Program

Massively parallel sequencing (MPS) is a revolutionizing technology to study eukaryotic diversities. This technology enables us to detect several hundreds of operational taxonomic units (OTUs) of eukaryotes from seawater samples, and facilitates the detection of low-abundance populations in complex eukaryote communities. A routine monitoring was conducted at 5 stations of O-line in Omaezaki in Shizuoka Prefecture, Japan (Kuroshiwo region) in August, 2015. Metagenome analysis based on amplicon sequences for 18S-rRNA gene (V4-5 regions) at 7-8 layers in each station was carried out by the Miseq 250PE platform. We detected 1,874 operational taxonomic units (OTUs) in total (1,690 OTUs in Rarefied). The numbers of OTUs fluctuated from 97 to 649 and 285 ± 167 (mean ± SD) (66–482, 230 ± 135, in rarefied). Relative abundances of identified OTUs at the supergroup levels were calculated. Alveolata (mainly dinoflagellates and ciliates) had the highest relative abundance in the samples, and the detected numbers of OTUs and MPSs were 47.4–62.9 (54.5 ± 4.1) % and 26.0–88.1 (57.0 ± 17.6) %, respectively. Stramenopiles (mainly diatoms) is one of the most dominant groups with Alveolata in coastal waters all over the worlds. However, surprisingly, the second largest group was Rhizaria, with relative abundances of OTUs and MPSs of 8.9–30.9 (19.6 ± 6.0) % and 0.8–73.4 (33.7 ± 23.2) %, respectively, showing the unique ecosystem in the oligotrophic offshore regions. The frequency of OTUs and MPSs, which were identified as a single species, at the supergroup levels were 9.6–34.0% and 4.5–56.9%, respectively. The taxonomic identification power was relatively high for Rhizaria, Opisthokonta, and Viridiplantae, but low for Alveolata and Stramenopiles, implying the insufficient registration of the taxonomic and genetic information on Alveolata and Stramenopiles in the regions at international nucleotide sequence databases (INSDs).

Photosynthetic picoeukaryotes (PPEs) are smaller size fractions of phytoplankton performing photosynthesis as important components in marine microbial food webs. However, PPEs have been marginally studied at a basin scale in the Ofunato Bay, an economically important zone located in the northeastern Pacific coast of Japan. The use of Ilumina MiSeq shotgun sequencing allowed us to estimate the seasonal occurrences of PPEs at three sampling stations, KSt. 1 (innermost bay area), KSt. 2 (middle bay area) and KSt. 3 (bay entrance area) at depths of 1 m (KSt. 1, KSt. 2 and KSt. 3) and 8 m (KSt. 1) or 10 m (KSt. 2 and KSt. 3) from January to December in 2015. Irrespective of the three sampling stations and depths, the relative abundances of the three genera of PPEs, Bathycoccus, Ostreococcus and Micromonas, all of which belong to Mamiellophyceae under the phylum of Chlorophyta, were 42-49%, 35-49% and 13-17%, respectively. Seasonal changes were revealed by high abundances of PPEs during winter and summer, while low abundances were observed during spring and autumn at blooming periods confirmed by chlorophyll a concentrations, where larger cells like diatom and dinoflagellate dominate the plankton community. Furthermore, there were significant negative correlations between the abundances of picoeukaryotes and chlorophyll a concentrations, especially at 1m depth on KSt. 1 and KSt. 3 (r= -0.66 and r= -0.58, P<0.05). Of the three genera assessed, Bathycoccus was most abundant in winter, and Ostreococcus showed a high abundance during summer. Another genus, Micromonas, was relatively low in abundance throughout the year, which appeared to be associated with a complex of environmental factors. These results imply that during winter and summer, when larger phytoplankton are not dominant, PPEs contribute significantly to the primary production in marine environments.

Ofunato Bay in Iwate Prefecture, Japan is the home of important aquaculture activities. Chlorophyll a (chl-a) concentration has been reported to be high in the bay that attracted buoy-and-rope-type oyster aquaculture facilities for Pacific giant oyster (Crassostrea gigas). Since oysters filter suspended material and excrete organic matters into the seawater, the bacterial communities residing in its vicinity may show dynamic seasonal changes. Accordingly we are studying bacterial composition and their functional traits in the seawater near oyster aquaculture facilities from September 2014 employing shotgun metagenomic technique and correlating them with water quality parameters including temperature, salinity, pH, DO, NO₂₊₃-N, NH₄-N, and chl-a. We previously reported that during 2015, diverse communities of free-living bacteria in seawater near the oyster aquaculture facilities exhibited intense inter-seasonal differences. It was also observed that during the same period the distribution and abundance of several functional traits including “amino acid metabolism”, “energy metabolism”, “metabolism of cofactors” and “translation” revealed by KEGG Ontology (KO) analysis were significantly different (p<0.05) between surface (1m) and deep (10m) waters near oyster aquaculture facilities. Functional abundance was also significantly different when second tier KO categories between seawater near oyster aquaculture facilities were compared to those far from there in Ofunato Bay. We are currently analyzing such changes of bacterial communities in 2016, and interested in identifying their temporal variations in relation to physical and biological environmental factors for two consecutive years.

