Toshiaki Miyadai

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.

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.

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.

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.

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.

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.

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.

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.

Milky hemolymph syndrome associated with an intranuclear bacilliform virus in snow crab Chionoecetes opilio from the Sea of Japan

Milky hemolymph syndrome (MHS) is a previously undescribed disease in snow crab Chionoecetes opilio. Outbreaks of this disease occurred in reared and wild populations from the Sea of Japan when the water temperature was 2–3°C. The common symptom of the disease was a distinct milky or opaque coloration of the hemolymph. Some severely affected crabs also showed yellow or ivory discoloration on the ventral shell and uncalcification of the arthrodial membranes of walking legs. Histopathological changes consisted of widespread cellular degeneration characterized by enlarged nuclei with marginal hyperchromatosis and basophilic intranuclear inclusions in the interstitial connective tissues of various organs. Electron microscopy analyses of degenerated cells revealed a nonoccluded, enveloped bacilliform virus within the nuclei. The virus morphologically resembles white spot syndrome virus (WSSV) or unassigned intranuclear bacilliform viruses (IBVs) reported from other crustaceans. However, we could not detect WSSV from diseased tissues by PCR using WSSV-specific primers, and the tissues targeted by the virus differed from those targeted by IBVs. This is the first report of a virus from the genus Chionoecetes. Thus, we have tentatively designated the virus presumably associated with MHS as Chionoecetes opilio bacilliform virus (CoBV) until its relationship with other crustacean viruses can be clarified.

Proliferation of kuchijirosho causative agent in a fugu-derived cell line

Kuchijirosho is a fatal disease of commercially cultured fugu, Takifugu rubripes. The transmissible nature of kuchijirosho strongly suggests that an infectious pathogen is the causative agent. Because it is filtrable, the agent is thought to be a virus; however, it has not yet been identified. The lack of a permissive cell line for the putative kuchijirosho-causing agent (KCA) has hindered research on the identification of this pathogen. We inoculated brain extract prepared from kuchijirosho-affected fugu onto an established fugu cell line, fugu eye, and observed that cytopathic effect appeared 7 days after inoculation. Injection of the culture medium of infected fugu eye cells into fugu resulted in the onset of kuchijirosho, indicating that fugu eye cells are able to proliferate KCA. An infectious fraction separated by sodium iottalamate density gradient centrifugation showed a density of 1.15 g mL(-1) equivalent to that of KCA derived from affected fugu brain. To determine whether the genome of KCA is RNA or DNA based, nucleotide synthesis inhibitors were applied to inoculated fugu eye cell line to influence the production of KCA. 5-Fluorouracil but not IUdR showed a concentration-dependent inhibition of KCA yield. These results suggest that KCA is an RNA virus.