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研究生:李京翰
研究生(外文):Ching-Han Lee
論文名稱:渦鞭毛藻Symbiodinium sp. 趨光性之分析及其在建立胞內共生中所扮演的角色
論文名稱(外文):The Microscopic Analysis of Symbiodinium Phototaxis: an implication for the recognition mechanism of coral endosymbiosis
指導教授:陳啟祥陳啟祥引用關係
指導教授(外文):Chii-Shiarng Chen
學位類別:碩士
校院名稱:國立東華大學
系所名稱:海洋生物科技研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
論文頁數:87
中文關鍵詞:共生藻珊瑚礁趨光性胞內共生
外文關鍵詞:Symbiodiniumcoral reefphototaxisendosymbiosis
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珊瑚與共生藻的胞內共生關係,雖然仍有許多機轉有待闡明,但能確定是其為維持整個珊瑚礁生態系統高生產力的重要基礎。珊瑚宿主與非共生狀態之共生藻之間的辨認及透過胞吞作用進入宿主內胚層細胞為建立共生的最初兩步驟。已有研究推測共生藻趨向某特定光照波長的特性可能和建立共生初期有關,但尚未完整確認。本篇研究利用顯微攝影分析,比較不同系群之共生藻在紫外線照後對特定波長及能量的螢光所表現有方向性的游動。結果顯示非共生型的共生藻系群E對波長470 ± 20nm,能量: 289.0 ± 32.6µW/cm2之藍光有表現出趨光性,在固定12小時黑暗/12小時光照的培養環境下,我們亦發現此趨光表現是受日夜週期調控,且只表現在白天。反觀其他四種有被記載可共生的系群皆不具趨光性。本篇研究證實透過趨光性已達到宿主和共生藻之間的辨認及後續的內化作用應是不可行的。
The coral-dinoflagellate endosymbiosis plays a pivotal role on maintaining the productivity of marine ecosystems. However the underlying its mechanism remains to be elucidated. The recognition between coral hosts and free-living Symbiodinium, followed by the internalization (or phagocytosis) of the latter into the host gastrodermal cell, are two initial steps that are required for this process. It has been suggested that free-living Symbiodinium demonstrate phototaxis towards specific irradiation wavelengths might be feasible. This hypothesis, however, remains to be conclusively validated. In the present study, a microscopic analysis was developed to measure the phototaxis in different Symbiodinium clades, and this system was based on directional cell mobility towards fluorescent lights of varying wavelengths after UV expulsion. The results show of these experiments revealed that the asymbiotic clade E Symbiodinium exhibits a positive phototaxis towards blue light (470±20 nm). Furthermore, this blue-light phototaxis is diel-regulated, and only occurs during the daytime for samples cultured under a 12:12 light:dark cycle. At the same irradiation energy (289.0±32.6 µW/cm2), all symbiotic Symbiodinium, including those of caldes A, B, C, and D, exhibited no phototaxis towards light irradiation across a spectrum of wavelengths. The present study provides partial evidence that the initial recognition-internalization process between the free-living Symbiodinium and coral hosts due to phototaxis may not be feasible.
中文摘要…………………………………………………………………………………………………………………………………………………………………I
ABSTRACT……………………………………………………………………………………………………………………………………………………………II
LIST OF TABLES AND FIGURES……………………………………………………………………………………………………………VI
LIST OF APPENDICES………………………………………………………………………………………………………………………………VII

1 INTRODUCTION
1-1 Phototaxis……………………………………………………………………………………………………………………………………1
1-2 Coral-dinoflagellate endosymbiosis……………………………………………………………………1
1-3 Coral sexual reproduction and Symbiodinium acquisition………………2
1-4 Cnidarian-Symbiodinium recognition……………………………………………………………………3
1-5 Goals of the proposed research………………………………………………………………………………4

