Yutaka Takeuchi

Production of Tiger Puffer Takifugu rubripes Offspring from Triploid Grass Puffer Takifugu niphobles Parents

The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3xa0years until maturation. Second, the parental tiger puffer has a body size (2–5xa0kg) much larger than average market size (0.6–1.0xa0kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1xa0day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.

Adjuvant effect of recombinant interleukin-12 in the Nocardiosis formalin-killed vaccine of the amberjack Seriola dumerili

Abstract Nocardiosis causes serious economic damage in the fish farming of Japanese yellowtail fish. Nocardia seriolae identified as pathogenic bacterium is an intracellular‐pathogen. In general, induction of cell‐mediated immunity (CMI) is effective in infection defense against intracellular‐pathogen. However, the conventional formalin‐killed N. seriolae (FKC) vaccine induces humoral immunity. Interleukin‐12 (IL‐12) is Th1 type heterodimeric cytokine and induces cell differentiation in mammals. Our previous study showed that recombinant amberjack IL‐12 has a role in CMI induction in vitro and could be a possible CMI inducing adjuvant. However, its adjuvant effect of fish IL‐12 was not studied. In the present study, six types of amberjack recombinant IL‐12 (rIL‐12) were mixed and injected into amberjack with FKC. Firstly, we analyzed Th1‐ and Th2‐ related gene expression and monitored Th1/Th2 status followed by investigation of antibody titer. As a result, Th1‐type immunity was induced in FKC + rIL‐12 vaccinated fish. Secondly, we checked Th1/Th2 status of vaccinated fish after 10 days of N. seriolae infection using the expression of related genes. High T‐bet/GATA‐3 ratio was observed in FKC + rIL‐12 vaccinated fish, suggesting that Th1 cells possesing antigen memory were induced against N. seriolae infection. Finally, the survival rate in challenge test showed that 88% of FKC + rIL‐12 vaccinated fish was survived at 34 days after N. seriolae injection whereas PBS (control) and FKC only were exterminated. These result suggest that i) rIL‐12 is viable CMI inducible adjuvant and ii) production of Th1 cells having antigen memory resulting from activation of IL‐12 signaling pathway is important for defense against N. seriolae infection. HighlightsN. seriolae FKC + recombinant IL‐12 vaccinated fish induced cell‐mediated immunity and suppressed humoral immunity.An induction of the cell‐mediated immunity is important against Nocardiosis.Recombinant IL‐12 were found to be a cell‐mediated immunity inducible adjuvant.

Flow-cytometric enrichment of Pacific bluefin tuna type A spermatogonia based on light-scattering properties

We previously established surrogate broodstock in which the donor germ cells transplanted into the peritoneal cavities of xenogeneic recipients were capable of developing into functional eggs and sperm in teleost fish. In this transplantation system, only the undifferentiated germ cells such as type A spermatogonia (ASG) or a portion of the ASG population were capable of being incorporated into the genital ridges of the recipients and undergo gametogenesis. Therefore, the use of enriched ASGs can be expected to achieve efficient donor-cell incorporation. Here, we established a method of isolation and enrichment of the ASG of Pacific bluefin tuna using flow cytometry. Whole testicular cell suspensions were fractionated by forward and side scatter properties, following which ASGs were enriched in a fraction in which the forward scatter signal was relatively high and side scatter signal was relatively low. The diameter of sorted cells using the fraction was identical to the size of ASGs observed in histological analysis, and these cells also expressed the vasa gene. In addition, we succeeded in applying this method to several maturation stages of Pacific bluefin tuna. Since this method was based on light-scattering characteristics of ASGs, it can potentially be applied to various teleosts. We expect that this method can contribute to the production of seeds of Pacific bluefin tuna using surrogate broodstock.

