the spiny eel Mastacembelus armatus.

Toll-like receptors (TLRs) play an important role in innate and adaptive immunity. Here, we identify two new TLRs from the spiny eel Mastacembelus armatus (TLR3 and membrane TLR5M). Both MaTLR3 and MaTLR5M were expressed in all tested tissues; expression was highest in liver and spleen, respectively. After infection with Vibrio parahaemolyticus, expression of both TLRs fluctuated and differed significantly from controls at several time points. The predicted three-dimensional model of the MaTLR3 and MaTLR5M proteins indicates that most sites under positive selection were located in the extracellular domains of TLRs. Evolutionary analysis detected positively selected sites in the ancestral lineages of vertebrates, amphibians and reptiles. Multiple ML methods recovered 10 positively selected sites in teleost TLR3 and 24 in TLR5M, and most sites were located in leucine-rich repeat domain, possibly related to an "arms-race" co-evolution with pathogens.

Galectins in the abdominal cavity of the conger eel Conger myriaster participate in the cellular encapsulation of parasitic nematodes by host cells.

Congerin is a proto-type galectin distributed on the skin and mucosal epithelia of the upper digestive tract of the Japanese conger eel Conger myriaster. It has at least 2 isotypes, namely, congerin I and II, and plays a role in bio-defense at the body surface. In the current study, we identified both isotypes in the peritoneal fluid and peritoneal cells of C. myriaster by western blot and mass spectrometry (MS)/MS analysis. Cucullanus nematodes parasitize the abdominal cavity of C. myriaster, and immunohistochemical analyses demonstrated that congerins can bind to both the body surface of the encapsulated nematodes and the encapsulating cells. Furthermore, adhesion of the peritoneal cells to Sepharose particles was greatly accelerated when the microspheres were coated with congerin. Indeed, this effect was significantly hampered by the addition of lactose. These results indicate that congerin participates in the cellular encapsulation of the Cucullanus nematode via the induction of cellular adhesion to the parasites depending on lectin-glycoside recognition.

LPS induces gene expression of interleukin-1beta in conger eel (Conger myriaster) macrophages: first cytokine sequence within Anguilliformes.

Our aim was to comprehensively analyze the immune response in Anguilliformes macrophages and to survey cytokine genes expressed from them. We therefore used suppression subtractive hybridization (SSH) to randomly clone molecules that are specifically expressed in conger eel (Conger myriaster) macrophages when cells are stimulated by LPS. As a result, we succeeded in identifying a conger eel IL-1beta. This is the first report on cytokines in Anguilliformes, which is the most ancient order in living teleosts.

Possible immune functions of congerin, a mucosal galectin, in the intestinal lumen of Japanese conger eel.

Congerin, a mucosal galectin of the Japanese conger eel, provides chemical fortification through its agglutinating and opsonizing activity. Congerin is produced in the epidermis, and the epithelia of the oral cavity to the esophagus, but not in the stomach or intestine. We hypothesized that congerin secreted from the upper digestive tract can reach and function in the intestinal lumen. We found that congerin possessed marked resistance against digestion by gastric and enteric enzymes of conger eel. It was not degraded until 6h of incubation with stomach extract or intestinal digestion juice. Western blotting demonstrated that congerin essentially remained in the intestinal mucus. The mucus agglutinated rabbit erythrocytes, and the agglutination was hampered by anti-congerin antibody. Furthermore, congerin could bind to some enteric bacteria. These results support the above hypothesis.

The effects of five different glycans on innate immune responses by phagocytes of hybrid tilapia and Japanese eels Anguilla japonica.

