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Kitasato-KAUST Joint

International Workshop

Marine Metagenomics: Comparative Study among Different Marine Resources

24-25 August, 2016

Sanriku Education and Research Center for Marine Biosciences, Kitasato University School of Marine

Biosciences Organized by Kitasato University and King Abdullah University of Science and Technology (KAUST, Saudi Arabia) Co-sponsored by Iwate Prefecture and Ofunato City

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Organizing Committees International Organizing Committee Co-chair

Shugo Watabe, Kitasato University

Takashi Gojobori, KAUST

Local Organizing Committee Chair

Shugo Watabe, Kitasato University

Vice Chair Hiroshi Tomoda, Kiasato University

Members Atsushi Kobiyama, Kitasato University

Md. Shaheed Reza, Kitasato University

Kazuho Ikeo, National Institute of Genetics

Kimiaki Naiki, Iwate Fisheries Technology Center

Katsuhiko Mineta, KAUST

Tomoko Sakami, Tohoku National Fisheries�Research

Institute

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Preface

To enhance scientific exchange and research activity, and to

endorse collaborative ongoing research work between Kitasato

University, Japan, and King Abdullah University of Science and

Technology (KAUST), Saudi Arabia, this workshop will focus on the

metagenomic analyses of microbial communities residing in the Red

Sea and in the Ofunato Bay to reveal their taxonomic affiliation as

well as their functional roles in the environment. Challenges involved

in such assignments and the comparative metagenomic approach

used to elucidate the role of microbes in marine ecosystem to

combat the possible affect of global warming will be discussed.

This workshop is supported in part by Grant for All Kitasato

Project Study and Grant from KAUST.

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Program 24 August

9:30-9:45 Opening remarks

Nobuhiro Kan-no (Kitasato University)

Takashi Gojobori (KAUST)

9:45-10:15 Vladimir Bajic (KAUST)

Text-mining support in predicting protein functions

10:15-10:45 Kazuho Ikeo (National Institute of Genetics)

Usage of shot gun sequence data for marine meta-genomics

10:45-11:00 Coffee break

11:00-11:30 Takashi Gojobori (KAUST)

New frontier of comparative marine metagenomics

11:30-12:00 John Archer (KAUST)

The picocyanobacterial adaptations to high temperature and high salinity

as revealed by genomic analyses

12:00-13:00 Lunch break

13:00-13:30 Kimiaki Naiki (Iwate Fisheries Technology Center)

Changes of seawater quality in the Ofunato Bay

13:30-14:00 Yuichiro Yamada (Kitasato University)

Seasonal variations of water quality and pico-plankton abundance in

Ofunato Bay

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14:00-14:30 Md. Shaheed Reza (Kitasato University)

Functional traits of bacterial communities associated with oyster farms in

the Ofunato Bay

14:30-14:45 Coffee break

14:45-15:15 Atsushi Kobiyama (Kitasato University)

Microbial community dynamics of free-living microbial assemblages in the

Ofunato Bay

15:15-15:45 Hiroshi Tomoda (Kitasato University)

Discovery of marine microbial natural products

16:00-17:00 Poster session

25 August

9:00-9:30 Haruko Takeyama (Waseda University)

Metagenomic analysis of invertebrate holobionts and supporting

technologies

9:30-10:00 Tsuyoshi Watanabe (Tohoku National Fisheries Research

Institute)

Seasonal dynamics of phytoplankton community in Sendai Bay studied by

ocean monitoring combined with metagenomic approach

10:00-10:30 Tomoko Sakami (Tohoku National Fisheries Research Institute)

Variation of bacterial community composition in relation to the

hydrographical profiles in Sendai Bay

10:30-10:45 Coffee break

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10:45-11:15 Satoshi Nagai (Central National Fisheries Research Institute)

Ocean current properties and changes of plankton biodiversity in Okhotsk

Sea

11:15-11:45 Ko Yasumoto (Kitasato University)

Mechanisms involved in carbon dioxide fixation by marine microorganisms

11:45-12:00 Closing remarks

Shugo Watabe (Kitasato University)

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Poster session 1. Akira Kuwata

THE EVOLUTIONARY LINK BETWEEN PARMALES AND THE SUCCESS OF DIATOMS IN MARINE ECOSYSTEMS

2. Akira Kuwata

SEASONAL AND GEOGRAPHICAL DISTRIBUTION OF NEAR-SURFACE SMALL PHOTOSYNTHETIC-EUKARYOTES IN THE WESTERN NORTH PACIFIC DETERMINED BY PYROSEQUENCING OF 18S RDNA

3. Kaoru Matsumoto

SEASONAL CHANGE OF BACTERIAL COMMUNITY IN SENDAI BAY, FROM A FUNCTIONAL PERSPECTIVE

4. Norikazu Kitamura

METAGENOMIC ANALYSIS OF BACTERIAL FRACTION DURING CHATTONELLA-KARENIA BLOOM

5. Shuichi Asakawa CHARACTERIZATION OF MARINE METAGENOME BASED ON THE PROTEIN MOTIF

6. Nanami Mizusawa AMPLICON ANALYSIS OF SEASONAL CHANGES IN FREE-LIVING BACTERIOPLANKTON FROM THE TAMA RIVER IN TOKYO

7. Rashid Jonaira

METAGENOMIC APPROACH ON THE CHANGES IN MICROFLORA DURING MAY-JUNE, 2016, IN THE OFUNATO BAY

8. Toshiaki Kudo SEASONAL CHANGE OF SOME BACTERIA AT THE OFUNATO BAY BY SHOTGUN METAGENOMICS

9. Yoshimasa Kaga

TEMPORAL CHANGE IN VERTICAL DISTRIBUTION OF Alexandrium tamarense IN THE OFUNATO BAY, JAPAN

10.Yuri Ikeda

COMPOSITION OF DINOFLAGELLATE CYSTS IN THE OFUNATO BAY, IWATE, JAPAN

11.Shigeru Sato

A NEW ELISA KIT AS A MONITORING TOOL FOR PARALYTIC SHELLFISH TOXINS

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12.Shigeru Sato A NEW POLYCLONAL ANTIBODY AGAINST TETRODOTOXIN

13.Hiroaki Kasai

POSSIBLE USE OF RESIDUAL BIOMASS OF OIL-PRODUCING MICROALAGE FOR AQUAFEED

14.Mitsuru Jimbo

A GALNAC-BINDING LECTIN COULD BE INVOLVED IN ACQUISITION OF SYMBIODINIUM BY A CORAL ACROPRA TENUIS LECTIN

15.Daisuke Ikeda

GLOBAL GENE EXPRESSION ANALYSIS ON MEDAKA MUSCLE TISSUES IN ASSOCIATION WITH TEMPERATURE ACCLIMATION

16.Yoshihiro Takahashi

AN APPROACH TO GENOME ANALYSIS OF JAPANESE SEAWEED, WAKAME Undaria pinnatifida

17.Shugo Watabe

16S AMPLICON ANALYSIS OF MICROBIAL FLORA IN THE SALTED AND FERMENTED SQUID PRODUCTS AIMING AT THEIR QUALITY CONTROL

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Lectures TEXT-MINING SUPPORT IN PREDICTING PROTEIN FUNCTIONS

Adil Salhi, Aleksandar Radovanovic, Tanvir Alam, Magbubah Essack, Vladimir B. Bajic

King Abdullah University of Science and Technology (KAUST)

Computational Bioscience Research Center (CBRC) Thuwal, Saudi Arabia

Not available due to the official reason(s).

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USAGE OF SHOT GUN SEQUENCE DATA FOR MARINE-META GENOMICS Kazuho Ikeo1)2), Kaoru Matsumoto2), Norikazu Kitamura2), Takehiko Ogata1), Katsuhiko

Mineta3), Takashi Gojobori3) and Shugo Watabe1) School of Marine Biosciences, Kitasato Univ., Japan1), Natiional Institute of Genetics2),

King Abdullah Univ. Sci. Technol., Saudi Arabia 3) Next Generation Sequencing (NGS) technology makes a big step of the life science in the many varieties of the field including environment, populations, and comparative genomics. Especially meta-genomics study gives important and useful information on the interaction between species or among species. Because of the performance of NGS, Now it became possible to analysis the sequences from wide area through continuous sampling. The approaches of the combination of meta-genomics data and any additional environmental information by using multiple analytical approaches are expected to give useful and new insight of the dynamics of biological systems and environments. Such information will also be interested in to understand evolution. For this purpose, we need to process the large sequence data from NGS and additional information to understand the phenomena. We are now analysis the data of meta-genomics to understand the dynamics of marine micro-biome and developing the data analysis system for the NGS data with simple interface for data analysis. Here, I will introduce the recent trial by using meta-genomics approaches to understand the dynamics of marine biological systems and also the introduction of our NGS data analysis system named Maser.

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NE FRONTIER OF COMP R TIVE M RINE MET GENOMICS

a i Mi a and Ta a i G j ri

Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology (KAUST), Saudi Arabia

Not available due to the official reason(s).

