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(1) The ethyl acetate fraction from Ecklonia cava expression of chemokine and cytokines via down-‐regulating NF-‐κB and MAPK pathway in TNF-‐α/IFN-‐γ-‐stimulated HaCaT human keratinocytes. Park, SY1, Kang, N1, Han, EJ2, Kim, HJ3, Lim, YH3, Jang, JT4, Jee, Y5, Jeon, YJ6 Ahn, G1,2,*. 1Department of Marine Bio-‐Food Sciences, Chonnam National University, 2Department of Food Technology and Nutrition, Chonnam National University, 3Seojin Biotech Co., Ltd, 4Aquagreen technology Co., Ltd., 5Department of Veterinary Medicine, Applied Radiological Science Research Institute, Jeju National University, 6Department of Marine Life Science, Jeju National University. Ecklonia cava, an edible brown seaweed is demonstrated to have various biological activities such as anti-‐inflammatory, anti-‐oxidant and cytoprotective effects. In this study, we evaluated the anti-‐inflammatory potential and mechanisms of the ethyl acetate fraction from E. cava (ECEF) in the tumor necrosis factor-‐α (TNF-‐α)/interferon-‐γ (IFN-‐γ)-‐stimulated inflammatory response in HaCaT human keratinocytes. To measure the effects of ECEF on chemokine and pro-‐inflammatory cytokine expression in HaCaT cells, we used the following methods: cell viability assay, reverse transcription-‐polymerase chain reaction, and western blotting. The result that ECEF did not show cytotoxicity to HaCaT cells at the tested concentrations. ECEF suppressed TNF-‐α/IFN-‐γ-‐stimulated mRNA expression of chemokines and pro-‐inflammatory cytokines. In addition, ECEF inhibited the activation of NF-‐κB and extracellular signal-‐related kinases (ERK)/p38 MAPK signaling pathways. ECEF exerts anti-‐inflammatory effects by suppressing the expression of TNF-‐α/IFN-‐γ-‐stimulated chemokines and pro-‐inflammatory molecules via a blocked NF-‐κB, ERK and p38 activation. Our results suggest that ECEF might be a useful therapeutic candidate for inflammatory skin diseases. (2) Venoms to Drugs. Alewood P*. Institute for Molecular Bioscience, University of Queensland, Brisbane Australia. Many organisms including snakes, spiders, scorpions and cone snails have evolved venom for prey capture or defence1. These venoms typically contain a complex cocktail of bioactive disulfide bond rich polypeptide toxins that target a wide range of receptors including enzymes, ion channels, GPCRs and transporters. Of interest to drug designers is their high potency and selectivity combined with their resistance to many proteases. Of particular interest are venoms from the marine Conidae2,3, with smaller polypeptide chains of 10-‐40 amino acids that are highly constrained by one to five disulfide bridges and are structurally well defined. Their high potency and exquisite selectivity for ion channels and

receptors has led to two drug candidates from our laboratories. In this presentation I will outline our program of discovery, describe the amazing diversity of molecular structures being discovered and the regioselective chemistry developed to facilitate disulfide bond formation. This has led to mimetics that have similar or improved potency to the native molecule plus exceptional stability when exposed to reducing environments and in plasma. Together, these results underpin the development of more stable and potent peptide mimetics suitable for new drug therapies, and highlight the application of this technology more broadly to disulfide bonded peptides and proteins. References: 1. Sébastien Dutertre, Ai-‐Hua Jin, Irina Vetter, Brett Hamilton, Kartik Sunagar, Vincent Lavergne, Valentin Dutertre, Bryan Fry, Agostinho Antunes, Paul F. Alewood and Richard J. Lewis. Nature Communications 5:3521, 2014. 2. Akondi KB, Muttenthaler M, Dutertre S, Kaas Q, Craik DJ, Lewis RJ, Alewood PF (2014). Chemical Reviews 114 (11) 5815. 3. I Sharpe, J Gehrmann, M Loughnan, L Thomas, D Adams A Atkins, DJ Craik, D Adams PF Alewood and RJ Lewis (2001). Nature Neuroscience, 4(9) 902. (3) Algal-‐biorefinery: challenges and opportunities. Angelidaki, I* and Alvarado-‐Morales, M. Department of Environmental Engineering, Technical University of Denmark. The recent years challenges such as climate change and environmental problems require action and strategic planning in the future to ensure access to food, feed and energy. Therefore, there is a need for alternative sources of biological-‐based products. Algal biomass is a not completely explored "treasure" and has been considered ad possibility to address these future challenges. The technological potential of the combined use of micro-‐and macro-‐algae for the production of various biological products is great. Macroalgae can be used directly as food, snack, as sprinkle in salads and as additive for flavor enhancing. Microalgae such as Spirulina and Chlorella are already used with great success as nutritional supplements. Food additives (such as alginate) may be extracted from macroalgae and the remainder of the biomass can then be used for bio-‐and fertilizer without any waste product. Micro-‐and macro-‐algae have a high content of biochemicals such as vitamins and antioxidants, which can be used to fortify foods and protect the more unsaturated fatty acid (omega-‐3) to be converted into saturated fatty acids or rancid.

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In a biorefinery concept algae can be used for the production of biomass, functional food, feed, biochemicals and bioenergy. In addition, algae can with advantage be used as fertilizer for crops. This applies both to the use of algal waste products from biofuel production, but also from blue-‐green algae that can grow in the wet rice fields and fix nitrogen from the air and thus provide plants with nitrogen, without the need for application of fertilizers. There is however a challenge to make the process economically feasible and environmentally sound. The main key challenges are economical algae cultivation and harvesting. Currently, technological development is taking place which is giving the algae biomass more opportunities. (4) Genomics of adaptation: relevance for biotechnology Agostinho Antunes1,2,*.1 CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos, s/n, 4450–208 Porto, Portugal. 2Department of Biology, Faculty of Sciences, University of Porto. Rua do Campo Alegre, Porto, Portugal. The completion of the human genome sequencing in 2003 opened a new perspective into the importance of whole genome sequencing projects, and currently thousand of species are having their genomes completely sequenced, from simple organisms, such as bacteria, to more complex taxa, such as mammals. This voluminous sequencing data generated across multiple organisms provides also the framework to better understand the genetic makeup of such species and related ones, allowing to explore the genetic changes underlining the evolution of diverse phenotypic and adaptive traits. Here, recent results from our group retrieved from comparative evolutionary genomic analyses of varied species will be considered to exemplify how gene novelty and gene enhancement by positive selection might have been determinant in the success of adaptive radiations into diverse habitats and lifestyles. The findings pinpoint unique molecular products of critical relevance in species evolution and diversification, but also highlight genomic novelties with biotechnological potential and importance for environmental and biomedical research. (5) Growth and trade-‐off effects in CRISPR/Cas9-‐mediated myostatin-‐mutated F4 medaka (Oryzias latipes). Takashi Aoki1,,

2 Ying-‐Chun Yeh1, Masato Kinoshita3, Tze Hann Ng1, Yu-‐Hsuan Chang1, Shun Maekawa1, Yi-‐An Chiang1, Haruko Takeyama4 , Han-‐Ching Wang1. 1 Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan,

Taiwan, and Institute of Biotechnology, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan. 2 Research Organization for Nano and life Innovation, Waseda University, Tokyo, Japan. 3 Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-‐8502, Japan.4

Faculty of Science and Engineering, Waseda University, Tokyo, Japan We used the powerful gene editing tool CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats and CRISPR-‐associated protein 9) to mutate the MSTN gene in medaka (Oryzias latipes) and evaluate the growth performance of the resulting mutants. Injection of a mixture of Cas9 mRNA and OlMSTN-‐gRNA into fertilized medaka eggs produced mutant F0 fish that carried different frameshifts in the OlMSTN coding sequence. To test the heritability of the mutant genotypes to the F1 generation, these mutant F0 fish were mated together to generate OlMSTN-‐CRISPR/Cas9 mediated MSTN-‐/-‐ F1 medaka. Two F1 fish with the same heterozygous frame-‐shifted genomic mutations (a 22 bp insertion in one allele; a 32 bp insertion in the other) were then crossbred to produce subsequent generations (F2~F5), all of which had either the same heterozygous mutation or else a homozygous mutation that consisted of the 22 bp insertion in both alleles. The MSTN protein expressed by these fish contained no active C-‐terminal domain and was non-‐functional. We found that the body length and weight of the MSTN-‐/-‐ F4 medaka were significantly higher than in the wild type fish. There was also a decrease in the density of the muscle fibers in the inner and outer compartments of the epaxial muscles, suggesting that MSTN null mutation induces muscle hypertrophy. We further show that from 3~4 weeks post hatching (wph), the expression of three major myogenic related factors (MRFs), MyoD, Myf5 and Myogenin, was significantly upregulated in the MSTN-‐/-‐ F4 medaka. Although a small number of MSTN-‐/-‐ medaka presented with a spinal deformity, and we also observed a trade-‐off between growth and immunity in MSTN-‐/-‐ F4 medaka, the fast-‐growth phenotypes with relatively normal body shape appeared to have normal reproductive ability. (6) Role of hemocyte homeostasis-‐associated protein (HHAP) in two species of crustaceans. Apitanyasai, K1*,

Charoensapsri, W2, Amparyup, P2, Noonin, C3, Söderhäll, I3, Söderhäll, K3, Tassanakajon, A1. 1Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 2National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA),

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3Department of Comparative of Physiology, Uppsala University. Hemocyte homeostasis-‐associated protein (PmHHAP) has been reported as a viral responsive protein that plays an important role in controlling hemocyte homeostasis in shrimp Penaeus monodon. In this study, the role of PmHHAP in regulating apoptosis in shrimp was investigated. In vivo gene silencing of PmHHAP could induce the high level of apoptosis in shrimp hemocytes. Moreover, PmHHAP was able to bind to PmCasp and the rPmHHAP protein could decrease apoptosis in the rPmCasp-‐treated hemocytes cells. These results indicated that PmHHAP is an anti-‐apoptosis protein that regulates hemocyte homeostasis by inhibiting apoptosis in P.monodon. Yeast two-‐hybrid and co-‐immunoprecipitation revealed that PmHHAP binds to an anti-‐apoptosis protein of white spot syndrome virus, WSSV134. The viral protein WSSV134 is a late protein of WSSV which is initially expressed after 24-‐hour post infection (hpi) and is likely involved in viral propagation. Co-‐silencing of PmHHAP and WSSV134 prior to WSSV infection showed significant increase of caspase activity, which was higher than silencing only PmHHAP or WSSV134, suggesting that these two proteins might work concordantly to control apoptosis during WSSV infection. In addition, a homolog of HHAP in crayfish Pacifastacus leniusculus with 47% similarity to PmHHAP was functional characterized. PlHHAP was detected in all examined tissues but highly expressed in intestine and gill. Surprisingly, PlHHAP was found to play important role in bacterial but not viral defense in contrast to PmHHAP. PlHHAP transcript remained unchanged upon WSSV infection but dramatically increased upon bacterial Aeromonas hydrophila B1 challenge. Furthermore, suppression of PlHHAP resulted in increasing the number of bacteria in intestine but did not affected hemocyte apoptosis suggested that HHAP from different crustacean species have different functions in defense against invading pathogens. (7) Breeding of alkenones producing haptophyte using heavy ion-‐beam irradiation. Araie H1*, Hase Y2, Ohno Y2, Suzuki I1 and Shiraiwa Y1. 1Faculty of Life and Environmental Sciences, University of Tsukuba, Japan, 2National Institutes for Quantum and Radiological Science and Technology, Japan. Biofuel production using algae is attracting keen attention as a next generation energy candidate because it has advantages such that production per unit area is higher than land plants and not competing with food production. However, in order to advance the algae biomass industry, it is necessary to improve oil-‐producing algae by mutation for making oil high accumulated strain or strain with high

growth rate, and so on. In this research, we focused on unique lipid called alkenones that are synthesized by only five marine haptophyte species since that are thought as one of the sources of fossil fuels. We selected one of alkenone-‐producing haptophytes, Tisochrysis lutea, as material to achieve efficient alkenone production. For the purpose, we have developed heavy ion-‐beam irradiation technology since obtained mutants can be used in open system as non-‐GMO. Heavy ion-‐beam irradiation was performed on T. lutea (T-‐Iso strain) placed on the agar plate with 0~320 Gy of carbon ions (26.7 MeV/u) at the TIARA ion beam irradiation facility, Japan. After 1~2 week, we counted the number of colonies for survival rate and picked up colonies for evaluation of alkenone productivity. Dose response curve showed that LD50 of the colony formation was around 30 Gy but no survival over 120 Gy. Seven of 32 strains at 80 Gy irradiation and 21 of 59 strains at 20 Gy irradiation showed approximately 1.5-‐fold higher alkenone productivity than wild type. These results suggested that irradiation dose at around LD50 is suitable for obtaining useful mutants. This is the first attempt to improve alkenone productivity by using heavy ion-‐beam irradiation. (8) Complementary chromatic acclimation (CCA) in marine cyanobacteria. Ariyanti, D1,2*, Sode, K1,2. 1Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan, 2JST, CREST, Tokyo, Japan. The authors’ research group has been engaged in the designing of microbial bioprocesses which can be controlled by the light signal. Cyanobacteria has the ability of sensing various light wave lengths, and responds to adapt its photosynthetic machinary by altering phycobilisome composition, known as complementary chromatic acclimation (CCA). We are interested in the utilization of CCA systems for the future bioprocess control. Synechococcus sp. PCC 7335 here after called PCC 7335, is one of unicellular marine cyanobacteria reported perform CCA, although the detail study of CCA level of this strain is yet to be done. In this study, we report molecular biology of CCA in PCC 7335 related to their phycobiliprotein changes. CCA condition were provided by various combination of red and green light. Phenotypic characterization were then confirmed by observing macroscopic changes of cells color, which was confirmed by biochemical analyses of phycobilisome properties component. CCA relating gene were screened using signature of bilin chromophore binding site via cystein covalent link. The putative genes responsible for CCA were further evaluated at transcriptional level. Confirmation of phenotypic and biochemicals properties analyses

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supported with transcriptional evaluation suggest that PCC 7335 closely related to type III CCA species. (9) A multi-‐omics approach for environmental health assessment. Bowden, JA1*, Schock, TB1, Ulmer, CZ1, Jones, CM1, Koelmel, JP2. 1National Institute of Standards and Technology, NIST, Hollings Marine Laboratory, Charleston, SC, 2University of Florida, Gainesville, FL. While omics-‐based approaches have been focused on studies related to human health, an increase in the occurrence of pathophysiological conditions in wildlife suggests that the application of omics for the assessment of environmental health presents a promising new direction. Strategies capable of increasing the number and chemical variability of compounds examined in a single sample and/or analysis need to be developed for improving the likelihood of environmental biomarker discovery and depth of description of disease etiology. A promising strategy we are developing in the Marine Biochemical Sciences Group at the Hollings Marine Laboratory involves performing multi-‐omics studies of chemical and biological processes in marine organisms (e.g., coral, marine mammals, fish), for example, performing lipidomics and metabolomics using ultra-‐high performance liquid chromatography coupled to high-‐resolution mass spectrometry on a single extraction. In this presentation, we aim to highlight the potential of employing this multi-‐omics strategy to several environmental health-‐related projects. The multi-‐omics approach has been applied to identify new disease biomarkers in South African Mozambique tilapia (Oreochromis mossambicus) afflicted with pansteatitis, a disease characterized by severe inflammation of adipose tissue. This disease is increasingly present across different species, trophic levels, and sites and may be the cause of large-‐scale mortality events in the region. A current obstacle to providing population-‐level surveys to determine the pervasiveness of pansteatitis is that current methods to assess disease status are lethal. As this disease largely affects adipose tissue, identified metabolites and/or lipids in the plasma of healthy and pansteatitis-‐affected fish could serve as markers for non-‐invasively classifying health status. The potential of multi-‐omics will be further applied to harmful algal blooms, soy-‐based aquaculture, the metabolic effects of anthropogenic contaminant exposure, and the establishment of omic baselines for sentinel species for future analysis. (10) The shrimp (Fenneropenaeus merguiensis) molt inhibiting hormone (MIH1) gene: molecular characterization and functional studies. Lau, Y, Liang, HF, Chan SF*. Fisheries College, Guangdong Ocean University.

The Molt Inhibiting Hormone gene and cDNA of the banana shrimp Fenneropenaeus merguiensis (FmMIH1) has been cloned and characterized. FmMIH1 possesses most of the characteristics of the eyestalk type-‐II CHH/MIH/GIH family neuropeptide. The open reading frame of FmMIH1 consists of 315 bp encoding for a protein of 105 amino acid residues. The mature peptide of FmMIH1 consists of 76 amino acid residues, a glycine residue at position 11 of the mature peptide and 6 cysteine residues located in the conserved position of the mature peptide. In addition to eyestalk, high levels of FmMIH1 transcript could also be detected in the intestine. FmMIH1 transcript level is low throughout the post-‐molt, early to mid-‐intermolt and premolt. However, a sharp increase could be observed in late intermolt (C3 stage). Both alignment and phylogenetic analysis reveal that FmMIH1 is most similar to the MIH1 of other shrimp species. For functional assay, RNA interference results show that a significant 2.3 days (P<0.05) reduction in molt cycle duration could be observed in shrimp receiving dsFmMIH1 injection. Surprisingly, injection of recombinant FmMIH1 could also cause a significant reduction of the molt cycle duration (average 1.9 days, P<0.05). We hypothesize that the recombinant protein is biological inactive but it competes with the endogenous MIH for carrier protein binding and consequently reduces the amount of biological MIH that could reach the targets. In conclusion, the result of this study will provide us new insight to manipulate molting/growth in crustaceans. (11) Transcriptomic analysis of host responses in shrimp with acute hepatopancreatic necrosis disease (AHPND). Chang, CC1,2*, Chang, WC3, Lin, SS4, Ng, TH1,2, Wang HC1,2. 1Institute of Biotechnology, National Cheng Kung University, 2Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, 3Institute of Tropical Plants Sciences, National Cheng Kung University, 4Institute of Biotechnology, National Taiwan University. Global shrimp aquaculture has had substantial production losses due to various diseases, including acute hepatopancreatic necrosis disease (AHPND), a recently emergent bacterial disease, caused by strains of Vibrio parahaemolyticus (VP) containing a large virulent plasmid (pVA1). Despite much research on AHPND, molecular pathogenesis of AHPND and host responses in AHPND-‐infected shrimp are not well characterized. Therefore, the objective was to use transcriptome sequencing, an NGS platform and bioinformatics to identify the key factor or pathway in the stomach of AHPND-‐infected shrimp. First, by detecting virulent gene Pir as an indicator of a pathogen marker, we monitored shrimp infected with AHPND. Subsequently, samples were subjected to NGS to

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obtain a transcriptome database to assess host responses after AHPND infection. Using a TSB group (only treated with medium) as a control, we compare different treatment groups to detect differential expression of genes (DEGs) in each group. Furthermore, a gene co-‐expression network between non-‐virulent and virulent VP-‐challenged groups was established using a ContigViews platform. From these networks, we proposed that AHPND infection affected cell junctions as well as immune responses in the stomach. We anticipate that this network will be a critical tool to understand the association between VP and shrimp, and elucidate key factors of AHPND infection. (12) Development of the oral delivery system for shrimp aquaculture using attenuated Listeria monocytogenes. Chen, LL*. Institute of Marine Biology, National Taiwan Ocean University. Viral and bacterial diseases still smite to shrimp aquaculture industry and cause serious economic less. In the present study, recombinant proteins were mostly applied to develop vaccine against diseases and animals were always vaccinated by injection. However, injection method is difficult to apply to aquaculture. Therefore, the development of oral delivery system will be the main objective for the future. Recently, Listeria monocytogenes was applied to human cancer treatment. The pathogenicity of L. monocytogenes was deleted and this system can be attempted to aquaculture. In this study, the mutated L. monocytogenes was used to develop oral delivery system. The results indicated that the expression of green fluorescent protein gene (GFP) increased in the primary cultured shrimp hemocytes which were treated with heat-‐killed L. monocytogenes containing GFP. Besides, GFP was also detected in the shrimps fed with heat-‐killed L. monocytogenes. It indicated that the inactivated L. monocytogenes can enter shrimp cells. Our results show that the attenuated L. monocytogenes is a good candidate to develop the oral delivery system in shrimp aquaculture. (13) Photobacterium damselae subsp. piscicida responds to antimicrobial peptides through phage-‐shock-‐protein A (PspA)-‐related extracytoplasmic stress response system. Wei-‐Jung Chen*, Tsun-‐Yung Kuo, and Wei-‐Chen Tsai. Department of biotechnology and animal science, National Ilan University, Taiwan. Objectives: An outbreak of fish photobacteriosis is recognized as one of the most threatening bacterial diseases in aquaculture worldwide due to its wide host range and massive mortality. Photobacterium damselae subsp. piscicida (Phdp) is a halophilic, facultatively anaerobic Gram-‐negative marine pathogen, causing

severe fish photobacteriosis. Antibiotics were applied to prevent bacterial infection, however, the extensive use has led to the growing emergence of many resistant strains. Evidence suggested that antimicrobial peptides (AMPs) are of greatest potential to replace classical antibiotics. In the current study, we intended to investigate whether Phdp can sense and directly respond to the presence of cationic AMPs. Methods and Results: We performed proteomic methodologies to investigate the responsive proteins of Phdp on exposure to AMP Q6. Proteins significantly altered were analyzed by two-‐dimensional gel electrophoresis and LC-‐ESI-‐Q-‐TOF MS/MS, thus resulting in 5 outer membrane proteins (OMPs), 7 inner membrane proteins (IMPs) and 17 cytoplasmic proteins (CPs) identified. Quantitative real-‐time PCR was also applied to monitor the mRNA expression level of these target proteins. Conclusions: COG analysis revealed that upon exposure to AMP Q6, the majority of the upregulated proteins were involved in signal transduction mechanism, carbohydrate transport and metabolism, post-‐translational modification and chaperones, while the downregulated proteins were mainly related to energy production and conversion. Among them, phage-‐shock-‐protein A (PspA)-‐related stress response system was considered to play a crucial role. Impact and significance: To the best of our knowledge, this is the first report elucidating Phdp AMP-‐response mechanism using proteomics approach. AMP-‐responsive proteins identified in this study could serve as attractive targets for developing more effective antimicrobial agents against Phdp and other marine bacterial pathogens. (14) Characterization of growth performance and muscle composition using immune-‐inhibition technology in orange-‐spotted grouper (Epinephelus coioides).Chen, YT1,3*, Lin, CF1,3, Chen, TY1,2,3,4. 1Institute of Biotechnology, 2Department of Biotechnology and Bioindustry Sciences, 3Translational Center for Marine Biotechnology, 4Agriculture Biotechnology Research Center, National Cheng Kung University, Tainan, Taiwan. Grouper is an important aquaculture fish species that contributes important economic profit in Taiwan. However, the main molecular factors that regulate grouper growth performance and its nutritional value remain unclear due to lack of the technology to establish grouper model organism. In present study, we characterized the growth performance and muscle composition of myostatin-‐inhibited groupers and secreted protein acidic and rich in cysteine (SPARC)-‐inhibited groupers. The immune-‐inhibition technology was applied to inhibit the negative growth factors, myostatin and SPARC, respectively. Our results showed that the groupers