The aim of this study was to evaluate the immune responses in hybrid tilapia (Nile tilapia Oreochromis niloticus x Mozambique tilapia O. mossambicus) and Japanese eels Anguilla japonica after treatment with five glycans: barley, krestin, MacroGard, scleroglucan, and zymosan. The effects of the glycans on the innate immune responses of the fish were investigated using the phagocytic index (PI), lysozyme activity, complement opsonization, and activation assay. The results of the lysozyme assay demonstrated that the lysozyme activities increased after treatment with glycans. Moreover, based on the PI, treatment with each of the five glycans resulted in increased phagocytic activities in anterior kidney and peripheral blood phagocytes in both tilapia and Japanese eels. The opsonic effect of complement on phagocytosis in tilapia and Japanese eels were investigated using baker's yeast, which served as the activator in the classical complement pathway (CCP) and in the alternative complement pathway (ACP). Tilapia and Japanese eel sera that were treated with glycans greatly enhanced phagocytosis. The classical pathway--hemolytic complement titer (CH50) of Japanese eels treated with glycans was slightly increased in vitro and in vivo. While glycan treatment enhanced the CCP of both species in vitro and in vivo, the alternative pathway-hemolytic complement titer (ACH50) was only increased in vitro and in vivo in glycan-treated tilapia. Thus, it follows that the ACP must have been activated in tilapia treated with glycans. However, in Japanese eels, the ACH50 of the ACP activation assay was undetected in vitro or in vivo due to possible unknown factors in the Japanese eel serum that caused lysis of the rabbit red blood cells. Our study investigated the effects of glycans used to enhance phagocytosis and activate both of the complement pathways involved in stimulating the innate immune responses of Japanese eels and tilapia.

Yeast-binding C-type lectin with opsonic activity from conger eel (Conger myriaster) skin mucus.

A mannose-specific lectin with agglutination activity against a yeast, Saccharomyces serevisiae, was purified from conger eel (Conger myriaster) skin mucus. The lectin, named conCL-s, has a tetrameric structure consisting of two non-covalently associated dimers whose constituent monomers with a molecular mass of about 16,000 Da are linked by a disulphide bond. conCL-s is composed of 173 amino acid residues including a 24-residue signal peptide, and belongs to the C-type lectin family, which is typically characterized by binding to sugar in a Ca2+-dependent manner. Nevertheless, conCL-s showed Ca2+-independent activity in its yeast-binding. The gene expression of the lectin was widely detected in external and internal mucosal tissues, i.e., skin, gills, tongue, buccal cavity wall and esophagus. In these tissues, mRNA of conCL-s was exclusively distributed in club cells, which are one of the major components of the epidermis and mucosal epithelium in Anguilliforme species. conCL-s showed agglutination activity to a bacterium, Escherichia coli. Furthermore, attachment of the lectin to microspheres significantly enhanced their phagocytosis in conger eel macrophages. These findings suggest that conCL-s acts as an opsonin and plays an important role in the innate immunity on the body surface in conger eels.

How parasitism and pollution affect the physiological homeostasis of aquatic hosts.

Parasitism poses a serious threat to hosts under certain circumstances, while the well-being of organisms is also negatively affected by environmental pollution. Little information is available on the simultaneous effects of parasites and pollutants on the physiological homeostasis of organisms. The present paper demonstrates that parasites: (i) may influence the metabolism of pollutants in infected hosts, (ii) interact with pollution in synergistic or antagonistic ways, and (iii) may induce physiological reactions in hosts which were thought to be pollutant-induced. Experimental studies on the uptake and accumulation of metals by fish reveal that fish infected with acanthocephalans have lower metal levels than uninfected hosts; e.g. Pomphorhynchus laevis reduces lead levels in fish bile, thereby diminishing or impeding the hepatic intestinal cycling of lead, which may reduce the quantity of metals available for fish. Alterations in pollutant uptake and accumulation in different intermediate and final hosts due to parasites are thus very important in the field of ecotoxicology. In addition to such alterations, there is a close interaction between the effects of pollutants and parasites which seems to be mediated at least partly by the endocrine system, which itself is closely related to the immune system in fish. Laboratory studies on eels experimentally infected with the swimbladder nematode Anguillicola crassus reveal that toxic chemicals such as polychlorinated biphenyls produce immunosuppressive effects which facilitate parasite infection. Similarly, an increase in serum cortisol concentration in eels due to chemical exposure and infection is correlated with decreasing levels of anti-A. crassus antibodies. Furthermore, parasites are able to elicit physiological changes which are attributed to chemicals with endocrine disrupting activity, e.g. the cestode Ligula intestinalis is known to suppress gonad development in roach. The most thoroughly documented examples of endocrine disruption in wild fish are in roach, and it is conceivable that this disruption is not only due to chemical activity but also to parasites such as L. intestinalis or species of the phylum Microspora.

