Program

Rapid progress in molecular biology has led to the identification of many unique metabolic features in aquatic organisms. These include the presence of foreign genes acquired by horizontal gene transfer (e.g., photosynthetic genes in sea slug) and limited ability of glycolytic proteins that has been predicted by a large number of nutritional studies. These findings will be strengthened by calculating metabolic flux, the rate of turnover of molecules through metabolic pathways. However, until recently, there has been no safe and simple method to quantitatively trace metabolites. Classically ¹⁴C and ³H have been used, but utilization of these radioactive nuclei requires special equipment. Introduction of innovative metabolite tracing technologies will significantly contribute to understanding the unique metabolic features of aquatic organisms. Here I present the application of a new technology to trace metabolites labeled with stable isotope ¹³C. This method achieved high sensitivity and specificity by observing signals from protons bound to ¹³C, and thus called proton-observed carbon-edited nuclear magnetic resonance (POCE NMR) spectroscopy. Branched-chain amino acid synthesis in a bdelloid rotifer Adineta vaga will be presented as an example of how we can apply POCE NMR in aquatic biochemistry.

The teleosts exhibit inefficient use of digestible carbohydrate and are generally considered to be glucose intolerant. Thus, most teleost fish are known to require high levels of dietary proteins because nutrients other than carbohydrates including proteins are preferentially utilized for biological energy production.
Amino acids have long been viewed simply as precursors for the synthesis of proteins and other N-containing compounds. In recent years, amino acids have been widely investigated as signaling molecules for the regulation of major metabolic pathways. Signaling through mammalian target of rapamycin (mTOR) is activated by amino acids, insulin, and growth factors, and impaired by nutrient or energy deficiency. mTOR is a protein kinase involved in the control of a diverse range of cellular processes including protein synthesis.
In fish, the activation of TOR by amino acids and insulin has been demonstrated in primary culture cells of rainbow trout. It is suggested that Leu might have an involvement in the activation of TOR pathway induced by amino acids in fish, as in mammals. However, the effects of each individual amino acid on TOR signaling remain unclear in fish.
Using fish cell lines CHSE-214 and RTG-2, we examined the effects of amino acid deprivation on TOR signaling pathway. We found that Arg deprivation was reduced the phosphorylation of not only TOR, but also p70 ribosomal protein S6 kinase, and eukaryotic initiation factor 4E-binding protein 1 in CHSE-214 and RTG-2 cells.
Although the regulation of the TOR pathway by amino acids are still not fully understood, Arg would be one of the potent effectors involved in the activation of TOR pathway in fish. The study of amino acids as signaling molecules will provide some novel and fundamental insights into fish biology and a framework for further studies in fish.

Teleosts require a higher level of dietary protein because they depend on proteins (and/or amino acids) for energy metabolism more than dietary carbohydrates. Teleost skeletal muscle is not just a locomotive organ but also a protein pool for biological energy. On the other hand, their unique physiological homeostasis systems enable them to keep muscular locomotive activity even though under severe fasting. Such unique regulatory system of proteolysis in teleost skeletal muscle is still unclear because no method has been established to investigate it.

Comprehensive profile of proteolysis is needed to reveal the details in proteolysis including related proteases, substrates, and its regulatory system. High resolution tandem mass spectrometry (MS2) has been recently developed and allows more comprehensive analysis of protein and peptides. The quantitative peptidomic analysis using MS2 spectrometry provides whole sequence data of proteolytic fragment peptides. Substrate proteins can be identified using a reference protein database constructed through transcriptomic analysis even though little reference data are available as in the case of teleosts.

Because of the complementary nature of the protease-active site with the residues surrounding the cleaved bond in the substrate, terminal residues of the resulting peptide give us the information for the protease substrate specificity. We have recently developed a novel simple multiple linear regression model on the basis of this concept; sequence characteristic of the whole peptide termini is expressed as the linear combination of cleavage-site specificity of each protease with each contribution parameter.

We will describe limitations associated with prior methods for investigating regulatory system of proteolysis; a methodological framework for a novel method based on peptidomics by MS2; analysis of the proteolytic response to severe stress in horse mackerel skeletal muscle.

This study was supported in part by a grant from the Project of the Bio-oriented Technology Research Advancement Institution, NARO.

Leopard coral groupers are desirable fish to consumers in Asian markets. Aquaculture has commenced to meet the consequent increasing demands. Feeding efficiency is a major concern in aquaculture; however, little information is available on grouper nutritional biorhythms at the comprehensive molecular level. Given this deficiency, we analyzed nutritional biorhythms using transcriptomic and metabolomic techniques. Moreover, gut microbiota is known to play an important role in symbiotic metabolism. In this study, microbiota composition was examined together with trans-omics data. Hatchery-reared leopard coral groupers were maintained under 11 L:13 D conditions in winter. The fish were fasted for the first two days and then fed for the next two days of the experiment. Muscle and feces samples were collected every four hours. Next-generation DNA sequencing-based transcriptomic analysis and NMR-based metabolomic analysis were performed. Subsequently, obtained transcriptomic and metabolomic data were integrated and analyzed using a correlation analysis-based program. In muscle, several genes were expressed with circadian rhythmicity, and hormone receptors related to fish growth varied with feed condition. Moreover, glycolysis and TCA cycles and purine metabolites exhibited circadian patterns, and gene expression also varied. The variation in gut microbiota composition exhibited longer biorhythm, namely at the day scale such as fasted and fed day periods. In the fasting period, Proteobacteria was the major microbiota component, thought to be derived from ingested seawater. In contrast, Firmicutes increased during the feeding period. In practical aquaculture, these results might provide information to help improve feed metabolic efficiency.