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generated anti-‐myostatin or anti-‐SPARC antibodies after 14 weeks with treatment and without immune tolerance up to 32 weeks with treatment. The growth performance of myostatin-‐inhibited grouper and SPARC-‐inhibited grouper is better than control in various parameters including body weight, body weight without viscera and fillet weight. Besides, there is no significant difference of fatty acid and amino acid composition between experimental group and control group. In conclusion, both of myostatin-‐inhibition and SPARC-‐inhibition improved growth performance without modification of muscle composition in grouper. More importantly, we used immune-‐inhibition technology to establish a grouper model organism that is very useful for further studying grouper in vivo biological processes in the future. (15) 16S metagenomics sequencing of microbes in Marine Red Alga, Pyropia yezoensis. Rieka Chijiiwa1,2*, Sonomi Yamaguchi1, Keigo Ide1,2, Toru Maruyama1,2, Masahito Hosokawa3,4, Hiroshi Saito5, Haruko Takeyama1,2,3. 1Department of Life Science and Medical Bioscience, Waseda University, 2CBBD-‐OIL, AIST, 3Research Organization for Nano & Life Innovation, Waseda University, 4PRESTO, JST, 5School of Marine Science and Technology, Tokai University. Susabi-‐nori (Pyropia yezoensis), Marine red alga, is one of the valuable aquatic resources in the world. Recent reports have suggested that there are symbiotic relationships between host and microbes from the marine environment, and a complex commensal microbial community. Moreover, these bacteria play an important role of development of P. yezoensis. Nevertheless, details in the relation to these mechanisms are still relatively unknown in molecular level, and needs to be elucidated for investigation of the microbial functions in P. yezoensis. In this work, we applied culture-‐independent methods based on 16S rDNA gene sequences (V3-‐V4 variable region) to characterize diversity and structure of microbial communities in P. yezoensis. Moreover, we conducted the comparative analysis of 16S rDNA gene sequences in differences of distributions, seasons and processing by statistical analysis. Our results suggested that there was a clear difference between the composition of microbial communities in hosts and that in seawater, so we found the unique microbial communities in P. yezoensis. In addition, we characterized unique composition of microbes in each location, and these microbes exhibited clear seasonal shifts in community structure. Based on these preliminary results, we hope further elucidate the role and nature of these microbes in P. yezoensis.

(16) Regulation and application of Toll-‐like receptor-‐9 pathway in grouper. Chiou, Pinwen Peter1*, Lee, Frank Fang-‐Yao1,2, Chuang, Hsiang-‐Chieh3, Chen, Shiang-‐Peng1, Peng, Ran-‐Hong1, Chou, Hsin-‐Yiu1. 1Department of Aquaculture, National Taiwan Ocean University, 2Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, Academia Sinica, 3Department of Life Science, National Taiwan University. Pattern Recognition Receptors (PRRs) are crucial in initiation innate immune response by recognizing the conserved molecular patterns associated with pathogens. Among the PRRs, Toll-‐like receptor 9 (TLR9) recognizes unmethylated CpG DNA presented in the genomes of bacteria and DNA viruses. In addition, synthetic unmethylated CpG oligodeoxynucleotides (ODNs) have been widely used in mammals as TLR9 agonists or to serve as vaccine adjuvant. We have shown that in response to CpG ODNs or bacteria the TLR9 pathway in orange-‐spotted grouper (Epinephelus coioides), an economically important teleost, can be orchestrated by a negative regulator gTLR9B isoform, whose production is mediated by RNA alternative splicing. We further showed that the ligand-‐induced activation of downstream NF-‐κB leads to phosphorylation of Pol II CTD Ser2, thereby biasing the gTlr9 alternative splicing for increasing production of gTLR9B as a means of self-‐limiting response. This regulatory mechanism has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish. To exploit the potential of CpG ODN as vaccine adjuvant in grouper, we have designed a series of class-‐A CpG ODNs capable of regulating immune response in the fish. When incorporated into a DNA or inactivated vaccine against viruses that infect grouper, these CpG ODNs enhanced the efficacy of the vaccines. Further analyses revealed that these ODNs facilitated a Th1 response in the host. These data demonstrate the benefit of CpG ODNs as vaccine adjuvant in the prophylactic control of viral diseases in grouper. (17) A bivalent DNA vaccine against two different iridoviruses induces specific protection in orange-‐spotted grouper (Epinephelus coioides). Chou, HY1,2*, Liu, HI1,Chu, YT1,3, Ho, LP2, Pai, TW2,4. 1Department of Aquaculture, National Taiwan Ocean University, 2Center of Excellence for the Oceans, NTOU, 3Mariculture Research Center, Fisheries Research Institute, Taiwan, 4Department of Computer Science and Engineering, NTOU. Outbreaks of viral disease along with high mortalities have threatened the marine fish farming in Asia Pacific region for years. Grouper iridovirus (GIV) of genus ranavirus and grouper iridovirus of Taiwan (TGIV) of genus megalocytivirus are two important grouper pathogenic

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viruses and proved to be of high virulence to different teleost fishes in aquaculture. Moreover, coinfection phenomenon of GIV and TGIV was found in moribund groupers in Taiwan. Due to vaccination remains the most successful strategy for preventing and controlling infectious diseases for fish aquaculture. A bivalent DNA vaccine against the two diseases would be a preferable option. We report here the cloning of TGIV and GIV major capsid protein (MCP). Through combining sequence and structural alignment results and surface structure validation, both grouper iridovirus MCP sequences possess at least one unique segment respectively was confirmed. Subsequently, each MCP gene was used to construct corresponding DNA vaccine, and treated with water in oil in water (W/O/W) emulsions and making into bivalent DNA vaccine. In vivo expression of both MCP genes were observed in grouper intestines after tube-‐feeding with single or bivalent DNA vaccine. Further, after oral vaccination groupers were challenged with either TGIV or GIV by intramuscular injection. The results indicate that bivalent DNA vaccine against two different iridoviruses induces specific protection in orange-‐spotted grouper. (18) Using genomics to guide marine pest mitigation strategies: insights from the crown-‐of-‐thorns starfish genome project. Bernard M Degnan*. School of Biological Science, University of Queensland, Brisbane QLD, Australia. The crown-‐of-‐thorns starfish (COTS, the Acanthaster planci species group) is a highly fecund predator of reef-‐building corals throughout the Indo-‐Pacific. Their mass spawning events predispose them to population outbreaks that result in a dramatic loss of live coral cover and associated biodiversity. These outbreaks have more impact on reef health and resilience than the combined effects of coral bleaching and disease, and increase the susceptibility of reefs to other potentially detrimental events, such as severe storms. Although a range of local in situ control measures have been applied with some success, mitigation of COTS outbreaks on the necessary regional scale requires mass-‐deployed, species-‐specific strategies. In this context, genome-‐encoded COTS-‐specific attractants that underpin spawning aggregations have significant potential as biocontrol agents. Here we sequenced genomes of COTS from the Great Barrier Reef, Australia and Okinawa, Japan to identify gene products present in chemical plumes released from aggregating COTS that make normally sedentary starfish highly active. Peptide sequences detected in these plumes by mass spectrometry are encoded in the COTS genome and expressed in external tissues. The exoproteome released by aggregating COTS is largely comprised of signalling

factors and hydrolytic enzymes, and includes an expanded and rapidly-‐evolving set of starfish-‐specific ependymin-‐related proteins. These secreted proteins may be detected via members of a large family of olfactory receptor-‐like G protein-‐coupled receptors that are expressed externally, sometimes in a sex-‐specific manner. This study provides the first insights into COTS-‐specific communication that may guide the fabrication of peptide mimetics for use on reefs with COTS outbreaks. More broadly, this approach suggests ways in which ‘omics can be applied to the mitigation of other aquatic pests. (19) Continuous mass spectrometry analysis for hydrothermal decomposition of glucose. Pattasuda Duangkaew1*, Shuhei Inoue2, Tsunehiro Aki3, Yutaka Nakashimada3, Yoshiko Okamura3, Takahisa Tajima3, Yukihiko Matsumura2. 1Department of Mechanical Science and Engineering, Graduate School of Engineering, Hiroshima University, 2Division of Energy and Environmental Engineering, Institute of Engineering, Hiroshima University, 3Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University. This is the first report to apply mass spectrometry to real time observation of hydrothermal reactions. A continuous mass spectrometry (continuous MS analysis) analysis system was developed and designed to elucidate products of glucose decomposition during hydrothermal pretreatment process. It was achieved by coupling a continuous flow reactor with a quadrupole mass analyzer via custom-‐built connection fittings. The continuous MS method investigated the products of glucose decomposition, and high-‐performance liquid chromatography (HPLC) analysis of cold effluent was compared. At 140 °C, continuous MS showed that the decomposition of glucose does not proceed, this was confirmed by comparison with the mass spectral database for glucose. At 180 °C or higher, a clear base fragmentation peak of fructose (m/z 131 and 149), ring compound products like 5-‐hydroxymethylfurfural (5-‐HMF, m/z 97, 126 ) and furfural (m/z 96, 67) were observed. Retro-‐aldol products like glyceraldehyde and glycolaldehyde were also observed which were confirm by m/z 90, 72, 61 and increasing of ion current at m/z 29 and 31, respectively. No fructose was observed at the 200 °C, 10 min and 220 °C, 5 min by using conventional HPLC. The effectiveness of continuous MS analysis is clear, compared to HPLC analysis. (20) Korean black rockfish (Sebastes schlegelii) peroxiredoxin family proteins are vital regulators and sensors of peroxide. G. I. Godahewa*, N. C. N. Perera, and Jehee Lee. Department of Marine Life Sciences,

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School of Marine Biomedical Sciences, Jeju National University, Jeju Self-‐Governing Province, Republic of Korea. Incomplete reduction of oxygen to water during respiration results in formation of the superoxide anions, which are spontaneously or enzymatically converted to H2O2. Hydrogen peroxide itself is not very reactive, but can be further reduced to the extremely damaging hydroxyl radicals. Peroxiredoxins (Prx) are the possible antioxidants that are commonly available regulators of peroxides. Here, six Prx isoforms (1-‐6) of Sebastes schlegelii were characterized at molecular, genomic, functional and transcriptional level. The recombinant proteins (rSsPrxs) were subjected to flow cytometric, MTT cell viability and insulin reduction assays. Protein sequences of SsPrx1-‐5 were resided peroxidatic and resolving Cys residues and SsPrx6 resided only the peroxidatic Cys residue. These Cys residues were crucial on their activity. All the members of rockfish Prxs were consisted with thioredoxin domain. Genomic structures of SsPrx1 (5 exons), SsPrx2 (6 exons), SsPrx3 (6 exons), SsPrx4 (7 exons), SsPrx5 (6 exons) and SsPrx6 (5 exons) were followed the AG/GT splicing rule. The utmost antioxidant function was shown by the 100 µg/mL of rSsPrxs in flow cytometry analysis. Besides, rSsPrxs were increased the human leukemia THP-‐1 cell viability under the H2O2 induced oxidative stress in a dose dependent manner. The rSsPrxs could catalyze the insulin reduction and catalytic activity was increased with the incubation time. The SsPrxs mRNA was ubiquitously expressed in different healthy tissues in different scales. Collectively, we confirmed that the SsPrxs are homologs of the peroxiredoxin family with the potential to involve in black rockfish antioxidant defense system and innate immune system. Therefore, present study broadens the knowledge of peroxiredoxin family. (21) Antimicrobial peptide hepcidin genes were highly amplified in the genome of Nile tilapia. Gong, HY1,2,3*, Ho, CH1, Wu, SH1, Pham, HN1, Tseng, PC3, Kuo, YH1, Huang, CW1,2, Wu, JL3,4. 1Department of Aquaculture, National Taiwan Ocean University, Taiwan, 2Center of Excellence for the Oceans, National Taiwan Ocean University, Taiwan, 3Doctoral Degree Program of Marine Biotechnology, National Taiwan Ocean University, Taiwan, 4Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan. We would like to search for disease-‐resistant genes and related molecular markers for marker-‐assisted selection of Nile tilapia (Oreochromis niloticus). Three Nile tilapia hepatic antimicrobial peptide hepcidin/HAMP cDNAs and genes, encoding 87-‐a.a. HAMP1, 90-‐ a.a. HAMP2 and 83-‐a.a. HAMP3, were identified from transcriptome of Taiwan

Nile tilapia NT1 strain infected by virulent Streptococcus iniae 89353. The secreted mature peptide sequences of 22-‐a.a. HAMP1 and 26-‐a.a. HAMP2 of Nile tilapia with four disulfide bonds are identical to previously identified strong AMP TH1-‐5 and TH2-‐3 of Mozambique tilapia, respectively but 19-‐a.a. HAMP3 is a novel HAMP with only three disulfide bonds. Three Nile tilapia HAMP genes were differentially activated not only in the liver but also in head kidney, spleen and gill by S. iniae 89353 infection in dose of LD50 (1.5x105 CFU/g BW) to contribute for defense against S. iniae infection. The 19-‐a.a. HAMP3 mature peptide was chemically synthesized to test its antimicrobial activity against critical pathogens S. iniae and S. agalactiae for tilapia. The 19-‐a.a. HAMP3 synthetic peptide exhibited inhibitory activity on the growth of both S. iniae 89353 (MIC= 100 mM, 99.7% inhibition) and S. agalactiae (100 mM HAMP3: 66% inhibition) after 24 hours incubation. Recently, the new and improved genome assembly created from 44X coverage of PacBio reads sequenced from double haploid homozygous XX female individual of Oreochromis niloticus isolate:F11D_XX was submitted to GenBank by University of Maryland in end of 2016. From the new Nile tilapia genome assembly (GCA_001858045.2), we found 18 hepcidin genes were highly amplified in Nile tilapia genome including 12 hepcidin genes in LG11 and 6 hepcidin genes in two unplaced contig825 and contig1099. Furthermore, we identified 22 microsatellites DNA markers associated with hepcidin genes and are evaluating these hepcidin-‐associated SSRs as potential molecular markers in marker-‐assisted selection for disease-‐resistance of Nile tilapia. (22) Cultivation of marine microalgae (diatom) Amphiprora sp. for biodiesel production. Natanamurugaraj Govindan*, Mashitah M. Yusoff, Mohd Hasbi Ab. Rahim, Gaanty Pragas Maniam. Algae Biotechnology Laboratory, Faculty of Industrial Sciences and Technology, Universiti Malaysia Pahang, Lebuhraya Tun Razak, Gambang Kuantan Pahang, Malaysia. The biodiversity of microalgae reveals that the out of 65 species of marine diatom isolated and identified from the East coast of Peninsular Malaysia with the lipid content optimization is an important step for the micro algal biodiesel production. The marine diatom Amphiprora sp. is used as a main feedstock for biodiesel production. The effect of different light intensities and the presence of nanomaterial in culture medium on total lipid content were examined gravimetrically. The result for nanomaterial consisting culture medium induce a high lipid content by displaying 52.94±0.42% while conventional F2 medium only can produce 16.32±0.15%. The results disclose that at the range of 24 μmol m-‐²s-‐¹

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light intensity microalga Amphiprora sp. produces higher lipid compared to other light intensities. The metyl ester content produced for light intensity of 24 μmol m-‐²s-‐¹ is 33.52±1.25%. The most efficient temperature (65oC) produces 81.47±1.59% of methyl ester content. KOH as a homogenous catalyst in the transesterification of triglycerides with methanol to produce methyl esters (biodiesel). The optimal reaction conditions were found to be: 2 wt. % catalyst amount (based on oil weight) and 1.5:1 methanol to oil volume ratio for all the catalysts and under these reaction conditions, over 81.47% methyl esters content was achieved in a 3 h reaction period at the reflux temperature of methanol. GC-‐FID chromatograph depicts fatty acid metyl ester such as propionic acid (C3:0), decanoic acid (C10:0), hexadecanoic acid (C16:0). Heptadecanoic acid (C17:0), oleic acid (C18:1) and pentacosanoic acid (C25:0) used as internal standard. (23) The chloroform fraction from Sargassum horneri inhibits TNF-‐α/IFN-‐γ-‐stimulated inflammatory mediators in human keratinocytes via inhibition of the NF-‐κB and MAPK signaling pathway. Han, EJ1,*, Park, SY2, Kang N2, Kim, HJ3, Lim, YH3, Jang, JT4, Jee, Y5, Jeon, YJ6, Ahn, G1,2. 1Department of Food Technology and Nutrition, Chonnam National University, 2Department of Marine Bio-‐Food Sciences, Chonnam National University, 3Seojin Biotech Co., Ltd, 4Aquagreen technology Co., Ltd., 5Department of Veterinary Medicine, Applied Radiological Science Research Institute, Jeju National University, 6Department of Marine Life Science, Jeju National University. In this study, we investigated the molecular mechanism underlying the anti-‐inflammatory effects of the chloroform fraction of the Sargassum horneri (SHCF), a brown seaweed, on tumor necrosis factor-‐α (TNF-‐α)/interferon-‐γ (IFN-‐γ)-‐stimulated inflammatory response in human keratinocytes (HaCaT cells). We measured cell viability by MTT assay; pro-‐inflammatory mediators mRNA expression by reverse transcription-‐polymerase chain reaction; and nuclear factor (NF)-‐κB and mitogen-‐activated protein kinases (MAPKs) signaling expression by western blotting in TNF-‐α/IFN-‐γ-‐stimulated HaCaT cells before and after SHCF treatment. The results that SHCF had no cytotoxicity to HaCaT cells at the tested concentrations. Pretreatment of HaCaT cells with SHCF suppressed TNF-‐α/IFN-‐γ-‐stimulated mRNA expression of pro-‐inflammatory mediators such as TARC, MDC, IL-‐6, IL-‐4 and IL-‐1β. In addition, CFSH inhibited NF-‐κB translocation into the nucleus, as well as phosphorylation and degradation of IκBα. Furthermore, SHCF suppressed the phosphorylation of the extracellular signal-‐related kinases (ERK)/p38 MAPK signaling pathway. These results suggest that SHCF exhibits anti-‐inflammatory effects by

suppressing the expression of TNF-‐α/IFN-‐γ-‐stimulated chemokines and pro-‐inflammatory cytokines via down-‐regulation of NF-‐κB and ERK/p38 MAPK activation, and SHCF may be a useful therapeutic candidate for inflammatory skin diseases, such as atopic dermatitis. Acknowledgement: This work (GrantsNo.D11507215H480000110) was supported by Korea Institute of Marine Science & Technology Promotion (KIMST) in 2015 and 2016. (24) An investigation on the altered gene expression profiles of genes in the innate/adaptive immunity pathways of disease resistant transgenic rainbow trout. Han, Yueh-‐chiang*, Lin, CM. Chen, TT. Department of Molecular and Cell Biology, University of Connecticut, USA. We previously developed several homozygous transgenic rainbow trout lines harboring cecropin P1 transgene (Chiou et al., 2013). These fish resist to infection by bacterial, viral and parasitic pathogens. Chiou et al. (2006) reported earlier that treatment of trout macrophage cells (RTS11) with a linear cationic a-‐helical antimicrobial peptide (e.g., cecropin B) resulted in elevated expression of two pro-‐inflammatory relevant genes (IL-‐1b and COX-‐2). We hypothesized that, in addition to the direct antimicrobial effect of cecropin P1 in the disease resistant transgenic trout, this peptide may also increase the expression of immune relevant genes in the host. To confirm this hypothesis, de novo assembly of rainbow trout transcriptomes in spleen, liver, and kidney tissues of transgenic and non-‐transgenic fish was conducted by Illumina second generation RNA sequencing analysis. A total of 131,671 unigenes were obtained, and annotated using the Gene Ontology (GO) and the Nucleotide Basic Local Alignment Search Tool (BLAST-‐nr) programs. Over 2000 differentially expressed genes (DEGs) were determined by normalizing ratio of Reads Per Kilobase of transcript per Million mapped reads (RPKM) among transgenic and non-‐transgenic fish in a tissue specific manner. Enrichment analysis using Gene Ontology Biological Process (GO: BP) and KEGG pathway analysis were performed to illustrate the altered immune-‐related functions in each tissue. By comparing the expression levels of DEGs that show consistency among three tissues and DEGs related to specific immune-‐related functions of each tissue, we found that the expression of genes relevant to the innate immune system such as phagocytosis, lysosomal processing, complement activation, leukocyte migration, and antigen processing/presentation is perturbed. Furthermore, perturbation of genes in other functional groups of biological significance that might contribute indirectly to the host immunity was also observed. [This work was