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GENETIC ND P SIO OGIC IG T D PT TION IN RED SE Synechococcus .

rc r, Noor A lin Mokhtar, iMei Ng, Suhai a Ahmad amhor

Microbial Technology roup, Computational Biosciences Research Center, Computer,

Electrical and Mathematical Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia

Not available due to the official reason(s).

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CHANGES OF SEAWATER QUALITY IN THE OFUNATO BAY, JAPAN

1Kimiaki Naiki, 1Shiho Watanabe, 1Satoshi Segawa, 1Yoshimasa Kaga, 1Yasushi Kuji, 2Shinnosuke Kaga, 3Yuichiro Yamada, 3Yuri Ikeda, 3Md. Shaheed Reza, 3Atsushi Kobiyama,

3Shigeru Sato, 3Takehiko Ogata, 3Daisuke Ikeda, 3Nanami Mizusawa, 3Kazuho Ikeo, 3Toshiaki Kudo, 4Katsuhiko Mineta, 4Takashi Gojobori, and 3Shugo Watabe

1Iwate Fisheries Technology Center, Japan (k-naiki@pref.iwate.jp); 2Iwate Prefectural Government, Japan; 3School of Marine Biosciences, Kitasato University, Japan; 4King

Abdullah University of Science and Technology, Saudi Arabia

The Sanriku Rias Coast is one of the most active aquaculture regions in the world. The Ofunato Bay is located on this Coast, where the aquaculture of oysters is flourishing. In this bay, sometimes aquaculture has been damaged by extreme low temperature water derived from the Coastal Oyashio water (COW). On the other hand, the COW transports nutrient rich water and contributes to phytoplankton blooms. Since oysters assimilate natural resources, abrupt environmental changes such as the introduction of the COW may have a significant influence on the filtration and excretion rate of oysters. We, therefore, monitored seawater quality around oyster culture facilities over a period of approximately two years. Seawater samples were collected every month at upper (1 m) and deeper (8 or 10 m) layer from three locations at the Ofunato Bay. Basic water quality parameters were recorded on the point using water quality profiler. As a result, water temperature was found to be highest in August (25.3 °C) and lowest in March (4.7 °C). Salinity ranged from 20.8-33.9 throughout the sampling period. Extremely cold temperature (<5 °C) and high concentration of nitrate + nitrite (5.2-12.1 μM) were observed in March 2015, which was due to the entry of the COW. Thereafter, chlorophyll a concentrations increased dramatically (6.5-20.3 μg/L) during spring in parallel to the decrease in nutrients. These results will give insights into seasonal changes in metagenomics profiles in this bay, which is currently under investigation in our group.

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SEASONAL VARIATIONS OF WATER QUALITY AND PICO-PLANKTON ABUNDANCE IN OFUNATO BAY

1Yuichiro Yamada, 2Shinnosuke Kaga, 2Yoshimasa Kaga, 2Shiho Watanabe, 2Kimiaki Naiki,

1Atsushi Kobiyama, 1Md. Shaheed Reza, 1Yuri Ikeda, 1Takehiko Ogata, 1Toshiaki Kudo and 1Shugo Watabe,

1School of Marine Biosciences, Kitasato Univ., Japan (yyamada@kitasato-u.ac.jp);

2Iwate Fish. Technol. Ctr., Japan

Ofunato Bay was a semi-closed area because of the breakwater effect at the entrance, however, the breakwater was destroyed by a massive tsunami generated by the 2011 Earthquake. Consequently, the physical environment of Ofunato Bay has been changed significantly, i.e., the modification of the stratified structure of seawater inside the bay and the intermittent intrusion of seawater outside the bay. These alterations of physical environment are considered to have an influence on the chemical and biological environment in Ofunato Bay. To elucidate the influence of the tsunami on the aquatic environment, we measured dissolved inorganic nutrients, chlorophyll a concentrations, heterotrophic bacteria and cyanobacteria abundance inside of Ofunato Bay from September 2014 to July 2016, and compared these data with those obtained before the earthquake (2009-2010). As compared with before earthquake, ammonium and phosphate concentrations decreased, and chlorophyll a concentrations and hetrotrophic bacteria abundances increased significantly. The collapse of breakwater and consequential enhanced water exchange were considered to have brought the decrease of nutrient concentration inside of the bay. Furthermore, washout of shellfish mariculture rafts by the tsunami decreased the shellfish biodeposits along with the elution of nutrients. Decrease of cultivated shellfish further caused a decline in feeding pressure on phytoplankton, subsequently, increasing the phytoplankton biomass that contributed to the decrease of nutrients inside of the bay. Increase of phytoplankton biomass induces increase an elution of dissolved organic carbon, and sequential increase of heterotrophic bacteria abundance.

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FUNCTIONAL TRAITS OF BACTERIAL COMMUNITIES ASSOCIATED WITH OYSTER FARMS IN THE OFUNATO BAY

1Md. Shaheed Reza, 1Atsushi Kobiyama,1Yuichiro Yamada, 1Yuri Ikeda,1Jonaira

Rashid,1Daisuke Ikeda, 1Nanami Mizusawa, 1Shigeru Sato, 1Takehiko Ogata, 1Kazuho Ikeo, 1Toshiaki Kudo, 2Shinnosuke Kaga, 2Shiho Watanabe, 2Kimiaki Naiki, 2Yoshimasa Kaga,

3Katsuhiko Mineta, 3Vladimir Bajic,3Takashi Gojobori and 1Shugo Watabe

1School of Marine Biosciences, Kitasato University, Japan (rezams@gmail.com); 2Iwate Fisheries Technology Center, Japan; 3King Abdullah University of Science and Technology,

Saudi Arabia

The Ofunato Bay in Iwate Prefecture, Japan, is the home of important aquaculture and economical activities. Chlorophyll a (chl-a) concentration has been reported to be high in the bay that attracted Buoy-and-Rope type scallop and oyster culture facilities particularly for Japanese oyster (Crassostrea gigas). Since these oysters filter suspended material and excrete organic matters into the seawater, bacterial community residing in its vicinity may also show dynamic seasonal changes. We, therefore, studied bacterial community structure in the seawater near oyster culture facilities over a period of one year employing shotgun metagenomic technique and correlated them with water quality parameters including temperature, salinity, pH, DO, NO2+3-N, NH4-N, and chl-a. A diverse community of free-living bacteria was observed in seawater near oyster culture facility that exhibited an intense inter-seasonal differences. Two alphaproteobacterial genera viz. Planktomarina and Ca. Pelagibacter involved in ocean biogeochemical cycle were most dominant at both water depths. They showed a sharp rise during spring and autumn with little fluctuation during other periods of the year, while three Bacteroidetes genera (Polaribacter, Flavobacterium and Cellulophaga) involved in degradation of organic matters and two Cyanobacteria genera (Synechococcus and Prochlorococcus) involved in primary production showed little or no fluctuation throughout the year. It was found that the distribution and abundance of seven functional traits including "amino acid metabolism", "energy metabolism", “metabolism of cofactors", "metabolism of terpenoids", "folding, sorting and degradation", "translation" and "signaling molecules and interaction" revealed by KEGG Orthology (KO) analysis were significantly different (p<0.05) between surface (1m) and deep (10m) waters near oyster aquaculture facilities. Functional abundance was also different when 2nd tier KO categories for seawater near oyster aquaculture facilities were compared to those far from oyster aquaculture facilities in the Ofunato Bay, particularly during autumn and summer near the surface (1 m), indicating some differences in functional traits of bacterial communities across the Ofunato Bay basin.

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MICROBIAL COMMUNITY DYNAMICS OF FREE�LIVING MICROBIAL ASSEMBLAGES IN THE OFUNATO BAY

1Atsushi Kobiyama, 1Md. Shaheed Reza, 1Rashid Jonaira, 1Yuichiro Yamada, 1Yuri Ikeda,

1Daisuke Ikeda, 1Nanami Mizusawa, 1Kazuho Ikeo, 1Shigeru Sato, 1Takehiko Ogata, 1Mitsuru Jimbo, 1Toshiaki Kudo, 2Shinnosuke Kaga, 2Shiho Watanabe, 2Kimiaki Naiki, 2Yoshimasa

Kaga, 3Katsuhiko Mineta, 3Vladimir Bajic, 3Takashi Gojobori and 1Shugo Watabe

1School of Marine Biosciences, Kitasato University, Japan (kobiyama@kitasato-u.ac.jp);

2Iwate Fisheries Technology Center, Japan; 3King Abdullah University of Science and Technology, Saudi Arabia

Sanriku Rias Coast in Japan is a typical ria coast and considered as one of the most productive areas, primarily due to tidal influences by the cold Oyashio and the warm Kuroshio Currents. The Ofunato Bay in Iwate Prefecture is located in the center of the Sanriku Rias Coast that possesses a complex physical structure, reflecting its complicated coastal line. Marine free-living bacterioplankton are key players of nutrient cycling in the oceans. Change in their community structure generally occur in relation to physical and chemical environments of seawater, and the understanding of the changes in microbial taxa and related microbial processes is a key step to ensure sustainability of the ocean. Accordingly, we tried to understand the ecosystem in the Ofunato Bay by employing shotgun metagenomic sequencing to identify genus- or species-level taxonomic profiles and diversities of microbial community.

