D-mannose-specific Immunoglobulin M in Grass Puffer (Takifugu niphobles), a Nonhost Fish of a Monogenean Ectoparasite Heterobothrium okamotoi, Can Act as a Trigger for its Parasitism.

Heterobothrium okamotoi, a monogenean gill parasite, exhibits high host specificity for the tiger puffer, Takifugu rubripes, and it has been experimentally verified that the parasite cannot colonize either closely related species such as the grass puffer Takifugu niphobles or distantly related fish such as the red seabream Pagrus major. Previously, we demonstrated in T. rubripes that immunoglobulin M (IgM) with d-mannose affinity induced deciliation of the oncomiracidia, the first step of parasitism, indicating that the parasite utilizes the molecule as a receptor for infection. In the present study, we purified mannose-specific IgM from 2 nonhost species, T. niphobles and P. major, by affinity and gel-filtration chromatography techniques and compared their deciliation-inducing activity against H. okamotoi oncomiracidia. The IgM of the former showed activity, whereas the latter had no effect, suggesting that in addition to d-mannose-binding ability, the crystallizable fragment domain of IgM, which is not part of the antigen-binding domain, plays an important role in host recognition by the oncomiracidia, such as direct binding to the parasites. It also suggests that the host specificity of H. okamotoi is relatively low upon initial recognition, and the specificity is established by exclusion in nonhosts during a later stage.

Original Ligand for LTßR Is LIGHT: Insight into Evolution of the LT/LTßR System.

The lymphotoxin (LT)/LTß receptor (LTßR) axis is crucial for the regulation of immune responses and development of lymphoid tissues in mammals. Despite the importance of this pathway, the existence and function of LT and LTßR remain obscure for nonmammalian species. In this study, we report a nonmammalian LTßR and its ligand. We demonstrate that TNF-New (TNFN), which has been considered orthologous to mammalian LT, was expressed on the cell surface as a homomer in vitro. This different protein structure indicates that TNFN is not orthologous to mammalian LTα and LTß. Additionally, we found that LTßR was conserved in teleosts, but the soluble form of recombinant fugu LTßR did not bind to membrane TNFN under the circumstance tested. Conversely, the LTßR recombinant bound to another ligand, LIGHT, similar to that of mammals. These findings indicate that teleost LTßR is originally a LIGHT receptor. In the cytoplasmic region of fugu LTßR, recombinant fugu LTßR bound to the adaptor protein TNFR-associated factor (TRAF) 2, but little to TRAF3. This difference suggests that teleost LTßR could potentially activate the classical NF-κB pathway with a novel binding domain, but would have little ability to activate an alternative one. Collectively, our results suggested that LIGHT was the original ligand for LTßR, and that the teleost immune system lacked the LT/LTßR pathway. Acquisition of the LT ligand and TRAF binding domain after lobe-finned fish may have facilitated the sophistication of the immune system and lymphoid tissues.

Teleost Basophils Have IgM-Dependent and Dual Ig-Independent Degranulation Systems.

Recently, mammalian basophils have been highlighted as having roles in allergy and antiparasitic immunity; however, there is little information about the functions and evolutionary origin of basophils, because they are the least abundant leukocyte in most vertebrates. In this study, we characterized the teleost basophils that are abundant in the peripheral blood of fugu (Takifugu rubripes). Fugu basophils have two distinct granules: reddish-purple and dark violet ones. Teleost fish do not have IgG and IgE, but we found that fugu IgM bound on the surface of the basophils, and the cross-linked IgM induced degranulation of both types of granules. This indicates that teleost basophils can be activated in an Ab-dependent manner. Furthermore, papain induced the degranulation of the reddish-purple granules, which contain histamine, and the released granules stimulated the migration of various leukocytes. In contrast, chitin elicited the degranulation of the dark violet granules, which resulted in CD4+ T cell-specific migration. Thus, fugu basophils control immune responses via two distinct Ab-independent mechanisms. In addition, fugu basophils endocytosed soluble Ag and expressed MHC class II and B7-H1/DC. These findings suggested that fugu basophils can interact with T cells as APCs. Thus, the Ab-dependent basophil activation predates the emergence of IgG and IgE, and fish basophils exhibit different dynamics and features of degranulation to distinct stimuli compared with mammalian basophils. Some features of teleost basophils are more similar to those of mammalian mast cells than to those of mammalian basophils.