Cross-reactivity to eel, eelpout and ocean pout in codfish-allergic patients.

Fish allergy is one of the most common food allergies in both children and adults and patients with allergic reactions to one fish species have in many cases been given the advice to avoid all fish, without further evaluation. The possible common reactivity between different fish species is not well studied. Because of this and a possible exploitation of fish species hitherto not much used in the Scandinavian diet ocean pout, eelpout and eel were evaluated. We examined the serological and biological cross-reactivity of these species in double-blind challenged-confirmed codfish-allergic patients using CAP, Maxisorp-radio allergosorbent test (RAST) inhibition, western blot, skin prick test (SPT) and histamine release (HR). All 18 codfish allergic patients had specific IgE to ocean pout, eelpout and eel determined by Maxisorp-RAST. All four fish species could induce basophil HR using blood from 16 of 18 patients and all patients tested reacted in SPT. This study demonstrates that patients with a verified clinical allergy to codfish in a high frequency express biological cross-reactivity to other fish species. By RAST inhibition this common reactivity was shown to be a true cross-reactivity.

Characterization of Japanese eel immunoglobulin M and its level in serum.

Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790,000; it contained an equimolar heavy chain and light chain with molecular weights of 72,000 and 25,000, respectively. While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains. Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by D-mannose and could be abolished by alpha-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses. The average IgM concentration in the sera of the healthy eels was 3.4 mg ml(-1); it amounted to 10.3% of the total serum protein.

The lipopolysaccharide O side chain of Vibrio vulnificus serogroup E is a virulence determinant for eels.

Vibrio vulnificus is a gram-negative bacterium capable of producing septicemic infections in eels and immunocompromised humans. Two biotypes are classically recognized, with the virulence for eels being specific to strains belonging to biotype 2, which constitutes a homogeneous lipopolysaccharide (LPS)-based O serogroup (which we have designated serogroup E). In the present study we demonstrated that the O side chain of this LPS determines the selective virulence of biotype 2 for eels: (i) biotype 1 strains (which do not belong to serogroup E) are destroyed by the bactericidal action of nonimmune eel serum (NIS) through activation of the alternative pathway of complement, (ii) biotype 2 strains (of serogroup E) are resistant to NIS, and (iii) rough mutants of biotype 2 lacking the O polysaccharide side chain are sensitive to NIS and avirulent for eels.

Influence of flumequine on in vivo mitogen responses of European eel (Anguilla anguilla L., 1758) lymphoid cells.

The influence of flumequine on mitogen induced lymphoid cell proliferation in European eels (Anguilla anguilla L., 1758) was studied. For this purpose an in vivo test, using peroral drug administration followed by successive intraperitoneal injections with concanavalin A (ConA) or bacterial lipopolysaccharides (LPS) and 5-bromo-2'-deoxyuridine, was applied. Direct counting of proliferated cells in blood smears revealed that flumequine possesses mitogenic properties. A synergistic and an antagonistic effect of the drug was observed after LPS and ConA stimulation, respectively. Flow cytometric analysis of peripheral blood lymphoid cells showed a significant reduction of the mean proportion surface immunoglobulin positive cells in the flumequine-treated animals. It is concluded that flumequine enhances proliferation of lymphoid cells (probably surface immunoglobulin negative cells) in eel under the present experimental conditions.