Seawater samples were collected every month as before at surface (1 m) and deeper (8 or 10 m) layers from three locations at the Ofunato Bay and corresponding water quality parameters were measured. DNA was extracted from 0.2 μm filter membranes and prepared for metagenomic analysis using Nextera XT DNA Sample Preparation Kit and MiSeq Reagent Kit V3. The products were then sequenced with an Illumina MiSeq. Acquired Illumina reads were uploaded to suitable metagenomic pipelines, e.g., Meta Genome Rapid Annotation using Subsystem Technology (MG-RAST) server for downstream analysis. Taxonomic analyses at the levels of genus or species were also performed using MEGAN ver 5.10.3 after parsing the BLAST output. It was found that the shotgun metagenomic reads were derived from bacteria, archaea, eukaryotes and viruses where the majority were composed of Proteobacteria, followed by Bacteriodetes and Actinobacteria. The microbial community in the Ofunato Bay changed seasonally and the association between some bacteria and water quality parameters showed at certain levels of correlations. It is expected that detailed analyses of these data could reveal seawater conditions in the Ofunato Bay.

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DISCOVERY OF MARAINE MICROBIAL NATURAL PRODUCTS

Hiroshi Tomoda and Takashi Fukuda

1Graduate School of Pharmaceutical Sciences, Kitasato University, Japan

(tomodah@pharm.kitasato-u.ac.jp)

Marine environments are totally different from terrestrial ones; therefore, microorganisms

living there are believed also different. Recently, marine-derived microorganisms have

potential to produce new biologically active compounds, whose number has increased for the

decade. Our group has been interested in isolation of microorganisms from samples collected

in marine environments and has searched their cultures for new biologically active

compounds in original screening systems. Particularly our group is focusing on discovery of

new leads effective on life style-related diseases and infectious diseases.

In this workshop, I will present two topics; dinapinones (new inhibitors of triacylglycerol

(TG) synthesis in mammalian cells) produced by a fungal strain isolated from soil collected

in Hawaii Island, USA and graphiumins (new inhibitors of yellow pigment (staphyloxanthin)

production in methicillin-resistant Staphylococcus aureus (MRSA)) produced by a fungal

strain isolated from the sea sediments collected at Ishigakijima, Japan.

Dinapinones

From the cell-based screening for inhibitors of TG synthesis, the fungus identified as

Talaromyces pinophilus was found to produce dinapinones. Dinapinone A, a homodimer of

monapinone A, was separated into atropisomers, dinapinones A1 and A2. Interestingly, the

fungus mainly produced monapinone A in the seawater-containing culture medium, while

dinapinone A in the tap water medium. A mixture of dinapinones A1 and A2 (1:1) showed

the most potent inhibitory activity of TG synthesis. Further study on the mechanism of action

revealed that dinapinone A (A1 and A2 (1:1) mixture) showed no effect on TG synthesis, but

enhanced the degradation of TG accumulated in the cells.

Graphiumins

Ten new graphiumins were isolated from a culture of the fungal identified as Graphium sp.

as inhibitors of yellow pigment production by MRSA, which allows the pathogen to persist in

the host cells. The absolute structures were determined by NMR experiments and chemical

reactions. Regarding the side chain extending from the central ring in the structures, these

graphiumins were the second diketopiperazines having one or two long acyl chains.

Graphiumins I and J inhibited yellow pigment production in MRSA.

References

1) Ohte S et al., J. Antibiot., 64, 489-94 (2011)

2) Kawamoto K. et al., J. Antibiot., 64, 503-508 (2011)

3) Fukuda T. et al., J. Antibiot., 68, 620-628 (2015)

4) Fukuda T. et al., Nat. Prod. Sci., 21, 251-260 (2015)

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METAGENOMIC ANALYSIS OF INVERTEBRATE HOLOBIONTS AND SUPPORTING TECHNOLOGIES

Haruko Takeyama

Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan

(haruko-takeyama@waseda.jp)

Since more than 99% of environmental bacteria are uncultivable, single-cell genomics is the powerful tool for obtaining their genome information without cultivation. For genome sequencing of single bacterial cells, whole genome amplification (WGA) is required because the quantity of DNA contained in a single cell is quite small. So far, we have explored genomic feature of single bacterial filaments isolated from marine sponges by combination of single-cell sorting and WGA (Wilson et al, Nature, 2014). However, conventional WGA usually resulted in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras. In addition, for understanding the environmental microbial diversity, ideal single-cell genomics methods should analyze thousands of single microbial cells in an efficient manner. Recently, our study aims to develop droplet-based microfluidics for massively parallel single-cell genomics (Nishikawa et al, PLoS ONE, 2015), to elucidate marine microbial diversity and its relationships with host organism at the single cell level. By compartmenting genome fragments or single microbial cells into individual picoliter droplets, DNA fragments are amplified uniformly, that minimizes amplification bias and the effect of contaminating DNA. Our results demonstrated that microfluidic-generated droplets show a potential for contamination-free reaction and enhancing sequencing coverage from single-cells. In addition, we have also developed the application of Raman microspectroscopy to in situ detection of bioactive compounds (Miyaoka et al, Marine drugs 2014). Our results indicate that Raman microspectroscopy has a potential for the efficient and non-destructive screening of producers of bioactive compounds from uncultivable bacteria. These advantages will greatly reduce the cost and labor investment required for single-cell study of environmental microbes toward the application of bioactive compounds.

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SEASONAL DYNAMICS OF PHYTOPLANKTON COMMUNITIES IN SENDAI BAY, JAPAN:

COMPARISON OF MICROSCOPIC AND METAGENOMIC APPROACHES

1Tsuyoshi Watanabe, 2Yukiko Taniuchi, 1Tomoko Sakami, 3Kazutoshi Yoshitake, 1Shigeho Kakehi and 1Akira Kuwata

1Tohoku National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Japan (tsuyoshiw@affrc.go.jp); 2Hokkaido National Fisheries Research Institute,

Japan Fisheries Research and Education Agency, Japan; 3Japan Software Management Company Limited, Japan.

Diatoms are the major primary producers of coastal waters. While most species can be identified by silica cell wall morphology, and enumerated using light microscopy (LM), the identification of some species may require either scanning electron microscopy (SEM) or DNA sequencing. To clarify the dynamics of diatom communities in coastal waters, we investigated seasonal changes in the phytoplankton community structure of Sendai Bay, the largest bay in Tohoku in northern Japan. This is an area with a shallow continental shelf that opens into the Pacific Ocean, and allows for the frequent intrusion of marine water. Freshwater from several rivers is also discharged into the bay. We also examined diatom species composition during the spring bloom of April 2012 using metagenomic analysis (whole metagenome, ribosomal small subunits [SSU] and large subunits [LSU] from metagenome, and amplicon of SSU V4 region) and compared those results with those obtained via microscopic identification. Phytoplankton community variation Total phytoplankton biomass, as estimated by Chlorophyll a levels, peaked during spring at a concentration of 8.8 μg/L. Diatoms were the predominant phytoplankton species between winter and the spring bloom. Cyanobacteria and picoeukaryote species became more abundant from summer to autumn. Within the diatom community, Chaetoceros spp. were the most abundant during the onset of the spring bloom, when water temperatures ranged from 6-10°C. Skeletonema costatum s.l., predominated as salinity and nutrient concentrations decreased. Leptocylindrus danicus was the predominant species in summer. It was later replaced by small Thalassiosira spp. in autumn and winter. Comparison between microscopic and metagenomic identification Despite diatoms being the most dominant taxon observed under LM during the spring bloom, dinoflagellates were the most abundant according to metagenomic analysis. Within the diatom community, Skeletonema, Chaetoceros and Thalassiosira were abundant according to LM enumeration. Metagenomic analysis revealed that Thalassiosira spp. occurred in particularly large numbers. We compared the results of microscopic observations and metagenomic identification for the species composition of the three afore-mentioned genera. Skeletonema species composition, according to SEM analysis, was different to that revealed by metagenomic analysis. With regard to the genus Thalassiosira, all of the species observed under LM were also detected using metagenomic analysis. Several species, such as Thalassiosira pseudonana, whose entire genome have been sequenced, were overestimated. Finally, the number and abundance of Chaetoceros spp., when determined using LM, exceeded those seen following metagenomic analysis. Of all the metagenomic analysis methods, abundances according to SSU sequences were similar to those revealed by LM. Generating a complete reference sequence database, with sufficient numbers of metagenome sequences (> 107 reads), would, however, be necessary to improve estimations of species composition based on SSU in metagenome data.