2 MATERIALS AND METHODS
2-1 Symbiodinium culture
2-1.1 Experimetal samples……………………………………………………………………………………………………………5
2-1.2 Media preparation…………………………………………………………………………………………………………………5
2-1.3 Dinoflagellate washing……………………………………………………………………………………………………6
2-1.4 Culture conditions………………………………………………………………………………………………………………7
2-2 Cell motility measurements
2-2.1 Sample preparation………………………………………………………………………………………………………………7
2-2.2 Image acquisition…………………………………………………………………………………………………………………8
2-2.3 Image analysis…………………………………………………………………………………………………………………………8
2-3 Phototaxis after radiation exposure (PARE)
2-3.1 Sample preparation………………………………………………………………………………………………………………8
2-3.2 Video/image acquisition…………………………………………………………………………………………………8
2-3.3 Light intensity setting…………………………………………………………………………………………………9
2-3.4 Video/Image analysis………………………………………………………………………………………………………10
2-3.5 Statistical analysis………………………………………………………………………………………………………11
2-4 Symbiodinium clade determination
2-4.1 Sample preparation……………………………………………………………………………………………………………11
2-4.2 Polymerase chain reaction (PCR)…………………………………………………………………………12
2-4.3 Ligation and transformation (blue/white screening)………………………12
2-4.4 Sequencing…………………………………………………………………………………………………………………………………13

3 RESULTS
3-1 Cellular motility………………………………………………………………………………………………………………15
3-2 Effect of cell density on the recovery of clade E Symbiodinium…………………………………………………………………………………………………………………………………………………15
3-3 Cellular phototaxis to different light wavelengths in different Symbiodinium clades……………………………………………………………………………………………………16
3-4 Effects of UV irradiation on the recovery of Symbiodinium…………………………………………………………………………………………………………………………………………………17
3-5 Phototaxis without UV irradiation……………………………………………………………………17
3-6 The diel pattern of clade E Symbiodinium phototaxis……………………18
3-7 Movement trajectory during phototaxis in clade E Symbiodinium…………………………………………………………………………………………………………………………………………………18

4 DISCUSSION
4-1 The role of phototaxis in the establishment of endosymbiosis………………………………………………………………………………………………………………………………………………21
4-2 Experimental techniques………………………………………………………………………………………………22
4-3 The effect of UV irradiation on cell density………………………………………23
5 SUMMARY & CONCLUSIONS……………………………………………………………………………………………………25
6 REFERENCES…………………………………………………………………………………………………………………………………27
TABLES & FIGURES………………………………………………………………………………………………………………………………………37
APPENDICES………………………………………………………………………………………………………………………………………………………59




















LIST OF TABLES AND FIGURES
Page
Table 1. Composition of Symbiodinium culture media……………………………………………35
Table 2. Irradiation parameters of the phototaxis experiments………………35
Figure 1A. Phototaxis after radiation exposure (PARE)……………………………………38
Figure 1B. Phototaxis without UV irradiation……………………………………………………………38
Figure 1C. A simple flowchart of PARE………………………………………………………………………………39
Figure 1D. Estimation of Phototaxis……………………………………………………………………………………39
Figure 2A. The cellular motility assay……………………………………………………………………………42
Figure 2B. The diel pattern of cellular motility…………………………………………………42
Figure 2C. The diel pattern of clade E Symbiodinium phototaxis……………43
Figure 3A. Effect of UV irradiation on the recovery of Symbiodinium44
Figure 3B. Effect of cell density on the recovery of Symbiodinium……44
Figure 4. Cellular phototaxis at different light irradiation in different Symbiodinium clades……………………………………………………………………………………………………47
Figure 5. Phototaxis without UV irradiation………………………………………………………………51
Figure 6. Movement trajectory during phototaxis in clade E Symbiodinim……………………………………………………………………………………………………………………………………………………53
Figure 7. Chloroplast partial 23s-rDNA-based during the clade identification……………………………………………………………………………………………………………………………………………55
Figure 8. BLASTn results against the National Center for Biotechnology Information (NCBI)…………………………………………………………………………………………………………………………………57

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