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supported by a grant from U.S. Department of Agriculture (CONTR 58-‐1930-‐0-‐009) to T.T.C.] (25) Transcriptomic analysis in the intestine of IL-‐17A/F1-‐knockout medaka, Oryzias latipes. Hikima, J1*, Ikeda, D2, Izumi, M1, Nagaoka, M1, Morimoto, N1, Kono, T1, Sakai, M1, Takeyama, H3, Aoki, T4, Mizusawa N2, Watabe S2, Kinoshita M5. 1University of Miyazaki Faculty of Agriculture, 2Kitasato University School of Marine Biosciences, 3Waseda University School of Advanced Science and Engineering, 4Waseda University Research Organization for Nano and Life Innovation, 5University of Kyoto Graduate School of Agriculture. In mammals, interleukin (IL)-‐17A and IL-‐17F are hallmark inflammatory cytokines, which are expressed by Th17 cells, and play important roles in protection against infection and in intestinal mucosal immunity. Although fish IL-‐17A and IL-‐17F homologs named as IL-‐17A/F have been identified, their functional aspects are still poorly understood, especially in intestinal mucosal immunity. In this study, IL17A/F1-‐mutated (IL17AF1-‐KO-‐) medaka (Oryzias latipes) was established using CRISPR/Cas system, and a 7-‐bp deletion was confirmed in the IL-‐17A/F1 gene. To understand the roles of IL-‐17A/F in the mucosal tissues of fish, the transcriptomic analysis (RNA-‐Seq) on the intestine of IL17AF1-‐KO-‐medaka was performed using a MiSeq next generation sequencer. In the intestine of unstimulated IL17AF1-‐ KO-‐medaka, expression levels of the genes encoding C1q-‐like protein (c1ql2), peptideglycan-‐recognition protein 2 (pglyrp2), nonspecific cytotoxic cell receptor 1 (nccrp1), chymotrypsin-‐like protease (ctrl), and carboxypeptidase A (cpa) were significantly decreased compared to those of the wild-‐type medaka. Furthermore, in the intestine of IL17AF1-‐KO-‐medaka stimulated with lipopolysaccharide, expression levels of the genes encoding c-‐type lysozyme (lyz), NLRC3-‐like (nlrc3l), transferrin A (tfa), and carboxyl ester lipase (cel) were significantly lower than those of the wild-‐type medaka. These results suggest that IL-‐17A/F1 is involved in expression of antimicrobial peptides and digestive enzyme genes in the medaka intestine. (26) Structural and functional analyses of the lectins from the green algae Codium subtubulosum and C. latum. Hirayama, M*, Shimazaki, H, Hori, K. Graduate School of Biosphere Science, Hiroshima University. Several lectins from the green algae of genus Codium have been isolated. They are divided into 3 types with their primary structures: C. fragile agglutinin (CFA) type, C. latum agglutinin (CLA) type, and C. barbatum agglutinin (CBA) type. CFA type lectins belong to the H-‐type lectin family, which are found in snails, a soft coral, and a slime mold. CLA and CBA types do not show any similarity to

known lectins, but CLA contains a fascin domain in its molecule, which is found in fascin, an actin bundling protein. In this study, we performed functional analyses of CFAsub1, a CFA type lectin from C. subtubulosum, and CLA, using their active recombinants. In a detailed oligosaccharide binding assay using 26 pyridylaminated-‐oligosaccharides by a centrifugal ultrafiltration HPLC method, CFAsub1 strictly bound to b1-‐4 linked N-‐acetyl-‐D-‐galactosamine (GalNAc) on a glycolipid type oligosaccharide. The oligosaccharide binding preference of CFAsub1 differed from those of CFA and CPA, a CFA type lectin from C. pugniforme; CFA recognizes a1-‐3 linked GalNAc, and CPA does b1-‐3 and 1-‐4 linked GalNAc on glycolipid type oligosaccharides. Even from the same genus, CFA type lectins are functionally differentiated in their sugar-‐binding specificity. Meanwhile, actin binding assay using F-‐actin was performed for CLA. Interestingly, CLA bound to F-‐actin as well as fascin. We considered, however, that the biological function of CLA was different from that of fascin for the following reasons; fascin did not show hemagglutination activity; hemagglutination activity of CLA was inhibited with glycoproteins; a cDNA encoding CLA contains a signal peptide-‐coding region whereas fascin cDNA does not; CLA consists of a single fascin domain while fascin does of 4 domains. (27) Assessment of the genetic diversity in the broodstock and development of the growth-‐related markers from the transcriptome for marker-‐assisted selection in giant grouper, Epinephelus lanceolatus. Huang, CW1,2*, Gong HY1,2, Chou HY1,2, Yeh SL3, Tai KT4. 1Department of Aquaculture, National Taiwan Ocean University, Taiwan, 2Center of Excellence for the Oceans, National Taiwan Ocean University, Taiwan, 3Mariculture Research Center, Fisheries Research Institute, COA, Taiwan, 4Long Diann Marine Bio Technology Co., Ltd. The giant grouper (Epinephelus lanceolatus) is a commercially important fish species. Currently, selective breeding for the genetic improvement of quality-‐based grouper is an urgent task in Southeast Asia. Genetics and broodstock management programs of molecular marker-‐assisted selection and breeding are important for promoting improved varieties of seeds. The purpose of this study is to develop growth-‐related quantitative trait loci by genomic DNA-‐based method to explore the correlation between genotype marker and growth trait using Radio-‐Frequency IDentification (RFID) technology to build broodstock database, supplied with selection breeding of giant grouper. A total of 610 alleles at 22 microsatellite loci were detected in 536 individuals from five different populations. The expected heterozygosity varied from 0.41 ± 0.18 to 0.6 ± 0.26 with a mean value of 0.54 ± 0.22, and the observed heterozygosity varied from

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0.72 ± 0.21 to 0.78 ± 0.14 with a mean value of 0.75 ± 0.16. The allelic richness and heterozygosity studies revealed that the genetic diversity of the broodstock population was not significantly different when compared to that of the wild population. Seven functional SSR markers based on RNA-‐seq data and associated significantly with the body weight (P < 0.05) were identified in 175 fish from two sibling groups. In addition, we are screening polymorphic microsatellite DNA markers associated with disease resistance of giant grouper from 164 SSRs in 126 functional genes related to immune cell activation, including B cell, T cell, NK cell, and mast cell activation for MAS of giant grouper with both disease resistance and fast growth, by multiple DNA markers of two economic traits. Our results will be useful to the grouper industry for a scientific management of the complete traceability and branding certification and to create a new approach for increasing productivity and providing a competitive advantage in the future. (28) Improvement of omega-‐3 polyunsaturated fatty acid biosynthesis and cold tolerance in zebrafish and tilapia by transgenesis. Shin-‐Jie Huang1*, Ching-‐Yu Huang2, Hong-‐Yi Gong3, Jyh-‐Yih Chen2, Chuian-‐Fu Ken4, Jen-‐Leih Wu1,2. 1Institute of Fisheries Science, National Taiwan University, Taiwan, 2Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan, 3Department of Aquaculture, National Taiwan Ocean University, 4Department of Biology, National Changhua University of Education. In this work, we investigated the effects of the single, double and multiple enzyme complex combinations on accumulating the high value omega-‐3 PUFA synthesis by liver specific promoter using the conventional Delta 6-‐pathway. Cold tolerance ability and omics profiles to evaluate the gene expression change were investigated. Until now, double enzyme complex on omega-‐3 PUFA synthesis has not been thoroughly studied. This was achieved by the generation of transgenic zebrafish with strong liver-‐specific expression of Fadsd5, Fadsd6, Fadsd4 and Elvol5a (driven by the zebrafish fabp10 promoter). Then double transgenic fish D56, D6E and D64 were inbred to augment the endogenous fatty acid biosynthetic pathway. Similarly, triple transgenic fish D456 and D64E were generated by inbreeding. Results show double and triple gene transgenic zebrafish exhibited enhanced ability to synthesize EPA, DPA, and DHA than single gene fish. The D6E double transgenic zebrafish increased the EPA and DHA content to 2.43-‐ and 3.07-‐fold, respectively. Similarly, D56 double transgenic zebrafish increased the EPA and DHA content to 2.78-‐ and 4.05-‐fold, respectively. In tilapia, two single Fadsd5, Fadsd6 and one double transgenic tilapia expressing in both muscle and liver were

established. Those transgenic tilapias also exhibited the enhanced ability to cold stress tolerance and synthesize high level omega-‐3. Our findings revealed for the first time that combinations of conventional Delta-‐6 omega-‐3 PUFA critical enzymes cause’s alterations in omega 3 PUFA level and improve survival rates under cold stress. (29) The diversity of marine Methanogens; Isolated from marine gas hydrate bearing sediments. Imajo, T1*, Kobayashi, T1, Terahara, T1, Imada, C1, Matsumoto, R2. 1Tokyo University of Marine Science and Technology (TUMSAT), 2Gas Hydrate Research Laboratory, Meiji University. In the eastern margin of Japan Sea, shallow gas (methane) hydrates are widely distributed. In these area, bacteria mats and methane seeps are often observed. It is suggested that formation of bacteria mats has relationship with the methane related archaea. Methane related archaea is consisted of Methanogenic archaea (Methanogen) and Anaerobic Methanotrophic archaea (ANME). In these area, we had conducted environment monitoring survey “NT cruse” for three years, and sampled the sediment with/without bacteria mat. In order to know whether there are “living” or “activating” methanogens in those mats, we tried to isolate methanogen by anaerobic incubation. As the result, we successfully isolated several methanogens, including genus Methanogenium. Some of them are expected to be novel species. We suggest that there are specific methanogen structures related with gas hydrates or active methane seeps in Japan Sea. This study was conducted under the commission from AIST as a part of the methane hydrate research project funded by METI (the Ministry of Economy, Trade and Industry, Japan). (30) Comprehensive omics analysis reveals regulation mechanism of wax ester production in Euglena gracilis under anaerobic condition. Kimura, M1,2, Ogawa, T1,2, Maruta, T1,2, Arakawa, K3,4, Mori, M3,4, Ishikawa, T1,2*. 1Department of Life Science and Biotechnology, Faculty of Life and Environment Science, Shimane University, 2JST, CREST, 3Institute for Advanced Biosciences, Keio University, 4SFC Graduate School of Media and Governance, Keio University. A unicellular phytoflagellated protozoan, Euglena gracilis, has an ability to produce and accumulate wax esters, which have a great potential as an alternative biofuel, instead of triacylglycerol under oxygen-‐limited conditions. In spite of the importance and usefulness of Euglena, the regulation mechanism of wax ester synthesis in response to anaerobic condition is still largely unknown. In this study, we performed a comprehensive omics analysis

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including RNA-‐Seq and proteome to provide some insights into the regulation of wax ester metabolism under anaerobic conditions. RNA-‐Seq analysis indicated that wax ester production is regulated at post-‐transcriptional level rather than the transcriptional level in response to anaerobic conditions. Treatment of some inhibitors for protein kinase and phosphatase indicated that phosphorylation is a critical regulation factor for anaerobically wax ester production. Phospho proteome analysis showed that phosphorylation level in at least 487 proteins significantly altered in response to anaerobic condition. Among 26 protein kinases that are involved in the phospho proteome analysis, we identified that one kinase is critical factor for wax ester production. (31) Seaweed transgenics and biotechnology: key technology development of a stable transformation system and breeding strategy for biofuel production. Izumi, H1*, Uji, T2, Fukuda, S1, Mizuta, H2, Saga, N1. 1Institute for Food Sciences, Hirosaki University, 2Faculty of Fisheries Sciences, Hokkaido University. Marine macro-‐algae (so called seaweeds), which do not compete with food resources in the bio-‐industry, are regarded as promising candidates for biofuel resources. Almost biofuel production studies in seaweeds focus on natural sugar which can be fermented to produce biogas or alcohol-‐based fuels. In contrast, the studies for lipid as source of biofuel in seaweeds have been limited since their biomass has lower lipid content compared to land plants or microalgae. Biofuel production from microalgae has reached commercial level due to high photosynthetic efficiency and ability to produce lipids. Efficient production of seaweed lipid expands the availability of their biomass as a biofuel feedstock, thus establishment of efficient breeding is required in seaweeds. Genetic transformation is a powerful tool for a molecular breeding study. To date, genetic transformation of microalgae has succeeded in some species. In contrast, a stable transformation system has not yet been established in seaweeds although transient gene expression has been reported in a few seaweeds. Recently, we first succeeded the stable gene expression system in a model marine plant Pyropia yezoensis (Rhodophyta), which can synthesize fatty acids promising as biofuel (e.g. palmitic acid). As an initial step in establishing stable transformation of P. yezoensis, we developed a transient gene expression system using codon-‐optimized reporter genes. Moreover, we showed hygromycin-‐resistance gene is useful for isolating stable transformants as selection marker. Finally, we observed co-‐expression of reporter genes and selection marker in the nuclear transformation system of whole P. yezoensis. Here, we review key

technology development of the genetic transformation and breeding strategy in seaweeds. (32) Combinatorial pearl-‐associated two-‐protein system to study functional prioritization and hydrogel regulation. Jain, G1*, Pendola, M1, Gebauer, D2, Johnson, S3, Evans, JS1. 1Basic Science and Craniofacial Biology, College of Dentistry, New York University USA, 2Department of Chemistry, Physical Chemistry, Universität Konstanz, Germany, 3Department of Electronics, University of York, UK. Nacre of the pearl is composed of multilayered calcium carbonate structures interspersed with proteins. Several approaches have been developed to understand protein-‐associated mechanisms of pearl nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. We characterized the interaction of two pearl nacre-‐associated recombinant proteins, rPFMG1-‐rPFMG2 (Japanese oyster pearl nacre, P. fucata) in a proportionally defined combinatorial system invitro. Electron microscopy data revealed that both proteins when present in 1:1 ratio formed orthogonally arranged calcite crystals and were synergistic in their function in later stages of nucleation. However, these proteins were found to be not synergistic during earlier stages using a potentiometric titration assay. Further, protein-‐mineral hybrid crystals showed intracrystalline porosities while hybrid particles showed dimensional and film thickness attenuation on mica surfaces at 1:1 ratio using AFM. Both proteins together formed detectable multimers in rPFMG2: rPFMG1 ratio 2:2, 4:3, 6:4 using mass spectrometry which may suggest the control exerted by rPFMG2 in co-‐aggregation process. To investigate intermolecular interactions, we conducted QCM-‐D studies that showed that in absence of Ca(II), rPFMG1-‐rPFMG2 binding is of weaker affinity compared to when Ca(II) is present possibly due to Ca(II) invoked conformational changes in rPFMG1. Thus, we conclude that combining two proteins in 1:1 molar ratio results in the formation of a hybrid hydrogel phase that selectively regulates both stages of mineral nucleation. These processes may affect the formation of the composite that may ultimately modulate the fracture resistance, strength, and luster of the nacre of the pearl. (33) Induction of p53-‐independent apoptosis and G1 cell cycle arrest by fucoidan in HCT116 human colorectal carcinoma cells. Jeong, JW1*, Park, C2, Choi, YH1, 3. 1Anti-‐Aging Research Center, 2Department of Molecular Biology, College of Natural Sciences, Dongeui University, 3Department of Biochemistry, Dongeui University College of Korean Medicine, Busan, Republic of Korea.

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It is well known that fucoidan, a natural sulfated polysaccharide present in various brown algae, mediates anti-‐cancer effects through induction of cell cycle arrest and apoptosis. Nevertheless, the role of p53 as a tumor suppressor on the mechanism of action of fucoidan remains unclear. In this study, we investigated the anticancer effect of fucoidan on HCT116 human colorectal carcinoma cells with different p53 status. The results of this study showed that inhibition of cell viability and induction of apoptosis by treatment with fucoidan were similar in two p53 isogenic HCT116 cell lines (p53+/+ and p53−/− HCT116). Increased DNA-‐damage, reflected by phosphorylation of ɣH2AX proteins, was also coped by fucoidan treatment in both cell lines. Flow cytometric analysis revealed that fucoidan resulted in G1 arrest in cell cycle progression, which correlated with inhibition of phosphorylation of retinoblastoma protein (pRB) and concomitant association of pRB with the transcription factor E2Fs. Furthermore, treatment with fucoidan obviously up-‐regulated the expression of cyclin-‐dependent kinase (CDK) inhibitors, such as p21WAF1/CIP1 and p27KIP1, which was paralleled by an enhanced binding with CDK2 and CDK4. These events also commonly occurred in both cell lines, suggesting that fucoidan triggered G1 arrest and apoptosis in HCT116 cells by a p53-‐independent mechanism. Taken together, our results clearly demonstrate that fucoidan exhibits similar anti-‐cancer effects in HCT116 cells with different p53 status. Thus, given the fact that most tumors exhibit functional p53 inactivation, fucoidan could be a possible therapeutic option for cancer treatment regardless of its p53 status. (34) Heat shock proteins play roles in protection against AHPND-‐causing strain of Vibrio parahaemolyticus by induction of shrimp immune system. Junprung, W1*, Supungul, P2, Tassanakajon, A1. 1Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, 2National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA). Acute hepatopancreatic necrosis disease (AHPND) caused by the bacterium Vibrio parahaemolyticus carrying toxin producing plasmid (VPAHPND), has led to severe mortalities in farmed penaeid shrimp throughout Asia. Previous studies reported that a chronic non-‐lethal heat shock (chronic-‐NLHS) could enhance Penaeus vannamei to be tolerant to VPAHPND infection. The results showed that the shrimp exposed to chronic NLHS had higher survival rate (>50%) than that of the non-‐heated shrimp control (20%) when they were challenged with VPAHPND. Furthermore, the qRT-‐PCR analysis revealed that the expression of heat shock proteins, LvHSP70 and LvHSP90, as well as other

immune-‐related genes, LvproPO1 and LvCrustin1, were induced upon exposure of shrimp to chronic NLHS. Moreover, gene silencing of LvHSP70 and LvHSP90 eradicated the VPAHPND tolerance in the chronic NLHS shrimp and decreased PO activity. The recombinant LvHSP70 (rLvHSP70) was successfully overexpressed in E. coli system and dose dependent (0.1 µmol) of rLvHSP70 injection induced the expression of several immune-‐related genes (LvIKKε, LvIKKβ, LvCrustin1, LvPEN2, LvPEN4, LvproPO1, LvproPO2, and LvTG1) in the shrimp immune system. Interestingly, rLvHSP70 enhances P. vannamei resistance to VPAHPND infection increasing survival rate from 20% (control group) to >75%. These results suggested that LvHSP70 plays crucial roles in bacterial defense by activating shrimp immune system. (35) Octaphlorethol A regulates obesity through leptin signaling pathway. Kang, N1, *, Ahn, G1, Byun, KH2, Jeon, YJ3. 1Department of Marine Bio-‐Food Sciences, Chonnam National University, 2Functional Cellular Networks Laboratory, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, 3Department of Marine Life Sciences, Jeju National University. One potential causative factor in obesity is leptin resistance. Leptin, an adiopocyte-‐produced hormone, suppresses appetite and increases metabolic rate, however, its effects are diminished in the obese state. In this study, we explored a leptin substitute from marine natural products and investigated the anti-‐obesity effects of the natural product. To select the candidates among the marine natural products through leptin signaling pathway, in silico analysis was performed using crystal structure of leptin receptor (PDB ID: 3V6O). Among them, octaphlorethol A (OPA) derived from Ishige sinicola, brown algae founded along the coast of Jeju island Korea, favorably docked to the leptin receptor. OPA (0.1 μg/ml) stimulated leptin signaling pathway including STAT5 in hypothalamic N1 neuron cell line. To investigate the anti-‐obesity effects of OPA through leptin signaling pathway, OPA (0.25 mg/kg) was oral administrated to C57BL/6J obese mice fed with high fat diet during 4 weeks and leptin signaling pathway was analyzed in brain, white adipose tissues, liver and muscle. C57BL/6J obese mice treated with OPA reduced amount of food intake and body weight. Also, OPA stimulated leptin receptor and activated p-‐STAT5 in ARC of hypothalamus. Moreover, OPA activated the leptin signaling in all peripheral tissues including white adipose tissues, liver, and muscle and reduced the fat size, hepatic steatosis and regulated glucose metabolism. These results indicated that OPA, marine bioactive compound, regulates the obesity through leptin signaling pathway in both appetite control in central nervous system and energy homeostasis in

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peripheral nervous system in obese mice fed with high fat diet. (36) Production of volatile fatty acids from alginate by bacterial consortium derived from marine sediment. Kita, A1,3*, Miura, T1,3, Okamura, Y1,3, Aki, T1,3, Matsumura, Y2,3, Tajima, T1,3, Kato, J1, Nakashimada, Y1,3. 1Department of Molecular Biotechnology, Hiroshima University, 2Division of Energy and Environment Engineering, Hiroshima University, 3CREST JST. Brown algae are abundant in the ocean and drawing attentions as third generation biomass. Alginate is a major component of brown algae. Therefore, alginate is promising renewable carbon source and has recently been used as substrate for fermentation of ethanol, pyruvic acid and volatile fatty acids (VFAs). However, the full potential of alginate utilization has not yet been realized because industrial microbes such as Escherichia coli or yeast are not able to metabolize the alginate. VFAs are short chain fatty acids consisting of 6 or fewer carbon atoms and can be widely applied to bioplastics, biogas, biodiesel production and so on. Commercial production of VFAs has been mainly carried out by chemical synthesis, but interest in biological production is increasing due to soaring oil prices and the like. In this study, we succeeded in obtaining halotolerant alginate degrading bacterial consortium from the marine sediment in Japan. The bacterial consortium mainly produced acetate and formate from alginate as a sole carbon source. Therefore, VFAs production from alginate by semicontinuous culture using bioreactor was attempted. (37) Development of technique for parallel single cell genome amplification of bacteria and sequence read cleaning for de novo assembly. Kogawa, M1,2*, Nishikawa, Y1, Mori, K2, Hosokawa, M3,4, Takeyama, H1,2,3. 1Department of Life Science and Medical Bioscience, Waseda University, 2CBBD-‐OIL, AIST, 3Research Organization for Nano & Life Innovation, Waseda University, 4PRESTO, JST. Single cell genomics is a powerful tool to reveal genomes of environmental bacteria which are mostly uncultivable. However, there are difficulties to acquire accurate genome information from amplified single-‐cell genome. One is the random amplification bias and the other is the chimeric reads. The uneven and inaccurate amplicons may degrade the quality of de novo assembly. We have developed the droplet-‐based single-‐cell genome amplification method. Approximately 10,000 single cells are encapsulated into microdroplets per minute, and their genomes are amplified in parallel. Using this method, multiple single-‐cell genome data were simultaneously acquired. In this study, we developed a novel analysis

method for cleaning of sequence reads by cross-‐reference of single-‐cell data. The chimeric or contaminated sequences are removed by comparison of multiple single-‐cell data. In this approach, multiple single-‐cell genome data obtained by droplet-‐based single-‐cell genome amplification showed only small contamination. Then, sequence reads were cleaned by cross-‐reference analysis and resulted in significant improvement of quality in de novo assembly. Accuracy of the single cell genome reached at the same level of that obtained from the cultured bacteria DNA. Droplet-‐based single cell genome analysis has been demonstrated to be a great tool for elucidating the function of microbes in environments. (38) Candidate markers for phagocytic hemocytes of kuruma shrimp Marsupenaeus japonicus identified by transcriptome analysis. Koiwai, K*, Kondo, H, Hirono, I. Tokyo University of Marine Science and Technology. Invertebrate hemocytes play important roles in innate immune responses. Conventionally, hemocytes of kuruma shrimp are characterized based on morphology into several sub-‐populations, and it has been considered different sub-‐populations function differently. Up to now, however, the hemocyte characterization have been performed based on its morphology by microscopic observation. Therefore, establishment of molecular markers to distinguish hemocytes is required for the further immunological studies in kuruma shrimp. In this study, we isolated phagocytic hemocytes of kuruma shrimp by magnetic microbeads, and the candidate markers for phagocytic hemocytes were identified by RNA-‐seq. To collect phagocytic hemocytes, shrimps were injected with 1-‐2 µm diameter magnetic microbeads. After collection of total hemocytes from that shrimp, phagocytic hemocytes which engulfed magnetic microbeads were isolated by magnetic force. Total RNA were extracted and cDNA libraries were constructed from both total and isolated hemocytes, and then RNA-‐seq was performed by Illumina Miseq. After de novo assembly by Trinity, 4-‐fold differentially expressed transcripts between total and isolated hemocytes were extracted as candidate markers for phagocytic hemocytes. qPCR analysis was conducted to validate the result of RNA-‐seq. The ratio of phagocytic hemocytes was increased from about 10% to about 70% by magnetic isolation. Eleven (11) differentially expressed transcripts were identified as candidate markers for phagocytic hemocytes, and whose expression were also confirmed through qPCR analysis. These 11 transcripts might potentially be used as markers for phagocytic hemocytes. (39) NGS analysis of cytokine gene expression profiles revealed the changes of absolute mRNA levels after

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polyI:C treatment in Japanese flounder Paralichthys olivaceus. Kondo, Hd*, Yoshikawa, T, Nozaki R, Hirono, I. Tokyo University of Marine Science and Technology. Cytokines function coordinately to regulate immune system in vertebrates. In fish, a number of cytokines have been identified and their expression profiles after various stimulation have been investigated. However, these analyses were normally performed by comparing between samples, and do not represent exact amount of the transcripts in the sample. Recently the whole genome sequence of Japanese flounder was published and a number of transcripts have been identified. Although the information is still preliminary, it paves the way for the understanding of the fish immune system. By using the genome data with our transcriptome data, we identified 64 cytokines, including 4 type I interferons (type I IFN), 25 chemokines, and many others, in Japanese flounder. In order to show how the mRNA levels of these cytokines changes after injection of polyI:C, a dsRNA analogue mimicking viral infection, differentially expressed gene (DEG) analysis using transcriptome data were conducted. The kidney and spleen were isolated from the fish injected with 100 μg polyI:C at 3 and 24 hours post injection (hpi). The normalized read number (TMM values) of 32 cytokines were increased after the injection. The TMM value of a type I IFN was significantly high at 3 hpi in the kidney and spleen, although the other type I IFNs were very low to nothing. On the other hand, the TMM values of certain chemokines also increased at 3 hpi and were 10~20 times higher than those of the type I IFN at 3 hpi. These results may reflect the number of cells producing these cytokines, and provide more detailed information on how the cytokines work together. (40) Genome editing of marine diatom Fistulifera solaris JPCC DA0580 using CRISPR/Cas9 system. Kushiyama, N1*, Nojima, D1, Matsumoto, M2, Yoshino, T1, Tanaka, T1. 1Institute of Engineering, Tokyo University of Agriculture and Technology, 2Electric Power Development CO, Ltd. Microalgal oil has been recognized as valuable source of biofuels and high-‐value substances. We have investigated microalgal oil production using the marine oleaginous diatom Fistulifera solaris JPCC DA0580. This strain was selected as the highest oil producer among the microalgae in our culture collections. The whole genome sequencing and comprehensive transcriptome analysis of this strain were completed so far. Furthermore, several transformants of F. solaris were successfully created by microparticle bombardment to enhance the oil productivity. However, the established methodology was based on random insertion of exogenous genes into the genome. Therefore, establishment of specific gene manipulation techniques is required to further improve

the performance of F. solaris. In this study, the CRISPR/Cas9 system, which is one of the genome editing tools for highly specific gene manipulation, was employed for the transformation of F. solaris. The genome editing technique for microalgae has been successfully established in several species, such as Chlamydomonas reinhardtii, Nannochloropsis oceanica, Phaeodactylum tricornutum and Thalassiosira pseudonana. The proof-‐of-‐concept study was performed by the knockout of the gfp gene in a GFP-‐expressing strain of F. solaris. The plasmid containing the Cas9 gene and gRNA targettingthe gfp gene was transferred into GFP-‐expressing cells by microparticle bombardment. As a result, no GFP fluorescence was observed in the resulting transformant by fluorescence microscopy. Western blotting analysis showed successful expression of Cas9 protein in the transformant of F. solaris. Furthermore, high resolution melting analysis and Sanger-‐sequencing revealed the deletion of gfp gene at the target site. These results indicate that the CRISPR/Cas9 system can be utilized for the specific gene manipulation in F. solaris. (41) Molecular analysis of growth-‐related genes using SPARC-‐immune-‐inhibition model in orange-‐spotted grouper (Epinephelus coioides). Lin, CF1,3*, Chen, YT1,3, Chen, TY1,2,3,4. 1Institute of Biotechnology, 2Department of Biotechnology and Bioindustry Sciences, 3Translational Center for Marine Biotechnology, 4Agriculture Biotechnology Research Center, National Cheng Kung University, Tainan, Taiwan. The development of molecular technology has helped to study the cellular mechanism in model organisms, but the studies of molecular mechanisms correlated with physiological properties on commercial species is still difficult. Groupers are economically valuable fishes in marine aquaculture. Here we established a model with knockdown of specific gene using immune-‐inhibition technology to study the growth-‐related genes in grouper. SPARC (secreted protein acidic and rich in cysteine) is a secreted glycoprotein which regulates interactions between cells and extracellular matrix. Previous researches reported SPARC is major in adipose tissue, where it has a role in adipocyte differentiation and adipogenesis. In this study, we used a SPARC-‐inhibited grouper to investigate the function of grouper SPARC. The tissue section analysis of abdominal and dorsal meat showed that fat distribution area was larger in the SPARC-‐inhibited grouper. Moreover, we found that the notable weight gain in the SPARC-‐inhibited grouper, which is similar to that in myostatin-‐inhibited grouper. Myostatin, a member of the TGF-‐β superfamily, is a negative regulator for skeletal muscle growth. We proposed that SPARC may interact with myostatin to regulate smad

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signaling pathway involved in cell growth. Here, we identified smad2, smad3, smad4 and smad7, transcripts in orange-‐spotted grouper. The phylogenetic analysis revealed the significant homologies to the other species and reflected the structure similarity of grouper smads. Tissue distribution of smad genes analyzed by qPCR showed the smad genes were detectable across the tissues. We also investigated the smads expression level in muscle tissue of SPARC-‐inhibited grouper, the results indicated elevated gene expression levels of smad7. These data implied the immune-‐inhibited model would be applied for further in vivo physiological function studies. (42) Effects of four physical environment factors on the movement and feeding behavior of sea cucumber Apostichopus japonicus. Lin, CG*, Zhang LB, Yang HS. Institute of Oceanology, Chinese Academy of Sciences. Sea cucumber (Apostichopus japonicus) is the largest aquaculture species in China by output value. It is crucial to solve the problems, such as choosing the bottom-‐sowing spot, high risk and low efficient of diving capture, to study for the characteristics of A.japonicus in behavioral ecology. We observed and recorded the effects of four physical factors on the movement and feeding behaviors, as well as on the habitat selection, growth of A.japonicus. The main results are as follows: 1) Magnets have a significant attractive effect to A.japonicus in experimental conditions. The mean attractive rate of large size A.japonicus in 800mT group was significantly higher than that in 0.05mT (P<0.05). However, after being tested in natural sea area for two months, the self-‐developed magnet trap device for sea cucumber did not show a significant attractive effect on capturing A.japonicus. 2) Low-‐frequency (100 Hz) sound waves attracted medium and small A.japonicus (<10 g/ind.), whereas high-‐frequency sound waves (10000 and 28000 Hz) repelled all sizes of A.japonicus. 3) With the help of the self-‐developed device, the pressure of water within the depth of 50m can be simulated indoors. A.japonicus can adjust to the pressure within the depth of 0-‐50m, by showing themselves to move and feed normally without significant physical damage. 4) With the slow flow (~5 cm/s), A.japonicus moved more distance than in the still water, and hardly moved in the riptide (~30 cm/s). And A.japonicus were able to attach the bottom after any attached time in the slow flow, after 10 s in the medium flow (~15 cm/s) and after 60 s in the riptide (~30 cm/s). (43) Comparative genomics of chloroplasts and mitochondria in brown algae. Feng Liu*. Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, P. R. China.

The Phaeophyceae (brown algae) are multicellular photosynthetic marine organisms and display great morphological and physiological diversity. After their own independent evolution for more than 200 million years, the current brown algal group consists of a multitude of taxa including 19 orders, 62 families, 473 genera, and more than 2000 species. However, the data on their chloroplast and mitochondrial genomes are limited so far. The known brown algal chloroplast genomes are 124.1-‐140.0 kb in size, and contain 173-‐185 genes including 6 rRNA, 28-‐31 tRNA, and 139-‐148 protein-‐coding genes (PCGs), and appear to be highly rearranged in genome architectures among the different orders but be highly conserved in order Fucales and Laminariales. Brown algal chloroplast genomes contain multiple small inverted repeats (SIRs) and tandem repeats (TRs). The mitogenome sizes of brown algae are 31.6-‐58.5 kb, and harbor 65-‐79 genes including 3 rRNA, 24-‐26 tRNA, and 37-‐52 PCGs. The mitogenome organization in order Ectocarpales, Laminariales, Desmarestiales, and Fucales (ELDF) has high similarity only varying in ORF number and one or two tRNA position, which are apparently different from that in Dictyotales representing a more ancestral brown algal lineage. The total spacer size is positively correlated with brown algal genome size. The chloroplast and mitochondrial genomes obtained provide important information for us to understand plastid and mitochondria evolution as well as phylogeny in brown algae. (44) Evaluate the effectiveness of interleukin-‐6 and interferon-‐γ as vaccine additive in Epinephelus coioides. Liu, YL1*, Chen HH1, Kuo WC1, Lin HY1,2. 1Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan, 2Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan. Epinephelus coioides is an economically important farmed fish in Asia. In the recent year, high density aquaculture has increased the incidence of diseases hence vaccine with higher efficiency is required to prevent disease outbreak. Although vaccine application in grouper is vastly studied, little is known about grouper specific immune responses. Theoretically, an ideal immune protection was mainly contributed by specific cytotoxicity or antibody. For example, more specific antibody is required to neutralize soluble antigen or toxin; and stronger specific cytotoxicity is needed to eliminate virus or intracellular pathogens. The direction of specific immunity is controlled by the specific cytokines such as interleukin-‐6 and interferon-‐γ. Therefore, the additive of specific cytokines in vaccine might help to induce an optimized specific immune reaction and enhance the efficacy of vaccine. To prove this hypothesis, grouper TH1 and TH2 differentiation cytokine, interferon-‐γ and interleukin-‐6, were selected as

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vaccine additive candidates and ELISA and flow cytometry assay were employed to evaluate the effectiveness of specific antibody secretion and T lymphocyte proliferation triggered by these cytokines. Results showed that in groupers, interleukin-‐6 activates specific antibody production and helper T cell proliferation whereas interferon-‐γ induces cytotoxic T cell proliferation. It suggested that interleukin-‐6 and interferon-‐ γ successfully alter the direction of specific immunity. Furthermore, in the challenge trial, an increased survival rate was observed in the groupers under the treatment of vaccine combined with cytokine additives. These results indicated that the direction of immune response can be controlled by the additive of different cytokines and these cytokine additives can further increase the efficacy of fish vaccine. (45) Science to the rescue: interventions that help shrimp in the arms race against WSSV. Lo, CF1,2*, Chen, IT2. 1Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, 2Center for Shrimp Disease Control and Genetic Improvement, National Cheng Kung University. White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD) is a very unique with over 90% of the WSSV ORFs that show no significant similarity to other known proteins. WSSV also has a very broad host range, and it has a lot of anti-‐host defense strategies. It is always a losing battle for shrimp infected with WSSV. When WSSV infects its crustacean host: host defenses such can be circumvented; defense proteins can even be co-‐opted by virus genes; the host can eventually be overwhelmed. WSD is a disease that is very difficult to control. Our laboratory is always seeking new knowledge to prevent or minimize effects of WSSV on shrimp. One intervention strategy currently being explored by us is genetic improvement of shrimp. We applied a systems-‐biology approach to elucidate critical host factors for WSSV infection and replication, and we used these genes to develop anti-‐WSSV genetic markers. Subsequently, these markers were used to develop a screening platform for shrimp (farmed or wild) with resistance to WSSV. Fortuitously, we have already identified four families of shrimp that are resistant to WSSV infection. When these shrimp were challenged with virus, the WSSV-‐resistant shrimp all survived, and no virus genomic DNA copies were present in the shrimp. In contrast, all susceptible shrimp died (with a high virus load), confirming the pathogenicity of the virus used. Building on this achievement, the next step will be to develop shrimp lines that are both disease resistant and fast growing for the use of shrimp industry.

(46) New strategies for grouper industry in nervous necrosis virus prevention. Ming-‐Wei Lu*. Department of Aquaculture, National Taiwan Ocean University, Taiwan. Nervous Necrosis Virus (NNV) is an aquatic virus that can infect more than 30 species including the grouper, which is a valuable fish species in Taiwan. NNV causes up to 90-‐100% mortality in the aquaculture industry and that results in great economic loss in Taiwan grouper culture industry. In present, the anti-‐NNV vaccines such as inactivated vaccines, subunit vaccines and recombinant vaccines which rely on adaptive immunity can only be applied to 30-‐days-‐post-‐hatching grouper larvae. However, the NNV acute infection is before adaptive immunity development. By using small interfering RNA (siRNA), specific degradation of viral mRNA could suppress the replication of virus effectively. The plasmid contained NNV RNA2 and EGFP reporter gene was co-‐transfected with siRNA plasmid into cells to examine the efficiency of siRNA. In previous study, we constructed the vector containing siRNA sequence against NNV and verified that it has effective suppression to the replication of NNV either in vitro or in vivo. Moreover, the successful breeding rate of grouper larvae was increased to 27-‐40% after feeding with RNAi vaccine in our field trial compared to 2-‐5% in traditional grouper aquaculture. By large scale experiment, we also tried to feed our siRNA to the grouper in outbreak farm. The survival rate also significantly increased from 10% to 50-‐75%. These techniques were already developed and suitable for grouper industry in the future. (47) IoT and biotechnology for development of precision aquaculture. Lu, J-‐K*. Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan,ROC. With the rapid global changes in the 21st century, aquaculture should be transformed into a knowledge-‐based, environment-‐friendly, high-‐tech industry in order to remain competitive and sustainable. In this paper we focus on employing biotechnology and IoT technology strategies to revolutionize aquaculture. 1) Advanced monitoring of aquaculture environment by IoT technology-‐ IoT+aquaculture system means extensive measuring, monitoring and control. Using ICT and IoT technology the operator has a full overview of water quality parameters such as dissolved oxygen, temperature, pH, salinity, NH3 etc. are constantly monitored. Early warning system and automatic control water quality conditioning equipment such as pumps, blowers, UV systems, pH and water temperature adjuster are included. 2) Automatic species recognition, weight determination and precise feeding

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system-‐the collection of biological data on individual length and weight of specimen has always been an important part of aquaculturists. We demonstrated that using a vision-‐based catch registration system can automatically recognize fish species and measure the body length and then converts to body weight. The concept of precision feeding system is using highly digestible feed sources in a controlled feeding environment with an emphasis on feed efficiency, reducing daily ration costs, and minimizing feces output. Different time-‐automatic feeder designs are available that can be programmed to feed hourly for extended periods. Through PLC or WNS to activate a peristaltic pump and solenoid valves for food distribution into the tanks. Utilized a submersible camera and image recognize analysis tools to determine the food quantity provided and intake. Using this system, the main benefits are saving food (reducing the feed conversion rate) and manpower, diminishing water pollution, thus avoiding economic loss. 3) Tele-‐diagnosis system for fish diseases -‐Fish disease is a serious problem due to its ability to spread rapidly through water to neighboring aqua-‐farms. Therefore, rapid and accurate diagnosis is required to control such diseases, prevent their spread and limit excessive use of antibiotics. To overcome traditional diagnosis limitations, IoT technology and a clinical sign-‐based diagnosis aid system is applied by sending infected animals images, text description through mobile phone to the diagnosis center. To implement the designed diagnosis process, fish disease database and disease cause/prevention/treatment database were established. Candidate disease by comparing the observed and selected clinical signs to the information on the remote database. Information such as cause, diagnosis, treatment and prevention method of candidate disease are provided as a medical advice through internet to user PC or mobile devices. This system will support fish farmers and veterinarians by provide easy and rapid diagnosis of fish diseases. Since ICT and IoT technology are used to collect information from farmers, biotechnology can provide the solution for the problems of commercial fish farming. Therefore, We have used biotechnology techniques to exploit water treatment agents, functional feed additives, molecular diagnostic kits, vaccine etc. (48) Biased contribution of the homoeologous subgenomes to lipid metabolisms in the marine allodiploid diatom Fistulifera solaris. Maeda, Y1*, Nomaguchi, T2, Yoshino, T1, Asahi, T2, Tanaka, T1. 1Institute of Engineering, Tokyo University of Agriculture and Technology, 2Graduate School of Advanced Science and Engineering, Waseda University.

Allopolyploidization is prevalent in botanical evolution. Allopolyploid organisms contain two or more genomes (termed subgenomes) derived from divergent progenitors, and frequently exhibit advantageous characteristics for agricultural and industrial use (e.g. broad environmental adaptability and high yield). Analysis of allopolyploid crop plants have suggested that each subgenome could asymmetrically contribute to transcriptome, and this biased contribution might be related to their advantageous characteristics. In contrast to terrestrial plants, allopolyploid microalgae have almost never reported despite their enormous diversity. As a rare example, we discovered the allodiploid diatom, Fistulifera solaris, which is a promising host for biofuel production owing to their high lipid content and growth rate. Whole genome analysis for F. solaris suggested that each subgenomes containing 9007 homoeologous gene pairs syntenically co-‐exist in this diatom. However, classification of these homoeologous genes into individual subgenomes remained to be determined because of the lack of reference genomic information of potential progenitor diatoms; it hampered the analysis of the biased contribution of each subgenome to transcriptome and the resulting metabolisms. In this study, we attempted to classify the homoeologous genes of F. solaris into 2 subgenomes without any reference genome information. We compared the relative content ratio of guanine and cytosine in genes (dGC) and codon usage preference between the homoeologous gene pairs. As a result, classifications based on dGC and codon usage preference were perfectly consistent. In general, both subgenomes (termed Fso_l and Fso_h with lower and higher dGC, respectively) tend to evenly contribute to global transcriptome, whereas Fso_l and Fso_h showed relatively higher contribution to lipid synthesis and degradation pathways, respectively. This result suggest that there was the biased contribution of the subgenomes to particular metabolic pathways in F. solaris. This outcome could provide insights into stable and highly efficient biofuel production using F. solaris. (49) Green oil production by mesophilic and cold tolerant marine oleaginous diatom adapt to seasonal condition in Japan. Matsumoto, M*. Electric Power Development CO, Ltd. “The GREEN OIL” created by microalgae, get worldwide interest as friendly production methodology for sustainability of environment. Because of these organisms can produce many of useful material from CO2 and solar energy such as high value and biofuel. The mesophilic and oleaginous marine diatom Fistulifera solaris JPCC DA0580 and cold tolerant and oleaginous marine diatom Mayamaea sp. JPCC CTDA0820 were

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isolated from Japan coastal line. F.solaris JPCC 0580 and Mayamaea sp. JPCC CTDA0820 are high neutral lipid producer up to 60wt% of 7~10days of cultivation under 25 °C or 10°C in laboratory condition. We installed the test facility of total process for green oil production in kitakyushu. (Facility area is 7,000m2, including mass culture, harvesting/dewatering and oil extraction process). Outdoor culture of both strains were have performed by D=5m(10,000L), D=10m(40,000L), D=20m(160,000L) and D=40m(640,000L) through the year since 2014, and then investigated biomass and oil productivity. We will here discuss about capability of large scale outdoor culture by F.solaris JPCC 0580 and Mayamaea sp. JPCC CTDA0820 obtained from our results.

(50) Process evaluation of macroalgae utilization system. Matsumura, Y*, Aki, T, Nakashimada, Y, Okamura, Y, Tajima, T. Hiroshima University. Macroalgae is easy to cultivate and harvest compared to microalgae, but its utilization is not always economic and energy productive. When macroalgae is used only for energy source, the cost of energy production is not competitive with the fossil fuel. When macroalgae is used only for value-‐added product, the process is energy consuming. To answer this dilemma, we proposed a process where macroalgae is first hydrothermally treated, and then methane fermentation and fermentation for value-‐added oil is made for the hydrothermally pretreated macroalgae. The former produces energy while the latter produces value-‐added product so that the total process can be energy producing and economically feasible. The remaining organics after these treatment was further converted into low molecular weight oils and removed. The objective of this study was to evaluate this proposed process from the viewpoint of energy and economy. Mass balance and energy balance for the process were developed based on the experimental results, and yield of the products as well as energy efficiency were determined. Using the heating value of product biogas and unit price of value-‐added oil product, the process evaluation was made. It was shown that the process was energy productive when all the feedstock was used for methane fermentation, but economic feasibility depended on the price of value-‐added oil. This result shows the effectiveness of hybridized process, or biorefinery, for utilization of macroalgae so that the process can be both energy producing and economically feasible. (51) Production of biofuel and useful material using marine microalgae. Matsunaga, T1,2*. 1Tokyo University of Agriculture and Technology, 2Waseda University. Vast marine environments support extremely diverse microorganisms. Scientists have long studied a number of

marine microorganisms that synthesize useful materials for various purposes including food and feed production, biofuel and bioenergy generation, and drug development. Among such microorganisms, marine microalgae have been recognized as key subjects for biotechnology researches owing to their diversity, CO2 fixation capacity through photosynthesis, and high biomass productivity. A variety of prokaryotic (cyanobacteria) and eukaryotic microalgae have been identified as promising candidate hosts for useful material production, and some of them are now commercially available. However, considerable efforts still need to be devoted for industrial success in microalgal biotechnology, because it is essential for the identified microalgae to be sustainable, scalable, and affordable sources for target compounds. To achieve this goal, we need to take broad cross-‐sectoral approaches including marine biology, microbiology, biotechnology, genomics, bioinformatics, and chemical engineering. CREST (Core Research for Evolutional Science and Technology) and PRESTO (Precursory Research for Embryonic Science and Technology) projects supported by JST (Japan Science and Technology Agency) are the ideal research platforms to promote such interdisciplinary collaborations in the field of microalgal biotechnology. Multiple CREST and PRESTO researchers actively and synergistically collaborated, and created breakthroughs for the last several years. In this lecture, I will introduce some epoch-‐making achievements that were obtained in the CREST and PRESTO projects. Microalgal production of biofuels (e.g., bioalcohol, biodiesel, and biohydrogen) and other useful materials (e.g., bioplastic) was intensively studied by means of multi-‐omics analyses and metabolic engineering. These achievements will pave the way for sustainable development of our society supported by marine microalgal biotechnology. (52) The Philippine Mollusk Symbiont ICBG: Integrating biology and chemistry for drug discovery. Miller, BW1*, Rosenberg, G2, Distel, D3, Schmidt, E1, Concepcion, GP4, Haygood, MG1. 1University of Utah Department of Medicinal Chemistry, 2Academy of Natural Sciences of Drexel University, 3Ocean Genome Legacy of Northeastern University, 4Marine Science Institute, University of the Philippines. The Philippine Mollusk Symbiont International Cooperative Biodiversity Group (PMS-‐ICBG) is a collaborative project dedicated to exploring the marine environment of the Philippines for bioactive secondary metabolites with applications in drug discovery. Mollusks are among the most biodiverse groups of eukaryotic organisms in the marine environment, and the diversity of associated microbial communities is equally vast, though largely unexplored for novel chemical entities.

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Bioinformatic analysis of these associated microbes has revealed an enormous capacity for secondary metabolite production, including species with upwards of 18 different biosynthetic gene clusters in the NRPS, PKS, terpene, or hybrid families. The PMS-‐ICBG has thus focused on integrating the basic biology research of mollusk-‐bacterial symbiosis with chemical extraction and bioactivity screening for the discovery of compounds with antimicrobial, anti-‐cancer, anti-‐HIV, and neuroactive properties. This platform led to the recent description of an exceptional symbiosis between chemoautotrophic microbes and the giant teredinid bivalve Kuphus polythalamia, collected and studied in the Philippines. Furthermore, 163 compounds have been isolated from mollusk-‐associated bacteria isolated during this ongoing project, including over 40 new structures with wide-‐ranging bioactivities. (53) Continuous methane production from volatile fatty acids for the second stage of two-‐stage methanization of undiluted brown algae. Miura, T1,3*, Kita, A1,3, Okamura, Y1,3, Aki, T1,3, Matsumura, Y2,3, Tajima, T1,3, Kato, J1, Nakashimada, Y1,3. 1Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 2Division of Energy and Environmental Engineering, Institute of Engineering, Hiroshima University, 3CREST, JST. Marine macroalgae, which contain a large amount of water in addition to salts, should be methanized under undiluted conditions in order to keep organic loading rate (OLR) at a certain hydraulic retention time (HRT). So far, undiluted brown algae have been methanized semi-‐continuously by using fed-‐batch acclimated marine sediment-‐derived culture. However, high-‐rate methanization of brown algae remained to be achieved. In this study, a continuous upflow anaerobic silt blanket reactor was developed under high salinity for the second methanogenic stage of two-‐stage methanization of undiluted brown algae by using the acclimated culture. First, organic overloading was conducted by using increasing concentrations of volatile fatty acids (VFAs) ranging from 100 mM acetate and 30 mM propionate to 400 mM sodium acetate and 120 mM sodium propionate in the presence of 3% NaCl at low OLRs to examine limitation of VFAs concentrations. Organic overloading increased methane production rate except in the case where 400 mM acetate and 120 mM propionate were used and salinity reached near 5%. The pH of effluent increased from below 8.0 to 8.4 during the overloading, indicating that methanization of a high concentration of VFAs increased pH. Next, hydraulic overloading was conducted by using 200 mM acetate and 90 mM propionate to increase OLR. The overloading produced

methane even at OLR of 25 g COD/L/day and HRT of 0.77 day without decrease in methane yield. Therefore, the reactor can be used for high-‐rate methanization of brown algae. (54) Reflectins: A protein machine driving an osmotic motor to dynamically tune color in squids. Daniel E. Morse*. Institute for Collaborative Biotechnologies. University of California, Santa Barbara, CA, USA. Dramatic changes in the skin color of Doryteuthid squids are produced for camouflage and communication by tunable control of pigment-‐containing chromatophore cells and iridescent reflective cells called iridocytes. Accordion-‐like pleats or invaginations of the iridocyte cell membrane form multiple lamellae of a tunable Bragg reflector that is the source of the iridescence. These lamellae are filled with 4 different reflectins – members of a family of proteins thus far unique to various reflective tissues in molluscs. Contrary to a popular misconception, we find that the reflectins are not the source of the reflected color, but act as a molecular machine that drive an osmotic motor to dynamically control changes in the refractive index, thickness and spacing of the Bragg lamellae, tunably controlling the brightness and color of light reflected from these subcellular, membrane-‐bound structures. Hanlon and colleagues first showed that the squid iridocytes are enervated by cholinergic neurons that regulate iridescence via muscarinic acetylcholine receptors. We confirmed that finding and showed that these receptors activate a G protein-‐phospholipase-‐calcium/calmodulin-‐mediated signal transduction cascade that culminates in activation of a protein kinase to phosphorylate each of the reflectins in a site-‐specific manner, and that this phosphorylation is essential for the activation of reflectance and the progressive tuning of its color. In vitro analyses with the purified, recombinant reflectins have now revealed their mechanism of action – a puzzle that had remained for the decade since their discovery. The tunable reflectin proteins are transparent, carry no chromophores, and have no known homologs. Their structures contain multiple, canonical “repeat” motifs of previously unknown function, separated by linker peptides that are highly positively charged. As a result of their positive charge, when in the “off” state, the reflectins are intrinsically unstructured, with Coulombic repulsion preventing intra-‐chain condensation, secondary folding and hierarchical assembly. ACh-‐activated phosphorylation adds negative charges that neutralize the reflectins, overcoming this Coulombic repulsion to permit intra-‐chain condensation, secondary folding of the conserved domains, and subsequent hierarchical assembly

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(increasing the intra-‐lamellar refractive index and thus activating reflectance). This assembly masks the reflectins’ surface charges, driving a net diffusion of small counterions across the lamellar membrane, with the resulting osmotic imbalance “pulling” water from the lamellae by Gibbs-‐Donnan-‐mediated reversible dehydration (in a process we monitored with D2O) This dehydration shrinks the lamellae and their spacing, thus progressively tuning the reflected color from red to blue. In vitro analyses with the purified, recombinant reflectins reveal further details of this mechanism, showing by dynamic light scattering, spectroscopy, fluorimetry, TEM and computational simulations that neutralization-‐driven secondary folding of the conserved domains results in the emergence of previously cryptic, bifacially phase-‐segregated hydrophobic surfaces that act like molecular Velcro™ to drive hierarchical assembly of the reflectin monomers to form large multimers. Using titration with increasing pH to progressively drive charge-‐neutralization of the recombinant reflectins as an in vitro surrogate for the neurotransmitter-‐activated phosphorylation in vivo, and related analyses of charge-‐substituted genetically engineered mutants, reveal that the degree of charge-‐neutralization directly determines the size of the assembled multimers, and thus, the brightness and color of light reflected from the Bragg lamellae. This process proves to be readily reversible and repeatedly cyclable in vitro, as it is in vivo, with the dimensions of the reflectin assemblies in the “off” and activated states measured in vitro by dynamic light scattering and TEM agreeing closely with measurements in vivo. (55) The engineering of the green-‐light sensor protein CcaS derived from Synechocystis sp. PCC 6803. Nakajima, M1*, Ferri, S2,3, Rögner, M4, Sode, K1,3,5, 1Institute of Global Research Innovation, Tokyo University of Agriculture and Technology, 2Department of Applied Chemistry and Biochemical Engineering, Shizuoka University, 3JST, CREST, 4Plant Biochemistry, Faculty of Biology and Biotechnology, Ruhr-‐Universität Bochum, 5Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology. Recent advances in our understanding of photosensing in biological systems have led to the use of photoreceptors as tools regulating gene expression in bioprocess models. Light-‐regulated microbial bioprocesses using photoreceptors have been reported. Among photoreceptors, we focused on the application of the chromatic acclimation sensor, CcaS, from the Synechocystis sp. PCC 6803 to construct light-‐regulated gene expression system in marine cyanobacterium Synechocystis sp. NKBG 15041c1 for their future

application to the marine bioprocess. The wild-‐type CcaS is composed of two-‐component regulatory system and induces gene expression under green-‐light illumination and represses it under red-‐light illumination. However, in the regulation of gene expression by CcaS, the background expression level under non-‐inductive condition is not negligible. In this study, we attempted to engineer CcaS. We focused on the domain structure of CcaS, which consists of an N-‐terminal transmembrane helix; a GAF domain, which serves as the sensor domain; a linker region (L1); two PAS domains; a second linker region; and a C-‐terminal histidine kinase (HK) domain. Truncated versions of the photoreceptor, “miniaturized CcaSs”, were constructed by removing the L1 linker region and the two PAS domains, and fusing the GAF and HK domains with a truncated linker region. Then, Escherichia coli transformants harboring light sensing system with engineered CcaSs and RFP gene as reporter gene were constructed and cultivated under red or green light, and fluorescence intensity of RFP was measured for screening the functional sensors. As a result, thus constructing miniaturized CcaSs capable of high expression levels under inductive conditions and strict repression of gene expression under non-‐inductive conditions2. In addition, miniaturized CcaSs were grouped into four distinct categories according to their responses toward light illumination. References: 1. A. Badary et al., Mar Biotechnol (NY)., 17(3):245-‐51 (2015). 2. M. Nakajima et al., Sci Rep., 6:37595 (2016). (56) Dissolution of the methane fermentation residue of macroalgae in ionic liquids. Nakamura, N1,2*. 1Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2JST-‐CREST. Our group is working on the process of producing methane from macroalgae as raw material. Although the efficiency of methane fermentation has been optimized considerably, the methane fermentation residue still contains organic compounds. We hope to recover and use the organic compounds in the residue. However, it is very difficult to recover the organic compounds because they are not soluble in ordinary organic solvents. Therefore, we attempted recovery of the organic compounds from the methane fermentation residue using ionic liquids as solvent. Dissolution tests of the methane fermentation residue were performed using various ionic liquids. It was found that 1-‐ethyl-‐3-‐methylimidazolium methylphosphonate ([C2mim][(MeO)HPO2]) was suitable. The ionic liquid, [C2mim][(MeO)HPO2], is high polar, which is known by dissolving a poorly soluble polymer such as

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cellulose. In addition, onium hydroxides which are strong bases were also found to be effective for dissolving the residue. We will also report on producing electric energy directly from the solution. A platinum nanoparticle-‐modified electrode was prepared by the casting method. The catalytic current obtained using the prepared platinum nanoparticle-‐modified electrode was about 100 times larger than that of an ordinal platinum electrode. The organic compounds dissolved in the ionic liquids were oxidized with the platinum nanoparticle-‐modified electrode. Electric energy can be obtained by fabricating a fuel cell by combining this electrode as an anode with an appropriate cathode. (57) Integrated process development for biorefinery of seaweed. Nakashimada, Y*1,4, Aki, T1,4, Okamura, Y1,4, Matsumura, Y2,4, Nakamura, N3,4. 1Department of Molecular Biotechnology, 2Department of Mechanical System Engineering, Hiroshima University, 3Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 4CREST, JST. Seaweeds have attracted the attention as a promising candidate of a renewable feedstock because they can be produced with a similar or higher yield compared to terrestrial energy crops and are less resistant to degradation than terrestrial lignocellulosic feedstocks. However, to develop energetically, economically, and environmentally feasible process for biorefinery of seaweed, integrated research approach including not only deep-‐understanding and improvement of elemental technology but also the process optimization in consideration of industrial use is needed. Hence, we have jointly developed several key technologies for complete utilization of macroalgae such as efficient pretreatment, biofuel and valuable chemical production with zero-‐emission of hazardous materials. In the presentation, we briefly introduce several achievements on bioenergy production from macroalgae by using high-‐rate biogas (methane) production with marine methanogenic microbial consortium, high-‐value added chemicals production by co-‐culture system of marine protists, thraustochytrids, Aurantiochytrium and the algal saccharide-‐converting bacteria, removal of hazardous heavy metal and valuable rare metals absorbed on seaweed, and wastewater treatment of fermentation residue with further recovery of energy and valuable materials. Furthermore, it is discussed that the possibility of economic and green use of seaweed by combination with developed technologies.

(58) Whole genome sequence of Pacific abalone, Haliotis discus hannai. Nam, BH1*, Park, EH 1, Kim, YO1, Kim, DG1, Park, JY1, Shin, YH2, Kwak, W3. 1National Institute of Fisheries Science, 2Insilicogen Inc., 3C&K genomics, Republic of Korea. Pacific abalone (Haiotis discus hannai) is one of the most valuable marine aquatic species in Korea, Japan and China. During the past few years, a great loss of abalones has been reported due to several factors such as polluted environments, global climate change, and the occurrence of diseases that are caused by microbial pathogens. As one of projects to develop a breeding program for genetic improvement of abalone, whole genome and transcriptome sequencings have been carried out by NIFS. We generated a total of 580 Gb and assembled 1.87 Gb of draft genome into 80,032 scaffolds with an N50 of 201 Kb. Gene predictions identified 29,449 genes in total. Using comparative analysis, we gain insight into the large genome size of abalone and gene family expansion. In addition, we sequenced the transcriptome of normal and bacterial challenged abalone tissues. A total of 10, 575 transcripts exhibiting the differentially expression at least one pair of comparison and the functional annotations highlight genes related to immune response, cell adhesion, immune regulators and redox molecules. (59) Expression of biomineralization-‐related genes in Emiliania huxleyi at different calcium concentrations. Nam, O1*, Chang, KS2, Jin, ES1,2. 1Department of Life Science, Hanyang University, 2Research Institute for Natural Sciences, Hanyang University. Phytoplanktons are an enormous group of unicellular aquatic organisms which are speculated to be responsible for worlds carbon cycling. Among them coccolithophores are a class of algae producing minute calcium carbonate structure called coccoliths. Emiliania huxleyi is the most abundant coccolithophore in the ocean nowadays. In addition, E. huxleyi occasionally forms massive blooms at temperate latitudes. Coccoliths are produced one at a time in an intracellular structure known as coccolith-‐vesicle (CV). Molecular mechanisms underlying this tightly controlled biomineralization process is still relatively unclear. To elucidate the putative biomineralization-‐related genes, we have sort out the candidate genes from the preliminary experiments by comparing the transcriptome of calcifying-‐strain (CCMP 371) and non-‐calcifying strain (CCMP 2090). Based on the contigs of the transcriptome analysis, 10 contigs which were relatively highly expressed in the calcifying strain, compared to the non-‐calcifying strain, were selected. By blast search, 10 putative genes which were expected to be the best hits for the contig sequences were chosen for quantitative reverse transcriptase PCR (qRT-‐PCR). Furthermore, to

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verify the putative genes in the calcifying strain, we have cultured calcifying strain in different calcium concentration which could alter the coccolith production. At different calcium concentrations 2 putative genes showed distinct expression level between calcifying state (Ca2+ 10 mM) and non-‐calcifying state (Ca2+ 0 mM, 0.1 mM). In this study, we have presented biomineralization related genes to understand the molecular basis of the intensely interesting coccolithophore algae E. huxleyi. (60) Alteration of stomach microbiota of Pacific white shrimp (Litopenaeus vannamei) that succumbed to an outbreak of AHPND. Ng, TH1, 2*, Liu, PY3, Yu, HT3, Lo, CF2, Wang, HC1,2. 1Institute of Biotechnology, National Cheng Kung University, 2Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, 3Department of Life Sciences, National Taiwan University. Digestive tract microbial communities have recently become a topic of increasing interest, especially in terms of the host microbiota and pathogenic bacteria interactions. Microbial balance is believed to be important for human health and diseases. These studies reveal cause-‐and-‐effect relationships which suggest that the microbiome influences, not only colonization by pathogenic bacteria, but also affects their growth and enhances their virulence. Acute hepatopancreatic necrosis disease (AHPND) is an emerging (since 2009) disease in Southeast Asia. It is generally accepted that this disease is caused by Vibrio parahaemolyticus (VP) with extra-‐chromosomal elements producing a virulent toxin, causing damage to shrimp hepatopancreas. In the case of AHPND-‐causing VP strains which replicate in the shrimp stomach, whether stomach microbiome change during the course of AHPND, which bacterial taxa are affected and the role of the environmental microbiome in the shrimp stomach microbiome remains unaddressed. We applied a standardized approach to monitor shrimp grow-‐out pond from the 21st day after stocking with postlarvae that ultimately succumbed to an outbreak of AHPND. We collecting 10 shrimp samples per day, all of which were tested for disease markers individually. We used a culture-‐independent metagenomics approach with next generation sequencing technology (NGS) to characterize the AHPND-‐associated microbiome. Our multivariate analysis examined the individual microbiomes from 37 shrimp and 25 culture pond water samples and determined a total of 4576 OTUs. The samples were clustered into two groups on PCA, with a cutoff date at 21st Sept., whereas the taxonomic richness of bacteria was decreased at the late time points in disease marker positive shrimp. Instead of VP abundance was the critical factor for AHPND pathogenesis, there were also third party microbiota that were potential AHPND biomarkers.

Key microbial community contributions to differences in this study may be a critical factor in controlling AHPND. (61) Identification and functional analysis of oil body-‐associated proteins of oleaginous diatom Fistulifera solaris JPCC DA0580. Nonoyama, T1*, Nojima, D1, Maeda, Y1, Matsumoto, M2, Yoshino, T1, Tanaka, T1. 1Institute of Engineering, Tokyo University of Agriculture and Technology, 2Electric Power Development CO, Ltd. Microalgae store triacylglycerols in certain organelles, often called oil bodies, and the accumulated microalga oils have attract broad interests because of their potential use in biofuels. It has long been know that several types of proteins are associated with the surface of oil bodies, and these oil body-‐associated proteins likely play important roles in lipid metabolisms. Functions of these proteins are partly elucidated in animal cells and higher plant cells, whereas those in microalgae remain largely unknown. We have identified the marine oleaginous diatom, Fistulifera solaris JPCC DA0580 as a promising candidate for microalgal biofuel production. In our previous study, whole genome of F. solaris was sequenced, and it allowed us to perform the oil body-‐proteomics study, in which several oil body-‐associated proteins were identified. However, considerable contamination of the oil body fraction by other protein fractions hampered the comprehensive identification of the oil body-‐associated proteins, and thus functional insights into lipid metabolisms were limited. Here we attempted to prevent the contamination of the oil body fraction obtained from F. solaris in order to identify novel oil body-‐associated proteins for better understanding of oil body biology in diatoms. Appropriate washing methods were investigated, and the proteins in the purified oil body fraction were identified using nano-‐liquid chromatography-‐mass spectrometry (nanoLC-‐MS). As a result, 32 proteins were newly identified, and a lot of the identified proteins were likely related to membrane trafficking. The inhibitors of these membrane trafficking proteins significantly accelerated the oil accumulation, suggesting that they might be involved in the degradation of oil bodies in F. solaris. These outcomes could provide new insights into oil body dynamics, as well as new clues to enhance the oil productivity in diatoms. (62) Exploration of the hypoxia sensor in marine annelid, Capitella teleta. Tetsuya Ogino1,2*, Haruhiko Toyohara1. 1Graduate school of Agriculture, Kyoto University, 2JSPS Research fellow. Oxygen is the essential material for almost all animals on earth. Marine annelids acquired their niches in the bottom of the ocean, where they are often exposed to severe hypoxic condition. Many researches about adaptation of

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marine invertebrates to the hypoxic condition showed that they overcome it by promoting anaerobic metabolism and escaping from hypoxic water. However, it remains unknown what sensor is involved in the detection of lowering dissolved oxygen (DO) concentration in marine annelids. Recently, in mammal transient receptor potential channel A1 (TRPA1) were reported to detect the changes oxygen concentration and regulate rapid response to hypoxia, such as respiratory rate. We showed the possibility of involvement of TRPA1 on the response to hypoxia in Capitella teleta, Annelida species with tolerance to hypoxic condition. The full length of Trpa1 ortholog from C. teleta was acquired and the deduced amino acid sequence of this gene implied the possession of oxygen degradation domain, which was hydroxylated in normoxia but not in hypoxia. The whole mount in situ hybridization analysis showed Trpa1 gene was transcribed at the prostomium, peristomium and the posterior region. They feed and dig into mud sediments. As a result, their posterior region is initially exposed to surrounding water. Our results implied that TRPA1 in the prostomium has a role of orientation to a habitat comfortable in DO concentration and that in the posterior region functions as a sensor for environmental DO concentration. (63) Oil production from organic acids derived from marine algal biomass by Nitratireductor sp. OM-‐1. Okamura, Y1,2*, Nakai, S1, Aki, T1,2, Nakashimada, Y1,2, Matsumura, Y1,2, Tajima, T1,2. 1Hiroshima University, 2CREST, JST. Nitratireductor sp. strain OM-‐1 can assimilate not only propionate but also volatile fatty acids (VFAs) found in methane fermentation process. Moreover, strain OM-‐1 can produce triacylglycerol when both propionic and acetic acids or glycerol are present. Further, nitrogen depletion in medium containing an acetic–propionic acid mixture resulted in the production of the light oil, but not triacylglycerol. Therefore, effects of nitrogen and carbon source species on oil accumulation were evaluated. When formic, acetic, propionic and butyric acids were assimilated, strain OM-‐1 accumulated 2-‐butenoic acid and its ester mainly. Valeric acid was converted into 2-‐pentenoic acid and its ester. The oil content was 100-‐200 mg/g DCW in the nitrogen-‐depleted conditions with any organic acids while 10-‐20 mg/g DCW of oil was obtained when the cells grown with 1.0g/L of ammonium chloride. Valeric acid is the most favorable carbon source for strain OM-‐1. In addition, pre-‐culture conditions and inoculum concentrations were also evaluated. As a result, the oil content reached up to 960 mg/g DCW when the cells grown in the model medium of VFA fermentation containing C2-‐C5 organic acids (4.0 g/L in total) for 10 days

with pre-‐culture at Day 1 and 10% of inoculum concentrations. Oil production by strain OM-‐1 needs the nitrogen-‐depleted conditions. The organic acids solutions obtained from algal biomass will contain the certain amounts of nitrogen source. This result suggests that new techniques for dissolved nitrogen removal from algal biomass solutions will be required for oil production by strain OM-‐1. (64) Marine-‐derived biosilicification: Silica-‐forming proteins. Ki Baek Yeo, Mi-‐Ran Ki, and Seung Pil Pack*. Department of Biotechnology and Bioinformatics. Korea University. Silica with nontoxic and highly biocompatible characteristics can be applied for tissue scaffolds, drug-‐delivery system, biosensors and imaging. For silica synthesis, conventional methods typically require a combination of high temperatures and extreme pH and also it is difficult to prepare controlled structures. However, the discovery of the critical molecules involved in biosilicifica found both in diatoms (silaffins and polyamines) and sponges (silicateins) brings out understandings about silica forming process in vivo and has presented a new paradigm for silica synthesis under ambient or mild conditions. The use of biosilicification machinery is based on the availability of enzymatically active recombinant proteins involved in silica formation. Recombinant silicatein (SIL), however, was expressed both in heterogeneous host systems, yielding limited quantities of the protein or inclusion body. To overcome these limitations, design of SIL or SIL-‐like protein should be required. In this study, two alternative silica-‐forming proteins are focused: CAT, hypothetical cathepsin-‐like protein from Nematostella vectensis; and SIL-‐like cathepsin (SLC), a mutant CAT obtained by mutating residues. (65) Lessons from the application of genomic selection to salmonids aquaculture. Yniv Palti*. National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, USDA, West Virginia, USA. Breeding technologies for Atlantic salmon and rainbow trout are arguably amongst the most mature and advanced in finfish aquaculture. Selective breeding programs to improve performance in economically important traits such as growth, disease resistance, slaughter traits and flesh quality are key to the success and sustainability of maturing aquaculture industries. The rate of genetic progress per generation has traditionally been impaired for traits like disease resistance and fillet characteristics that cannot be measured directly in the selection candidates, as salmonid breeders have typically relied on phenotype records from siblings of the breeding

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candidates. This has limited progress to family-‐based selection that does not exploit within family variation. Recent advances in molecular biology technologies coupled with sophisticated bioinformatics and statistical modeling have facilitated the use of molecular information in selective breeding programs to take advantage of within family variation and accelerate the rate of genetic gains. The most widely used approaches are marker assisted selection and whole-‐genome enabled selection, or genomic selection in short. The efficacy of genomic selection approaches to enhance genetic and economic gains is dependent upon the level of genetic variation and the genetic architecture of the trait in the specific breeding population. Examples from recent salmonids breeding research will be presented, and a systematic research approach with a decision tree model that is based on those empirical examples will be described and discussed. (66) Comparative analysis of selected four antioxidant genes from Big Belly Seahorse (Hippocampus abdominalis) and black rockfish (Sebestes schlegelii); revealing their putative significance in host antioxidant defense system. N. C. N. Perera*, G. I. Godahewa, and Jehee Lee. Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-‐Governing Province, Republic of Korea. Four SOD genes including; Copper Zinc SOD (Cu/ZnSOD), Manganese SOD (MnSOD) from seahorse (HaCuZnSOD, HaMnSOD) and rockfish (RfCuZnSOD, RfMnSOD) were characterized. Bioinformatics tools were used to analyze the characteristic features. qPCR was conducted with the mRNA transcripts. XOD assay, MTT and FACS assays were performed with recombinant proteins. Domain analysis of two CuZnSOD aa sequences discovered a conserved CuZnSOD domain, two ion binding sites and two polypeptide binding sites which are involved in detoxification function. The two conserved domains; SOD Fe-‐N and SOD Fe-‐C, are important for the detoxification mechanism of HaMnSOD and RfMnSOD. The highest antioxidant activity of the HaCuZnSOD and HaMnSOD were given at pH 9 and 25 °C and the highest antioxidant activities of RfCuZnSOD and RfMnSOD were observed in pH 8 and 25 °C. Inhibition assay was given more or less similar results for four genes and the inhibition rate was as, KCN>DDC>NaN3>EDTA. The results showed that HaCuZnSOD and RfCuZnSOD have the peroxidase ability with the presence of HCO3

-‐ and it was dose dependent. The highest expression of HaCuZnSOD was observed in blood where the HaMnSOD was in the ovary. Both RfCuZnSOD and RfMnSOD were given their highest expressions in blood. The SOD genes from the two species were shown their potential antioxidant activities thus may

conclude that they play main role in their host defense system. These for genes can be useful biomarkers in aquaculture to evaluate fish health and environmental management and related studies. (67) Regulation of double-‐stranded RNA-‐specific deaminase (ADAR) gene by interferon and Nervous Necrosis Virus in Malabar Grouper (Epinephelus malabaricus). Thirunavukkarasu Periyasamy*, Ming-‐Wei Lu. Department of Aquaculture, National Taiwan Ocean University. ADARs are RNA editing catalysts that bind double-‐stranded RNA and convert adenosine to inosine, a process that can lead to destabilization of dsRNA structures. In mammals, the ADAR1 gene is involved in a number of cellular pathways and the regulation of interferon (IFN)-‐mediated response. However, the function of fish ADAR1 remains to be elucidated. We report here the cloning of an ADAR1 homologue in Malabar grouper and its response to various immune stimulants. The MgADAR1 cDNA is 5371-‐bp long, consisting of an open reading frame encoding a putative protein of 1381 amino acids, a 235-‐nt 5'-‐terminal untranslated region (UTR), and a 990-‐nt 3'-‐UTR. The deduced amino acid sequence exhibits characteristic features of a chitin synthesis regulation domain, two Z-‐DNA-‐binding domains (Zalpha), three dsRNA binding motifs (DSRM) and one tRNA-‐specific and dsRNA adenosine deaminase domain (ADEAMc). MgADAR1 mRNA was expressed ubiquitously in tissues of healthy Malabar grouper, with elevated levels in the brain, gills, eye and skin. The MgADAR1 mRNA level was significantly up-‐regulated in the brain in response to poly (I: C), but not LPS. Interestingly, in spleen and head kidney the induction of interferon-‐inducible Mx by poly (I: C) was evident but the expression of MgADAR1 was not significantly affected. Upon nervous necrosis virus (NNV) infection, the expression of MgADAR1 was up-‐regulated in the brain and spleen. Similar response to poly (I: C) and NNV was observed in the grouper kidney GK cell line. In addition, the expression of MgADAR1 was upregulated by recombinant grouper IFN in the GK cells. These data hence indicate interplay between MgADAR1 and NNV infection. The data also indicate that MgADAR1 might be regulated by IFN in a tissue-‐specific manner. (68) Genetic diversity and family assignment of giant tiger shrimp Penaeus monodon and Pacific white shrimp Litopenaeus vannamei. Prasertlux, S1*, Wongtripop, S1, Menasveta, P2, Klinbunga, S1,2, Khamnamtong, B1,2

1National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agenct (NSTDA), 2Center of Excellence for Biotechnology, Faculty of Science,

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Chulalongkorn University, 3Department of Marine Science, Faculty of Science, Chulalongkorn University. The basic information on genetic diversity of propagated stocks is important for the construction of appropriate breeding programs of penaeid shrimp. To implement genetic improvement of Penaeus monodon and Litopenaeus vannamei based on the family selection scheme, rapid genotyping and family assignment of these shrimp are required. In P. monodon, multiplex analysis platform for simultaneously amplification of seven microsatellite loci (Pm02, PmMS6, PmMS8A2, CuPmo13, PmMS11AH, PmMS9 and PmMS9GG) was developed and preliminary applied to examine genetic diversity of the 5th

generation of domesticated P. monodon (N = 60) at Shrimp Genetic Improvement Center (SGIC). Subsequently, fluorescent multiplex microsatellite analysis was used for genotyping of parents (18 pairs, 6th generation) and 23 full-‐sib families of the progeny cultured in 6 concrete pond (4 families for 1S7 -‐ 5S7 samples and 3 families for the 6S7 samples). A phylogenetic tree correctly allocated each progeny group with their parents. Family assignment of a genetically improved stock of a commercial company was correctly determined using this analysis platform. In L. vannamei, fluorescent multiplex amplification of seven microsatellite loci (TumxLv10.208, TumxLv5.27, TumxLv9.103, TumxLv10.312, TumxLv10.220, TumxLv9.178 and TumxLv8.256) was also developed. A total of 65 groups of samples including imported broodstock and local shrimp from various commercial farms in Thailand were collected and genotyped. A UPGMA dendogram indicated that all imported SIS broodstock from Hawai were allocated to the cluster C while two groups of SIS broodstock from Singapore were allocated to cluster D. Almost all of the Kona Bay broodstock from Hawaii were allocated to clusters A+B. Local samples were allocated in each of these clusters. Accordingly, crosses between local samples from different phylogenetic groups should be carried out. In addition, samples with high genetic diversity within samples should also be taken into the account. (69) Seaweed biorefinery: for bio-‐based commodities and marine bio-‐economy. Reddy, CRK1*, Baghel, RS1, Trivedi, N2, Reena, P2, Annamma, A2, Lali, AM2. 1CSIR-‐Central Salt and Marine Chemicals Research Institute, Bhavnagar, 2DBT-‐ICT Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai. Marine macroalgae (seaweeds) which are historically farmed and traded in few Asian countries, gaining prominence world over as a promising feedstock for sustainable production of various commodity products including bioenergy. Seaweeds being marine living forms don’t require agricultural inputs such as fertilizer and

pesticides for their farming. Also, their farming doesn’t compete with agricultural crops for land and water. High productivity, efficient assimilation of nutrients, absence of lignin in their cell wall renders seaweeds a distinct advantage over terrestrial feedstocks in terms of farming and processing. The recent advancements made in biomass conversion technologies have led to the development of innovative process in biorefinery model for integrated extraction of the major components of algal biomass as commodity products such as sap (rich of plant nutrients and plant growth regulators), pigments, lipid, agar, protein and energy-‐dense substrate-‐cellulose. The computed yield data from small scale biorefinery trials of red algal feedstock suggest that a ton of fresh biomass can yield 0.3-‐0.7 kg of R-‐phycoerythrin, 0.1-‐0.3 kg of R-‐phycocyanin, 1.2-‐4.8 kg of lipids, 28.4-‐94.4 kg of agar, 4.4-‐41.9 kg of cellulose and 300-‐500 lit of sap. The advantages of biorefinery process include efficient utilisation of feedstock, densification of product results into minimum input costs, enabling recovery of stream of products. The latter enhance the value for feedstock with environmentally safe effluents. The estimated value of products recovered from biomass processing technology is 8-‐10 folds greater than that of the direct sale of seaweed crop in the wholesale market. There is an initiative in India to establish decentralized biomass processing units for promoting seaweed enterprise. This would not only strengthen the coastal rural economies but would also encourage large scale farming of seaweeds ‘sea agriculture’ offering sustainable additional income to the stakeholders. (70) Aerobic selenate reduction by Shewanella sp. strain KND-‐1 was isolated from the intestines of a searobin, Lepidotrigla guentheri which was captured in East China Sea. Sakaguchi, T1*, Tanaka, S1, Umakoshi, T1, Okamura, Y2, Ishikawa, A3, Taguchi, K3. 1Prefectural University of Hiroshima, 2Hiroshima University, 3Mie University. Selenium (Se) is an important element for various organisms, which widely distributed over the geosphere and biosphere on the earth. Se is also be understanding that it is an essential element as a nutrient to maintain bioactivities of animals and microorganisms. So far, several specific biological processes regarding selenium and/or its compounds metabolisms, have been found and suggested in various organisms. Microbes also have the specific dissimilative pathway on anaerobic respiration of selenium oxyanions such as selenate and selenite as terminal electron acceptor for growth. Other hands, it is reported that a gram negative bacterium, Pseudomonas stutzeri, can aerobically reduce selenate into elemental selenium without anaerobic treatment. Microorganism without requiring such process for selenate reduction is

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expected to be able to utilize to the selenium bioremediation, biofabrication and biomineralization. In this study, we report the isolation and characterization of marine Shewanella, designated strain KND-‐1, capable of growing aerobically by selenate reduction into elemental selenium. Marine fishes inhabiting the sand mud sea bottom where is deeper than depth of the water 70 m, were harvested by a trawl fishing in T-‐4 point (31-‐6.1N 127-‐43.02E) from depth of the water 137m in East China sea during the SE1512 cruise of T/S Seisui-‐maru (Mie Univeristy). Strain KND-‐1 was isolated from the intestines of a searobin, Lepidotrigla guentheri, which was collected at T-‐4 point. KND-‐1 could grow both aerobically and anaerobically in marine broth with generating specific red precipitations by elemental selenium formation due to selenate reduction. The phylogenetic analysis showed strain KND-‐1 belongs in genus Shewanella. (71) Metagenomic approaches to exploit antiinfectives from marine sponge microbiomes. 1Anuj Nishanth L, 2G. Seghal Kiran, 1R. Meenatchi, 1Saqib Hassan, 1*Joseph Selvin. 1Department of Microbiology, Pondicherry University, Puducherry, India. 2Department of Food Science and Technology, Pondicherry University, Puducherry, India. Metagenomics is a culture independent technique involve sequencing of whole microbiome, creation of shotgun libraries, high throughput screening and sequence-‐based data analysis. Metagenome mining of microbiomes reveals unique and novel biosynthetic gene clusters of marine natural products. Cloning and expression of these biosynthetic gene clusters are not obviously successful. Recent emergence of Metagenomic applications evolved genome mining and synthetic biology approaches which deals with pre-‐designed semi-‐synthetic genomes and whole genome transfer, now it is known as genome engineering. Marine environment is having unique niches of microbiomes particularly endosymbionts associated with sponges and corals. The wider biosynthetic potential of marine sponges are associated with the symbiotic microorganisms. Bacterial endosymbionts occupy 30% to 60% of the mesohyl in marine sponges. A major problem in studying these endosymbionts is failure in cultivating them under laboratory conditions. Bacterial symbionts are now proven to be true producers of bioactive molecules isolated from marine sponges. Our recent metagenomic explorations of marine sponge microbiome evident of advancing next-‐generation antiinefctive molecules. To combat mutidrug resistance and increasing emerging / remerging infectious agents, novel drug leads essentially required to contain outbreaks. In this report, metagenomic library was constructed using pCC1FOSTM fosmid vector from a marine sponge Mycale mytilorum

collected from southwest coast of India. Approximately 52,000 clones which were then screened for protease activity on skim milk agar showed one clone (f81) with zone of clearance after 28 hours of incubation at 37°C. The PCR amplified protease gene was digested with PagI and HindIII (Fermentas) and ligated to pBAD/Myc-‐HisA expression vector (Invitrogen) that has been digested with PagI and HindIII. Recombinants were transferred into E. coli BL21 cells and clones were assayed on skim milk agar plates for protease activity. Antagonistic effectiveness of purified protease was examined against various fungi such as Aspergillus niger, Candida albicans, Trichoderma longibrachiatum and Macrophomina phaseolina. Mycelial invasion inhibition was assessed by the crescent of retarded growth around the discs impregnated with Sp24 protease. Significant antifungal activity was exhibited by Sp24 protease against T. longibrachiatum and M. phaseolina. Research progress in our laboratory evidenced that metagenomic approaches are highly promising to develop peptide antibiotics which are being recognized as target-‐specific and least of chance of developing resistance. Metagenome guided exploitation of marine sponge microbiomes could be a promising approach in bioprospecting of marine natural products. (72) Priority research needs for profitable and sustainable aquaculture. Brian S. Shepherd*. USDA/ ARS/ School of Freshwater Sciences, University of Wisconsin-‐Milwaukee, Milwaukee, WI USA. The definition of sustainable aquaculture varies substantially, depending on the stakeholder groups that define it. Consequently, the needs vary when attempting to define priority research areas for action (ecological, industrial or research). The aim of the sustainable aquaculture session is to focus on perceived research needs and current efforts in this area. Feeding, feed formulations, and availability of cost-‐effective aquafeeds are the most pressing issues facing aquaculture at this time. To address aquafeed issues, research entities must increase their investment in nutritional research and technologies that result in sustainable feeds that meet nutritional demands of various species and their life-‐stages. Researchers must also identify improved feeding strategies to reduce waste and improve organism well-‐being. Continued and improved access to open waters is critical to aquaculture operations; however, concerns regarding the environmental impacts of aquaculture impede industry expansion and limit access to economic opportunities for new entrepreneurs. Environmental concerns could be allayed if policy makers, regulators, and stakeholders had a defined set of monitoring parameters for the aquaculture industry to meet. Once a desired set of parameters are in place, then governmental, industrial,

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and private entities could collaborate to develop and deploy remote monitoring and sensing platforms to assess environmental parameters. There are still areas of concern that involve extension, marketing, planning, and impact of invasive species. However, diseases and control of pathogens continue to negatively impact the aquaculture production. Given the limited number of approved and cost-‐effective chemotherapeutics and vaccines, and a limited understanding of the best time for vaccination in many species, enhanced efforts are needed to identify and implement innovative methods to improve disease resistance in aquaculture species. (73) Biofuel and biorefinery production by marine haptophytes. Shiraiwa, Y1,2*, Araie, H1,2, Hanawa, Y1,2, Endo, H1,2, Suzuki, I1,2, Nakamura, H2,3 and Sawada, K2,3. 1University of Tsukuba, 2The Shiraiwa Team of CREST/JST, 3Hokkaido University, Japan. Major biomass producers of marine phytoplankton are cyanobacteria, diatoms and haptophytes. Huge blooms of those phytoplanktons are frequently observed in the ocean and produce huge biomass including lipids such as triacylglycerol (TAG) or unique lipid compounds such as long chain-‐ketones (alkenones). Haptophytes, secondary symbiotic eukaryotic microalgae, are physiologically categorized into two groups because of neutral lipids produced, namely either TAG or alkenones. Interestingly, only alkenone-‐producing species are known as oceanic bloom-‐producers. Among them, the coccolithophore Emilainia huxleyi (Haptophytes) has been known to produce huge amount of biomass and calcium carbonate crystals as cell-‐covering and then transported into deep oceans by biological pump and precipitated as sediments. Those sediments are considered to become sources of petroleum and limestones. Our previous studies suggested that crude oils and natural gasses could be produced by pyrolysis of alkenone-‐producing haptophytes. However, metabolic pathway of neither alkenone nor DHA biosynthesis is known yet. The present talk will present our research results on the mechanism how alkenones are metabolically produced in the cells and how such compounds can be used for biofuels. Those are: (1) How much carbons are fixed by photosynthesis and distributed into metabolites such as polyunsaturated fatty acid, docosahexaenoic acid (DHA, 22:6ω-‐3), alkenones and neutral polysaccharides? (2) How intracellular oil droplets (named as “alkenone body”) are formed and which materials are involved as components? (3) What are metabolic pathways for alkenone and DHA productions? (4) How such unique lipids can be processed to direct usable biofuel and biorefinery candidates by cellular metabolic engineering? (5) How haptophytes can be transformed genetically to produce adequate biofuel and

biorefinery? Finally, we will present our recent progress on the novel biosynthetic pathway of alkenones and alkenes and unique metabolic pathway of DHA and will discuss on future prospects on biofuel and biorefinery production by haptophytes. (74) Two major Penaeus monodon antilipopolysaccharide factor isoforms (ALFPms) are regulated by Toll and IMD pathways. Kamsaeng, P, Tassanakajon, A, Somboonwiwat, K*. Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. The antimicrobial peptides from Penaeus monodon, ALFPm3 and ALFPm6, belonging to antilipopolysaccharide factor family, play an important role in shrimp immunity against pathogen invasions. However, only ALFPm3 but not ALFPm6 has been reported for the range of antimicrobial activity. We demonstrated here that the synthetic cyclic ALFPm6#29-‐52 peptide (cALFPm6#29-‐52) corresponding to ALFPm6 LPS-‐binding domain exhibited bacterial killing activity on a Gram-‐negative bacterium, Escherichia coli 363 as well as Gram-‐positive bacteria, Bacillus megaterium, Aerococcus viridans, and Micrococcus luteus. Its bactericidal effect is mediated by bacterial agglutination. ALFPm6 and ALFPm3 are different in terms of antibacterial activity and gene expression pattern upon pathogen infections. Herein, the regulation of ALFPm3 and ALFPm6 gene expression was studied. Upon identification of the 5ʹ′-‐upstream and promoter sequences, their putative transcription factor (TF)-‐binding sites were predicted. Narrow down assay indicated that ALFPm3 and ALFPm6-‐promoter active regions located at nucleotide position (-‐814/+302) and (-‐282/+85), respectively. Mutagenesis at the conserved nucleotides of selected TF-‐binding sites revealed that Rel/NF-‐κB (-‐280/-‐270) of ALFPm3 as well as C/EBPβ (-‐88/-‐78) and Sp1 (-‐249/-‐238) sites for ALFPm6 were the activator-‐binding sites. RNAi knockdown of MyD88 and Relish genes in V. harveyi-‐infected shrimp suggested that ALFPm3 gene expression might be regulated by both Toll and IMD pathways, while ALFPm6 gene expression might be regulated by Toll pathway. These finding provide insight into regulation of expression of different active ALF isoforms in shrimp. (75) Genetic breeding and cultivation of Gracilariopsis lemaneiformis (Rhodophyta). Sui, ZH*, Zhou W, Hu, YY, Wang JG, Fu F, Chang LP. Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, China. Gracilariopsis lemaneiformis is an economical important macroalga, which has become the second cultivated

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species referring to the biomass and annual output in China. The species is also suggested as a genetic study model based on many biological characters and advanced knowledge of its genetic background. To maintain the sustainable development of cultivation industry and promote genetic studies, a series studies were conducted in this alga. Mutagenic breeding was used in this study for screening strains with thermal tolerance. The results demonstrated that the two mutants grew at significantly higher rates than wild strains and similar to that of cultivar 981 both in the laboratory and the sea. Other physiological parameters were measured to elucidate the basis for the difference between mutants and control. The mutagenesis and selection process may help to develop heat-‐tolerant G. lemaneiformis cultivars in the future.

A genome survey of Gp. lemaneiformis using next-‐generation sequencing (NGS) technologies were conducted. A basic knowledge of the genome structure of this alga were obtained. This is the first report of genome-‐wide characterization within this taxon. 7737 simple sequence repeats (SSRs) were identified. Subsequently, the polymorphic primers out of 120 pairs of SSR primers were used to screen 82 samples from three geographical populations: Qingdao Sculpture Garden, Qingdao Zhanshan Bay and Weihai Stone Island. The genetic differentiation coefficient (Gst) between populations was below 0.05, revealing that the degree of differentiation between populations was very low, which was related to its special living environment and reproduction mode. A new cultivar named "Lu Long No. 1" was developed based on some of the genetic approaches and cultivated broadly in China. The new strain has many superior traits favored by industry. The large scale cultivation and developing of this strain will benefit for the aquaculture of Gracilariaceae species. (76) Construction of an artificial toluene sensor functioning in cells of the cyanobacterium Synechocystis sp. PCC 6803. Yu, Inaba1 and Suzuki, I2*. 1Graduate School of Life and Environmental Sciences, University of Tsukuba, 2Faculty of Life and Environmental Sciences, University of Tsukuba. Production of useful compounds by microalgae is attracted attention. However, when the synthesis of a target compound causes a concomitant decrease in the growth rate of the cells, because of the limitation of carbon partitioning to the synthesis of cellular components by means of allocation to the target compound and the toxicity of them. Due to these factors, the productivity by the algal cells may be restricted. If we introduce the switch to modify gene expression from the growth phase of cells to the production phase, it might eventually improve the productivity. In this study, we

attempted to develop an artificial sensory protein, which can respond to insoluble gaseous signal and regulate gene expression in the model cyanobacterium Synechocystis sp. PCC 6803. The insoluble gaseous signal can modulate the cellular metabolism by changing gene expression under exposure of the gas, after stopping the exposure the gas immediately eliminated from the medium and return to the initial metabolic conditions. We constructed a chimeric histidine kinase by fusing the signal-‐input domain of TodS, a toluene sensor, from Pseudomonas putida and the kinase domain of SphS, a phosphate-‐deficient sensor, from Synechocystis. We introduced gene for the chimeric sensor into Synechocystis cells and evaluated the response to toluene by alkaline phosphatase (AP) activity whose expression is, in nature, regulated via SphS. The cells harboring the chimeric sensor showed approximately 3.7-‐fold higher AP activity after 16 h exposure of toluene than those without toluene exposure. This is the first attempt to regulate gene expression by the gaseous signal in the model microalgae. (77) Challenge for commercial production of salmon trout in Japan. Suzuki, K1*, Maeda, Y2, Fukuda, S3, Izumi, H3, Okamura, K1, Saga, N3. 1Okamura Foods Co., Ltd., 2Inland Water Fisheries Research Institute, Aomori Prefectural Industrial Technology Research Center, 3Institute for Food Sciences, Hirosaki University. Salmon trout, large rainbow trout Oncorhynchus mykiss farmed seawater, is one of the most popular seafood in Japan, although most of the salmon trout come from foreign countries. In order to respond to domestic demand, furthermore looking the world market, commercial and sustainable production of salmon trout is needed in Japan. Use of triploid fish can be advantageous in salmon trout aquaculture due to sterility and the consequent superior growth. Additionally, saving space of farming area such as high-‐density rearing is effective for reduction of production costs. The use of triploid fish and high-‐density rearing can provide efficient production of salmon trout. However, the hatchery performance of Japanese triploid trout under the high-‐density condition is unknown. In this study, we compared the hatchery performance (growth, feed conversion, and survival) between Japanese triploid trout at high-‐density and diploid fish at low-‐density in freshwater farming. Donaldson strain cultivated in Japan for long period was used, triploidy was induced by thermal shock. Triploid and diploid fish (body weight: 1.2–1.3 g) were reared in freshwater (flow rate: 20–72 L/min, dissolved oxygen concentration: 5.9–11.5 mg/L) for 36 days at high-‐density (>30 kg/m3) and low-‐density (<20 kg/m3), respectively. There was no significant difference in growth, feed conversion and survival rate between triploid fish at high-‐

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density and diploid fish at low-‐density. This result suggests that Japanese triploid trout can be reared at the high-‐density without impair their growth in freshwater farming. This research was supported by grants from the Project of the BRAIN (the special scheme project on regional developing strategy). (78) Efficient conversion of carbohydrate derived from brown seaweed by psychrophile-‐based simple biocatalyst. Tajima, T1,3*, Tomita, K1, Miyahara, H1, Watanabe, K1,3 , Aki, T1,3, Okamura, Y1,3, Matsumura, Y2,3, Nakashimada, Y1,3, Kato, J1. 1Graduate School of Advanced Sciences of Matter, Hiroshima University, 2Graduate School of Engineering, Hiroshima University, 3Core Research of Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST). Bioconversion by conventional biocatalysts have some problems in the yields and productivities. Metabolic modifications or fermentation regulations will be required for whole cell biocatalysts to enhance the metabolic fluxes. Extracted enzymes can directly reacted with the substrate, however, these approach are often extremely laborious and costly. To overcome the circumstances we constructed the Psychrophile-‐based Simple biocatalyst (PSCat) for efficient conversion of bioresources. PSCat has significant advantages in the product yields and preparing the catalysts. The constructed catalysts can fully convert the chemicals because the simple heat treatment at moderate temperature inactivate the host psychrophile metabolism formed various metabolites as byproducts. Polyunsaturated fatty acids (PUFA) producer Aurantiochitrium sp. cannot grow with major carbohydrate such as mannitol or alginate derived from brown seaweed Konbu. To supply the sugar substrates to the PUFA producers PSCat was constructed by expressing mannitol dehydrogenase (Mdh) oxidized the mannitol to fructose and NAD+ regeneration enzymes. Mannitol almost converted to fructose by PSCat without fructose consumption. However, the conversion rate was relative lower using high concentration of pure mannitol as a substrate. PSCat also converted Konbu extracts containing mannitol to fructose with high conversion rate. Aurantiochitrium sp. could grow well with fructose converted by PSCat. PSCat can be useful as a sugar converter. (79) Paramylon production by Euglena gracilis under glucose regulated cultivation condition. Takamatsu, S1,2*, Kojima, K1,2, Ishikawa, T2,3, Sode, K1,2. 1Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2JST, CREST, 3Department of Life Science and

Biotechnology, Faculty of Life and Environmental Science, Shimane University. Euglena gracilis (E. gracilis) has been paid attractive attention as a candidate for the algal strain producing biodiesel, focusing its ability to accumulate wax esters. E. gracilis cells accumulate high quantities of β-‐1,3-‐glucan (paramylon) as excess carbon source storages, under aerobic conditions. When the cells with paramylon are transferred into anaerobic conditions, E. gracilis cells synthesize and accumulate wax esters from the paramylon intracellularly. Therefore, intracellular accumulation of paramylon is the crucial step to design and optimize the bioprocess for wax esters production using E. gracilis. In this study, we attempted to enhance the production of paramylon in E. gracilis, by optimizing culture conditions, especially using glucose as the carbon source. E. gracilis was cultivated either photoautotrophically, heterotrophically on glucose, or mixotrophically. Cell growth and paramylon accumulation of E. gracilis under various conditions were monitored. Simultaneously medium glucose concentration was measured by enzymatic glucose sensor. Under heterotrophic and mixotrophic conditions, E. gracilis showed high cell growth and accumulation of paramylon compared to photoautotrophically grown culture. Although heterotrophically cultivated E. gracilis stop growing after completely consumption of glucose in medium, mixotrophically cultivated E. gracilis cells kept their growth even after glucose depleted. The results indicated that paramylon production in E. gracilis could be controllable by regulating medium glucose concentration. (80) Marine microbiome analysis with the technologies for single-‐cell microbiology. Takeyama, H1,2,3*, Nishikawa, Y1, Kogawa, M1,2, Miyaoka, R1, Ando, M3, Hosokawa, M3,4. 1Department of Life Science and Medical Bioscience, Waseda University, 2CBBD-‐OIL, AIST, 3Research Organization for Nano & Life Innovation, Waseda University, 4PRESTO, JST. Single cell microbiology is the powerful analytical method for obtaining their genome information without cultivation at the single-‐cell level. We have explored genomic feature of single bacterial filaments isolated from marine sponges by single-‐cell genome sequencing (Wilson et al, Nature, 2014). However, conventional technique for single-‐cell genome sequencing usually resulted in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras. Our study aims to develop droplet-‐based microfluidics for precise and massively parallel single-‐cell genomics (Nishikawa et al, PLoS ONE, 2015), to elucidate marine microbial diversity and its relationships

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with host organism at the single cell level. Our droplet technique 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 marine bacteria. These techniques would be useful for single-‐cell study of environmental microbes toward the application of bioactive compounds. (81) Analysis of antibody diversity in the lymph fluids of goldfish, Bubble Eye. Momose, N1 , Nukada, N1, Tamaru, Y1,2*. 1Department of Life Sciences, Graduate School of Bioresources, Mie University 2Department of Bioinformatics, Advanced Science Research Center, Mie University. Development of antibody against membrane-‐associated proteins such G-‐protein coupled receptors (GPCRs) is necessary for diagnostic treatment and drug discovery. In general, mammals are used as host animals to produce antibody. However such host animals have never produced several specific antibodies against GPCR because of immune tolerance. To overcome these problems, we attempted to produce the antibodies using fish which is localized in the origin of vertebrates and have an acquired immune system in addition to the innate immune system. In this study, we have developed to establish antibody production system using goldfish (Carassius auratus), in particular we used “Bubble eye” which has blisters filled with lymph liquids, as an immune animal. First, the anti-‐goldfish IgM (gIgM) polyclonal rabbit antibody was targeted with the CH3 region of gIgM heavy chain. Next, the recombinant GFP as an antigen were expressed and purified in E. coli. The purified EGFP was directly injected into lymph liquids of Goldfish. After immunization, lymph liquids including gIgM were collected to detect whether antibody was raised against GFP. As a result, the detection by ELISA showed anti-‐GFP antibody existed in their lymph fluids. Moreover, mRNA was extracted from the lymph liquids and cDNAs were synthesized. Total 91 clones of gIgL were obtained and sequenced. As a result, the clones were classified into 3 groups and 19 subgroups between before and after immunization. (82) Potential of marine oleaginous diatom for biomass, biofuel and biochemical production. Tanaka, T*. Institute of Engineering, Tokyo University of Agriculture and Technology.

Microalgae have been recognized as promising hosts of biomass, biofuel and biochemical productions owing to their high growth rate, enormous diversity and broad environmental adaptability. Marine microalgae are culturable at vast ocean area using seawater. Therefore they do not compete with terrestrial crop plants, and it is beneficial for sustainable production of these useful materials. Towards the practical biofuel production using marine microalgae, we have screened oleaginous microalgae from our our culture collections consisting of thousands strains of marine microalgae. Among the promising candidates selected, the marine diatom Fistulifera solaris exhibited the highest lipid productivity. Outdoor large-‐scale cultivation of F. solaris was demonstrated using a natural seawater-‐based culture medium, indicating that this diatom could take advantage of marine strains culturable in seawater. We also performed the multi-‐omics studies for better understanding of the lipid metabolism in F. solaris, as well as for further enhancement of its superior oleaginous phenotype by genetic manipulation. In this presentation, outcomes of the multi-‐omics analyses including genomics, transcriptomics, proteomics and lipidomics are first introduced. Subsequently, successful demonstrations of the genetic manipulations for improved biofuel production are presented. Based on the multi-‐omics analyses, we identified the attractive targets of genetic manipulation. By overexpressing such target genes involved in lipid biosynthesis, the accelerated lipid accumulation was confirmed. Easy and rapid cell harvest was also demonstrated by modifying the surface of the diatom cells, on which silica cell wall-‐associated proteins fused with an affinity peptide were displayed. Furthermore, potential of F. solaris in the production of high-‐value added materials such as polyunsaturated fatty acids is also discussed in this presentation. According to these researches, we believe that both multi-‐omics studies and genetic engineering efforts based on the basic researches are necessary to fully exploit the potential of marine microalgae for biomass, biofuel and biochemical production. (83) Masquerade-‐like protein is involved in TSV resistance in Pacific white shrimp Penaeus vannamei. Supungul, P1, Tang, S1*, Junprung, W2, Tassanakajon, A2

. 1Aquatic Molecular Genetics and Biotechnology Laboratory, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Phathum Thani, Thailand, 2Molecular Biology and Genomics of Shrimp Laboratory, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.

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The shrimp industry contributes the export revenue of $US 2 billion annually to Thai economy. However, outbreak of infectious diseases is a major problem for shrimp aquaculture. Selective breeding program of Penaeus vannamei to improve resistance to Taura syndrome virus (TSV) has been established. However, a lack of basic knowledge on molecular mechanisms of shrimp immune against TSV infection limits the application of molecular markers to assist selective breeding program in P. vannamei. Here, a DNA marker linked with TSV-‐resistant/susceptible phenotypes of P. vannamei were developed. Masquerade-‐like protein (PvMas) which is one of the candidate genes possibly associated with TSV-‐resistance in shrimp was characterized. Correlation between the expression level of PvMas transcripts and susceptibility/resistance to TSV was examined. The result showed higher expression of PvMas in the TSV-‐resistant line than that in the susceptible line. Moreover, a TSV-‐challenged test further indicated that the TSV capsid gene (CP2) was expressed higher in PvMas knocked-‐down shrimp than the control. The findings suggested that PvMas should involve in TSV defense mechanisms of P. vannamei. In addition, an 82-‐bp insertion was found in an intron of PvMas of the TSV-‐resistant line. Genotype frequencies of the PvMas gene in susceptible and resistant groups were analyzed (N = 120 covering 2 families of each line). A significant difference in genotype distribution frequency between these lines was observed (p<0.05). The information on association between expression levels and the occurrence of an insertion in PvMas and resistance/susceptible phenotypes suggested that PvMas plays a role in the defense mechanisms against TSV. The 82-‐bp insertion marker can be applied in breeding programs of TSV-‐resistant P. vannamei. (84) Cloning and functional analysis of type 1 interferon receptor 1 (IFNR1) response to immune-‐stimulators in orange-‐spotted grouper (Epinephelus coioides). Tang, ZZ1,3*, Wang, TY1,3, Chen, TY1,2,3,4. 1Institute of Biotechnology, 2Department of Biotechnology and Bioindustry Sciences, 3Translational Center for Marine Biotechnology, 4Agriculture Biotechnology Research Center, National Cheng Kung University, Tainan, Taiwan. Aquaculture industry rising as one of most profitable industry in the world, specifically in marine aquaculture field, grouper has always known to bring high economic profits to fish farmers, however, large outbreak of virus infection during larvae stage threatened their harvest period. Understanding how teleosts activate their first line of defense mechanism during virus infection can help increase their survivability subsequently increase grouper yield rate. Our previous research showed recombinant

type 1 interferon (I-‐IFN) protein able to activate grouper Mx, a downstream antiviral protein, involve in activity against nodavirus infection. However, the mechanism of cytokine binding for signal transport through receptor interactions to activate downstream pathway in orange-‐spotted grouper is still currently unclear. Thus, in present study, we firstly cloned and identified type 1 interferon receptor (IFNR1) of orange-‐spotted grouper with nucleotides length of 1444 bp encoding ORF with 433 amino acids length. Bioinformatic analysis indicated osgIFNR1 is clustered within IFNR1 subunit with high structural homology similarities with other teleosts. Gene expression level varied according to LPS and Poly I:C treatments, to mimic microbial and virus infection respectively in their immune organs, head kidney and spleen. Highest gene expression of osgIFNR1 is observed during 12 hours of LPS treatment while in Poly I:C treatment, highest expression level is detected during 6 hours post-‐stimulation, this indicated elevated activity of osgIFNR1 expression from different immunostimulant in early stimulation treatments. This study reports on early insights of type 1 interferon receptor role in interferon system in preventing virus diseases. (85) Innate immunity and disease resistance in shrimp. Anchalee Tassanakajon1*, Piti Amparup2, Kunlaya Somboonwiwat1 and Wisarut Junprung1. 1Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand, 2National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani, Thailand. Diseases have caused tremendous economic losses and become the major problem threatening the sustainable development of shrimp aquaculture. The knowledge of host defense mechanism against invading pathogen is essential for the implementation of effective measures to prevent disease outbreaks. Like other invertebrates, shrimp rely on the innate immune system to defend themselves against a range of microbes by recognizing and destroying them through cellular and humoral immune responses. In the presentation, we review a recent progress on the two most powerful and the best -‐studied shrimp humoral responses: antimicrobial peptides (AMPs) and melanization. Shrimp AMPs are mainly cationic peptides with sequence diversity which endues them the broad range of activities against microorganisms. Melanization, regulated by the prophenoloxidase activating cascade, also plays a crucial role in killing and deposition of invading pathogens. The progress and emerging research on mechanisms and functional characterization of components of these two

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indispensable humoral responses in shrimp immunity will be summarized and discussed. Interestingly, the pattern recognition receptor (PRR) crosstalk is evidenced between the proPO activating cascade and the AMP synthesis pathways in shrimp, which enables the innate immune system to generate efficient immune response network to combat the invading pathogen. More recently, we have shown that exposure of Litopeneaus vannamei to non-‐lethal heat shock (NLHS) could enhance resistance to a strain of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VPAHPND) by inducing shrimp innate immunity. This knowledge can be exploited to reduce the impact of diseases in shrimp aquaculture. (86) Microbe-‐sponge symbiosis: New discoveries of molecular and metabolic interactions. Torsten Thomas*. Centre for Marine Bio-‐Innovation & School of Biological, Earth and Environmental Sciences, The University of New South Wales, Sydney, NSW, Australia. Sponges form close and essential symbiotic interaction with complex communities of microorganisms. These interactions allow for the recycling of waste products, the acquisition of nutrients and defense against predation. Due to the poor culturability of microbial symbionts of sponges, many details of these processes have remained unclear. However, recent metagenomic studies have now unlocked the genetic and functional potential of microbial symbionts and have provided new insight into their molecular and metabolic interactions with the sponge host. Here I will present some recent discoveries in this field. These include the surprising finding that microbial symbionts have acquired genes from eukaryotes and use these genes to influence feeding processes of sponges. We have recombinantly expressed these eukaryotic genes to demonstrate that they can act as a general modulator of eukaryotic phagocytosis. Furthermore genome reconstruction and transcriptomics has revealed how multiple symbionts work together to recycle nutrients and acquire carbon sources within the sponges. Such facilitative processes contrast antagonist activities that also occur between symbionts and which are based on new defense chemistries to control potential pathogens. These fundamental discoveries have potential biotechnology applications, which will be also further discussed.

(87) Double knockout of myostatin a and b genes causes muscle enhancement in zebrafish by CRISPR/Cas9 technology. Tsai, YT1*, Wu, SH1 and Gong, HY1,2. 1Department of Aquaculture, National Taiwan Ocean University, Taiwan, 2Center of Excellence for the Oceans, National Taiwan Ocean University, Taiwan.

Myostatin is a potent negative regulator of skeletal muscle growth. Mutation of myostatin gene causing double muscle phenotype had been reported in many domesticated animals. In contrast to only one myostatin gene in the genome of mammals, two myostatin genes exist in the majority of teleost. In this study, we used CRISPR/Cas9 targeted genome editing technology to knock out (KO) two distinct myostatin genes, mstna and mstnb in zebrafish to understand their roles in muscle and non-‐muscle tissues. Here we demonstrated that co-‐injection of two gene-‐specific gRNAs targeting exon 1 of two zebrafish mstna & mstnb genes (4 alleles), respectively and Cas9 mRNA made variant small insertion/deletion genotypes leading to in-‐frame stop codon to disable myostatin genes in individual F1 zebarfish. We had obtained F2 offspring of single mstna KO、mstnb KO and mstna/mstnb double KO zebrafish strains to compare their phenotypes with wild-‐type strain. We found that the enhanced muscle growth was significantly observed in 2-‐month mstna/mstnb double KO zebrafish, which exhibiting obviously bulging muscle on the back due to increased muscle cell numbers (hyperplasia) when compared with that of mstnb or mstna single KO and wild-‐type zebrafish. The phenotypes and molecular mechanisms of muscle growth, fat distribution and immunity are being investigated and compared in F3 offspring of mstna, mstnb single KO and mstna/mstnb double KO zebrafish lines with wild-‐type zebrafish. The CRISPR-‐mediated targeted genome editing technology is a powerful tool to improve desired traits of important aquaculture species to speed up the selective breeding program. (88) A metabolic flux model of glycolysis and glutaminolysis in shrimp hemocytes after white spot syndrome virus infection. Tung, CY1,2*, Lee, DY3, Wang, HC1,2. 1Institute of Biotechnology, National Cheng Kung University, 2Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, 3Graduate Institute of Integrated Medicine, China Medical University.

Since the white spot syndrome virus (WSSV) causes serious economic losses, it is important to elucidate its pathogenesis. We reported that WSSV induced a Warburg-‐like effect and glutaminolysis in shrimp at 12 h after infection (hpi), although these phenomena had ceased by 24 hpi. In this study, 13C metabolic flux analysis was used to characterize cellular metabolic states and metabolic fluxes in WSSV-‐infected hemocytes. At 12 and 24 hpi, white shrimp were treated with uniquely labeled isotopic tracers, hemocytes were collected and metabolites analyzed with liquid chromatography–electrospray ionization-‐tandem mass spectrometry (LC–ESI-‐MS). At the genome replication

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stage (12 hpi), using [U-‐13C]-‐glucose as the tracer, amounts of isotopic labeled glucose-‐derived intermediates in glycolysis were increased and corresponding production rates were faster than in the control group. However, there was no enhancement of the TCA cycle, whereas using [U-‐13C]-‐ glutamine as the tracer, amounts and production rates of isotopic-‐labeled intermediates in TCA cycle were both increased. At the late stage (24 hpi), using [U-‐13C]-‐glucose as the tracer, enhancement of glycolysis was no longer apparent. Taken together, at the WSSV genome replication stage (12 hpi), we concluded that high levels of glycolytic intermediates were causing a Warburg effect by triggering glycolysis. In addition, glutaminolysis was also enhanced, thereby providing an alternate carbon source for a glucose-‐independent TCA cycle. (89) Applying riboregulator to knock down chromosomal gene cyabrB2 in Synechocystis sp. PCC 6803 for higher glycogen production. Ueno, K1*, Sakai, Y1,2, Shono, C1,2, Sakamoto, I1,2, Tsukakoshi, K1, Hihara, Y2,3, Sode, K1,2, Ikebukuro, K1,2. 1Department of Biotechnology and Life Science Graduate School of Engineering Tokyo University of Agriculture and Technology, 2CREST Japan Science and Technology Agency, 3Graduate School of Science and Engineering Saitama University. Cyanobacteria are one of the attractive hosts for biofuel production but genetic approach to regulate the certain chromosomal gene in the cyanobacteria is still limited. To develop a new method to regulate chromosomal gene in cyanobacteria, we focused on a post-‐transcriptional gene regulation system, riboregulator1. Riboregulator consists of cis-‐repressed mRNA and trans-‐activating RNA and their interactions can lead to the expressional regulation of the target gene. As a target gene, we focused on the cyabrB2 gene, encoding global transcription regulator in cyanobacteria. It was reported that accumulation of glycogen granules in the cells was observed when the cyabrB2 was disrupted2. In this study, we chromosomally inserted riboregulator in the upstream of the cyabrB2 and evaluated the transcription and expression level of the target gene. Also, we evaluated the effect of the expression level of cyAbrB2 on the glycogen accumulation and transcription level of other genes regulated by cyAbrB2 were determined. In the integrated mutant, the repression of the cyabrB2 expression was observed under riboregulator inducing condition. Also, the higher amount of glycogen accumulation was observed in the cyabrB2 expression repressed mutant. Additionally, the transcription levels of other genes, ftsQ, ftsZ, sigE were affected by cyAbrB2 translation level. Altogether, it was showed that riboregulator could be applied to a new knockdown tool. References:

1. F. J. Issacs, et al., Nature Biotechnology, 22, 841-‐847 (2004) 2. Y. Kaniya, et al., Plant Physiology, 162, 1153-‐1163. (2013) (90) De novo assembly of large and small genomes in aquaculture. Geoffrey C Waldbieser*. Warmwater Aquaculture Research Unit, USDA, Agricultural Research Service, Stoneville, Mississippi, USA. The concentration of terrestrial animal production in only a few species enabled large research consortia to produce high quality genome sequence assemblies for a worldwide community. Production of genome assemblies from aquacultured species lagged due to a large number of species, each with smaller research groups with fewer resources. Recent advances in DNA sequencing technology and sequence assembly algorithms have made whole genome sequencing and assembly more cost efficient. This presentation will compare advantages and limitations of assemblies produced through second and third generation sequencing technologies for both vertebrate and microbial genomes, and will discuss advantages of whole genome assemblies for aquaculture. (91) A shrimp virus, WSSV, changes the balance of host metabolism during infection to complete its replication. Wang, HC*. Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University. Shrimp aquaculture is a global industry that incurs severe economic losses from several shrimp diseases. One of the most damaging diseases is WSD (white spot disease), which initially swept through the cultured shrimp industry with devastating effects in 1995. The shrimp industry continues to seek an effective strategy against this disease, which is caused by the shrimp virus WSSV (white spot syndrome virus). However, due to the uniqueness and complexity of the WSSV, the pathogenesis of this disease is still far from being completely understood. In our current research, we have used an approach based on human cancer systems biology to determine global metabolic changes induced by WSSV at various stages of viral replication. In this talk, we review some studies about metabolic changes in WSSV-‐infected shrimp, including aerobic glycolysis, lipid synthesis and glutaminolysis. Most interestingly, at the genome replication stage (12 hpi), WSSV induces a metabolic rerouting known as the invertebrate Warburg effect. This effect, common in cancer cells, increases availability of energy and biosynthetic building blocks in the host cell. It is noteworthy that WSSV is the first invertebrate virus reported to cause special metabolic rerouting in its host. This groundbreaking discovery in invertebrate metabolism

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and viral pathogenesis potentially provides a range of new ways to design and/or select for virus-‐resistant shrimp. (92) Developmental toxicity of triazole pesticide, Paclobutrazol, on zebrafish. Wen-‐Der Wang*. Department of Bio-‐Agricultural Sciences, National Chiayi University, Chiayi, Taiwan. Paclobutrazol (PBZ), a trazole-‐containing fungicide, is globally used on food crops. Wide and frequent usage of PBZ contaminates our environment and increases the health risk of organisms. Although the chronic effects of PBZ exposure on reproductive, antioxidant defense, and liver metabolism systems in rockfish have been reported, however the acute toxic effects of PBZ on aquatic embryos are unknown. Here, we report that embryonic PBZ exposure decreases their survival and hatching rates, developmental defective heart, craniofacial cartilage, digestive system and retinal photoreceptor cells. PBZ affects the normal process of cardiac looping, which may lead to a slower heart beat accompanied by pericardia edema and apoptotic myocytes. PBZ also decreases the population of migratory neural crest cells, which give rise to craniofacial cartilage. On the other hand, we reveal that PBZ altered the homeostasis of AHR and retinoic acid signaling, which result in the developmental defects on digestive system and retinal photoreceptor cells, respectively. Our results reveal high embryonic toxicity of PBZ on aquatic organisms, and thus hold significance for the impact of fungicides on public health and ecology. In addition, we are currently focusing on studying the potential toxic effects on embryonic organogenesis and the molecular mechanism of the toxic pathology. We believe our findings have implications for understanding the potential toxicity of PBZ during embryogenesis, and thus the potential impact of fungicides on public health and the environment. (93) Value-‐added lipid production by Aurantiochytrium sp. using sugars derived from macroalgae. Watanabe, K*, Arafiles, KHV, Okamura, Y, Tajima, T, Matsumura, Y, Nakashimada, Y, Aki, T. Hiroshima University. Labyrinthula Aurantiochytrium is eukaryotic microorganism living in the ocean and produces various high value-‐added lipids such as polyunsaturated fatty acids, unsaturated hydrocarbons and carotenoids, which are important compounds in health food, pharmaceutical, cosmetic and chemical industries. Aurantiochytrium can grow rapidly and accumulate lipids in simple medium and cultural conditions and, therefore, it is expected to be the source of high value-‐added lipids alternative to fish and plant oils. In order to supply lipids produced by Aurantiochytrium inexpensively and sustainably, we

attempted to use macroalgae, which is unutilized biomass abundant in the ocean, as culture substrate. The major components of brown algae, kombu, are mannitol and alginate, but these saccharides are not assimilated by Aurantiochytrium. Therefore, we used extra microorganisms, Gluconobacter oxydans and Dysgonomonas alginatilytica HUA-‐2, as intermediary biocatalysts to convert mannitol and alginate into fructose and succinate, respectively, and the resultant sugars had encouraged the growth and lipid production of Aurantiochytrium. Additionally, we analyzed the intracellular metabolites of Aurantiochytrium assimilating the macroalgae-‐derived sugars. Mass spectrometry-‐based metabolomic analysis revealed the significant metabolic fluctuations in citrate cycle and amino acids metabolism according to each carbon source. Based on these metabolic information, we improved the productivity of docosahexaenoic acid (DHA) of Aurantiochytrium through the activation of citrate cycle. The two-‐stage fermentation system constructed in this study demonstrates the usability of macroalgae as a substrate for high value-‐added lipid production and the availability of Aurantiochytrium for the conversion of unutilized biomass into valuable compounds. (94) Tug-‐of-‐war of sexual development in ovotestis in the protandrous black porgy. Wu, GC1,2*, Li, HW1, Chang, CF1,2. 1Department of Aquaculture, 2Center of Excellence of the Oceans, National Taiwan Ocean University. Controlling the development of the sexes is critically important for the broodstock management in aquaculture. Sex steroids are widely used for sex control of gonochoristic fish. Hermaphroditism occurs in 6% of teleosts which have a plastic sex, and a stable sex is difficult to maintain with sex steroids. In protogynous grouper, blockage Cyp19a1a activities (aromatase inhibitor administration) or methytestosterone administration and low endogenous estradiol (E2) induced the development of the testis development and the regression of the ovary and resulted in an unstable phase of maleness in grouper. This characteristic resulted to an unstable sex ratio in broodstock. We used the digonic black porgy (Acanthopagrus schlegelii) as a model to understand the mechanism of sexual fate decision. In black porgy, exogenous E2 induced the development of the ovary development and the regression of the testis and resulted in an unstable phase of femaleness. These data demonstrated that an unstable sexual phase is found in the younger fish. However, a stable femaleness (precocious female function) induced by the removal of testicular tissue in black porgy. Our data also demonstrated that the male-‐to-‐female sex change is blocked by the maintenance of male function with

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gonadotropin-‐induced dmrt1 expression in the testis. Furthermore, our data also indicated that ovarian genes (cyp19a1a and fshr) expression is regulated by the testis through epigenetic modifications. Therefore, the primary male guides the femaleness in the protandrous black porgy and the transition of sexual fate from male to female is determined by the status of the testicular tissue through the brain (Gnrh)-‐pituitary (Gths)-‐testes (Dmrt1) axis. (95) An engineered microbial platform for direct biofuel production from brown macroalgae. Yoshikuni, Y*. DOE Joint Genome Institute. The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-‐scale fuel ethanol production, however, requires extensive re-‐engineering of the alginate and mannitol catabolic pathways in the standard industrial microbes such as Escherichia coli and Saccharomyces cerevisiae. Here we present the discovery of enzymes for alginate degradation, transport, and metabolism in both prokaryote and eukaryote species. The genomic integration of these ensemble generated microbial platforms that can simultaneously degrade, uptake, and metabolize alginate. When these platforms were further engineered/adapted, they were capable of ethanol fermentation from carbohydrates in brown macroalgae very efficiently. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-‐added renewable chemicals in a manner that is comparable to traditional arable-‐land-‐based feedstocks. (96) Production of viable trout offspring derived from germ cells cultured in vitro. Yoshizaki, G*, and Iwasaki Y. Tokyo University of Marine Science and Technology. We previously established a germ cell transplantation technology that would provide a powerful tool for the conservation of endangered species and the efficient production of commercially valuable seeds. In vitro amplification of donor germ cells prior to transplantation, would produce seedlings of endangered fish or valuable fish species simply by using recipient fish, which are easy to raise, and donor cells harvested from a petri dish instead of using live donor fish. In this study, in order to produce live fish derived from in vitro cultured cells, we first cultured the rainbow trout germ cells in vitro and then transplanted the resulting cells into recipient fish. vasa-‐GFP transgenic and dominant albino rainbow trout were used as materials. The initial germ cells were enriched by differential plating from whole testicular cells

and used for the cell culture. The enriched germ cells (mainly type-‐A spermatogonia labeled with GFP) were cultured in DMEM/F12 media supplemented with various concentrations of rainbow trout serum and fetal bovine serum. Whenever the cultures reached 80% confluence, subcultures were created at a dilution rate of 1:2. After a month, the resulting amplified cells were intraperitoneally transplanted into wild-‐type, triploid rainbow trout hatchlings. The cell culture medium containing 1% of rainbow trout serum and 10% of fetal bovine serum demonstrated high growth rates. Remarkably, the amplified cells still displayed a clear green fluorescence in cultures after a month culture. The transplanted germ cells were incorporated into the recipient gonads and resumed gametogenesis. One year after the transplantation, some recipient males precociously matured. By performing a mating study, we confirmed that the offspring carrying the donor-‐derived phenotype (albino and GFP-‐positive) were produced. Thus we could successfully establish a culture condition of trout germ cells in vitro for at least a month without losing the potential to differentiate into functional sperm. (97) Vortex fluid device-‐intensified direct transesterification of microalgae Chloroparva pannonica biomass to biodiesel. Sitepu, EK1,2, Luo, X1,2, Pye, S3, Tang, YH3,4, Leterme, S5, Raston, C1,3, Zhang, W1,2*. 1Centre for Marine Bioproducts Development; 2Department of Medical Biotechnology, 3Centre for NanoScale Science and Technology; 4School of Computer Science, Engineering and Mathematics; 5School of Biological Sciences, Flinders University, South Australia, Australia. A novel room temperature vortex fluid device (VFD) intensified direct transesterification process of microalgae Chloroparva pannonica biomass to biodiesel was developed, using sulfuric acid or sodium hydroxide as the catalyst in confined and continuous flow mode of operation of the device. The operating parameters were optimized by varying the ratio of biomass to methanol (1:3.7 – 1:8, wt./v), catalyst concentration in methanol (1 – 10%, wt./v), reaction time (15 – 75 min) and rotational speed (4000 – 8000 rpm), for a 20 mm OD VFD tube, with the biodiesel conversion yield confirmed using 1H-‐NMR. The highest conversions for the confined mode of operation of the VFD, 83.3% and 96.4%, were obtained at 1:6.7 (wt./v) ratio of biomass to methanol, 30 min reaction time, 6000 rpm rotational speed and catalyst concentration of 10% and 3% (wt./v) for acid and base, respectively. For continuous flow scale up using the VFD, 3% (wt./v) of sodium hydroxide was used as the catalyst, with the influence of the ratio of biomass to methanol, flow rate, rotational speed and tilt angle of the VFD tube investigated. A 99.2% conversion was obtained using 1:10

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(wt./v) ratio of biomass to methanol, 0.5 mL/min flow rate, 6000 rpm rotational speed and 450 angle. The findings presents a novel energy efficient process to convert microalgae biomass directly into biodiesel at room temperature within short reaction times, with higher yield using VFD-‐mediated processing compared with the conventional batch method.

(98) Deep-‐sea adaptations of the symbiotic Bathymodiolus mussels. Ping Zheng*1,2, Minxiao Wang1,3, Chaolun Li3, Yan Sun1 and Song Sun1,4. 1Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Deep Sea Research Center, Institute of Oceanology, Chinese Academy of Sciences, 4Jiaozhou Bay Marine Ecosystem Research Station, Chinese Academy of Sciences. Mussels of subfamily Bathymodiolinae are found to be one of the most dominant species in a number of deep-‐sea habitats including hydrothermal vents and cold seeps, and generally depend on the chemosynthetic endosymbionts in their gills to obtain nutrients. Our phylogenetic analysis supports the hypothesis that the deep-‐sea Bathymodiolin mussels are recent colonizer to vents and seeps. And many transposase-‐related genes are lineage specific or expanded in Bathymodiolus mussels, which might contribute to the genome rearrangement and lead to speciation. Compared with coastal mussels, the most extraordinary trait of Bathymodiolus is their endosymbiosis. According to our metagenome and electron microscopy results, Bathymodiolus platifrons houses type I methanotrophic endosymbiosis in their specialized gill epithelial cells called bacteriocytes. During the cultivation of B. platifrons, although the mussels can survive in both methane-‐present and methane-‐absent environments under the atmospheric pressure, the abundance of the symbionts are significantly different after a certain time during the acclimation. The loss of symbionts induced various changes related with immune response, apoptosis and lysosomal activities in B. platifrons. As many other symbiotic systems, nutrient interactions linked the mussels and symbionts. Our comparative transcriptomic results suggest that nutrition fixed by the symbionts can be absorbed in a ‘farming’ way wherein the symbionts are digested by lysosomes. Correspondently, the host can transfer and even enrich some metabolic substrate for their endosymbionts. Our studies enriched our knowledge of the origin and adaptations of the deep-‐sea Bathymodiolus mussels.