Distribution and molecular forms of C-type natriuretic peptide in plasma and tissue of a dogfish, Triakis scyllia.

A radioimmunoassay (RIA) to measure C-type natriuretic peptide (CNP) of a dogfish, Triakis scyllia, was established, and plasma and tissue levels of CNP were measured. Molecular forms of CNP in plasma and tissues were also examined using a combination of the RIA and cation-exchange high-performance liquid chromatography (HPLC). The antibody used in the assay cross-reacted with all forms of Triakis CNP, as well as eel CNP, against which the antibody was raised. The antibody exhibited no cross-reaction with any atrial, brain (B-type), and ventricular natriuretic peptides examined and showed a weak cross-reaction with porcine CNP. The detection limit of this assay was 0.8 fmol/tube of Triakis CNP-22 which was used as standard. The CNP level in the Triakis plasma was 1.97 +/- 0.38 pmol/ml (n = 5) which far exceeded the physiological levels of any natriuretic peptides in other species. Among various tissues examined, the highest concentration of CNP was measured in the atrium, followed by the ventricle, brain, and pituitary. Low levels were detected in the kidney, liver, and digestive tracts. HPLC analyses revealed that the major form of CNP in the brain was CNP-22, while it was proCNP (CNP-115) in the heart. In contrast to other species from teleosts to mammals thus far examined, the majority of CNP in dogfish plasma was prohormone instead of processed, mature forms.

Purification and characterization of a galactose-binding lectin from the skin mucus of the conger eel Conger myriaster.

1. A galactose-binding lectin was purified from the skin mucus of the conger eel Conger myriaster by affinity chromatography and HPLC. 2. The lectin was a simple protein having the same two subunits with a mol. wt of 12,500 and a N-terminal amino acid of phenylalanine. 3. Electrofocusing suggested that the purified lectin was composed of several isolectins. 4. From the ultraviolet difference spectra attributable to tryptophanyl residues in the binding site, the binding constant of the lectin for D-galactose was estimated to be 5.3 x 10(3)/M.

Purification and properties of agglutinins from conger eel, Conger Myriaster (Brevoort), skin mucus.

The skin mucus of the conger eel, Conger myriaster, contains galactose-specific agglutinins. Hemagglutinating activity is independent of divalent cations and is destroyed by heating at 50 degrees C for 15 min. The mucus agglutinins, named congerins, are a mixture of proteins with different electrical charges. Three of these molecules were isolated by affinity chromatography on acid-treated Sepharose 4B and by ion-exchange chromatography. They are simple proteins with the same molecular weight of 30,000 and consisting of two subunits (each 13,000 daltons). The agglutinins inhibited the normal embryonic development of the starfish Asterina pectinifera, and lysed the fertilized eggs at a concentration of 25 micrograms protein/ml. They also agglutinated but did not inhibit the growth of a marine bacterium, Vibrio anguillarum.

Specificities of antibody to acetylcholine receptor in rabbits with experimental myasthenia gravis.

The injection of acetylcholine receptor (AChR) purified from Narke electroplax japonica induced 'experimental autoimmune myasthenia gravis' (EAMG) in rabbits. Serial measurements of anti-AChR antibody titre using Narke AChR and rabbit AChR as antigen revealed that the intensity of myasthenia had a rather closer correlation with titre to Narke AChR than that to rabbit AChR. Antibody reacting to rabbit AChR was almost completely adsorbed out with torpedo receptor conjugated to agarose. Serum concentrations of antibody protein measured by affinity chromatography ranged from 54 to 896 microgram/ml serum and were well correlated with the intensity of myasthenia. The results suggested that in rabbits immunized with heterologous AChR the cross-reaction of anti-heterologous AChR antibody with rabbit AChR caused myasthenia rather than an immune response specific to rabbit AChR dose.