The Ofunato Bay is a type of an enclosed bay in the Sanriku Rias Coast, where seawater enviroments rich in nutrients are affected by both warm and cold ocean currents along with river, providing good aquaculture fields for marine bivalves. We started the metagenomic survey on microbial communities in the Ofunato Bay as a KAUST project since 2014 and here we summarize our obtained results. Sampling stations are KSt. 1 at the innermost, KSt. 2 at the center and KSt. 3 at the bay entrance. Seawaters were collected every month at 1 m and 8 or 10 m depths from the three stations. Water quality was monitored with a profiler for several parameters including temperature and chlorophyll a concentrations. Collected seawaters were filtered sequentially through 5, 0.8 and 0.22-μm filters, while DNA was extracted only from the 0.22 μm-filters mainly targeting free-living bacteria and subsequently applied to a MiSeq sequencer to obtain the whole genome sequencing reads. Most abundant bacteria belonged to Alphaproteobacteria which consist of Planktomarina, Canditatus Pelagibacter, and Bartonella, irrespective of the three stations and two seawater depths. Discrimination analysis with ROC-AUC, targeting the two depth, three stations and 12 months from January to December 2015 for seasonality (12 months to adjacent continuous 2 seasons) revealed that the period of July to August were significantly different from other months. The responsible genera responsible for this discrimination were unclassified Verrucomicrobia, unclassified Proteobacteria, and Coraliomargarita. This discrimination was further confirmed by the hierarchal cluster dendrogram. ROC-AUC for local combination clearly discriminated KSt. 2 from KSt. 1 plus KSt. 2 at the KEGG matrix, whereas the bacterial community in KSt. 3 at 1 m depth was different from others as revealed by PCA on the relative abundances of bacterial genera. Further analysis is under progress on the effects of environmental factors.

The offshore of Sanriku in Japan is known as a prominent fishing ground because of the collision of the Oyashio and Kuroshio currents. The Ofunato Bay at the Iwate Prefecture, which is famous for oyster and seaweed farming, is influenced by these currents. Analyses on the microbial community is essential to understand the marine environments and ensure sustainable utilization of ecosystem services from the ocean. Accordingly, we have been performing metagenomic analyses on the microbial community in the Ofunato Bay in collaboration with King Abdullah University of Science and Technology. Seawater samples were collected every month from 1 m and 8 or 10 m layers of three locations in the Ofunato Bay in 2015 and 2016. The water quality was measured with a water quality data logger. DNA was extracted from filters with a pore size of 0.2 µm, and subjected to preparation for shotgun metagenomic analysis using commercially available kits. The preparations were then sequenced using an Illumina MiSeq. The obtained Illumina reads were uploaded to metagenomic pipelines including MG-RAST and MEGAN. It was found that the shotgun metagenomic sequences were organized from bacteria, eukaryotes, archaea, and viruses. When the results of our analyses were focused on bacteria, bacteria in the sea water collected from the Ofunato Bay in 2015 and 2016 were found to comprise mainly Candidatus Pelagibacter and Planktomarina, and their community was different between the two years. Moreover, the abundance of Synechococcus in 2016 was higher than that in 2015. It is expected that continuous and detailed analyses on the metagenomic data could reveal the changes in the microbial communities on the material cycling system for the primary production in the Ofunato Bay.

The Sanriku Ofunato Bay locates in the northeastern Pacific Ocean area of Japan where marine organisms show the highest biodiversity in the world, primarily due to tidal influences by t the cold Oyashio Currents, the warm Kuroshio ,Currents and the Tsugaru Warm Current.
Our previous results by shotgun metagenomics indicated that both Candidatus Pelagibacter ubique and Planktomarina temperata were dominated in bacteria (Reza et al., under review). It is reported that these bacteria have abilities to catabolize dimethylsulfoniopropionate (DMSP) to dimethyl sulfide (DMS) or methanethiol (MeSH). The present study focused on seasonal change in the abundance of bacterial genes (dddP, dmdA) related to DMSP catabolism in the seawater at the Ofunato Bay by blast+ analysis using a shotgun metagenomic data.
Seasonality was observed in catabolite gene dynamics of DMSP metabolism in Sanriku Ofunato Bay. Namely peaks of Candidatus Pelagibacter ubique HTCC1062 dmdA gene were observed between March and June, suggesting their relation with spring bloom. Peaks of Candidatus Pelagibacter ubique O19-dddP and O19-dmdA homologs were observed between Sep and Dec (Autumn), respectively. We found seasonal changes among Candidatus Pelagibacter ubique strains, that is, HTCC1062-type strains and the Red Sea-type strains. On the other hand, peaks of Planktomarina temperata dddP gene were observed in July.
These bacteria have the gene homologs to catabolite DMSP produced by phytoplankton, suggesting that these bacteria locate next to phytoplankton in marine material cycle system. DMSP, DMS and MeSH are reported as signalling molecules, suggesting that they have roles to attract bacteria, alga, animal plankton, fishes and seabirds. These facts suggest that the molecules play a role to trigger fishery formation.