SJL-1, a C-type lectin, acts as a surface defense molecule in Japanese sea cucumber, Apostichopus japonicus.

The surface defense molecules of aquatic invertebrates against infectious microorganisms have remained largely unexplored. In the present study, hemagglutinins were isolated from an extract of body surface layer of Japanese sea cucumber, Apostichopus japonicus, by affinity chromatography with fixed rabbit erythrocyte membranes. The N-terminal sequence of a 15-kDa agglutinin was almost identical with that of SJL-1, a C-type lectin formerly identified in this species. Because cDNA sequence and tissue distribution of SJL-1 have not been reported, we performed cDNA sequencing, gene expression analysis, and western blotting and immunohistochemical evaluation with anti-recombinant SJL-1 (rSJL-1) antibodies. The hemagglutinin gene was transcribed mainly in the integument, tentacles, and respiratory tree. Western blotting revealed that SJL-I is present in a body surface rinse, indicating that SJL-1 is secreted onto the body surface. SJL-1-positive cells scattered beneath the outermost layer of the integument were detected by immunohistochemistry. Furthermore, rSJL-1 agglutinated Gram-positive and Gram-negative bacteria, and yeast. These results indicate that SJL-1 acts as a surface defense molecule in A. japonicus.

Light- and circadian-controlled genes respond to a broad light spectrum in Puffer Fish-derived Fugu eye cells.

Some cell lines retain intrinsic phototransduction pathways to control the expression of light-regulated genes such as the circadian clock gene. Here we investigated the photosensitivity of a Fugu eye, a cell line established from the eye of Takifugu rubripes, to examine whether such a photosensitive nature is present. Microarray analysis identified 15 genes that showed blue light-dependent change at the transcript level. We investigated temporal profiles of the light-induced genes, as well as Cry and Per, under light-dark, constant light (LL), and constant dark (DD) conditions by quantitative RT-PCR. Transcript levels of Per1a and Per3 genes showed circadian rhythmic changes under both LL and DD conditions, while those of Cry genes were controlled by light. All genes examined, including DNA-damage response genes and photolyase genes, were upregulated not only by blue light but also green and red light, implying the contribution of multiple photopigments. The present study is the first to identify a photosensitive clock cell line originating from a marine fish. These findings may help to characterize the molecular mechanisms underlying photic synchronization of the physiological states of fishes to not only daily light-dark cycles but also to various marine environmental cycles such as the lunar or semi-lunar cycle.

Mucosal IgM Antibody with d-Mannose Affinity in Fugu Takifugu rubripes Is Utilized by a Monogenean Parasite Heterobothrium okamotoi for Host Recognition.

How parasites recognize their definitive hosts is a mystery; however, parasitism is reportedly initiated by recognition of certain molecules on host surfaces. Fish ectoparasites make initial contact with their hosts at body surfaces, such as skin and gills, which are covered with mucosa that are similar to those of mammalian guts. Fish are among the most primitive vertebrates with immune systems that are equivalent to those in mammals, and they produce and secrete IgM into mucus. In this study, we showed that the monogenean parasite Heterobothrium okamotoi utilizes IgM to recognize its host, fugu Takifugu rubripes Oncomiracidia are infective larvae of H. okamotoi that shed their cilia and metamorphose into juveniles when exposed to purified d-mannose-binding fractions from fugu mucus. Using liquid chromatography-tandem mass spectrometry analysis, proteins contained in the fraction were identified as d-mannose-specific IgM with two d-mannose-binding lectins. However, although deciliation was significantly induced by IgM and was inhibited by d-mannose or a specific Ab against fugu IgM, other lectins had no effect, and IgM without d-mannose affinity induced deciliation to a limited degree. Subsequent immunofluorescent staining experiments showed that fugu d-mannose-specific IgM binds ciliated epidermal cells of oncomiracidium. These observations suggest that deciliation is triggered by binding of fugu IgM to cell surface Ags via Ag binding sites. Moreover, concentrations of d-mannose-binding IgM in gill mucus were sufficient to induce deciliation in vitro, indicating that H. okamotoi parasites initially use host Abs to colonize host gills.