The present study investigates the role of slc15a1a and b, genes that encode oligopeptide transporters, in the nutrient absorption processes in the intestine of Mozambique tilapia. We elucidated tissue distribution of those gene transcripts along distinct segments of the intestine (the hepatic loop, HL; proximal major coil, PMC; gastric loop, GL; distal major coil, DMC; terminal segment, TS), and effects of nutrient condition on slc15a1a and b expressions through fasting and refeeding. Results showed that slc15a1a was predominantly expressed in the absorptive epithelia of the anterior part of the intestine (HL and PMC). Short-term and long-term fasting resulted in significant upregulation and downregulation, respectively, thereby producing a biphasic effect on slc15a1a expression. The gene expression was also examined at a refeeding phase after 14-day fasting. Refeeding significantly stimulated slc15a1a expression at day 3, although the expression did not exceed the pre-fasted level. The slc15a1b was also expressed in the anterior intestine, although the expression is about 10 times lower than that of slc15a1a, suggesting that slc15a1a is the major oligopeptide transporter in the intestine of tilapia. The slc15a1b expression was continuously downregulated by fasting, and was stimulated at the 3rd day of refeeding. Observed changes in response to fasting and refeeding suggest that slc15a1a serves as a sensitive indicator of changes that may occur in altering nutritional conditions. These findings contribute to a better understanding of oligopeptides and amino acid absorption mechanisms and to the development of possible means to manipulate these processes for the improvement of growth and other metabolic and physiological conditions in fish.

Microalgae are a potential source of novel food and feed ingredients due to their high protein contents and omega-3 fatty acid composition. In this work, an Antarctic microalga, Koliella antarctica was studied in controlled bioreactor conditions, to optimize its growth and to determine its biomass and lipid productivity. Optimization studies in bubble-tube photobioreactors showed that growth was optimal under low salinity conditions (<8 ‰). The microalga was then tested for its ability to utilize extracellular sources of organic carbon (photoheterotrophy), with glucose, acetate and glycerol supplied in the growth medium. The effects of different stresses (nitrogen and phosphorus deficiency, plus osmotic shock) were studied by measuring changes in the fatty acid composition and protein content. Polar and neutral lipids (triacylglycerides, TAGs) were analyzed and changes in omega-3 fatty acids were measured. Subsequently, the microalga was cultivated in flat plate photobioreactors with a short light path length. The effects of different incident light intensities (70, 250 and 500 micromol photons /m²/s) on growth, light utilization efficiency and TAG production were studied. The highest light utilization efficiency measured 0.8 g/mol, while the highest growth rate showed 0.9 g/L/d, which is approximately equivalent to that of temperate strains at >25℃. Koliella antarctica and similar coldwater microalgae could be a novel resource that are rich in omega-3 fatty acids, and potentially more productive in colder climates or during the winter.

Aim: Fishmeal is a major component of fish feed used in aquaculture and we are now switching protein ingredients from fish to plant from the viewpoint of resource sustainability. On the other hand, plant substitution introduces phytosterols in the feed. Phytosterols are reported to alter gene expression of farnesoid X receptor (FXR) in mammalian liver. FXR is responsible for various metabolic regulations including bile acid synthesis, lipid metabolism and glucose metabolism. One of the major functions is to express the gene of PLTP and UGT2b4 Effect of phytosterol on fish FXR cascades is however undisclosed. In this study, we have investigated the effects of phytosterols on fish FXR functions.
Materials and methods: Wild type zebrafish (Danio rerio) was used for the following experimentations. Fishes were fed fish feeds; no sterol, 3 % cholesterol, and 3 % β-sitosterol. Total RNA was extracted from liver and transcripts of proteins were detected by real time PCR.
Results and discussion: Cholesterol administration did not change FXR gene expression, whereas β-sitosterol intake enhanced the gene expression. On the other hand, gene expression of phospholipid transfer protein (PLTP) increased in the cholesterol-fed group, but did not change in the β-sitosterol-fed group. Because physiological FXR ligands are bile acids, reduced dietary cholesterol might contribute to the FXR functions. Plant protein substitution in fish feed and the resulting administration of β-sitosterol might affect cholesterol metabolisms and physiological FXR functions in fish.

