Daisuke Ikeda

Early development of medaka Oryzias latipes muscles as revealed by transgenic approaches using embryonic and larval types of myosin heavy chain genes

We cloned three full‐length cDNAs encoding myosin heavy chains (MYHs) previously found to be expressed in embryos or larvae of medaka Oryzias latipes. Based on cDNA sequence information, the three medaka MYH genes, mMYHemb1, mMYHL1 and mMYHL2, were localized on the chromosomes. In vivo promoter assay using the gene encoding green or red fluorescent protein and linked to the 5′‐flanking region of mMYH demonstrated that the transcripts of fast‐type mMYHemb1, first expressed in embryos but belonging to the adult type in phylogenetic analysis, were located in the horizontal myoseptum. On the other hand, embryonic fast‐type mMYHL1 and mMYHL2 were expressed in the whole myotomes. Interestingly, cells expressing mMYHemb1 were localized together with engrailed, and cyclopamine, which blocks hedgehog signaling, inhibited mMYHemb1 expression as well as the formation of the horizontal myoseptum, suggesting that muscle pioneer cells express mMYHemb1 as a key protein in the formation of the horizontal myoseptum. Developmental Dynamics 239:1807–1817, 2010.

A 5'-flanking region of embryonic-type myosin heavy chain gene, MYHM743-2, from torafugu Takifugu rubripes regulates developmental muscle-specific expression ☆

The myosin heavy chain gene, MYH(M)₇₄₃₋₂, is highly expressed in fast muscle fibers of torafugu embryos. However, the regulatory mechanisms involved in its expression have been unclear. In this study, we examined spatio-temporal expression patterns of this gene during development by injecting expression vectors containing the GFP reporter gene fused to the 5-flanking region of MYH(M)₇₄₃₋₂ into fertilized eggs of zebrafish and medaka. Although the -2.1kb 5-flanking region of torafugu MYH(M)₇₄₃₋₂ showed no homology with the corresponding regions of zebrafish and medaka orthologous genes on the rVISTA analysis, the torafugu 5-flanking region activated the GFP expression which was detected in the myotomal compartment for both zebrafish and medaka embryos. The GFP expression was localized to fast and slow muscle fibers in larvae as revealed by immunohistochemical analysis. In addition to the above tissues, GFP was also expressed in jaw, eye and pectoral fin muscles in embryos and larvae. These results clearly demonstrated that the 2.1 kb 5-flanking region of MYH(M)₇₄₃₋₂ contains essential cis-regulatory sequences for myogenesis that are conserved among torafugu, zebrafish and medaka.

An alpha-1-acid glycoprotein-like protein as a major component of the ovarian cavity fluid of viviparous fish, Neoditrema ransonnetii (Perciformes, Embiotocidae)

Developing fetuses of surfperch (Neoditrema ransonnetii, Perciformes; Embiotocidae) are retained in the ovarian cavity until birth, where they are surrounded by ovarian cavity fluid (OCF). Expecting the OCF to have key roles in maintaining pregnancy, we purified and characterized a major glycoprotein of 51 kDa in the OCF of surfperch. On the basis of the N-terminal amino acid sequence, we cloned and sequenced a full-length cDNA. The deduced sequence comprises 214 amino acids (aa) including a signal peptide of 20 aa and a mature protein of 194 aa. This protein had an extremely low pI (below 2.8) and extraordinarily high glycosylation rate (more than 50%), characteristics being shared with alpha-1-acid glycoprotein (AGP), a member of the lipocalin superfamily. A homology search and phylogenetic analysis indicated that the 51 kDa protein and tributyltin-binding protein found in Japanese flounder are the closest known relatives of AGP. We therefore named the protein nrF-AGP. Messenger RNA of nrF-AGP was expressed intensively in the liver, but not at all in the ovarian tissue. Because nrF-AGP is the most salient component in OCF but not in plasma, we reasoned that it was selectively sequestered from blood to the ovarian cavity in pregnant females, and consequently, plays crucial roles in pregnancy.

Taxonomic profiles in metagenomic analyses of free-living microbial communities in the Ofunato Bay