Genomic DNA variation confirmed Seriola lalandi comprises three different populations in the Pacific, but with recent divergence

Captive breeding programs and aquaculture production have commenced worldwide for the globally distributed yellowtail kingfish (Seriola lalandi), and captive bred fingerlings are being shipped from the Southern Hemisphere to be farmed in the Northern Hemisphere. It was recently proposed that Pacific S. lalandi comprise at least three distinct species that diverged more than 2 million years ago. Here, we tested the hypothesis of different “species” in the Pacific using novel genomic data (namely single nucleotide polymorphisms and diversity array technology markers), as well as mtDNA and DNA microsatellite variation. These new data support the hypothesis of population subdivision between the Northeast Pacific, Northwest Pacific and South Pacific, and genetic divergence indicates restriction to the gene flow between hemispheres. However, our estimates of maximum mtDNA and nuclear DNA divergences of 2.43% and 0.67%, respectively, were within the ranges more commonly observed for populations within species than species within genera. Accordingly our data support the more traditional view that S. lalandi in the Pacific comprises three distinct populations rather than the subdivisions into several species.

Gonadal Transcriptome Analysis of Pacific Abalone Haliotis discus discus: Identification of Genes Involved in Germ Cell Development

Little is known about the molecular mechanisms governing gonadal developmental processes in abalones. Here, we conducted transcriptome analysis of Pacific abalone Haliotis discus discus for gene discovery in the brain, ovary, testis, and unfertilized eggs. Among the annotated unigenes, 48.6% of unigenes were identified by Venn diagram analysis as having universal or tissue-specific expression. Twenty-three genes with gonad-biased gene ontology (GO) terms were first obtained. Secondly, 36 genes were found by screening known gene names related to germ cell development. Finally, 17 genes were obtained by querying the annotated unigene database for zygotically expressed gonadal genes (ovary and testis) and maternally expressed gonadal genes (ovary, testis, and unfertilized eggs) using keywords related to reproduction. To further verify tissue distribution pattern and subcellular localization of these genes, RT-PCR and in situ hybridization were performed using a unigene encoding a germ cell marker, vasa, as control. The results showed that vasa was expressed mainly in the early developmental stages of germ cells in both sexes. One of the candidate genes, vitelline envelope zona pellucida domain protein 12 (ZP12), was expressed in the primordial germ cells of immature gonad and early developmental stages of germ cells of the adult female. The results obtained from the present study suggest that vasa and ZP12 are involved in germ cell development of Pacific abalone and that ZP12 is an especially useful germ cell-specific marker in immature adults. The current gonadal transcriptome profile is an extensive resource for future reproductive molecular biology studies of this species.

Spawning induction of blue mackerel Scomber australasicus and eastern little tuna Euthynnus affinis by oral administration of a crude gonadotropin-releasing hormone analogue

Scombridae species, such as tunas and mackerels, often do not spawn in land-based fish tanks without hormone treatment. To induce spawning in various fishes, a gonadotropin-releasing hormone analogue (GnRHa) is often administered by pellet implantation. Noninvasive administration is desired to induce spawning in scombrids that are sensitive to handling stress. Spawning induction by oral administration has been reported in several fishes, yet this method has not been put into practice in the aquaculture industry since a considerable amount of GnRHa is needed. Utilization of peptide synthesizers is widespread, and antigen-grade GnRHa (AgGnRHa) produced by a custom-peptide supplier is approximately 100-fold cheaper than conventional reagent-grade GnRHa (RgGnRHa), although the purity of AgGnRHa is lower. Here, we confirmed that the spawning induction potency of AgGnRHa was similar to that of RgGnRHa by pellet implantation in blue mackerel Scomber australasicus. Oral administration of AgGnRHa [6.0xa0mg/kg body weight (BW) per day] showed an equivalent ability to induce spawning of the mackerel as pellet implantation (0.1xa0mg/kg BW). We could also induce spawning of eastern little tuna Euthynnus affinis by oral administration of the AgGnRHa. Further, the obtained eggs showed higher survival. Thus, the oral delivery of AgGnRHa could be a powerful tool to induce spawning in Scombridae.