【Objective】 Regulation of muscle lipid content is an important concern in aquaculture fish in terms of fish meat quality. Triglyceride (TG) is synthesized in the liver and then transported to muscles. Recently, it is reported that gangliosides are involved in TG accumulation in mouse liver. However, roles of gangliosides in TG accumulation are still not clear in vertebrates. In this study, the influence of desialylation of ganglioside on lipid metabolism was examined with fish hepatocyte. Especially, we focused on sialidase Neu3 which is a ganglioside specific sialidase.
【Materials and Methods】 mRNA levels of neu3a and lipid accumulation related genes in the liver of fasting medaka were analyzed by real-time PCR. To examine Neu3a influences toward lipid metabolism, neu3a-stable transfectant cells were established with fish hepatic Hepa-T1 cell. After the cells were exposed to oleic acid, lipid droplet formations were observed by Oil Red O staining. Alteration of ganglioside composition in stable transfectants were assessed by thin layer chromatography (TLC).
【Results and Discussion】 Hepatic neu3a gene expression was up-regulated under deficient energy conditions, accompanied with pparg and lpl1 genes expression. By bioinformatics analysis, several binding sites of lipid metabolism-related transcription factors were predicted in the upstream of neu3a transcription start site. After oleic acid exposure, newly synthesized TG was detected as lipid droplet in the cell. Bigger size of lipid droplets were observed in neu3a-stable transfectant cells compared with vector. By TLC analysis, decrease of ganglioside GM3 and increase of lactosylceramide were detected in neu3a-stable transfectant cells. These results suggested that alteration of ganglioside composition by Neu3a would regulate TG accumulation in fish liver.

Amino acids are important for not only protein components but energy source in fish, and play various roles in metabolic regulations. Previous studies have shown that insulin secretion is induced by some amino acids intake in fish, and some signaling pathways regulating energy metabolism are directly or indirectly affected by amino acids. Our previous studies indicated that dietary amino acid-imbalance drastically affect lipid metabolism in fish. In this study, we investigated the effects of short-term administration of lysine (Lys)-deficient diet on lipid metabolism in rainbow trout Oncorhynchus mykiss. Corn gluten meal was used as main protein source in the Lys-deficient diet. Combination of soybean and fish oils provided similar fatty acid compositions both in the control and Lys-deficient diets. Fish were fed with the Lys-deficient diet for 4 days after adaptation into experimental conditions by control diet feeding for 5 days. The Lys-deficient group showed a significant increase in total lipid content in muscle and liver for 4 days. Total n-3 fatty acid amounts in muscle of the Lys-deficient group significantly increased in the day 4. In particular, 22:5n-3 and 22:6n-3 (DHA) were accumulated in the Lys-deficient group. Saturated and mono-unsaturated fatty acids increased in liver of the Lys-deficient group in the day 4. Increases in gene expression levels of fatty acid synthase, fatty acid elongase 2 (Elovl2), and delta 5 and delta 6 desaturases were observed in Lys-deficient fish liver. These results suggest that synthesis, desaturation, and elongation of fatty acids in the Lys-deficient group were up-regulated through the activation of transcription factors, such as sterol regulatory element-binding protein 1 and peroxisome proliferator-activated receptors.
Although details in the regulatory mechanism is still veiled, previous studies and these results strongly suggest that ingested amino acids should be closely linked with fish lipid metabolism.

Rubber seed is suitable for aquafeed ingredients due to its relatively high nutrient value (33.8 % protein, 15.1% fat, 11.0 % water content, 15.0 % fiber, and 5.1% ash) and abundant availability since it was considered as a co-product and is not used for human consumption. However, the use of rubber seed meal in the aquafeed ingredients is hindered by the presence of cyanide as anti-nutrient factor, and imbalance amino acid profile. Thus, this research was conducted to evaluate lysine supplementation on defatted hydrolyzed rubber seed meal (DHRSM) as protein source in Nile tilapia diets.
A completely randomized design with five treatments that used combination of different percentage of protein supplied from DHRSM and lysine supplementation (ctrl: control treatment, 40D: 40% of DHRSM, 40DL: 40% DHRSM + 0.15% lysine, 60D: 60% of DHRSM and 60DL: 60% DHRSM + 0.3% lysine). Growth study was conducted using Nile tilapia at an initial body weight of 6.29±0.12 gram on 175 L aquaria for 40 days. During rearing period fish was fed 3 times a day until apparent satiation at 08.00 am, 12.00 pm, and 4.00 pm. After final sampling, Fish survival, feed intake, feed efficiency and daily growth rate were determined and whole body amino acid from 3 fish per treatment was analyzed using high precision liquid chromatography method.
Although higher inclusion of DHRSM was found to produce lower growth performance and retention of several essential amino acid (40D vs 60D), supplementation with lysine seems to alleviate this poor growth performance and improves the retention of histidine, threonine, methionine, leucine, and phenylalanine. Thus, it can be concluded that lysine supplementation is beneficial when using DHRSM as aquafeed ingredient at high inclusion level.