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VARIATION OF BACTERIAL COMMUNITY COMPOSITION IN RELATION TO HYDROGRAPHICAL PROFILES IN SENDAI BAY

1Tomoko Sakami, 1Tsuyoshi Watanabe, 2Yukiko Taniuchi, 1Akira Kuwata, 1Shigeho

Kakehi, 3Kazutoshi Yoshitake, and 4Takashi Gojobori

1Tohoku National Fisheries Research Institute, Japan Fisheries Research and Education Agency (sakami@affrc.go.jp); 2Hokkaido National Fisheries Research Institute, Japan Fisheries Research and Education Agency; 3Japan Software Management Company Limited; 4King Abdullah University of Science and Technology, Saudi Arabia

Metagenome analysis will become a useful tool to monitor biological diversities in marine environments. However, environmental conditions fluctuate widely in coastal areas. Therefore, we examined the bacterial community compositions in coastal seawater from Sendai Bay for two years to clarify their spatial and temporal variation in relation to physical and biological environmental factors. A fragment analysis method of 16S rRNA gene amplicons indicated that the spatial variation of a bacterial community in the bay was often coordinated with the hydrographic profiles indicated by temperature and salinity. This suggests that physical events such as a stratification/mixing of seawater and an oceanic–seawater intrusion can affect bacterial community compositions. Concomitantly, phytoplankton abundance and nutrient concentrations were also related to community compositions. Nevertheless, bacterial communities of surface water from the central part of the bay showed obvious seasonal variation. We also examined shotgun metagenomes in the same water samples. Using an assembling analysis, the metagenomes indicated that microbial communities in the bay had a clear seasonal variation that was clustered into three major groups: the phytoplankton blooming period from February to May, the stratified period from June to August, and the mixing period from September to January. These results also indicated that spring phytoplankton bloom and collapse of stratification were the important events that affected the bay’s microbial community. Sequencing of the 16S rRNA gene amplicons revealed that the most dominant OTU (operational taxonomic unit) was assigned to an oceanic bacterium, SAR 11. An OTU assigned to Rhodobacteraceae was also dominant during the phytoplankton blooming period. OTUs assigned to Cyanobacteria or Alteromonadaceae were increased in the warm-water period after phytoplankton blooming, from June to September. Of the top 20 OTUs, five OTUs, which occupied 31% of the total abundance, did not show any correlation with environmental factors. The SAR 11 OTU was included in this category. This implied that they were derived from outside the bay. Eight OTUs (24%) had a significant correlation with water temperature, indicating that their occurrences fluctuated seasonally. Two OTUs (2%) had a significant correlation with salinity, indicating that a freshwater discharge influenced the bacterial community in the bay. Our results indicate that hydrographical conditions are very important factors to determine microbial community composition in coastal seawater.

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WEEKLY MONITORING OF EUKARYOTE BIODIVERSITY USING MASSIVELY PARALLEL SEQUENCING (MPS)-BASED TECHNOLOGY IN OKHOTSK SEA,

JAPAN

1Satoshi Nagai, 2Shingo Urushizaki, 1Chen Hungyen, 1Atushi Fujiwara, 1Motoshige Yasuike, 3Seiji Katakura

1National Research Institute of Fisheries Science, Japan (snagai@affrc.go.jp), 2AXIOHELIX Co. Ltd., Japan, 3Mombetsu city office, Japan

We have conducted a monitoring of eukaryote biodiversity by using MPS-based technology one a week for nearly four years in Mombetsu city, Hokkaido, Japan (��������ƍ1�����°�����ƍ().Water temperature, salinity and chlorophyll-fluorescence were -1.6–22.4 oC, 18.3–33.7 PSU, <0.0-–9.8 ȝJ�/-1 during the survey periods of 2012–2014, respectively. In this study, we analyzed the biodiversity of seawater samples (n = 116) and succeeded in detecting over twenty hundreds of operational taxonomic units (OTUs) from 500 mL seawaters. In the NMDS analysis, samples taken at the same month but in different years were more closely plotted and also the samples of closest months were located in the closest positions, showing the consecutive transition of species composition and the clear seasonal pattern of eukaryote species in Okhotsk Sea. In the relative abundance of identified OTUs at the supergroup levels, Alveolata, Opisthokonta, Stramenopiles were27.3, 22.5 and 21.5%, respectively and followed by Rhizaria (12.9%) and Viridiplantae (12.9%), indicating the typical pattern in Japanese coastal waters. Interestingly, the heatmap and cluster dendrogram indicated the samples were divided into two large different nodes, i.e. samples obtained in Jun–Nov and remaining samples obtained in Dec–May, showing the strong influence by Tsushima warm current (= Souya warm current) in summer and East Sakhakin current in winter season. The MPS-based technology also enabled to detect conspicuous diatom spring blooms and sudden blooms, which occurred irregularly, caused by dinoflagellate, ciliate or metazoa species, strongly suggesting the effectiveness of the monitoring by the MPS-based technology. As the first record, we detected the genus Alexandrium, Azadinium, Chattonella, Cochlodinium, Karenia, Karlodunium,Pseudochattnella, Takayama including toxic and fish killing species, indicating that these HAB species were carried by the Tsushima warm current in summer seasons. These data suggest the increasing possibility of settlements in northern Japan.

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MECHANISMS INVOLVED IN CARBON DIOXIDE FIXATION BY MARINE MICROORGANISMS

1Ko Yasumoto, 2Mina Yasumoto-Hirose, 3 Jun Yasumoto, 4Takenori Kusumi,

5Kanami Mori-Yasumoto, 1Mitsuru Jimbo and 1Shugo Watabe

1School of Marine Biosciences, Kitasato University, Kanagawa, Japan

(�������������� �� ��); 2Tropical Technology Plus, Okinawa, Japan; 3 Faculty of Agriculture, University of the Ryukyus, Okinawa, Japan, 4 Graduate School of

Science and Engineering, Tokyo Institute of Technology, Tokyo, Japan, 5Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Kagawa,

Japan Marine bacteria, including cyanobacteria are known to deposit calcium carbonate (CaCO3) extracellularly in calcium-containing artificial medium. Despite extensive investigation, the mechanisms involved in extracellular formation of CaCO3 by bacteria have remained unclear. The ability of synthetic amines to remove carbon dioxide (CO2) from natural gas led us to examine the role of biogenic polyamines in CaCO3 deposition by bacteria. Here, we demonstrated that biogenic polyamines such as putrescine, spermidine, and spermine were able to react with atmospheric CO2 and the resultant carbamate anion was characterized by using nuclear magnetic resonance (NMR) analysis. Biogenic polyamines accelerated the formation of CaCO3, and we artificially synthesized the dumbbell-shaped calcites, which had the same form as observed with bacterial CaCO3 precipitates, under nonbacterial conditions by using polyamines. The reaction rate of calcification increased with temperature with an optimum of around 40 °C. Our observation suggests a novel scheme for CO2 dissipation that could be a potential tool in reducing atmospheric CO2 levels and, therefore, global warming.

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��������THE EVOLUTIONARY LINK BETWEEN PARMALES AND THE SUCCESS OF

DIATOMS IN MARINE ECOSYSTEMS

1Akira Kuwata, 2Mutsuo Ichinomiya, 2Kazumasa Yamada, 3Shinya Yoshikawa, 4Mary-Hélène Noël ,4Masanobu Kawachi, 5Kenji Saitoh, 5Yoji Nakamura, 6 Naoki Sato,

6Naoyuki Tajima,; 7Ken Sawada, 8Adriana Lopes, 8Daniel Vaulot.

1Tohoku National Fisheries Research Institute (FRA), Japan (akuwata@affrc.go.jp); 2Prefectual University of Kumamoto, Japan; 3Prefectural University of Fukui, Japan;

4National Institute for Environmental Studies, Japan; 5National Research Insitute of Fisheries Science (FRA), Japan; 6University of Tokyo, Japan; 7Hokkaido University, Japan; 8 Station

Biologique de Roscoff (CNRS), France. Parmales is a group of pico-sized eukaryotic marine phytoplankton with cells surrounded by several silica plates. Parmales has been found widely in the world’s oceans, from polar to subtropical regions, and is frequently abundant in polar and subarctic regions. Based on similarities in cell wall structure, Parmales has been proposed as a close relative to diatoms, which are the most successful phytoplankton group in modern oceans. However, there was very little biological information on Parmales, because no cultures were available for laboratory study. Recently, we successfully established the first ever culture of Parmales from the Oyashio water in the Western Subarctic Pacific. SEM and TEM observations, molecular phylogenetics and photosynthetic pigments analyses of this culture indicated that Parmales belongs to Heterokonta and is positioned within Bolidophyceae, a sister group of diatoms, which are pico-sized autotrophic naked flagellates mainly living in subtropical waters. It can be hypothesized that parmalean and bolidophycean algae (or their common ancestor) have a life cycle that switches between silicified non-flagellated and non-silicified flagellate stages. This hypothetical life cycle has similarities to centric diatoms, which is an ancient lineage that has a vegetative stage that switches to production of naked flagellated male gametes (spermatozoa) for sexual reproduction. Because the origin of the silica cell wall and the early evolution of diatoms are not yet fully understood, Parmales may present important information in answering these questions. We present our ongoing study of the evolutionary close relationship between Parmales, Bolidophycea and diatoms using ecological, physiological, genomic and biogeochemical approaches.�

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No.1

SEASONAL AND GEOGRAPHICAL DISTRIBUTION OF NEAR-SURFACE SMALL

PHOTOSYNTHETIC-EUKARYOTES IN THE WESTERN NORTH PACIFIC

DETERMINED BY PYROSEQUENCING OF 18S RDNA

1,*Takafumi Kataoka, 1Haruyo Yamaguchi, 1Mayumi Sato, 2Tsuyoshi Watanabe,

2,3Yukiko Taniuchi, 2Akira Kuwata and 1Masanobu Kawachi

1Center for Environmental Biology and Ecosystem Studies, National Institute for

Environmental Studies, Japan; 2Tohoku National Fisheries Research Institute, Japan; 3Hokkaido National Fisheries Research Institute, Japan *Faculty of Marine Bioscience, Fukui

Prefectural University Japan (kataoka@fpu.ac.jp)

In this study, we investigated the distribution of small photosynthetic eukaryotes in the near-

surface layer of western North Pacific. Samples were collected by seasonal sampling at four

stations, i.e., two oceanic stations where the subarctic Oyashio and subtropical Kuroshio

currents influence a transition region and the bay mouth and head of the Sendai Bay from

April 2012 to May 2013. Flow cytometry was applied to sort cells of small photosynthetic

eukaryotes (< 5µm) targeting to chlorophyll fluorescence and side angle scatter, and high-

throughput sequencing (GS Jr. pyrosequencer) was used to determine sequences of V4 region

in 18S rDNA amplified with eukaryotes universal primer. Among the 20 samples collected in

five seasons at four sites, 572,390 high quality reads (26,620 reads/sample in average) were

obtained. Among the 362 operational taxonomic units (OTUs), which was constructed based

on 98% similarity, in total, 195 OTUs (53.9%) were assigned to taxa known to

photosynthetic eukaryotes. The results obtained by taxonomic analysis showed that 19/195

OTUs composed of eight Bacillariphyceae, five Cryptophyceae, two Prymnesiophyceae, and

one OTU each from Pyramimonadales, Dictyochophyceae, Dinophyceae, and

Chlorarachniophyceae were frequently distributed among all sites. Composition analysis of

SIMPROF showed that the OTUs had characteristic patterns and that they were divided into

four main groups (p<0.05). SIMPER analysis showed that two groups reflected the low-saline

water and winter season where the characteristic OTUs were diatoms; Chaetoceros and

Leptocylindrus were characteristic of low saline water, and two diatoms (Minidiscus and

Minutocellus) and Cryptomonadales-related OTUs were common in winter. Our results

indicate that the community compositions of small photosynthetic eukaryotes change

seasonally but in a more dynamic manner according to variations in the water properties and

the season-specific environments in this area.

No.2

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SEASONAL CHANGE OF BACTERIAL COMMUNITY IN SENDAI BAY, FROM A FUNCTIONAL PERSPECTIVE

1Kaoru Matsumoto, 1Kazuho Ikeo, 2Tsuyoshi Watanabe, 2Yukiko Taniuchi, 2Shigeho Kakehi, 2Tomoko Sakami, 2Akira Kuwata, 3Engkong Tan, 3Shigeharu Kinoshita, 3Shuichi

Asakawa, 2Takanori Kobayashi, 4Yoshizumi Ishino, 1Takashi Gojobori

1National Institute of Genetics, 2Japan Fisheries Research and Education Agency, 3The University of Tokyo, 4Kyushu University

Marine microbial communities change seasonally, as with the seasonal cycle of the environmental condition. Here we checked seasonal changes of the bacterial community in Sendai bay based on functional gene, as well as taxonomy. We used shotgun metagenomic data of 40 seawater samples collected from April 2012 to

June 2013. After prediction of gene-coding sequences, the sequences were annotated based on blastp against GeneBank nr database and MEGAN software. Annotated sequences of Bacterial group (except for Cyanobacteria) were retained for functional analysis based on KEGG database.The functional gene profile also showed seasonality, though the difference was much smaller

than that of taxonomic profile. In comparison between spring bloom (high chlorophyl-aconcentration) and autumn (low chlorophyl-a concentration), genes related to several amino acid metabolism, as well as ABC transporters, were more abundantly observed in spring. On the other hand, genes related to carbon fixation, carbohydrate metabolism, or transcription were more abundant in autumn.

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No.3

METAGENOMIC ANALYSIS OF BACTERIAL FRACTION DURING CHATTONELLA-KARENIA BLOOM

Norikazu Kitamura1, Kazuho Ikeo1, Yoshizumi Ishino2, Kosuke Tashiro2, Satoru Kuhara2, Takeshi Masuda3, Yoji Nakamura4, Motoshige Yasuike4, Atsushi Fujiwara4, Satoshi Nagai4,

Takanori Kobayashi4, Takashi Gojobori1

1National Institute of Genetics, 2Kyushu University, 3Mie Prefecture Fisheries Research Institute, 4Japan Fisheries Research and Education Agency

Some marine bacteria are associated with phytoplankton. In general, phytoplankton bloom produces organic matters, resulting in proliferation and increase of productivity of bacteria. Since types of generated organic matters change with phytoplankton species, bacterial assemblages during phytoplankton bloom are thought to be different for blooming phytoplankton species due to differences in metabolic ability among bacterial strains. Blooms of Chattonella, raphidophytes, and Karenia, dinoflagellates, are classified as noxious red tides, sometimes leading to large economic damage for farmed fishes. To clarify bacterial assemblage transition in a duration from onset of these harmful algal bloom to end of the event, we extracted DNA from free living bacterial fraction (1-0.2μm filter) of coastal water time-series samples from Ago Bay (Japan, Mie prefecture) in summer 2015, including Chattonella and Karenia blooms, performed shotgun-sequencing with Miseq, and investigated taxonomic and gene function abundance. Comparison between bloom and normal samples showed significant differences in bacterial composition. In contrast, there were not significant differences in gene function between bloom and normal samples. Based on the preliminary results, effects of the phytoplankton bloom on bacterial assemblage will be discussed.

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No.4

CHARACTERIZATION OF MARINE METAGENOME BASED ON THE PROTEIN MOTIF

1Yoji Igarashi, 1Engkong Tan, 1Daisuke Mori, 1Yusuke Osaki, 1Susumu Mitsuyama,

1Shigeharu Kinoshita, 2Shugo Watabe, and 1Shuichi Asakawa 1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan (asakawa@mail.ecc.u-tokyo.ac.jp); 2School of Marine

Biosciences, Kitasato University, Japan; Marine metagenomic DNA sequencing by using next generation sequencer (NGS) is a powerful tool that reveal expansive biodiversity of microorganisms. However, most of the sequence has been shown to have no similarity with known DNA/RNA sequence in the databases by the metagenome analysis based on the current homologous search. On the contrary, some software of protein motif prediction predict protein motifs from the sequence with no similarity to known sequence in the database, indicating such software may complement the metagenomic analysis based on sequence homology. It may be useful to utilize protein motif as a basic unit in order to characterize environment. In this study, we developed a new metagenomic analysis method based on the information of the protein motifs. Obtained nucleotide sequences were translated into amino-acid sequence, then those are subjected to several protein motif prediction software. Each predicted motif was counted for individual metagenomic data to form a matrix. Baaed on the matrix, several algorithms such as heat map, a cluster analysis and principal component analysis (PCA) were performed to characterize the metagenomic data. We will show our initial result of these analyses and discuss the effectiveness of these analysis methods.

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No.5

16S AMPLICON ANALYSIS OF SEASONAL CHANGES IN FREE-LIVING

BACTERIOPLANKTON FROM THE TAMA RIVER IN TOKYO

Nanami Mizusawa, Md. Shaheed Reza, Chiharu Oikawa, Daisuke Ouchi, Ayaka Kumano, Atsushi Kobiyama, Yuichiro Yamada, Yuri Ikeda, Daisuke Ikeda, Kazuho Ikeo, Shigeru Sato,

Takehiko Ogata, Ko Yasumoto, Mitsuru Jimbo, Toshiaki Kudo, and Shugo Watabe

School of Marine Biosciences, Kitasato University, Japan (nmizusawa@kitasato-u.ac.jp)

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 inter-seasonal differences of the bacterial community in a non-tidal, urban-impacted part of this river to better understand its ecology. Water samples (~2L) were collected from the surface to 12.5 cm depth every month from September 6, 2015, to July 10, 2016, with sterile beakers from 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 fraction, DNA was only extracted from 0.2 μm filter membranes and we prepared 16S amplicon products targeting V1-V3 regions. The products were then sequenced with an Illumina MiSeq using MiSeq Reagent Kit V3. Acquired Illumina reads were uploaded to a Basespace server and analyzed using the Illumina 16S Metagenoimc App (v1.0.1) for taxonomic classification. Five prokaryotic groups were found to be dominant in the Tama River, viz., Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria and Firmicutes. The relative abundance of Bacteroidetes decreased from 62.3% in September to 11.3% in January, while those of Proteobacteria, Actinobacteria and Firmicutes in September increased gradually from 27.4 to 53.9% in January, from 3.0 to 6.4% in December and from 1.4% to 13.8% in February, respectively. A marked increase of Cyanobacteria was observed in October. A very strong negative correlation against water temperature was observed with the relative abundance of Proteobacteria (r = 0.91, p < 0.001), while a positive correlation observed with Bacteroidetes (r = 0.83, p < 0.005). These results indicate versatile adaptation strategies of free-living bacterioplankton in relation to environmental factors.