Serum GlcNAc-binding IgM of fugu (Takifugu rubripes) suppresses the growth of fish pathogenic bacteria: a novel function of teleost antibody.

N-acetyl-d-glucosamine (GlcNAc) is one of the components of peptidoglycan, a biopolymer in the bacterial cell wall. We purified a novel GlcNAc-binding protein, designated as fGBP-78, from sera of fugu (Takifugu rubripes). The fGBP-78 is a heteromer of 78- and 25-kDa subunits. Moreover, fGBP-78 exerted remarkable inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria, including ones virulent for marine fish species as well as non-pathogenic Escherichia coli. These results suggest that fGBP-78 contributes to bacterial clearance in fugu. Furthermore, the nanoLC-MS/MS and Western blotting analyses reveal that the 78-kDa subunit is the fugu IgM heavy chain. In addition, the molecular mass of the other subunit (25 kDa) was equal to that of the Ig light chain. Overall, results indicate that fGBP-78 is an IgM molecule presumably acts as a natural antibody. This paper reports a novel function of teleost IgM as a significant suppresser against bacterial growth.

A trans-species missense SNP in Amhr2 is associated with sex determination in the tiger pufferfish, Takifugu rubripes (fugu).

Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.

Teleost IL-6 promotes antibody production through STAT3 signaling via IL-6R and gp130.

Teleost IL-6 is upregulated after antigen stimulation; therefore, we hypothesized that fish IL-6 contributes to antibody production during immune responses against infections. To verify this hypothesis, we first cloned IL-6R and gp130 in fugu (Takifugu rubripes) in the present study. The membrane and soluble forms of IL-6R were identified by the identification of cDNA clones of IL-6R homologues. Three STAT3-docking sites were found in the intracellular region of fugu gp130. Expression analysis showed that fugu IL-6R and gp130 were expressed in mIgM(+) B cells, suggesting that fugu B cells are stimulated by IL-6. Recombinant fugu IL-6 (rfIL-6) increased the gene expression of secretory antibodies by mIgM(+) B cells in vitro. The rfIL-6 and soluble form of rfIL-6R activated STAT3 phosphorylation in the B cells and a cultured cell line transfected with fugu gp130. These results indicate that fugu IL-6 enhances antibody production in the B-cell lineage via gp130 and STAT3 signaling.

The plasmablast-like leukocyte in the kidney of fugu (Takifugu rubripes).

In teleosts, the kidney is the major immune organ. From the kidney of fugu (Takifugu rubripes), we isolated a unique leukocyte population. This population shows properties similar to those of mammalian plasmablasts. First, adherent cells expressing IgM protein on their surface were obtained from the fugu kidney. Flow cytometry (FCM) showed that these cells were mainly composed of two cell populations: IgM+CD8α⁻ cells and IgM+CD8α+ cells. Further characterization of the IgM+CD8α⁻ population by RT-PCR demonstrated that the cells expressed secretory-type IgM as well as Bcl-6 and Blimp-1, developmental marker genes for the B cell lineage. Western blotting also showed that the cells secreted IgM protein. These results indicate that the IgM+CD8α⁻ cells are similar to cells at the plasmablast stage in mammals. This is the first report isolating plasmablast-like leukocytes in fish species. Our data also suggests that the teleosts kidney is a organ where B cells terminally differentiate into the plasma cells.

Functional analysis of fish BCL-6 and Blimp-1 in vitro: transcriptional repressors for B-cell terminal differentiation in fugu (Takifugu rubripes).

The transcriptional repressors BCL-6 and Blimp-1 are key regulators of B-cell terminal differentiation in mammals. We have previously identified the BCL-6 gene and Blimp-1 gene in fugu (Takifugu rubripes). In the present report, we conducted a functional analysis of fugu BCL-6 and Blimp-1 by using a one-hybrid reporter assay with Gal4 fusion proteins and Gal4DBD luciferase reporter gene. Results from the reporter assays in mammalian cell lines (HeLa, HEK-293, CV-1 and NIH3T3) and the fish cell line EPC show that Gal4-BCL6 and Gal4-Blimp1 strongly repress the transcription of the luciferase gene in all cell lines. Furthermore, deletion analyses show that the N-terminal region of BCL-6 has transcriptional repression activity; the BTB/POZ domain is an especially potent repression domain. In contrast to BCL-6, although the N-acidic domain and PR domain are insufficient for repression, most functional motifs of Blimp-1 are associated with transcriptional repression. These results suggest that BCL-6 and Blimp-1 are functional transcriptional repressors in fugu and that they regulate B-cell terminal differentiation in fugu.

