臺灣博碩士論文加值系統

近年來白棘三列海膽因大量捕撈導致捕撈量逐年下，須利用養殖的手段提高產量，目前仍無研究探討其適合生長的光波長環境，且不同光環境對其腸道菌相的影響仍然未知。本次實驗首先利用不同波長 (白光:全光譜、紅光: 630 nm、藍光：450 nm)發光二極體 (LED)燈具來確認海膽養殖水體在不同波長光環境下含菌量的變化，以光照週期12小時光亮及12小時黑暗 (12L：12D)的條件持續照射7週後，630 nm組海膽的存活率最高且水體含菌量顯著大於全光譜組及450 nm組，再以次世代定序的方式將照射不同波長LED一週後的海膽腸道細菌透過16S rRNA上的V3-V4高度變異區進行總基因體分析，樣本內多樣性分析的結果顯示海膽照射630 nm的光線後腸道細菌的總物種量最多且演化上的分歧最少，照射450 nm後的海膽腸道細菌總物種量最少且演化上的分歧最高，再透過樣本間多樣性分析確認全光譜組與630 nm組的腸道菌相組成相近，而450 nm組的腸道菌相組成與其他組差異最大，最後透過不同波長LED照射的方式從飼養水體中篩選並分離出兩株對不同波長LED有不同生長反應的細菌Vibrio mediterranei與Vibrio hangzhouensis，且Vibrio mediterranei對不同光波長的反應在海膽腸道內也有同樣的趨勢。綜合上述結果顯示，以波長630 nm的LED飼養之海膽其環境水體與腸道內的細菌最豐富且穩定，此時海膽存活率最高；以波長450 nm的LED飼養之海膽其環境水體與腸道內的細菌最少且菌相改變劇烈，海膽存活率較低；全光譜飼養之海膽其水體含菌量介於上述兩組之間，腸道內菌相與630 nm組最相似，但由於此組內其中一優勢菌種Vibrio fortis為已知海膽病原菌，導致存活率較低。本次實驗證實630 nm的光環境最適合白棘三列海膽的養殖，能促進飼養水體及海膽腸道內菌相穩定有助於海膽存活。

In recent years, sea urchin Tripneustes gratilla has decreasing fishing amount due to over harvesting, aquaculture of T. gratilla seems to be a solution to support the insufficient production, but the effect of different light condition in T. gratilla rearing have not been well studied. In the current study, we analyzed the total bacteria number of three T. gratilla breeding tanks’ water under different light-emitting diodes (LED) (full spectrum, 630 nm, 450 nm) for seven weeks, group 630 nm has 3.4 x 104 colony forming unit per milliliter (cfu/ml), significantly higher than full spectrum group 0.9 x 104 cfu/ml and 450 nm group 0.4 x 104 cfu/ml, and the survival rate of group 630 nm is higher than full spectrum group and group 450 nm. To characterize the composition of T. gratilla gut microbiota under different wavelength of light, we analyzed the metagenomics of sea urchin gut by using 16S rRNA next generation sequencing (NGS) targeting the V3-V4 region. A total of 2,206 individual bacterial operational taxonomic unit (OTU) were identified, belonging to 121 family-, 205 genus- and 249 species level OTU group. Alpha diversity analyses based on observed species, phylogenetic diversity whole tree, Chao1 and Shannon indices, showed that the microbiota of sea urchin gut is more stable under 630 nm group. Beta diversity analyses (Principle coordinate analysis, PCoA) showed the 630 nm group and full spectrum group have the shortest Unifrac distance, suggest the similarity of these two groups. Using different light condition we separated two bacteria Vibrio mediterranei and Vibrio hangzhouensis, which have different growth rate under blue light and red light. These results have proved that the 630 nm LED is effective to T. gratilla aquaculture by stabilizing the microbiota of water and sea urchin gut.

目錄
謝辭 i
中文摘要 ii
Abstract iii
目錄 iv
表目錄 vi
圖目錄 vii
第一章、 前言 1
1.1 海膽與其養殖產業 1
1.2 發光二極體 (Light emitting diodes, LEDs)對水生生物之影響 2
1.3 腸道微生物 3
1.4 生物膜 4
1.5 消化道細菌多樣性研究 5
1.6 研究動機與實驗設計 6
第二章、 材料與方法 7
2.1 實驗動物處理與採樣 7
2.2 總菌數檢測 8
2.3 生物膜形成能力檢測 8
2.4 光敏感性細菌鑑定 9
2.5 海膽腸道細菌次世代定序 10
2.6 生物資訊分析 11
2.7 統計分析 12
第三章、 實驗結果 13
3.1 不同光波長影響海膽的生長情形 13
3.2 不同光波長造成海膽飼養水體總菌量與生物膜形成差異 13
3.3 不同光波長短期照射對海膽環境中與腸道內菌量的影響 14
3.4 光敏感性細菌的篩選 15
3.5 不同光波長影響海膽腸道內菌相：次世代定序結果 15
3.6 海膽腸道細菌樣本內多樣性分析 (α-diversity) 17
3.7 海膽腸道細菌樣本間多樣性分析 (β-diversity) 18
第四章、 討論 19
4.1 不同光波長對海膽生長的影響 20
4.2 不同光波長對養殖水體菌量的影響 20
4.3 不同光波長影響海膽腸道內菌相 22
第五章、 結論 25
參考文獻 26
附錄 33

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