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No.6

METAGENOMIC APPROACH ON THE CHANGES IN MICROFLORA DURING MAY-JUNE, 2016, IN THE OFUNATO BAY

1Rashid Jonaira, 1Md. Shaheed Reza, 1Atsushi Kobiyama, 1Yuichiro Yamada, 1Yuri Ikeda,

1Daisuke Ikeda, 1Nanami Mizusawa, 1Shigeru Sato, 1Takehiko Ogata, 1Kazuho Ikeo, 1Toshiaki Kudo, 2Shinnosuke Kaga, 2Shiho Watanabe, 2Kimiaki Naiki, 2Yoshimasa Kaga,

3Katsuhiko Mineta, 3Vladimir Bajic, 3Takashi Gojobori, and 1Shugo Watabe

1School of Marine Biosciences, Kitasato University, Japan (df16006@kitasato-u.ac.jp); 2Iwate Fisheries Technology Center, Japan; 3King Abdullah University of Science and

Technology, Saudi Arabia

Marine metagenomics is a promising area of genomic studies to investigate the entire microbial communities in the marine environments. However, our understanding of these complex communities has remained limited. Next generation sequencing (NGS) is the core technology for metagenomics. The aim of this study was therefore to investigate microbial diversity in the Ofunato Bay, Japan, renowned for its adventure location and richness of aquaculture resources. Seawater samples were collected from three locations (KSt.1, 2 and 3) at two depths (1m and 10m or 8m) in the Bay during May-June, 2016. DNA was extracted from 0.2 μm-filtered seawater samples. While Illumina NGS was employed to acquire metagenomic data, the entire datasets were compared to the NCBI-NT database using BLAST and annotated by MEGAN software (ver. 5.11.3). The average GC content, base pair length, base count and sequence count were ranged from 40.27±9.91 to 44.98±10.71%, 265.50±97.35 to 316.92±98.44bp, 170 to 284Mb and 0.54 to 1.02M, respectively. The highest GC content was found at KSt.3-1m depth in May, whereas the highest sequence count was found at KSt.2-10m depth in the same month. The taxonomic profile showed that the Ofunato Bay was dominated by Proteobacteria, Bacteroidetes, Chlorophyta, Firmicutes, Actinobacteria and Cyanobacteria phyla. In particular, the abundance of the prominent species, Planktomarina temperata, was remarkably high at 1m depth (KSt.3-May, n=19133 and KSt.3-June, n=18439), in comparison to 8-10m depths. On the other hand, the second dominant species, Candidatus Pelagibacter ubique, was higher at 8m-10m depths (KSt.3-May, n= 11463 and KSt.2-June, n=4795). In addition, we identified eukaryotic sequences where the major identified species were Ostreococcus 'lucimarinus', Micromonas sp. RCC299 and Bathycoccus prasinos. The unicellular picoplankoton, Ostreococcus 'lucimarinus', was significantly increased fourfold in June. On the other hand, there was about twofold decrease in the abundance of Micromonas sp. RCC299 in June compared to May irrespective of sites and depths. The most abundant viral genome, which was identified as that of Micromonas pusilla virus 12T (Chlorophyta with cytoplasmic virus), was higher in number at 8-10m (KSt.2-May, n= 574 and KSt.1-June, n=525) than 1m depth. Overall, the species abundance of the major microflora was found to have remained quite constant throughout the study period.

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No.7

SEASONAL CHANGE OF SOME BACTERIA AT THE OFUNATO BAY BY SHOTGUN METAGENOMICS

1Toshiaki Kudo, 1Md. Shaheed Reza, 1Rashid Jonaira, 1Atsushi Kobiyama, 1Yuichiro Yamada, 1Yuri Ikeda, 1Daisuke Ikeda, 1Nanami Mizusawa, 1Shigeru Sato,

1Takehiko Ogata, 1Mituru Jinbo, 1Kazuho Ikeo, 2Shinnosuke Kaga, 2Shiho Watanabe,2Kimiaki Naiki, 2Yoshimasa Kaga, 3Katsuhiko Mineta, 3Vladimir Bajic, 3Takashi Gojobori,

and 1Shugo Watabe

1School of Marine Biosciences, Kitasato Univ., Japan (kudot@kitasato-u.ac.jp); 2Iwate Fish. Technol. Ctr., Japan;

3King Abdullah Univ. Sci. Technol., Saudi Arabia

The Ofunato Bay in the Iwate Prefecture locates in the northeastern Pacific Ocean area of Japan where marine organisms show the highest biodiversity in the world. The present study was undertaken to determine seasonal change of some bacteria in the seawater around oyster aquaculture facilities at the Ofunato Bay by utilizing a shotgun metagenomic technique.

Seawater samples were collected every month from October 2014 to December 2015(KST2) and January to December 2015 (KSt1 and KSt3) at upper (1 m) and deeper (8 or 10 m)columns from three points, inner part (KSt1), middle part (oyster aquaculture facilities) (KSt2) and entrance (KSt3) of WKH�2IXQDWR�%D\��'1$�ZDV�H[WUDFWHG�IURP�����ȝP�ILOWHU�membranes, prepared for metagenomic analysis, and each extracted DNA (1ng) was sequenced on an Illumina MiSeq. Acquired reads were then uploaded to Meta Genome Rapid Annotation using Subsystem Technology (MG-RAST) server and annotated into different categories. rRNA reads were selected and analyzed further.

Each sample (rRNA reads) contained 0.11 – 0.60 million sequences. These reads were applied to taxonomical analysis through Local BLAST using DDBJ 16S database. Each read was annotated by top-hit, and more than 97% identities and more than 100 bp reads were selected for further analysis. Many reads in this study showed high identity with CandidatusPelagibacter sp. partial 16S rRNA genes isolated from Red sea water surface. Seasonal change of Candidatus Pelagibacter was observed as total read numbers /g seawater.“Total read numbers /g seawater” is “read numbers mentioned above” multiplied by “amount of DNA (ng /g seawater)” in each sample. The results suggested that higher peaks were observed during Jun and Dec in Candidatus Pelagibacter. Candidatus Pelagibacter is marine oligotrophic bacterium dominant in ocean surface waters and simultaneously catabolizes dimethylsulfoniopropionate to the climatically active gases dimethyl sulfide and methanethiol (Jing Sun Nature microbiol. 2016). The functions will be discussed.

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No.8

TEMPORAL CHANGE IN VERTICAL DISTRIBUTION OF Alexandrium tamarense IN THE OFUNATO BAY, JAPAN

1Yoshimasa Kaga, 1Kimiaki Naiki, 1Shiho Watanabe, 1Satoshi Segawa,

2Shinnosuke Kaga, and 1Yasushi Kuji

1Iwate Fisheries Technology Center, Japan; 2Iwate Prefectural Government, Japan An accumulation of paralytic shellfish poisoning (PSP) toxins in bivalves causes severe damages almost annually to the fishery industry as well as public health along the Pacific coast of northern Japan (Oshima et al. 1982). In these area, the foodborne illness by PSP toxins occurred in the next year of the Chilean earthquake tsunami (1960). Since the PSP event in the Ofunato Bay, Iwate Prefecture, in 1961, the toxicity of shellfishes and the abundance of toxic dinoflagellates have been monitored (Ogata et al. 1982) along the coast of Iwate Prefecture. Our studies revealed the presence of several Alexandrium species in this area; bivalve contamination with PSP toxins has been caused by A. tamarense in the Ofunato Bay. After the Eastern Japan great earthquake in March 2011, the cell number of A. tamarense (100,600cells/l) was observed at a depth of 8-m in May 2011. Therefore, we have strengthened the observation of A. tamarense in the Ofunato Bay. Sampling was carried out at the Shizu station in the Ofunato Bay from January 2013 to July 2016. Water samples for monitoring cell numbers of A. tamarense were collected four to five times per month at 2-m depth intervals from the water surface to the bottom (12 layers). The abundance of A. tamarense reached a maximum from April to June each year, the peak bloom was followed by a sharp decrease. The maximum cell number (105,100 cells/l) recorded at a depth of 8-m in April 2015. These results indicate that further field investigations of A. tamarense are needed. These results indicate field investigations of A. tamarense by each water depth are needed.