Amberjack Seriola dumerili interleukin-10 negatively suppresses host cell-mediated immunity

Interleukin-10 (IL-10) is an anti-inflammatory cytokine and negatively regulates cell-mediated immunity (CMI) induction by inhibiting cytokine production in type 1 T helper cells. IL-10xa0genes have been isolated from several fish, and inflammatory cytokine inhibition by IL-10 has been well examined. However, a CMI regulator of IL-10 in fish has not yet been identified. In this study, we cloned the IL-10 gene in amberjack Seriola dumerili and analyzed its function using its recombinant protein (rIL-10). In an in vitro culture experiment, gene expression of inflammatory cytokines was suppressed in leukocytes incubated with rIL-10 compared with cells that only received Nocardia seriolae stimulation. This result suggests amberjack IL-10 has conserved function as an inflammatory cytokine inhibitor. Bactericidal activity of amberjack cells against intracellular pathogen stimulation was decreased in a rIL-10 dose-dependent manner. Furthermore, a significant reduction in the T-bet/GATA-3 ratio was observed in N. seriolae living cell (LC)u2009+u2009rIL-10-injected fish. Taken together, these results suggest amberjack rIL-10 suppresses CMI induction both in vitro and in vivo. In addition, the number of IgM+ cells among spleen leukocytes in N. seriolaeu2009+u2009rIL-10-injected fish was higher than in only N. seriolae LC, suggesting that Th2-dominant immunity was induced by adding rIL-10.

Adjuvant effect in aquaculture fish of cell-wall glycolipids isolated from acid-fast bacteria

ABSTRACT Mycobacteriosis and nocardiosis in cultured fish caused by infections with acid‐fast bacteria, are responsible for large economic losses globally. In this study, we suggest a novel adjuvant using glycolipids that activates host immune systems. The immune response to glycolipids stimulation was investigated using ginbuna crucian carp. Ginbuna vaccinated with FKC (formalin‐killed cells) + glycolipids isolated from Mycobacterium sp., upregulated inflammatory‐ and Th1‐related cytokines, and a DTH (delayed‐type hypersensitivity) response was confirmed only in ginbuna vaccinated with FKC + glycolipids. These observations suggest that glycolipids activated host innate and cell‐mediated immunity. Subsequently, we evaluated the adjuvant effect of glycolipids against amberjack nocardiosis. In a challenge test, a higher survival rate was observed in amberjack vaccinated with FKC + glycolipids emulsified with conventional oil adjuvant than in fish vaccinated with FKC + oil adjuvant without glycolipids. Therefore, glycolipids potentially could be used as a practical, economical and safe adjuvant for aquaculture fish. HighlightsGlycolipids isolated from Mycobacterium sp. activate ginbuna crucian carp innate immune system.Th1‐dominant immunity in ginbuna crucian carp is induced by glycolipids isolated from Mycobacterium sp.Glycolipids isolated from N. seriolae could be used as practical adjuvant against nocardiosis in yellowtail aquaculture industry.

Transcriptional response to low temperature in the yellow drum (Nibea albiflora) and identification of genes related to cold stress

The yellow drum (Nibea albiflora) is an economically important maricultured fish in China, but the aquaculture of this species is severely affected by overwinter mortality associated with cold stress. Characterization of the molecular mechanisms underlying the susceptibility of the yellow drum to cold might increase our understanding of how this fish adapts to environmental challenges. Here, the transcriptional response of the yellow drum to cold stress (7.5u202f°C) was investigated with RNA-Seq analysis. We compared brain and muscle transcriptomes among cold-tolerant (Tol) fish that survived the cold treatment, cold-sensitive (Sen) fish that were killed by the cold treatment, and control (Con) fish that were not subjected to cold. Our analysis recovered 233,245 unigenes. The genes (DEGs) differentially expressed in the brain and muscle of the Tol versus Con group, the Sen versus Con group, and the Tol versus Sen group had tissue-specific expression patterns. Gene ontology, enrichment, and pathway analyses indicated the most highly enriched pathways in the DEGs were signaling molecules and interaction, signal transduction, carbohydrate metabolism, lipid metabolism, digestive system, and endocrine system pathways. These pathways were all associated with biological functions relevant to cold adaptation in the yellow drum, including transduction of stress signals, energy metabolism, and stress-induced cell membrane changes. We identified genes likely to be involved in cold-susceptibility and -tolerance as those differentially expressed in the Tol group as compared to the Sen group. Further investigation and characterization of these candidate genes might improve our understanding of the mechanisms underlying cold adaptation in the yellow drum.