Genetic variation and geographic distribution of megalocytiviruses.

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae have caused mass mortalities in marine and freshwater fish in Asian countries. In this study, partial major capsid protein (MCP) gene of seven Japanese and six Korean megalocytiviruses was sequenced and compared with the known megalocytiviruses to evaluate genetic variation and geographic distribution of the viruses. Comparison of MCP gene nucleotide sequences revealed sequence identity of 92.8% or greater among these 48 isolates. A phylogenetic tree clearly revealed three clusters: genotype I including nine Japanese isolates, thirteen Korean isolates, one Chinese isolates, one Thailand isolate and one South China Sea isolate; genotype II including five freshwater fish isolates in Southeast Asian countries and Australia; and the remaining genotype III mainly consisted of flatfish isolate in Korea and China. This suggests that viruses belonging to the genotype I widely distribute among various fish species in many Asian countries. Conversely, the epidemic viruses belonged to genotype II and III are may be still locally spreading and constrained in their prevalence to the limited host fish species, i.e., genotype II viruses mainly distribute in Southeast Asian countries, whereas genotype III viruses distribute in flatfish species in Korea and China.

Effects of tributyltin on the immune system of Japanese flounder (Paralichthys olivaceus).

Effects of tributyltin (TBT) which has been used for antifouling paint of ship's hulls and fishing nets on the immune system in Japanese flounder (Paralichthys olivaceus) were investigated. After short-term exposure to a high level of TBT, leucocytes in the head kidney from 1-year-old flounder were examined for the proportion of neutrophils in total leucocytes. Also examined were their respiratory burst activities using flow cytometry, the reduction of nitroblue tetrazolium (NBT) and lysozyme activities. Furthermore, long-term exposures to a relatively low level of TBT using young flounder were also carried out. The proportion of neutrophils in total leucocytes prepared from head kidney in each fish exposed to TBT at 20 microg/L for 5 days and the reduction of NBT by leucocytes prepared from the same experimental conditions increase compared to the control group. The contents were 42.0+/-6.8 and 52.5+/-6.3%, respectively. Significant differences of the NBT reduction were observed between 0 and 20 microg/L TBT exposure groups. On the other hand, the respiratory burst activity of cells in the exposure group clearly showed a tendency to decrease compared to the control group. Furthermore, high level of TBT also inhibited lysozyme activity which plays an important role for the bacteriocidal procedures. However, similar results were not obtained in the exposure group with a relatively low level of TBT. To determine the immunotoxic effects of TBT, infection experiments using pathogens which are naturally occurring should be further investigated.

B-lymphocyte-induced maturation protein-1 (Blimp-1) gene of torafugu (Takifugu rubripes).

Homologous gene of B-lymphocyte-induced maturation protein-1 (Blimp-1) of torafugu (Takifugu rubripes) was identified by tblast search analysis. RT-PCR and 5'RACE clearly defined the sequence of the UTR and coding region which has been ambiguously determined by tblast analysis. Fugu Blimp-1 was mainly expressed in the lymphoid organs. These finding imply that Blimp-1 would take a major role in the terminal differentiation of B-cells to plasma cells in fish.

Molecular cloning of the BCL-6 gene, a transcriptional repressor for B-cell differentiation, in torafugu (Takifugu rubripes).

