在海洋環境中，共生現象普遍存在，許多如珊瑚、海綿等海洋無脊椎生物體內都有細菌共生的情形。澳洲球形海綿共生細菌初步研究，依據16S rRNA基因序列的分析結果，其中有85%的細菌，和深海熱泉環境中雙殼類原鰓目Solemya reidi的化學自營性硫氧化共生細菌有很高的相似度 (88.65%) 。本研究利用PCR增幅澳洲球形海綿中自營性共生細菌的RubisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase)基因，並以其序列來研究海綿中自營性共生細菌的親緣關係，以及藉由螢光原位雜化 (FISH) 探討化學自營性共生細菌在海綿中的分布。依據經由PCR從海綿放大出的RubisCO基因序列，從隨機挑選出的26株選殖株中，大致可分成二群，其中9個選殖株其RubisCO基因屬於真核藻類及藍綠細菌所持有的green-like type IB的型式，推測可能是進行PCR時亦將海綿共生藻類及附著於海綿表面藻類的RubisCO基因同時增幅出來所得到的；而另一群 (17個選殖株) 的RubisCO基因序列皆非常相似，屬於化學性共生細菌所持有的green-like typeIA的型式，證實澳洲球形海綿中確實含有化學自營性共生細菌。進一步研究，利用螢光原位雜化，則觀察到化學自營性硫氧化共生細菌存在於海綿中質層的海綿細胞中，屬共生關係中細胞內共生的形式。

Symbiosis is commonly present in marine invertebrates. Many corals and sponges have symbiotic algae or bacteria. In the previous studies of the sponge Cinachyra australiensis, 85% of the bacteria associated with the sponge have high similarity (88.65%) with the symbiotic chemoautotrophic sulfur-oxidizing bacteria of the deep-sea hydrothermal vent mussel, Solemya reidi. This study aims to investigate the localization of the chemoautotrophic sulfur-oxidizing bacteria associated with Cinachyra australiensis. The Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) large-subunit genes for autotrophic organisms were amplified by polymerase chain reaction from the sponge samples. The phylogenetic relationship of the RubisCO large subunit genes was analyzed. A total of 26 clones were selected and sequenced. They could be divided into two groups. One (9 clones) belongs to form I type IB (cynobacteria and green algae). The other (17 clones) belongs to form II type IA (chemoautotrophic symbiotic bacteria). The location of the sulfur-oxidizing chemoautotrophic bacteria was shown to be intracellular symbiosis within the mesoglial cells by fluorescence in situ hybridization.

ㄧ、 前言••••••••••••••1

二、 材料方法••••••••••••11

三、 結果••••••••••••••29

四、 討論••••••••••••••44

五、 參考文獻••••••••••••52

Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410.

Amann, R., W. Ludwig, and K. H. Schleifer. 1995. Phylogenetic
identification and in situ detection of individual microbial cells without
cultivation. Microbiol. Rev. 59:143–169.

Beer, S., and M. Ilan. 1998. In situ measurements of photosynthetic irradiance responses of two Red Sea sponges growing under dim light conditions. Mar. Biol. 131:613–617.

Bergquist, P. R. 1978. Sponges. University of California Press, Berkeley.

Bewley, C. A., N. D. Holland, and D. J. Faulkner. 1996. Two classes of
metabolites from Theonella swinhoei are localized in distinct populations of bacterial symbionts. Experientia 52:716–722.

Bradford, M. M. 1976. A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

Brock, D. T., M. T. Madigan, J. M. Martinko, and J. Parker. 1994. Biology of microorganisms, 7th edn. p. 85-86, 645-647, 748-749. Prentice-Hall, Inc., Englewood Cliffs, New Jersey.

Brusca, R. C., and G. J. Brusca. 1990. Phylum Porifera: the sponges, p.
181–210. In A. D. Sinauer (ed.), Invertebrates. Sinauer Press, Sunderland,
Mass.

Campbell, N. A. 1996. Biology, 4th edn. p. 513, 810, 720-722. Benjamin/Cummings, Inc., Menlo Park, California.

Chase, M. W. 1993. Phylogenetics of seed plants: an analysis of nucleotide sequences from the plastid gene rbcL. Ann. Mo. Bot. Gard. 80:528–580.

Curtis, S. E., and R. Haselkorn. 1983. Isolation and sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase from the cyanobacterium Anabaena 7120. Proc. Natl. Acad. Sci. USA 80:1835–1839.

Distel, D. L., D. J. Lane, G. J. Olsen, S. J. Giovannoni, B. Pace, N. R. Pace, D. A. Stahl, and H. felbeck. 1988. Suifur-Oxidizing bacterial endosymbiotics analysis of phylogeny and specificity by l6S rRNA sequences. J. Bacteriol. 170:2506–2510.

Eisen, J. A., S. W. Smith, and C. M. Cavanaugh. 1992. Phylogenetic relationships of Solemya velum Say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis. J. Bacteriol. 174:3416–3421.

Elyakov, G.B., T. Kuznetsova, V. V. Mikhailov, I. I. Maltsev, V. G. Voinov, and S. A. Fedoreyev. 1991. Brominated diphenyl ethers from a marine bacterium associated with the spong Dysidea sp. Experientia 47:632-633.

Faulkner, D. J., HY. He, MD. Unson, and C. A. Bewley. 1993. New metabolites from marine sponges: are symbionts important? Gazz. Chim. Ital. 123: 301–307.

Felsenstein, J. 1985. Confidence limits on phylogenies: a approach using
the bootstrape. Evolution. 39: 783-791.

Friedrich, A. B., H. Merkert, T. Fendert, J. Hacker, P. Proksch, and U. Hentschel. 1999. Microbial diversity in the marine sponge Aplysina cavernicola (formerly Verongia cavernicola) analyzed by fluorescence in situ hybridization (FISH). Mar. Biol. 134:461–470.

Friedrich, A. B., I. Fischer, P. Proksch, J. Hacker, and U. Hentschel. 2001. Temporal variation of the microbial community associated with the Mediterraneansponge Aplysina aerophoba. FEMS Microbiol. Ecol. 38:105–113.

Giovannoni, S. J., and M. S. Rappe. 2000. Evolution, diversity and molecular ecology of marine prokaryotes, p. 47–84. In D. L. Kirchman (ed.) Microbial ecology of the ocean. John Wiley & Sons, Inc., New York, N.Y.

Giovannoni, S. J., T. B. Britschgi, C. L. Moyer, and K. G. Field. 1990.
Genetic diversity in Sargasso Sea bacterioplankton. Nature 345:60–63.

Hadzi, J. 1963. The Evolution of the Metazoa I–XII, 1st edn. Pergamon
Press, Oxford.

Hosam, E., and N. Takeshi. 2001. Phylogenetic diversity of Ribulase-1,5-bisphosphate carboxylase/oxygenase large-subunit genes from deep-sea microorganisms. Appl. Environ. Microbiol. 67:1751-1765

Jordan, D. B., and W. L. Ogren. 1981. Species variation in the specificity of ribulose bisphosphate carboxylase/oxygenase. Nature 291:513–515.

Jordan, D. B., and W. L. Ogren. 1983. Species variation in kinetic properties of ribulose 1,5-bisphosphate carboxylase/oxygenase. Arch. Biochem. Biophys. 227:425–433.

Jukes, T. H. and C. R. Cantor. 1969. Evolution of protein molecules. In: H. N. Munro. (ed). Mammalian protein metabolism. Vol. 3. Academic Press., Inc. New York.

Kellogg, E. A., and N. D. Juliano. 1997. The structure and function of
RuBisCO and their implications for systematic studies. Am. J. Bot. 84:413–428.

Kusano, T., T. Takeshima, C. Inoue, and K. Sugawara. 1991. Evidence for two sets of structural genes coding for ribulose bisphosphate carboxylase in Thiobacillus ferrooxidans. J. Bacteriol. 173:7313–7323.

Kusian, B., and B. Bowien. 1997. Organization and regulation of cbb CO2 genes in assimilation autotrophic bacteria. FEMS Microbiol. Rev. 21:135–155.

Maeda, N., K. Kitano, T. Fukui, S. Ezaki, H. Atomi, K. Miki, and T.
Imanaka. 1999. Ribulose 1,5-bisphosphate carboxylase/oxygenase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 is composed solely of large subunits and forms a pentagonal structure. J. Mol. Biol. 293:57–66.

Manz, W., G. Arp, G. Schumann-Kindel, U. Szewzyk, and J. Reitner. 2000. Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue. J. Microbiol. Methods. 40:125-134

Margot, H., C. Acebal, E. Toril, R. Amils, and J.L. Fernandez Puentes. 2002. Consistent association of crenarchaeal Archaea with sponges of the genus Axinella. Mar. Biol. 140:739-745.

McFadden, B. A., and F. R. Tabita. 1974. D-ribulose-1,5-diphosphate carboxylase and the evolution of autotrophy. BioSystems 6:93–112.

McFadden, B. A., J. Torres-Ruiz, H. Daniell, and G. Sarojini. 1986. Interaction, functional relations and evolution of large and small subunits in RuBisCO from prokaryota and eukaryota. Philos. Trans. R. Soc. Lond. Biol. Sci. 313:347–358.

Miziorko, H. M., and G. H. Lorimer. 1983. Ribulose-1,5-bisphophate carboxylase-oxygenase. Annu. Rev. Biochem. 52:507–535.

Nargang, F. E., L. Mcintosh, and C. Somerville. 1984. Nucleotide sequence of the ribulose bisphosphate carboxylase gene from Rhodospirillum rubrum. Mol. Gen. Genet. 193:220–224.

Nishihara, H., T. Yagushi, S. Y. Chung, K. Suzuki, M. Yanagi, K. Yamasato, T. Kodama, and Y. Igarashi. 1998. Phylogenetic position of an obligatory chemoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus, on the basis of 16S rRNA gene sequences and two form I RuBisCO gene sequences. Arch. Microbiol. 169:364–368.

Pechenik, A. J. 1996. Biology of the invertebrates, 3rd edn. p. 65-79. McGraw-Hill, Inc., New York, N.Y.

Peruski Jr, L. F. and A. H. Peruski. 1997. Analysis of aequence data, p.206~252. In: Peruski Jr, L. F. and A. H. Peruski (ed.). The Internet and the new biology. American Society for Microbiology. Washington, D.C..

Rappe, M. S., D. A. Gordon, K. Vergin, and S. J. Giovannoni. 1999. Phylogeny of actinobacteria small subunit rRNA (SSU rRNA) gene clones recovered from diverse sea water samples. Syst. Appl. Microbiol. 22:106–112.

Rappe, M. S., K. Vergin, and S. J. Giovannoni. 2000. Phylogenetic comparisons of a coastal bacterioplankton community with its counterparts in open ocean and freshwater systems. FEMS Microbiol. Ecol. 33:219–232.

Reiswig, H. 1974. Water transport, respiration and energetics of three tropical marine sponges. J. Exp. Mar. Biol. Ecol. 14:231–249.

Robinson, J., J. Stein, and C. Cavanaugh. 1998. Cloning and sequencing of a form II ribulose-bisphosphate carboxylase/oxygenase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila. J. Bacteriol. 180:1596–1599.

Rowan, R., and D. A. Powers. 1991b. A molecular genetic classification of zooxanthellae and the evolution of animal-algal symbioses. Science. 251:1348–1351.

Rowan, R., and D. A. Powers. 1992. Ribosomal RNA sequences amd the diversity of symbiotic dinoflagellates (zooxanthellae) . Proc. Natl. Acad. Sci. USA. 89:3639–3643.

Rutzler, K. 1985. Associations between Caribbean sponges and photosynthetic organisms. In: Rutzler K (ed) New perspectives in sponge biology. Smithsonian Institution Press, Washington DC, pp 455–466.

Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new method for construction phylogenetic trees. Mol. Biol. Evol. 4:406-425.

Sambrook, V. et al., 1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory.NY.

Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-termination inhibitors. Rroc. Natl. Acad. Sci. U.S.A. 74:5463–5467.

Santavy, D. L., P. Willenz, and R. R. Colwell. 1990. Phenotypic study of bacteria associated with the Caribbean sclero-sponge, Ceratoporella nicholsoni. Appl. Environ. Microbiol. 56:1750–1762.

Sara, M., L. Liaci. 1964. Symbiotic association between zooxan-thellae and Cliona. Nature 203: 321–323.

Sara, M. 1971. Ultrastructural aspects of the symbiosis between two
species of the genus Aphanocapsa (Cyanophyceae) and Ircinia variabilis
(Demospongiae). Mar. Biol. 11:214–221.

Schmidt, E. W., C. A. Bewley, and D. J. Faulkner. 1998. Theopalauamide, a bicyclic glycopeptide from filamentous bacterial symbiosis of the lithistid sponge Theonella swinhoei from Palau and Mozambique. J. Org. Chem. 63: 1254–1258.

Schmidt, E. W., A. Y. Obraztova, S. K. Davidson, D. J. Faulkner, and M. G. Haygood. 2000. Identification of the antifungal peptide-containing symbiont of the marine sponge Theonella swinhoei as a novel Delta-Proteobacterium Candidatus Entotheonella palauensis. Mar. Biol. 136:969–977.

Shimada, A., S. Kanai, and T. Maruyama. 1995. Partial sequence of ribulose bisphosphate carboxylase/oxygenase and the phylogeny of Prochloron and Prochlorococcus (Prochlorales). J. Mol. Evol. 40:671–677.

Shinozaki, K., C. Yamada, N. Takahata, and M. Sugiura. 1983. Molecular cloning and sequence analysis of the cyanobacterial gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc. Natl. Acad. Sci. USA 80:4050–4054.

Shively, J. M., W. Devore, L. Stratford, L. Porter, L. Medlin, and S. E. Stevens. 1986. Molecular evolution of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). FEMS Microbiol. Lett. 37:251–257.
Stein, J., M. Haygood, and H. Felbeck. 1990. Nucleotide sequence and
expression of a deep-sea ribulose-1,5-bisphosphate carboxylase gene cloned from a chemoautotrophic bacterial endosymbiont. Proc. Natl. Acad. Sci. USA 87:8850–8854.

Stierle, A. C., J. H. I. Cardellina, and F. L. Singleton. 1988. A marine Micrococcus produces metabolites ascribed to the sponge Tedania ignis. Experientia 44:1021.

Tabita, F. R. 1988. Molecular and cellular regulation of autotrophic carbon dioxide fixation in microorganisms. Microbiol. Rev. 52:155–189.

Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through aequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic. Acids. Research. 22:4673-4680.

Unson, M. D., N. D. Holland, and D. J. Faulkner. 1994. A brominated
secondary metabolite synthesized by the cyanobacterial symbiont of a marine sponge and accumulation of the crystalline metabolite in the sponge tissue. Mar. Biol. 119:1–11.

Ute H., M. Schmid, M. Wagner, L. Fieseler, C. Gernert, and J. Hacker. 2001. Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola. FEMS. Microb. Ecol. 35:305-312

Ute, H., H. Jorn, H. Matthias, F. B. Anja, W. Michael, H. Jorg, and M. S. Bradley. 2002. Molecular evidence for a uniform microbial community in sponges from different oceans. Appl. Environ. Microbiol. 56:1750-1762.

Vacelet, J. 1971. Etude en microscopie electronique de l'association
entre une cyanophycee chroococcale et une eponge du genre Verongia. J. Microscopie 12: 363–380.

Vacelet, J. 1975. Etude en microscopie e´lectronique de l’association entre bacte´ries et spongiaires du genre Verongia (Dictyoceratida). J. Microsc. Biol. Cell. 23:271–288.

Vacelet, J., and C. Donadey. 1977. Electron microscope study of the association between some sponges and bacteria. J. Exp. Mar. Ecol. 30:301–314.

Vacelet, J., N. Boury-Esnault, A. Fiala-Medioni, and C. R. Fisher. 1995. A methanotrophic carnivorous sponge. Nature 377:296.

Vogel, S. 1977. Current-induced flow through living sponges in nature. Proc. Nat. Acad. Sci. USA 74:2069–2071.

Watson, G. M. F., and F. R. Tabita. 1996. Regulation, unique gene organization, and unusual primary structure of carbon fixation genes from a marine phycoerythrin-containing cyanobacterium. Plant. Mol. Biol. 32:1103–1115.

Watson, G. M. F., and F. R. Tabita. 1997. Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: a molecule for phylogenetic and enzymological investigation. FEMS Microbiol. Lett. 146:13–22.
Watson, G. M. F., J. Yu, and F. R. Tabita. 1999. Unusual ribulose 1,5-
bisphosphate carboxylase/oxygenase of anoxic archaea. J. Bacteriol. 181:
1569–1575.

Webster, N. S., and R. T. Hill. 2001. The culturable microbial community of the Great Barrier Reef sponge Rhopaloeides odorabile is dominated by a α–proteobacterium. Mar. Biol. 138:843–851.

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

Wilkinson, C. R. 1978a. Microbial associations in sponges. I. Ecology, physiology and microbial populations of coral reef sponges. Mar. Biol. 49:161–167.

Wilkinson, C. R. 1978b. Microbial associations in sponges. II. Numerical analysis of sponge and water bacterial populations. Mar. Biol. 49:169–176.

Wilkinson, C. R. 1978c. Microbial associations in sponges. III. Ul-
trastructure of the in situ associations in coral reef sponges. Mar. Biol. 49: 177–185.

Wilkinson, C. R. 1984. Immunological evidence for the Precambrian origin of bacterial symbioses in marine sponges. Proc. R. Soc. B 220: 509–517.

Wilkinson, C. R. 1992. Symbiotic interactions between marine sponges and algae. In: Algae and symbioses. Biopress, Bristol, pp 112–151.

Wilkinson, C. R., P. Fay. 1979. Nitrogen fixation in coral reef sponges with symbiotic cyanobacteria. Nature 279:527–529.

Wilkinson, C. R., M. Nowak, B. Austin, and R. R. Colwell. 1981. Specificity of bacterial symbionts in Mediterranean and Great Barrier Reef sponges. Microb. Ecol. 7:13–21.

Wilkinson, C. R., G. Garrone, and J. Vacelet. 1984. Marine sponges discriminate between food bacteria and bacterial symbionts: electron microscope radioautography and in situ evidence. Proc. R. Soc. Lond. B 220:519–528.

Woese, C. R. 1987. Bacterial evolution. Microbiol. rev. 51:221–271.

Yagushi, T., S. Y. Chung, Y. Igarashi, and T. Kodama. 1994. Cloning and sequence of the L2 form of RuBisCO from a marine obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110. Biosci. Biotech. Biochem. 58:1733–1737.

Yatsu, M., T. Yamazaki, Y. Meguro, A. Endo, and M. Murata. 1987. Production of tetradotoxin and its derivatives by Pseudomonas sp. Isolated from the skin of a pufferfish. Toxicon. 25: 225–228.

王憶凱，1996。澳洲球形海綿共生細菌之研究。國立中山大學海洋資源研究所。碩士論文。

方力行，1989。珊瑚學∼兼論台灣的珊瑚資源。黎明文化事業有限公司。台北。

杜啟安，1993。組織病理技術手冊。國興出版社。新竹。

陳勇輝，1988。澳洲球形海綿芽體與成體形態之研究。國立中山大學海洋生物研究所。碩士論文。