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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.

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.

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.

The tiger pufferfish (fugu) is one of the most important food fishes in East Asia. Since its testes are regarded as a delicacy, sex determination is economically relevant. Previous studies have identified a missense single-nucleotide polymorphism (SNP) in the Amhr2 ( anti-Müllerian hormone receptor type II ) gene as a strong candidate for a master sex-determining polymorphism. To distinguish genotypic sex efficiently, we developed a high-resolution melting (HRM) assay for this SNP site. By screening 396 fish from two independent crosses reared under controlled conditions, we observed perfect concordance between the SNP genotype and phenotypic sex. Thus, this method holds great potential for use in high-throughput sexing. When analyzing 293 progeny from a third cross reared under unknown conditions, we unexpectedly found that 25 % of phenotypic males exhibited female genotype. These results suggest that environmental factors such as rearing conditions could influence the sex-determination pathway in pufferfish. Alternatively, genetic modifiers might override the signals from Amhr2 . This finding raises a concern regarding enhanced stock management of this species, because sex-reversed fish could compromise the sex ratio in subsequent generations. The HRM assay will also be useful for monitoring the degree of sex reversal before release.

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.

Milky hemolymph syndrome (MHS) has been observed in both reared and wild populations of the snow crab Chionoecetes opilio from the Sea of Japan. The virus presumably associated with MHS and detected with electron microscopy was designated ‘Chionoecetes opilio bacilliform virus’ (CoBV), but little is known about its infectivity or pathogenicity in the snow crab. In this study, we performed an infectivity experiment in which snow crabs were injected with a viral inoculum prepared from MHS-affected crabs and then maintained at 2 °C or 2–8 °C. Cumulative mortality increased more rapidly at 2 °C than at 2–8 °C, reaching approximately 80% in both groups at the termination of the experiment. This result demonstrates the temperature-dependent pathogenicity of CoBV in the snow crab. CoBV-infected crabs displayed the clinical and histopathological signs of MHS. Mortality tended to increase as the viral load in the hemolymph increased at both 2 °C and 2–8 °C. The viral load in the hemolymph was significantly higher in dead crabs than in surviving crabs and was also elevated in various other organs. We conclude that CoBV is the causative agent of MHS and that severely affected crabs develop viremia that leads to death.

The distribution of sulfate-reducing bacteria (SRB) was investigated in the sediments below fish cages used for torafugu (Takifugu rubripes) aquaculture along the coast of southern Fukui Prefecture, Japan. A quantitative competitive PCR targeting the gene coding for a portion of the alpha-subunit of the dissimilatory (bi)sulfite reductase (dsrA) gene was used to determine the distribution of SRB in the sediments. In unpolluted fish farm sediments, SRB were detected at levels of 0.5-8.4xl0E8 cells/g dry sediment; whereas higher densities of SRB were found in sediments rich in organic matter and sulfide at levels of 0.8-4.4x10E9 cells/g dry sediment as determined by competitive PCR. We detected differences in SRB densities in fish farm sediments with different pollution levels, or organic contamination and sulfide content, suggesting SRB cell density may be used as an indicator of pollution levels in fish farm sediments as an adjunct to chemical analyses such as measurements of chemical oxygen demand (COD) or chemically determined total sulfide (TS).

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 ( Co BV) until its relationship with other crustacean viruses can be clarified.

Several lines of evidence have suggested that ganglioside GM1 stimulates neuronal sprouting and enhances the action of nerve growth factor (NGF), but its precise mechanism is yet to be elucidated. We report here that GM1 directly and tightly associates with Trk, the high-affinity tyrosine kinase-type receptor for NGF, and strongly enhances neurite outgrowth and neurofilament expression in rat PC12 cells elicited by a low dose of NGF that alone is insufficient to induce neuronal differentiation. The potentiation of NGF activity by GM1 appears to involve tyrosine-autophosphorylation of Trk, which contains intrinsic tyrosine kinase activity that has been localized to the cytoplasmic domain. In the presence of GM1 in culture medium, there is a >3-fold increase in NGF-induced autophosphorylation of Trk as compared with NGF alone. We also found that GM1 could directly enhance NGF-activated autophosphorylation of immunoprecipitated Trk in vitro. Monosialoganglioside GM1, but not polysialogangliosides, is tightly associated with immunoprecipitated Trk. Furthermore, such tight association of GM1 with Trk appears to be specific, since a similar association was not observed with other growth factor receptors, such as low-affinity NGF receptor (p75NGFR) and epidermal growth factor receptor (EGFR). Thus, these results strongly suggest that GM1 functions as a specific endogenous activator of NGF receptor function, and these enhanced effects appear to be due, at least in part, to tight association of GM1 with Trk.