The Ofunato Bay in Iwate Prefecture, Japan is a deep coastal bay located at the center of the Sanriku Rias Coast and considered an economically and environmentally important asset. Here, we describe the first whole genome sequencing (WGS) study on the microbial community of the bay, where surface water samples were collected from three stations along its length to cover the entire bay; we preliminarily sequenced a 0.2u202fμm filter fraction among sequentially size-fractionated samples of 20.0, 5.0, 0.8 and 0.2u202fμm filters, targeting the free-living fraction only. From the 0.27-0.34 Gb WGS library, 0.9u202f×u202f106-1.2u202f×u202f106 reads from three sampling stations revealed 29 bacterial phyla (~80% of assigned reads), 3 archaeal phyla (~4%) and 59 eukaryotic phyla (~15%). Microbial diversity obtained from the WGS approach was compared with 16S rRNA gene results by mining WGS metagenomes, and we found similar estimates. The most frequently recovered bacterial sequences were Proteobacteria, predominantly comprised of 18.0-19.6% Planktomarina (Family Rhodobacteraceae) and 13.7-17.5% Candidatus Pelagibacter (Family Pelagibacterales). Other dominant bacterial genera, including Polaribacter (3.5-6.1%), Flavobacterium (1.8-2.6%), Sphingobacterium (1.4-1.6%) and Cellulophaga (1.4-2.0%), were members of Bacteroidetes and likely associated with the degradation and turnover of organic matter. The Marine Group I Archaea Nitrosopumilus was also detected. Remarkably, eukaryotic green alga Bathycoccus, Ostreococcus and Micromonas accounted for 8.8-15.2%, 3.6-4.9% and 2.1-3.1% of total read counts, respectively, highlighting their potential roles in the phytoplankton bloom after winter mixing.

Global gene expression analysis of the muscle tissues of medaka acclimated to low and high environmental temperatures

Medaka (Oryzias latipes) is a temperate eurythermal fish that is able to survive over a wide range of water temperatures ranging from near zero to over 30°C throughout the year; it maintains its normal physiological and biochemical processes through temperature acclimation. To determine the mechanisms involved in temperature acclimation of fish, the fast skeletal muscle tissues of medaka underwent global gene expression analysis using next-generation sequencing. Ten individuals were placed into two aquariums at 24°C. While the water temperature of one aquarium was decreased to 10°C, that of the other aquarium was increased to 30°C; these temperatures were subsequently maintained for 5weeks. RNA sequencing (RNA-Seq) analyses revealed that 11 genes involved in energy metabolism and muscle atrophy were significantly highly expressed in the 10°C-acclimated fish. Meanwhile, significantly higher expression levels were observed for 20 genes encoding myofibrillar proteins and heat shock proteins in the 30°C-acclimated fish. Moreover, 1103 genes had at least fourfold differential expression between the acclimation groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses provided important information: although the expression of genes related to metabolic processes were activated, muscle atrophy occurred in the 10°C-acclimated fish, and muscle cells divided actively in the 30°C-acclimated fish and avoided thermal stress by expressing heat shock proteins. Therefore, RNA-Seq analyses with the available genome database will be useful for better understanding the molecular mechanisms involved in the temperature acclimation of fish.

Seasonal changes in the communities of photosynthetic picoeukaryotes in Ofunato Bay as revealed by shotgun metagenomic sequencing

Small photosynthetic eukaryotes play important roles in oceanic food webs in coastal regions. We investigated seasonal changes in the communities of photosynthetic picoeukaryotes (PPEs) of the class Mamiellophyceae, including the genera Bathycoccus, Micromonas and Ostreococcus, in Ofunato Bay, which is located in northeastern Japan and faces the Pacific Ocean. The abundances of PPEs were assessed over a period of one year in 2015 at three sampling stations, KSt. 1 (innermost bay area), KSt. 2 (middle bay area) and KSt. 3 (bay entrance area) at depths of 1u202fm (KSt. 1, KSt. 2 and KSt. 3), 8u202fm (KSt. 1) or 10u202fm (KSt. 2 and KSt. 3) by employing MiSeq shotgun metagenomic sequencing. The total abundances of Bathycoccus, Ostreococcus and Micromonas were in the ranges of 42-49%, 35-49% and 13-17%, respectively. Considering all assayed sampling stations and depths, seasonal changes revealed high abundances of PPEs during the winter and summer and low abundances during late winter to early spring and late summer to early autumn. Bathycoccus was most abundant in the winter, and Ostreococcus showed a high abundance during the summer. Another genus, Micromonas, was relatively low in abundance throughout the study period. Taken together with previously suggested blooming periods of phytoplankton, as revealed by chlorophyll a concentrations in Ofunato Bay during spring and autumn, these results for PPEs suggest that greater phytoplankton blooming has a negative influence on the seasonal occurrences of PPEs in the bay.

Lampreys Have a Single Gene Cluster for the Fast Skeletal Myosin Heavy Chain Gene Family

Muscle tissues contain the most classic sarcomeric myosin, called myosin II, which consists of 2 heavy chains (MYHs) and 4 light chains. In the case of humans (tetrapod), a total of 6 fast skeletal-type MYH genes (MYHs) are clustered on a single chromosome. In contrast, torafugu (teleost) contains at least 13 fast skeletal MYHs, which are distributed in 5 genomic regions; the MYHs are clustered in 3 of these regions. In the present study, the evolutionary relationship among fast skeletal MYHs is elucidated by comparing the MYHs of teleosts and tetrapods with those of cyclostome lampreys, one of two groups of extant jawless vertebrates (agnathans). We found that lampreys contain at least 3 fast skeletal MYHs, which are clustered in a head-to-tail manner in a single genomic region. Although there was apparent synteny in the corresponding MYH cluster regions between lampreys and tetrapods, phylogenetic analysis indicated that lamprey and tetrapod MYHs have independently duplicated and diversified. Subsequent transgenic approaches showed that the 5′-flanking sequences of Japanese lamprey fast skeletal MYHs function as a regulatory sequence to drive specific reporter gene expression in the fast skeletal muscle of zebrafish embryos. Although zebrafish MYH promoters showed apparent activity to direct reporter gene expression in myogenic cells derived from mice, promoters from Japanese lamprey MYHs had no activity. These results suggest that the muscle-specific regulatory mechanisms are partially conserved between teleosts and tetrapods but not between cyclostomes and tetrapods, despite the conserved synteny.

