細菌群落會影響水族系統中水生動物的健康與福祉。我們卻對這些生態系統中細菌群落的組成和結構以及季節性的動態變化了解甚少。在本研究中，我們比較了台灣國立海洋生物博物館(NMMBA)的兩個展示水池(大洋池和白鯨池)以及原水(海水入口)的季節性細菌群落變化。細菌群落使用高通量次世代定序擴增16S ribosomal RNA基因的高變區來分析。吾人總共獲得1,569,906條序列，依其相似度可分為4,163個操作分類單元(OTUs；97%序列相似性)。海水中的細菌群落總共有21個門，其中以Proteobacteria (58.11%)、Bacteroidetes (14.94%)、其他(14.52%)和Cyanbacteria (8.23%)最多。我們發現原水的細菌豐度和多樣性皆高於兩個水池。OTUs數和Chao1指數顯示，秋季比其他季節擁有更高的細菌豐度。主座標分析(principal coordinate analysis)與群聚分析(cluster analysis)顯示原水池和兩個展示水池的細菌群落明顯不同。本實驗研究結果顯示，各水池中的細菌群落主要是其環境因素(如溫度或營養鹽)的影響，而不是來自原水加入的影響。

It is believed that the bacterial community influences the health and welfare of the aquatic animals in aquarium system. However, bacterial community composition, structure and seasonal dynamics within these ecosystems are poorly understood. In the present study, we compare seasonal variation of bacterial communities in the water column from two tanks (the Open Ocean tank and the Whale tank), as well as a seawaters inlets, in National Museum of Marine Biology and Aquarium (NMMBA) in Taiwan. Members of the bacterial communities in inlet seawater and two aquarium tanks were characterized by means of next generation high-throughput amplicon sequencing of 16S ribosomal RNA gene hypervariable regions. A total of 1,569,906 sequence tags were obtained and clustered to 4163 operational taxonomic units (OUTs; 97% sequence similarity). The bacterial communities in seawater were assigned to 21 bacterial phyla and revealed abundant of phyla Proteobacteria (58.11%), Bacteroidetes (14.94%), others (14.52%), and Cyanbacteria (8.23%). We found that inlet seawater had higher bacterial richness and diversity than two tank seawater. OTUs and Chao1 estimator supported that autumn had higher bacterial richness than other seasons. Principal coordinate and cluster analysis revealed obvious differences among the bacterial communities in inlet and two aquarium tanks. Our results suggest that the bacterial community in the aquarium is the consequence of its own environmental factors (such as temperature or nutrients), not from the adjacent water body coming through inlets.

第一章 緒論 1

1.1 前言 1
1.2 海洋與微生物 2
1.3 養殖環境中的微生物 4
1.3.1 水產養殖環境中的微生物 4
1.3.2 水族館展示池環境中的微生物 7
1.4 宏基因體學(metagenomics)分析 9
1.4.1宏基因體學 9
1.4.2 可操作性分類單元(operational taxonomic units, OTUs) 10
1.5 次世代定序(next generation sequencing, NGS) 10
1.6 16S rRNA基因與細菌 11
1.7 指標微生物(indicator bacteria) 12
1.8 研究動機與目的 13

第二章 實驗材料與方法 17

2.1 藥品與儀器 17
2.1.1 藥品 17
2.1.2培養基 18
2.1.3培養基配製 18
2.1.4 實驗儀器 20
2.2樣本的採集與處理 20
2.2.1水質和指標微生物的採樣與分析 20
2.2.2 Metagenomics樣品採集與處理 21
2.2.3 展示水池組態與操作參數 21
2.3 16S rRNA序列鑑定 22
2.3.1 萃取海水樣本基因體DNA (genomic DNA) 22
2.3.2聚合酶連鎖反應(polymerase chain reaction, PCR) 23
2.3.3 DNA電泳分析 24
2.3.4 凝膠內DNA之純化 25
2.4 海水細菌群落宏基因體(metagenomics)定序分析 26
2.4.1萃取與純化海水細菌基因體DNA與Illumina定序 26
2.4.2 metagenomics序列生物資訊分析 26
2.4.3 OTUs分析 27
2.4.4稀釋曲線分析(rarefaction curve analysis) 27
2.4.5多樣性分析(diversity analysis) 28
2.4.6分類學分析(taxonomic analysis) 30

第三章 結果與討論 37

3.1展示池海水檢測 37
3.1.1水質檢測 37
3.1.2微生物檢測 38
3.2展示池海水宏基因體學分析 39
3.2.1 Raw read前處理 39
3.2.2 OTUs分析 39
3.2.3稀釋曲線分析 40
3.2.4多樣性分析 40
3.2.5分類學分析 41

第四章 結論與建議 63

參考文獻 65

附錄 77

Alekseyev, Y.O., Fazeli, R., Yang, S., Basran, R., Maher, T., Miller, N.S., and Remick, D. (2018) A next-generation sequencing primer—how does it work and what can it do? Academic Pathology 5: 1-11.

Ansorge, W.J. (2009) Next-generation DNA sequencing techniques. New Biotechnology 25: 195-203.

AWA (2017) Animal Welfare Act and animal welfare regulations: US Dept. of Agriculture, Animal and Plant Health Inspection Service.

Balter, M. (2012) Taking stock of the human microbiome and disease. Science 336: 1246-1247.

Bekaert, K., Devriese, L., Maes, S., and Robbens, J. (2015) Characterization of the dominant bacterial communities during storage of Norway lobster and Norway lobster tails (Nephrops norvegicus) based on 16S rDNA analysis by PCR-DGGE. Food Microbiology 46: 132-138.

Bentzon‐Tilia, M., Sonnenschein, E.C., and Gram, L. (2016) Monitoring and managing microbes in aquaculture–Towards a sustainable industry. Microbial Biotechnology 9: 576-584.

Blanch, A., Cerdà‐Cuéllar, M., and Hispano, C. (2001) Diversity of Vibrio spp. populations in several exhibition aquaria with a shared water supply. Letters in Applied Microbiology 33: 137-143.

Blanch, A., Van den Sande, P., Bulto, P., and Hispano, C. (1999) Health management in exhibition aquaria. Bulletin of the European Association of Fish Pathologists 19: 282-283.

Boaventura, C., Coelho, F., Martins, P., Pires, A., Duarte, L., Uetanabaro, A., Cleary, D., and Gomes, N. (2018) Micro-eukaryotic plankton diversity in an intensive aquaculture system for production of Scophthalmus maximus and Solea senegalensis. Aquaculture 490: 321-328.

Bowman, J.S., Rasmussen, S., Blom, N., Deming, J.W., Rysgaard, S., and Sicheritz-Ponten, T. (2012) Microbial community structure of Arctic multiyear sea ice and surface seawater by 454 sequencing of the 16S RNA gene. The ISME Journal 6: 11-20.

Breitbart, M. (2011) Marine viruses: truth or dare. Annual Review of Marine Science 4: 425-448.

Cabello, F.C., Godfrey, H.P., Tomova, A., Ivanova, L., Dölz, H., Millanao, A., and Buschmann, A.H. (2013) Antimicrobial use in aquaculture re‐examined: its relevance to antimicrobial resistance and to animal and human health. Environmental Microbiology 15: 1917-1942.

Chao, A. (1984) Nonparametric estimation of the number of classes in a population. Scandinavian Journal of Statistics 11: 265-270.

Chao, A., Hwang, W.-H., Chen, Y., and Kuo, C. (2000) Estimating the number of shared species in two communities. Statistica Sinica 10: 227-246.

Chao, A., and Lee, S.-M. (1992) Estimating the number of classes via sample coverage. Journal of the American Statistical Association 87: 210-217.

Coenye, T., and Vandamme, P. (2003) Intragenomic heterogeneity between multiple 16S ribosomal RNA operons in sequenced bacterial genomes. FEMS Microbiology Letters 228: 45-49.

Colwell, R., Brayton, P., Grimes, D., Roszak, D., Huq, S., and Palmer, L. (1985) Viable but non-culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms. Nature Biotechnology 3: 817-820.

Cottrell, M.T., and Kirchman, D.L. (2000) Community composition of marine bacterioplankton determined by 16S rRNA gene clone libraries and fluorescence in situ hybridization. Applied and Environmental Microbiology 66: 5116-5122.

Cruaud, P., Vigneron, A., Lucchetti-Miganeh, C., Ciron, P.E., Godfroy, A., and Cambon-Bonavita, M.-A. (2014) Influence of DNA extraction method, 16S rRNA targeted hypervariable regions, and sample origin on microbial diversity detected by 454 pyrosequencing in marine chemosynthetic ecosystems. Applied and Environmental Microbiology 80: 4626-4639.

Edgar, R.C. (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nature Methods 10: 996-998.

Edgar, R.C. (2018) Updating the 97% identity threshold for 16S ribosomal RNA OTUs. Bioinformatics: DOI: 10.1093/bioinformatics/bty1113.

Fan, L.M., Barry, K., Hu, G.D., long Meng, S., Song, C., Wu, W., Chen, J.Z., and Xu, P. (2016) Bacterioplankton community analysis in tilapia ponds by Illumina high-throughput sequencing. World Journal of Microbiology and Biotechnology 32: 1-11.

FAO (2009) The state of world fisheries and aquaculture. Rome, Italy: FAO, Electronic Publishing Policy and Support Branch, Communication Division.

Fernandes Da Silva, C., Ballester, E., Monserrat, J., Geracitano, L., Wasielesky Jr, W., and Abreu, P. (2008) Contribution of microorganisms to the biofilm nutritional quality: protein and lipid contents. Aquaculture Nutrition 14: 507-514.

Glibert, P.M., Landsberg, J.H., Evans, J.J., Al-Sarawi, M.A., Faraj, M., Al-Jarallah, M.A., Haywood, A., Ibrahem, S., Klesius, P., and Powell, C. (2002) A fish kill of massive proportion in Kuwait Bay, Arabian Gulf, 2001: the roles of bacterial disease, harmful algae, and eutrophication. Harmful Algae 1: 215-231.

Handelsman, J. (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiology and Molecular Biology Reviews 68: 669-685.

Hansen, G., and Olafsen, J. (1999) Bacterial interactions in early life stages of marine cold water fish. Microbial Ecology 38: 1-26.

Heck, K.L., van Belle, G., and Simberloff, D. (1975) Explicit calculation of the rarefaction diversity measurement and the determination of sufficient sample size. Ecology 56: 1459-1461.
Huchette, S.M., Koh, C., and Day, R.W. (2003) Growth of juvenile blacklip abalone (Haliotis rubra) in aquaculture tanks: effects of density and ammonia. Aquaculture 219: 457-470.

Hugerth, L.W., Muller, E.E., Hu, Y.O., Lebrun, L.A., Roume, H., Lundin, D., Wilmes, P., and Andersson, A.F. (2014) Systematic design of 18S rRNA gene primers for determining eukaryotic diversity in microbial consortia. PLoS One 9: e95567.

Hunter-Cevera, J., Karl, D., and Buckley, M. (2005) Marine microbial diversity: the key to earth’s habitability. San Francisco, California: American Academy of Microbiology.

Kim, Y., Van Bonn, W., Aw, T.G., and Rose, J.B. (2017) Aquarium viromes: viromes of human-managed aquatic systems. Frontiers in Microbiology 8: 1231.

Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, C., Horn, M., and Glöckner, F.O. (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Research 41: 1-11.

Kobiyama, A., Ikeo, K., Reza, M.S., Rashid, J., Yamada, Y., Ikeda, Y., Ikeda, D., Mizusawa, N., Sato, S., and Ogata, T. (2018) Metagenome-based diversity analyses suggest a strong locality signal for bacterial communities associated with oyster aquaculture farms in Ofunato Bay. Gene 665: 149-154.

Kolbert, C.P., and Persing, D.H. (1999) Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Current Opinion in Microbiology 2: 299-305.

Kozich, J.J., Westcott, S.L., Baxter, N.T., Highlander, S.K., and Schloss, P.D. (2013) Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and Environmental Microbiology 79: 5112-5120.

Lane, D.J., Pace, B., Olsen, G.J., Stahl, D.A., Sogin, M.L., and Pace, N.R. (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proceedings of the National Academy of Sciences of the United States of America 82: 6955-6959.

Leu, M.-Y., Li, J.-J., Ju, Y.-M., Hsiao, C.-M., Chang, C.-W., Meng, P.-J., Tew, K.S., and Wang, W.-H. (2015) Transportation, husbandary, and release of a whale shark (Rhincodon typus). Journal of Marine Science and Technology 23: 814-818.

Lin, J., Ju, B., Yao, Y., Lin, X., Xing, R., Teng, L., and Jiang, A. (2016) Microbial community in a multi-trophic aquaculture system of Apostichopus japonicus, Styela clava and microalgae. Aquaculture international 24: 1119-1140.

Liu, L., Li, Y., Li, S., Hu, N., He, Y., Pong, R., Lin, D., Lu, L., and Law, M. (2012) Comparison of next-generation sequencing systems. BioMed Research International 2012: 1-11.

Lušić, D.V., Kranjčević, L., Maćešić, S., Lušić, D., Jozić, S., Linšak, Ž., Bilajac, L., Grbčić, L., and Bilajac, N. (2017) Temporal variations analyses and predictive modeling of microbiological seawater quality. Water Research 119: 160-170.

Martínez‐Porchas, M., and Vargas‐Albores, F. (2017) Microbial metagenomics in aquaculture: a potential tool for a deeper insight into the activity. Reviews in Aquaculture 9: 42-56.

Moriarty, D.J. (1997) The role of microorganisms in aquaculture ponds. Aquaculture 151: 333-349.

Murray, A.G., and Peeler, E.J. (2005) A framework for understanding the potential for emerging diseases in aquaculture. Preventive Veterinary Medicine 67: 223-235.

Nayak, S. (2010) Probiotics and immunity: a fish perspective. Fish & Shellfish Immunology 29: 2-14.

Neave, M.J., Rachmawati, R., Xun, L., Michell, C.T., Bourne, D.G., Apprill, A., and Voolstra, C.R. (2017) Differential specificity between closely related corals and abundant Endozoicomonas endosymbionts across global scales. The ISME Journal 11: 186-200.

Not, F., del Campo, J., Balagué, V., de Vargas, C., and Massana, R. (2009) New insights into the diversity of marine picoeukaryotes. PLoS One 4: e7143.

Pabba, S.K., Samatha, B., Prasad, M.R., Nidadavolu, S.H., and Charya, M.S. (2017) Isolation and screening of marine bacteria for antimicrobial activity along Vishakapatanam Coast. Journal of Microbiology and Biotechnology Research 1: 86-89.

Patin, N.V., Pratte, Z.A., Regensburger, M., Hall, E., Gilde, K., Dove, A.D., and Stewart, F.J. (2018) Microbiome dynamics in a large artificial seawater aquarium. Applied and Environmental Microbiology: DOI : 10.1128/AEM.00179-00118.

Peeler, K.A., Opsahl, S.P., and Chanton, J.P. (2006) Tracking anthropogenic inputs using caffeine, indicator bacteria, and nutrients in rural freshwater and urban marine systems. Environmental Science & Technology 40: 7616-7622.

Portune, K.J., Pérez, M.C., Álvarez-Hornos, F.J., and Gabaldón, C. (2015) Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing. Applied Microbiology and Biotechnology 99: 3-18.

Qian, P.-Y., Wang, Y., Lee, O.O., Lau, S.C., Yang, J., Lafi, F.F., Al-Suwailem, A., and Wong, T.Y. (2011) Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing. The ISME Journal 5: 507-518.

Qin, Y., Hou, J., Deng, M., Liu, Q., Wu, C., Ji, Y., and He, X. (2016) Bacterial abundance and diversity in pond water supplied with different feeds. Scientific Reports 6: 35232.

Qiu, T., Liu, L., Gao, M., Zhang, L., Tursun, H., and Wang, X. (2016) Effects of solid-phase denitrification on the nitrate removal and bacterial community structure in recirculating aquaculture system. Biodegradation 27: 165-178.

Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., and Glöckner, F.O. (2012) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Research 41: D590-D596.

Raja, K., Fernando, O.J., Thavasi, R., Jayalaksmi, S., and Balasubramanian, T. (2006) Diversity of Bacterial Populations in Recirculating Marine Aquarium. Research Journal of Microbiology 1: 448-452.

Raja, K., Fernando, O.J., Thavasi, R., Jayalaksmi, S., and Balasubramanian, T. (2006) Diversity of bacterial populations in recirculating marine aquarium with different marine ornamental fishes. Research Journal of Microbiology 5: 1170-1174.

Reis-Filho, J.S. (2009) Next-generation sequencing. Breast Cancer Research 11: 12-19.

Resende, J.A., Silva, V.a.L., Cesar, D.E., Del'Duca, A., Fontes, C.O., and Diniz, C.G. (2015) Seasonal dynamics of microbial community in an aquaculture system for Nile tilapia (Oreochromis niloticus). Aquaculture Research 46: 1233-1240.

Revetta, R.P., Matlib, R.S., and Santo Domingo, J.W. (2011) 16S rRNA gene sequence analysis of drinking water using RNA and DNA extracts as targets for clone library development. Current Microbiology 63: 50-59.

Rodsæther, M.C., Olafsen, J., Raa, J., Myhre, K., and Steen, J.B. (1977) Copper as an initiating factor of vibriosis (Vibrio anguillarum) in eel (Anguilla anguilla). Journal of Fish Biology 10: 17-21.

Sakami, T., Abo, K., Takayanagi, K., and Toda, S. (2003) Effects of water mass exchange on bacterial communities in an aquaculture area during summer. Estuarine, Coastal and Shelf Science 56: 111-118.

Schloss, P.D., and Westcott, S.L. (2011) Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Applied and Environmental Microbiology 77: 3219-3226.

Schreier, H.J., Mirzoyan, N., and Saito, K. (2010) Microbial diversity of biological filters in recirculating aquaculture systems. Current Opinion in Biotechnology 21: 318-325.

Sepkoski, J.J. (1988) Alpha, beta, or gamma: where does all the diversity go? Paleobiology 14: 221-234.

Shannon, C.E., and Weaver, W. (1949) The mathematical theory of communication. University of Illinois Press 19: 1.

Simpson, E.H. (1949) Measurement of diversity. Nature 163: 688.

Sogin, M.L., Morrison, H.G., Huber, J.A., Welch, D.M., Huse, S.M., Neal, P.R., Arrieta, J.M., and Herndl, G.J. (2006) Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proceedings of the National Academy of Sciences of the United States of America 103: 12115-12120.

Sokal, R., and Sneath, P. (1963) Principles of numerical taxonomy San Francisco, California: W. H. Freeman and Company.

Spoon, T.R., and Romano, T.A. (2012) Neuroimmunological response of beluga whales (Delphinapterus leucas) to translocation and a novel social environment. Brain, Behavior, and Immunity 26: 122-131.

Streit, W.R., and Schmitz, R.A. (2004) Metagenomics– the key to the uncultured microbes. Current Opinion in Microbiology 7: 492-498.

Suttle, C.A. (2007) Marine viruses—major players in the global ecosystem. Nature Reviews Microbiology 5: 801-812.

Tew, K.S., Chang, Y.C., Meng, P.J., Leu, M.Y., and Glover, D.C. (2016) Towards sustainable exhibits–application of an inorganic fertilization method in coral reef fish larviculture in an aquarium. Aquaculture Research 47: 2748-2756.

Van Bonn, W., LaPointe, A., Gibbons, S.M., Frazier, A., Hampton‐Marcell, J., and Gilbert, J. (2015) Aquarium microbiome response to ninety‐percent system water change: Clues to microbiome management. Zoo Biology 34: 360-367.

Van Dijk, E.L., Auger, H., Jaszczyszyn, Y., and Thermes, C. (2014) Ten years of next-generation sequencing technology. Trends in Genetics 30: 418-426.

Venkat, H.K., Sahu, N.P., and Jain, K.K. (2004) Effect of feeding Lactobacillus‐based probiotics on the gut microflora, growth and survival of postlarvae of Macrobrachium rosenbergii (de Man). Aquaculture Research 35: 501-507.

Watson, M. (2014) Illuminating the future of DNA sequencing. Genome Biology 15: 108.

Weisburg, W.G., Barns, S.M., Pelletier, D.A., and Lane, D.J. (1991) 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173: 697-703.

Whitman, W.B., Coleman, D.C., and Wiebe, W.J. (1998) Prokaryotes: the unseen majority. Proceedings of the National Academy of Sciences of the United States of America 95: 6578-6583.

Whittaker, R.H. (1960) Vegetation of the Siskiyou mountains, Oregon and California. Ecological Monographs 30: 279-338.

Wik, T.E., Lindén, B.T., and Wramner, P.I. (2009) Integrated dynamic aquaculture and wastewater treatment modelling for recirculating aquaculture systems. Aquaculture 287: 361-370.

Yao, Z., Wang, A., Li, Y., Cai, Z., Lemaitre, B., and Zhang, H. (2016) The dual oxidase gene BdDuox regulates the intestinal bacterial community homeostasis of Bactrocera dorsalis. The ISME Journal 10: 1037-1050.

Zheng, X., Tang, J., Zhang, C., Qin, J., and Wang, Y. (2016) Bacterial composition, abundance and diversity in fish polyculture and mussel–fish integrated cultured ponds in China. Aquaculture Research 48: 3950-3961.

Zhou, T., Wang, Y., Tang, J., and Dai, Y. (2013) Bacterial communities in Chinese grass carp (Ctenopharyngodon idellus) farming ponds. Aquaculture Research 45: 138-149.

Zohar, Y., Tal, Y., Schreier, H.J., Steven, C.R., Stubblefield, J., and Place, A.R. (2005) Commercially feasible urban recirculating aquaculture: addressing the marine sector. Cambridge, Massachusetts: CABI Publishing.