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No.9

COMPOSITION OF DINOFLAGELLATE CYSTS IN THE OFUNATO BAY, IWATE, JAPAN

1Yuri Ikeda, 1Takehiko Ogata, 1Md. Shaheed Reza, 1Atsushi Kobiyama,1 Yuichiro Yamada,

1Daisuke Ikeda, 1Nanami Mizusawa, 1Kazuho Ikeo 1Shigeru Sato, 1Toshiaki Kudo, 2Shinnosuke Kaga, 2Shiho Watanabe, 2Kimiaki Naiki, 2Yoshimasa Kaga, and 1Shugo Watabe

1School of Marine Biosciences, Kitasato University, Japan (yikeda@kitasato-u.ac.jp);

2Iwate Fisheries Technology Center, Japan

The composition of resting cysts of dinoflagellate was investigated at the levels of genus or species on the sediment samples in the Ofunato Bay where a high toxicity of paralytic shellfish poisoning (PSP) toxin in bivalves has been recorded almost every year since 1961. The sediment samples were collected from two stations (Shizu and Sango-jima) with the Ekman-Birge grab sampler once a month from October to December, 2014, and sectioned every 1 cm from the top to the depth of 5 cm. The samples for cyst analysis were treated using the method of Fukuyo and Matsuoka (2000), observed for 1 ml samples under a light microscope, and identified morphologically at the species or genus levels. In this study, at least 33 cyst types were recorded in a total of both sampling stations and 28 types could be identified at the species level. In Sango-jima samples, the cysts belonging to either of Alexandrium tamarense and A. catenella which are the major causative organisms of PSP toxin in the Bay were dominant, followed by those belonging to genus Protoperidinium and Gonyaulax. Alexandrium cysts were also confirmed by PCR amplification using DNA solutions extracted from the sediments. The PCR results also suggested that the dominant cysts in any samples obtained belong to not A. catenella but A. tamarense.

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No.10

A NEW ELISA KIT AS A MONITORING TOOL FOR PARALYTIC SHELLFISH TOXINS

1Shigeru Sato, 1Hidenobu 1Kosugi, 1Suzuka Takaishi, 2Shinnosuke Kaga, 2Yoshimasa Kaga, 2Shiho Watanabe, 2Kimiaki Naiki, 1Atsushi Kobiyama, 1Md. Shaheed Reza, 1Yuichiro Yamada, 1Yuri Ikeda, 1Daisuke Ikeda, 1Nanami Mizusawa, 1Toshiaki Kudo and 1Shugo Watabe

1School of Marine Biosciences, Kitasato University, Japan (shigeru@kitasato-u.ac.jp); 2Iwate

Fisheries Technology Center, Japan

Contamination of paralytic shellfish toxins (PST) in bivalves causes serious damages on

shellfish industries in all over the world. In the areas where the contamination is suspected, regular monitoring on shellfish toxicity and causative microalgae are essential to avoid the food poisoning. So far, the density of causative dinoflagellate in culture area is monitored by microscopic observation. Shellfish toxicity is determined by mouse bioassay, though it has negative aspects accompanied with ethics. Several excellent methods to detect PSP toxins, such as fluorometric HPLC, LC-MS and receptor binding assay, have been developed to quantitate PSP toxins. Among them, enzyme-linked immunosorbent assay (ELISA) is the primary candidate as an alternative to mouse bioassay for its simplicity, convenience, high sensitivity and specificity. Recently, we have developed a new antigen in which C11 position of STX was coupled to carrier proteins using 1,2-ethaneditiol as a linker. The polyclonal antibody obtained by immunization with the new antigen reacted with most of all PSP toxin components, but still the reactivity to N1-OH toxins such as neoSTX, gonyautoxin (GTX) 1 and GTX4, and N-sulfo-carbamate toxins such as B and C toxins was insufficient. Koehn et al. (1982) reported that carbamoyl N-sulfo groups of B and C toxins are easily hydrolyzed with mild HCl treatment. Recently, while working on metabolism of PSP toxins, we found that N1-OH toxins are reduced to corresponding N1-H toxins using hemin as a catalyst. The combinations of these conversions transforms quantitatively all PSP toxin components commonly found in contaminated shellfish to N1-H/carbamoyl toxins such as GTX2, GTX3 and/or STX. In combination with these two pretreatments, toxin contents in shellfish can be determined by the ELISA with high accuracy. Because of its high sensitivity, the ELISA can detect also the occurrence of causative phytoplankton in seawater. In this poster, the application of ELISA kit as a monitoring tool for shellfish toxicity, as well as for detection of toxic microorganisms in seawater, will be presented.

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No.11

A NEW POLYCLONAL ANTIBODY AGAINST TETRODOTOXIN

Suzuka Takaishi, Shigeru Sato, Ko Yasumoto, Atsushi Kobiyama, Yuri Ikeda, Toshiaki Kudo, and Shugo Watabe

School of Marine Biosciences, Kitasato University, Japan (shigeru@kitasato-u.ac.jp)

Puffer fish have long been a food delicacy in Japan. Puffer fish, however, possess a potent

neurotoxin, tetrodotoxin (TTX), and has caused serious food poisonings including lethal cases. Toxicity of puffer fish varies with the species, the area and season; the same species caught in the same area at the same season shows a wide range of toxicity from non-toxic to extremely highly toxic. In contrast, cultured puffer fed with non-toxic diet is always non-toxic. These facts suggest that puffer fish accumulate TTX through food web. High concentration of TTX was also found in several aquatic organisms, such as xanthid crab, horseshoe crab, freshwater amphibian etc, but the mechanisms underlying the accumulation of TTX in these organisms has not been clear yet. To investigate the mechanisms involved, we decided to develop a new ELISA kit to detect TTX. Yotsu-Yamashita et al. (2005) reported that 4,9-anhydro-tetrodotoxin (4,9-anhTTX), a derivative of TTX found in puffer fish, reacts with glutathione (GSH) to form 4-S-GSH adduct of TTX. Not only GSH, but also various thiol compounds react with 4,9-anhTTX. These findings enable us to design a new haptenic antigen essential to develop immuno-detection of TTX. Sulfhydryl moiety was introduced to tetrodotoxin (TTX) by a reaction between 4,9-anhTTX and (+) dithiothreitol (DTT). A new haptenic antigen was obtained from the mixture of a bifunctional coupling reagent (GMBS)-treated bovine serum albumin (BSA) and the DTT –TTX adduct. The ratio of TTX coupled to the carrier protein was estimated to be 14 % (w/w) by fluorescent intensity after sodium hydroxide treatment. In addition, 4,9-anhTTX was treated with 1,2-ethanedithiol (EDT) and introduced to GMBS-treated keyhole limpet hemocyanin. These antigens were inoculated biweekly to rabbits, and relatively high titer was observed within 3 - 4 months. In this session, properties of the polyclonal antibodies as well as the prototype ELISA kit for TTX will be presented.

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No.12

POSSIBLE USE OF RESIDUAL BIOMASS OF OIL-PRODUCING MICROALAGE FOR AQUAFEED

Hiroaki Kasai

School of Marine Biosciences, Kitasato University, Japan (hkasai@kitasato-u.ac.jp)

Aquaculture, the farming of aquatic organisms, accounted for 40% of the yield of wild fisheries in the year 2000 and is expected to surpass the yield of wild fisheries by 2020–2025. Almost 40% of all aquaculture production is now firmly dependent on commercial feed, which contains large amounts of fish meal and fish oil as the primary sources of protein and oil. Because fish meal and fish oil are costly and of limited availability, plant-based sources have been receiving considerable attention in the aquaculture feed industry over the past few decades as partial or total substitutes. Microalgae have recently attracted considerable attention as possible sources of biofuel, and major research efforts to increase productivity will likely raise the possibility of microalgae use in aquaculture feed.

In order to evaluate oil-producing microalgae as a resource for aquaculture feed, we constructed an outdoor mass microalgae culture using a closed bioreactor, harvested the biomass, and extracted triglycerides using an organic solvent. The residual biomass containing feeds were used in a feeding experiment with juvenile ayu, Plecoglossus altivelis. The growth and physiological states of the tested fish were comparable to the commercial feed. Body surface color was yellower because the residual biomass contained lutein and zeaxanthin. We also compared the composition of gastro-intestinal tract microbes using 16S rRNA gene amplicon sequencing. The results suggest that residual biomass of oil-producing microalgae is suitable as a source of functional ingredients such as vitamins in aquaculture feed.

