Gluconeogenesis in the extraembryonic yolk syncytial layer of the zebrafish embryo.

Yolk-consuming (lecithotrophic) embryos of oviparous animals, such as those of fish, need to make do with the maternally derived yolk. However, in many cases, yolk possesses little carbohydrates and sugars, including glucose, the essential monosaccharide. Interestingly, increases in the glucose content were found in embryos of some teleost fishes; however, the origin of this glucose has been unknown. Unveiling new metabolic strategies in fish embryos has a potential for better aquaculture technologies. In the present study, using zebrafish, we assessed how these embryos obtain the glucose. We employed stable isotope (13C)-labeled substrates and injected them to the zebrafish embryos. Our liquid chromatography-mass spectrometry-based isotope tracking revealed that among all tested substrate, glutamate was most actively metabolized to produce glucose in the zebrafish embryos. Expression analysis for gluconeogenic genes found that many of these were expressed in the yolk syncytial layer (YSL), an extraembryonic tissue found in teleost fishes. Generation 0 (G0) knockout of pck2, a gene encoding the key enzyme for gluconeogenesis from Krebs cycle intermediates, reduced gluconeogenesis from glutamate, suggesting that this gene is responsible for gluconeogenesis from glutamate in the zebrafish embryos. These results showed that teleost YSL undergoes gluconeogenesis, likely contributing to the glucose supplementation to the embryos with limited glucose source. Since many other animal lineages lack YSL, further comparative analysis will be interesting.

Hemocyte migration and expression of four Sox genes during wound healing in Pacific abalone, Haliotis discus hannai.

In molluscs, migration of hemocytes and epithelial cells is believed to play central roles in wound healing. Here, we assessed cellular and molecular mechanisms of wound healing in Pacific abalone, a marine gastropod. Light and electron microscopy in the wounds showed early accumulation of putative hemocytes, collagen deposition by fibroblasts, and further coverage of this tissue by migration of adjacent epithelial cells. Cell labelling technique allowed us to track hemocytes, which migrated to wound surface within 24 h. The migrated cells first expressed PCNA and SoxF weakly, and then the epithelial cells expressed abundant PCNA and SoxB1, SoxB2, and SoxC. These findings imply that abalone SoxF is involved in hemocyte migration or their differentiation into fibroblasts, and suggest that the migrated epithelia acquire stem cell-like property and undergo active proliferation. This study is the first to show direct evidence of hemocyte migration to wounds and expression of Sox genes in molluscan wound healing.

Gluconeogenesis and glycogen metabolism during development of Pacific abalone, Haliotis discus hannai.

In lecithotrophic larvae, egg yolk nutrients are essential for development. Although yolk proteins and lipids are the major nutrient sources for most animal embryos and larvae, the contribution of carbohydrates to development has been less understood. In this study, we assessed glucose and glycogen metabolism in developing Pacific abalone, a marine gastropod mollusc caught and cultured in east Asia. We found that glucose and glycogen content gradually elevated in developing abalone larvae, and coincident expression increases of gluconeogenic genes and glycogen synthase suggested abalone larvae had activated gluconeogenesis and glycogenesis during this stage. At settling, however, glycogen sharply decreased, with concomitant increases in glucose content and expression of Pyg and G6pc, suggesting the settling larvae had enhanced glycogen conversion to glucose. A liquid chromatography-mass spectrometry (LC/MS)-based metabolomic approach that detected intermediates of these pathways further supported active metabolism of glycogen. Immunofluorescence staining and in situ hybridization suggested the digestive gland has an important role as glycogen storage tissue during settlement, while many other tissues also showed a capacity to metabolize glycogen. Finally, inhibition of glycolysis affected survival of the settling veliger larvae, revealing that glucose is, indeed, an important nutrient source in settling larvae. Our results suggest glucose and glycogen are required for proper energy balance in developing abalone and especially impact survival during settling.

Genetic structure of Japanese sea cucumbers (Apostichopus japonicus) along the Sanriku coast supports the effect of earthquakes and related tsunamis.

The Japanese common sea cucumber (Apostichopus japonicus) is a major marine product from Sanriku, Japan, but its populations were severely affected by the 2011 Tohoku earthquake, possibly decreasing its genetic diversity and increasing its extinction risk. In this study, we estimated the genetic structure and diversity of sea cucumbers from Touni and Yamada Bays of Sanriku over 4 years after the earthquake. The between-population genetic structure was estimated using two mitochondrial DNA regions (cytochrome c oxidase subunit I and 16S rDNA). Genetic differentiation (as measured by pairwise FST) was not significant between locations. Thus, even after the tsunami, gene flow and genetic diversity among the two sea cucumber populations were maintained. Our data also suggested that sea cucumbers in Sanriku experienced population expansion of about 0.20-0.24 million years ago, during the stable Mindel-Riss interglacial period. We conclude that A. japonicus populations in Sanriku could maintain their genetic structure throughout multiple disastrous tsunamis over the past several 1000 years. The high dispersal ability of planktonic larvae may enable the entrance of new recruits, thereby reducing risks associated with genetic structure and diversity changes stemming from mass die-offs in a given body of water from the past to the present.

Changes in glycogen concentration and gene expression levels of glycogen-metabolizing enzymes in muscle and liver of developing masu salmon.

Glycogen, as an intracellular deposit of polysaccharide, takes important roles in energy balance of many animals. In fish, however, the role of glycogen during development is poorly understood. In the present study, we assessed changes in glycogen concentration and gene expression patterns of glycogen-metabolizing enzymes in developing masu salmon (Oncorhynchus masou masou), a salmonid species inhabiting west side of North Pacific Ocean. As we measured glycogen levels in the bodies and yolk sacs containing the liver separately, the glycogen concentration increased in both parts as the fish developed, whereas it transiently decreased in the yolk sac after hatching, implying glycogen synthesis and breakdown in these tissues. Immunofluorescence staining using anti-glycogen monoclonal antibody revealed localization of glycogen in the liver, muscle and yolk syncytial layer of the pre-hatching embryos and hatched larvae. In order to estimate glycogen metabolism in the fish, the genes encoding homologs of glycogen synthase (gys1 and gys2) and glycogen phosphorylase (pygma, pygmb and pygl) were cloned, and their expression patterns were assessed by quantitative PCR and in situ hybridization. In the fish, gys1 and gys2 were robustly expressed in the muscle and liver, respectively. Also, expression of pyg isoforms was found in muscle, liver and yolk syncytial layer during hatching. With changes in glycogen concentration and expression patterns of relevant genes, our results suggest, for the first time, possible involvement of glycogen in energy balance of fish embryos, especially during hatching.

Mitogenic Activity of a Water-Soluble Adjuvant (Bu-WSA) Obtained from Bacterionema matruchotii: IV. Synergistic Effects of Bu-WSA on Concanavalin A-Induced Proliferative Response of Human Peripheral Blood Lymphocytes.

Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens.