海洋無脊椎動物體內普遍存在與細菌共生之現象，例如水母、珊瑚以及海綿。
海綿為一最簡單的多細胞生物，許多研究指出其體內通常都含有豐富且多樣的共
生細菌與之共生。文獻中指出澳洲球形海綿體內有 85%的細菌其 16S rRNA基因序
列的分析與生長在深海熱泉的底棲生物 Solemya reidi (雙殼類原鰓亞綱)體內之化
學自營性硫氧化共生細菌有高達 88.65%的相似度。本研究利用樹酯切片技術透過
光學顯微鏡、穿透式電子顯微鏡的影像初步觀察澳洲球形海綿組織細胞內部及表
面之微細結構，細胞內、外及中膠層中的確存在許多不同型態的共生細菌。進一
步利用文獻中針對優勢及次要化學自營性共生菌的 16S rRNA 和 RuBisCO 基因所
設計的不同專一性探針，以及針對澳洲球形海綿粒線體中的 COXI 基因所設計的
專一性探針進行以及螢光原位雜交技術，透過螢光顯微鏡觀察優勢化學自營性共
生菌以及粒線體的位置分布，再進一步利用免疫金定位法證實此化學自營性硫氧
化共生菌存在海綿組織中的變形原始細胞中，屬於穩定的細胞內共生。

Symbioses between chemoautotrophic bacteria and invertebrates are widespread in marine environments. Many jellyfishes, corals, and sponges often contain bacterial symbionts. Sponges are by far the simplest multicellular animals, which have been shown to harbor dense and diverse microbial communities within the intercellular matrix. The previous studies indicated that the 16S rRNA sequence of one dominant bacteria (85%) associated with sponge, Cinachyrella australiensis, has remarkable similarity (86.65%) with the symbiotic chemoautotrophic sulfur-oxidizing bacteria of the deep-sea hydrothermal vent mussel, Solemya reidi. The aim of this study is to investigate the ultrastructure and localization of the symbiotic bacteria in the C. australiensis by utilizing fluorescence in situ hybridization and immuno-gold labeling. Various morphological symbiotic bacteria in the C. australiensis were observed by electronicmicroscopical method (TEM). The specific probes designed based on 16S rRNA and RuBisCO gene were chosen to identify the localization of this chemoautotrophic sulfur-oxidizing bacteria within the mesoglial cell. The fluorescent and TEM image data demonstrated that the location of the chemoautotrophic sulfur-oxidizing bacteria is within amoeboid archaeocytes in the mesohyl, suggest that the presence of this symbiotic bacteria is stable .

謝辭 ................................................................................................................................... i
摘要 .................................................................................................................................. ii
Abstract .......................................................................................................................... iii
目次 ................................................................................................................................. iv
圖次 ................................................................................................................................. vi
表次 ................................................................................................................................ vii

壹、 緒論 ...................................................................................................................... 1
1 海綿簡介 .............................................................................................................. 1
2 海綿與微生物的共生關係 .................................................................................. 2
3 共生細菌的分布 .................................................................................................. 4
4 共生細菌的傳遞 .................................................................................................. 5
5 共生形式與構造-Bacteriocyte ............................................................................. 6
6 澳洲球形海綿共生菌的分布 .............................................................................. 9
貳、 材料與方法 ........................................................................................................ 12
1. 樣品來源及採集 ................................................................................................ 12
2 海綿組織液高壓冷凍法 .................................................................................... 12
3. DNA探針 (DNA probe) ................................................................................... 13
4 螢光原位雜合法 (fluorescence in situ hybridization (FISH)) .......................... 15
5 穿透式電子顯微鏡 (transmission electron microscopy) .................................. 16
6 免疫金標定法 (immuno-gold labeling) ............................................................ 17 v

參、 結果 .................................................................................................................... 19
1 澳洲球形海綿的形態及分布 ............................................................................ 19
2 澳洲球形海綿細胞型態學觀察 (光學顯微鏡) ............................................... 19
3 螢光原位雜合 (fluorescence in situ hybridization (FISH)) .............................. 20
4 穿透式電子顯微鏡 (transmission electron microscopy (TEM)) ...................... 22
5 免疫金定位 (immuno-gold labeling) ................................................................ 24
肆、 討論 .................................................................................................................... 25
伍、 參考文獻 ............................................................................................................ 30
陸、 附錄 .................................................................................................................... 74

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