Metagenomic studies have enabled us to identify taxonomic profiles of bacterioplankton and also elucidate ecological functions of the residing microbial communities. As the Tama River is regarded as a life line of urban Tokyo, we examined seasonal differences of the bacterial communities in a non-tidal, urban-impacted part of this river to better understand their ecological functions. Water samples (~2L) were collected every month from September 2015 to December 2016 with sterile beakers from the surface to 12.5 cm depth at a middle reach of the Tama River in Tama City, following the guidelines of the Bureau of Sewage, Tokyo Metropolitan Government (2012). Of the collected water, 500 ml were filtered sequentially through Isopore™ membranes with a 45 mm diameter each of 5, 0.8 and 0.2-µm pore sizes. As we targeted free-living bacterial fraction, DNA was extracted only from 0.2-µm filter membranes and we prepared shotgun metanogemic libraries using Nextera XT library preparation kit (Illumina). The libraries were then sequenced with an Illumina MiSeq using MiSeq Reagent Kit v3. Acquired Illumina reads were assigned to their taxonomic affiliations by BLASTN comparisons to the GenBank nonredundant nucleotide database. Betaproteobacteria and Flavobacteriia were the most abundant groups in the Tama River. The relative abundance of Flavobacteriia decreased from 51.4 % in May to 1.9 % in December, while that of Gammaproteobacteria increased gradually from 1.1 % in August to 12.1 % in November. A marked increase of Bacteroidia was observed in February. Negative correlations against water temperature were observed with the relative abundances of Gammmaproteobacteria, Bacilli, Mollicutes, Chlamydiia and Chlorobia. A positive correlation against dissolved oxygen was observed with Flavobacteriia, while a negative correlation observed with Gammaproteobacteria. These results suggest that seasonal changes in environmental variables contribute to the dynamic changes in the bacterial communities in the Tama River.

At the pacific side of Tohoku area in Japan, nutrients such as nitrogen and phosphorus are abundant in seawater and they cause a dense bloom of plankton in spring. Since the spring bloom feeds fishes, the area is rich in marine resources and is called as the world's three major fishing grounds. The key organisms of the bloom has been investigated by observing large planktons using a microscope, but microorganisms such as viruses and bacterias could not be investigated enough though they might be related to the occurrence and disappearance of the bloom. Therefore we began to investigate whole marine microorganisms from virus to large phytoplankton using shotgun metagenomics to comprehensively grasp their behavior. We collected seawater samples at the Tohoku area for three years, and collected shotgun metagenome sequence data. The seawater was filtered serially through three kinds of membrane filters having different pore size to fractionate microorganisms into bacterial, pico-phytoplankton, and large phytoplankton sizes. As a result, about 500 sequence data sets were obtained. Species composition was determined using the NCBI nt or SILVA database from the shotgun metagenome data. The analysis results are registered in a database as interactive time series graphs and interactive 3D graphs. We also extracted 748 probe sequences that can distinguish characteristics of seawater. By using this probe set, it became possible to estimate sampling season, area, nutrient abundance, and filter size used for the fractionation from shotgun metagenome data.

The shell of Pinctada fucata consists of calcium carbonate and organic matrices. The outer layer of the shell contains prismatic calcite crystals surrounded by organic frameworks. Calcite in the prismatic layer of P. fucata is extremely tough and stiff due to small-angle grain boundaries and localized organic networks inside of calcites to prevent their fracture. Although electron microscopy observations have suggested that such small crystal defects are caused by the organic networks during calcite formation, the components of organic matrices and their effect on the formation of crystal defects remain unclear. In this study, we used infrared spectroscopy and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify chitin as the main component of organic networks and chitinolytic enzymes that bind to chitin. To investigate the effects of chitin and chitinolytic enzymes on the formation of calcites, calcites were synthesized in chitin gel after treatment with chitinolytic enzymes. The crystal defects became larger as the chitin fibers became more degraded and thinned by chitinolytic enzymes in a dose-dependent manner. To determine the function of chitinolytic enzymes in vivo, allosamidin, a specific inhibitor of family 18 chitinases, was injected into living P. fucata. Examination of the prismatic layer of allosamidin-injected individuals revealed altered prism shapes and that the network was thicker and wider. These results suggest that the thinness of chitin, which is regulated by the degradation of chitinolytic enzymes, contributes to the formation of small-angle grain boundaries.

Abiological precipitation of calcium carbonate seems to be very limited, even though the surface seawaters in tropic oceans are about three to seven times supersaturated with respect to calcium carbonate. Indeed, a significant portion of the carbonate sediments are of a biogenic origin. There are many marine organisms synthesizing a variety of skeletons containing calcium carbonate crystals. Their crystal structures and formation mechanisms have attracted attention from many researchers, and various types of novel organic molecules involved in the formation of calcium carbonate have been reported. Recently, we reported biogenic polyamines having multiple amino groups were able to react with carbon dioxide and accelerate calcium carbonate formation in seawater (Yasumoto et al., 2014). For example, aragonite crystals were easily formed when polyamines were added to seawater and the mixture was allowed to stand for a few days at room temperature. We also found the effects of polyamines in an aqueous solution containing calcium ion on the formation of calcium carbonate crystals. Thus, we hypothesized that the biogenic polyamines play important roles in the formation of diverse calcareous skeletons in marine organisms. To prove such hypothesis, we are investigating the molecular mechanisms involved in the calcification of primary polyps of Acroporid coral. Moreover, we could observe the adhesion between crystals in the process of the calcium carbonate formation in marine bacteria and primary polyps of Acroporid coral. The mechanisms involved in adhesion between calcium carbonate crystals are now under investigation. If we could recover a large amount of calcium carbonate from seawater using polyamines, this novel method helps reduce atmospheric CO₂ levels and further supply raw cement materials, which are worried about the depletion of natural resources.

Molluscan smooth muscles can maintain tension for long periods with little energy consumption. This phenomenon is called ‘catch’. Twitchin—a giant myosin-associated protein of 530 kDa—controls the catch contraction through phosphorylation/dephosphorylation. Catch muscle starts developing its tension after an increase of intracellular [Ca²⁺] induced by a secretion of acetylcholine, and then enters into the catch state after a decrease of [Ca²⁺]. In the catch state the twitchin D2 phosphorylation site in an unphosphorylated form binds both to myosin and actin, tethering thick filaments to thin filaments. Thus, the D2 site contributes to the tension maintenance. There is the other phosphorylation site, D1, in the twitchin N-terminal region. The function of D1 is still obscure. In the present study, we constructed the D1 site polypeptide (TWD1) and analyzed its interaction with other muscle proteins to reveal how the D1 site works in the catch contraction. Overlay and solid-phase binding assays against catch muscle proteins using TWD1 as a probe revealed that TWD1 binds to tropomyosin in a phosphorylation-sensitive manner. These findings indicate that the D1 site might participate in the regulation of the catch contraction together with the D2 site. Now we are identifying the TWD1 binding regions on tropomyosin.

The pearl oyster, Pinctada fucata is one of the most important species for pearl farming in Japan and East Asia. Because pearl formation is a physiological process regulated by the animal, the genotype of the oyster affects the quality of the pearls produced. Molecular characteristics of pearls and the nacreous structure of the shell have been studied for more than 20 years, and dozens of genes and proteins that are involved in nacre formation have been identified. The whole genome has been sought in order to thoroughly understand the molecular process, and by using genetic information, it is possible to develop an effective breeding program to yield superior pearl oysters.
In 2012, we decoded the draft genome assembly of the pearl oyster, Pinctada fucata. The genome decoding project showed that the genome is highly heterozygotic, which made it challenging to obtain a haploid assembly. In order to cope with the complex genome, we developed a method for unifying heterozygotic sequences. Long-read sequences were applied to create a more continuous genome assembly. High-throughput SNP detection based on RAD-seq technology was conducted in order to generate a genetic map, which was utilized to improve the assembly.
This genome assembly strategy successfully reduced redundant sequences in the assembly. The improved genome assembly (P. fucata genome version 3.0), along with proteomic and transcriptomic data are analyzed to identify a comprehensive catalog of proteins involved in nacre formation. Genes responsible for tolerance to environmental changes, such as water temperature are also examined. The P. fucata genome provides basic biological information about the oyster, as well as an opportunity to improve the new breeding scheme, using techniques such as marker-assisted selection and genomic selection, which will facilitate development of excellent pearls.