B-cell lymphoma-6 (BCL-6) is a transcriptional repressor that prevents the terminal differentiation of mature B-cells to plasma cells, and is essential for germinal center formation in the primary lymphoid organs of mammals. In this study, we identified the BCL-6 gene in torafugu (Takifugu rubripes) using the torafugu genome database, and analyzed the expression of BCL-6 mRNA in various tissues of torafugu, using RT-PCR. The BCL-6 gene consisted of eight exons and seven introns spanning a genome of ca. 3.3 kb. BCL-6 mRNA contained a 2112 bp open reading frame encoding 703 amino acids, with a predicted protein size of 78.8 kDa. The predicted torafugu BCL-6 primary structure contains two conserved specific motifs, the BTB/POZ domain at the N-terminus and the sixC2H2-type zinc finger motifs at the C-terminal region. The homology of torafugu BCL-6 to those of zebrafish (Danio rerio), Xenopus laevis, mouse (Mus musculus) and human (Homo sapiens) is 76, 59, 60 and 60%, respectively. RT-PCR analysis revealed that BCL-6 mRNA is highly expressed in pronephros, thymus, intestine, ovary, brain, nasal cavity and muscle. These results imply that torafugu BCL-6 is involved in regulation of B-cell differentiation in torafugu.

Identification of genes encoding critical factors regulating B-cell terminal differentiation in torafugu (Takifugu rubripes).

Many transcription factors, and associated co-factors, are involved in the regulation of B-cell terminal differentiation in mammals. In the teleost and cartilaginous fish, although evidence has strongly suggested the existence of B-cell like lymphocytes, the mechanism of terminal differentiation of B-cells remains to be elucidated. In the present study, we searched for the nucleotide and amino acid sequences similar to the critical regulatory factors facilitating the terminal differentiation of B-cells using the fugu BLAST server. We cloned the following cDNAs from Takifugu rubripes: (1) B-lymphocyte-induced maturation protein-1 (Blimp-1), which plays a major role in promoting plasma cell differentiation by repressing the transcription of many genes that participate in maintaining the differentiation of mature B-cells; (2) Bcl-6, which facilitates germinal center formation and represses Blimp-1 expression; (3) X-box binding protein-1 (XBP-1), which operates Ig secretion by activating transcription of the ER-stress responsible genes; (4) Pax-5, which suppresses XBP-1 and enhances the expression of activation-induced cytidine deaminase (AID), an inducer of somatic hypermutation and class-switch recombination of the immunoglobulin gene; and (5) TLE-3, one of the Groucho family proteins, a co-factor for Blimp-1. We also identified other co-factors and many target genes of Blimp-1 by in silico and/or cDNA cloning. These finding indicates that the basal process of B-cell terminal differentiation in fish is controlled by factors identical to those in mammals.

Monoclonal antibodies recognising serum immunoglobulins and surface immunoglobulin-positive cells of puffer fish, torafugu (Takifugu rubripes).

Immunoglobulin of the torafugu, Takifugu rubripes, was purified by a combination of precipitation by low ionic strength dialysis and gel filtration. The Ig was used to immunise mice for the production of monoclonal antibody (MAb). Supernatants of hybridoma cultures were screened by enzyme-linked immunosorbent assay using purified-torafugu Ig-coated plates, and two stable hybridomas producing MAbs against torafugu Ig were obtained. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions and Western blotting indicated that one MAb (16F3) was specific for the deglycosylated heavy chain of torafugu, and the other MAb (4H5) did not bind to the reduced Ig, suggesting that 4H5 recognised the higher-order structure of Ig. Under non-reduced conditions, both MAbs recognised mainly a 750 kDa band and also minor bands of 672, 410 and 205 kDa. MAb 16F3- and 4H5-primed magnetic beads (Dynabeads) adsorbed 84.9+/-3.3% and 63.6+/-4.4% of the torafugu Ig, respectively. The Ig adsorbed by MAb 16F3-primed Dynabeads was reactive to 4H5 on immunoblotting, and vice versa, indicating that the epitopes for both MAbs are held on the same Ig molecule. Both of these MAbs cross-reacted extensively with the Ig of other Takifugu species, but not with other genus. The MAbs were used to identify surface Ig-positive lymphocytes in the spleen, pronephros, peripheral blood and thymocytes of torafugu by flow cytometry. Flow cytometric analysis of the cells in the lymphocyte-enriched fraction revealed that 50.2+/-6.9% in the PBL, 11.8+/-1.7% in the mesonephros, 13.3+/-2.1% in the pronephros, 42.5+/-4.3% in the spleen and 3.2+/-0.6% in thymus were reactive to 4H5 or 16F3.