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No.13

A GALNAC-BINDING LECTIN COULD BE INVOLVED IN ACQUISITION OF SYMBIODINIUM BY A CORAL ACROPRA TENUIS LECTIN

1Mitsuru Jimbo, 1Nami Kuniya, 1Ko Yasumoto, 1Shugo Watabe

1School of Marine Biosciences, Kitasato University, Japan (mjinbo@kitasato-u.ac.jp); 2

Many corals often harbor symbiotic dinoflagellates, Symbiodinium spp., and their symbiosis is indispensable for the survival of corals. However, they often acquire Symbiodinium around their environment, and this acquisition was inhibited by some carbohydrates, suggesting that a carbohydrate binding proteins may play a role in this step. So we purified a lectin using a hard coral Acropora tenuis. The juvenile polyps of A. tenuis acquire Symbiodinium strain NBRC102920, and this acquisition was inhibited by galactose, N-acetyl-galactosamine (GalNAc), and N-acetyl-glucosamine. By GalNAc-affinity chromatography, two components at 29.0 kDa and 14.6 kDa were purified. De novo sequencing by MALDI-TOF MS spectrometry suggests that 29.0 kDa component is similar to a GalNAc/GlcNAc binding lectin tachylectin-2 (TL-2) purified from a horseshoe crab, which is involved in immunity. Based on the sequence, the corresponding cDNA was cloned. The deduced amino acid sequence was composed of 267 amino acids, and had a similarity to TL-2 and some putative proteins from A. digitifera genome database, so the protein was named to AtTL-2. Immunohistochemistry of A. tenuis tissue suggests that AtTL-2 distributed a kind of nematosysts. Moreover, Anti-TL-2 antibody inhibited Symbiodinium acquisition by juvenile polyps. These results let us to hypothesize that A. tenuis may acquire Symbiodinium near themselves by releasing AtTL-2 from nematocysts. The recombinant AtTL-2 attracted the Symbiodinium, and the attraction was inhibited by GalNAc, suggesting that AtTL-2 attract Symbiodinium by binding to carbohydrates on the cell surface and the attraction results in the acquisition of Symbiodinium..

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No.14

GLOBAL GENE EXPRESSION ANALYSIS ON MEDAKA MUSCLE TISSUES IN ASSOCIATION WITH TEMPERATURE ACCLIMATION

1Daisuke Ikeda, 2Hiroki Koyama, 1Nanami Mizusawa, 1Tomoko Kagaya, 1Reina Ushijima,

3Engkong Tan, 3Shuichi Asakawa, 1Nobuhiro Kan-no, and 1Shugo Watabe

1School of Marine Biosciences, Kitasato University, Japan (adikeda@kitasato-u.ac.jp); 2Graduate School of Biosphere Science, Hiroshima University, Japan; 3Department of

Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan

To disclose the mechanisms involved in temperature acclimation of fish, global gene expression analysis using next generation sequencing (NGS) was carried out with medaka Oryzias latipes, one of typical temperate eurythermal fish, which is able to survive over a wide range of water temperature ranging from near zero to over 30ºC throughout the year, while maintaining their normal physiological and biochemical processes by temperature acclimation. About 10 individuals were introduced into two aquariums at 24ºC. While water temperature of one aquarium was decreased to 10ºC within 1 day, that of another aquarium was raised to 30ºC and subsequently maintained for a minimum of 5 weeks under a 12:12-h light-dark photoperiodic regime. Total reads were 4.8 and 4.2 million (>25 bases) from the muscle tissues of the 10 and 30ºC-acclimated fish, respectively. Subsequently, the collected reads were mapped to the publicly available medaka genome database based on the TopHat program and further analyzed by Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts. We revealed that 11 genes including FBXO32 and GLS2, and 20 genes including larval type MYHs and HSP70 showed significantly higher expression levels in the 30 and 10°C-acclimated fish, respectively.

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No.15

AN APPROACH TO GENOME ANALYSIS OF JAPANESE SEAWEED, WAKAME Undaria pinnatifida

1Yoshihiro Takahashi, 1Md. Shaheed Reza, 1Nanami Mizusawa, 1Kazutoshi Yoshitake, 1Ko

Yasumoto, 1Misturu Jimbo, 1Nobuyoshi Nanba, 2Hiroki Koyama, 3Takanori Kobayashi, 4Yoichi Sato, and 1Shugo Watabe

1School of Marine Biosciences, Kitasato University, Japan (fm11111d@st.kitasato-u.ac.jp);

2Graduate School of Biosphere Science, Hiroshima University, Japan; 3Japan Fisheries Research and Education Agency, National Research and Development Agency, Japan; 4Riken

Food Co. Ltd., Japan

Wakame is one of seaweeds frequently consumed in Japan. The most part of wakame production in Japan is derived from aquaculture, where the production in Iwate Prefecture is the largest. The Great East Japan Earthquake in March 11th, 2011, hit Iwate Prefecture and subsequent tsunami brought about enormous damages around coastal area. There are several major, regional productions of wakame and it is important for people engaged in wakame production in Iwate Prefecture to reinforce the brand strength for recovery from such disaster. The present study was initiated based on the above-mentioned background. Alive specimens of wakame were obtained from Iwate Prefecture and kept frozen at -30˚C until use. Meanwhile, wakame gametophytes were prepared from alive specimens by a conventional method. DNA extraction and purification from leaf tissues were performed using NucleoSpin Plant II kit (Takara). Shotgun genomic DNA library was constructed using IonXpress Plus gDNA Fragment Library Preparation Kit (Thermo Scientific). Sequencing was performed with an Ion PGM next generation sequencer (Thermo Scientific) and obtained data were subjected to the Maser analysis pipeline provided by National Institute of Genetics in Japan. This pipeline automates the processes of quality check with FastQC, counting k-mer frequency with JELLYFISH and sequence assembly with MIRA. Furthermore, we compared a few methods using cetyl-trimethylammonium bromide (CTAB) and TNES-urea buffer to extract DNA. In the case of gametophytes, ISOPLANT kit (Nippon Gene) with alginate lyase (Nippon Gene) and GM quicker 2 (Nippon Gene) were employed to purify DNA. A total of 5,066,379 reads were generated by IonPGM on the DNA library from leaf tissues. The assembly produced 278,281 contigs with a GC content of 48%, N50 contig size of 613, and k-mer coverage of 3x. As for an attempt to establish a suitable method for DNA extraction from leaf tissues, we found the CTAB method to be best. Higher purity of DNA was obtained when gametophytes were subjected to DNA extraction using ISOPLANT kit with alginate lyase and GM quicker 2.

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No.16

16S AMPLICON ANALYSIS OF MICROBIAL FLORA IN THE SALTED AND FERMENTED SQUID PRODUCTS AIMING AT THEIR QUALITY CONTROL

1Shugo Watabe, 1Nanami Mizusawa, 1Daisuke Ouchi, 1Yoshiaki Ida, 1Md. Shaheed Reza,

1Ko Yasumoto, 1Mitsuru Jimbo, 2Atsuko Nagaoka, and 2Jianrong Wan

1School of Marine Biosciences, Kitasato University, Japan (swatabe@kitasato-u.ac.jp), 2Research and Development Center, Suzuhiro Kamaboko Honten Co. Ltd

Salted and fermented squid is one of the traditional Japanese seafood products. While

various factors affects the maturation of these products during fermentation, microbial flora on the surface and in surrounding liquid part is one of them, producing various proteases which provide the products with taste-active compounds such as free amino acids and peptides from squid proteins. The present study was undertaken to determine microbial flora in the liquid part of salted and fermented squid for its possible control during processing.

The salted and fermented squid product commercially available was used as a starting material. A liquid part of the product was subjected to DNA extraction after diluting with 10 volumes of sterilized water, 0.1% NaCl, or 0.2% NaCl. Subsequently, these different types of diluted liquids were passed through 5.0, 0.8, and 0.2 μm, successively. Then DNA extracted from a 0.2 mm filter paper using PowerWater® DNA Isolation Kit (MO BIO Laboratories) was used as a template for amplification of 16S rDNA amplicons with a primer set targeting a V1-V3 region. After agarose electrophoresis, the amplicon products were cut out from the gel and subjected to PCR amplification using Ion PGM Hi-Q Seaqencing Kit (Thermo Fisher Scientific), whereas determined DNA nucleotide sequences were analyzed by SILVAngs pipelines.

Microbial profiles in the salted and fermented squid were characterized by the major bacterial genera consisting of Carnobacterium, Pseudomonas, Skermanella and Staphylococcus. However, these profiles changed depending on the DNA extraction conditions and the processed products supplied. For example, the major bacteria in DNA extracted from 3 g with sterilized water were composed of Staphylococcus and Methylobacterium and at 88 and 3%, respectively, whereas those from 5 g were 67 and 20%, respectively, suggesting that further investigation is necessary to obtain reproducible results.

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No.17

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Ofunato International Workshop 2016 Secretariat 1-15-1, Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan School of Marine Biosciences, Kitasato University Tel 042-778-8007, Fax 042-778-5010, kaust@kitasato-u.ac.jp http://www.kitasato-u.ac.jp/mb/kaust/en_index.html © Organizing Committee of Ofunato International Workshop 2016