Radiocesium Contamination of Marine Fish Muscle and Its Effective Elimination

High concentrations of radiocesium (134Cs, 136Cs, and 137Cs combined) were detected in several fish species such as nibe croaker, Pacific cod, and brown hakeling, which were collected from the Pacific Ocean off the coast of the Fukushima and Ibaraki Prefectures. High levels of radiocesium accumulated in fish muscle, but the radioactivity levels of naturally occurring radioactive K in some contaminated fish exceeded the levels of 134Cs and 137Cs. Three washes with 0.1% NaCl solution effectively removed the radiocesium from contaminated fish meat. This can be applied in the production of surimi-based products and other processed seafood such as boiled, dried, or seasoned products.

Changes in free amino acid concentrations and associated gene expression profiles in the abdominal muscle of kuruma shrimp Marsupenaeus japonicus acclimated at different salinities

ABSTRACT Shrimps inhabiting coastal waters can survive in a wide range of salinity. However, the molecular mechanisms involved in their acclimation to different environmental salinities have remained largely unknown. In the present study, we acclimated kuruma shrimp (Marsupenaeus japonicus) at 1.7%, 3.4% and 4.0% salinities. After acclimating for 6, 12, 24 and 72u2005h, we determined free amino acid concentrations in their abdominal muscle, and performed RNA sequencing analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after 24 h of acclimation. Glutamine and alanine concentrations were markedly increased following the increase of salinity. In association with such changes, many genes related to amino acid metabolism changed their expression levels. In particular, the increase of the expression level of the gene encoding glutamate-ammonia ligase, which functions in glutamine metabolism, appeared to be associated with the increased glutamine concentration at high salinity. Furthermore, the increased alanine concentration at high salinity was likely associated with the decrease in the expression levels of the the gene encoding alanine-glyoxylate transaminase. Thus, there is a possibility that changes in the concentration of free amino acids for osmoregulation in kuruma shrimp are regulated by changes in the expression levels of genes related to amino acid metabolism. Summary: Kuruma shrimp, Marsupenaeus japonicus, change free amino acid concentrations and associated gene expression levels in their muscle to adjust effectively to different salinities.

Metagenome-based diversity analyses suggest a strong locality signal for bacterial communities associated with oyster aquaculture farms in Ofunato Bay

Ofunato Bay, in Japan, is the home of buoy-and-rope-type oyster aquaculture activities. Since the oysters filter suspended materials and excrete organic matters into the seawater, bacterial communities residing in its vicinity may show dynamic changes depending on the oyster culture activities. We employed a shotgun metagenomic technique to study bacterial communities near oyster aquaculture facilities at the center of the bay (KSt. 2) and compared the results with those of two other localities far from the station, one to the northeast (innermost bay, KSt. 1) and the other to the southwest (bay entrance, KSt. 3). Seawater samples were collected every month from January to December 2015 from the surface (1u202fm) and deeper (8 or 10u202fm) layers of the three locations, and the sequentially filtered fraction on 0.2-μm membranes was sequenced on an Illumina MiSeq system. The acquired reads were uploaded to MG-RAST for KEGG functional abundance analysis, while taxonomic analyses at the phylum and genus levels were performed using MEGAN after parsing the BLAST output. Discrimination analyses were then performed using the ROC-AUC value of the cross validation, targeting the depth (shallow or deep), locality [(KSt. 1u202f+u202fKSt. 2) vs. KSt 3; (KSt. 1u202f+u202fKSt. 3) vs. KSt. 2 or the (KSt. 2u202f+u202fKSt. 3) vs. KSt. 1] and seasonality (12u202fmonths). The matrix discrimination analysis on the adjacent 2 continuous seasons by ROC-AUC, which was based on the datasets that originated from different depths, localities and months, showed the strongest discrimination signal on the taxonomy matrix at the phylum level for the datasets from July to August compared with those from September to June, while the KEGG matrix showed the strongest signal for the datasets from March to June compared with those from July to February. Then, the locality combination was subjected to the same ROC-AUC discrimination analysis, resulting in significant differences between KSt. 2 and KSt. 1u202f+u202fKSt. 3 on the KEGG matrix. These results suggest that aquaculture activities markedly affect bacterial functions.