臺灣博碩士論文加值系統

疾病為水產養殖產業所面臨的重要問題，傳統的治療方法容易產生藥物殘留或抗藥性，對環境及食安造成影響。若能利用生物防治則可以免除這些問題，但目前水產養殖的生物防治大多以被動式抑制病原的方式為主，缺乏有效主動減少病原菌的方式。本研究擬利用具有攝食細菌特性的寡毛綱急游屬纖毛蟲 (Oligotrichea)，評估將此生物應用於水產養殖病原弧菌防治的可行性。在目前已建立的 Strombidium parasulcatum 穩定培養條件下，利用生米加海水的培養基，可將纖毛蟲在 18 天內由 1隻/mL 增加 500 倍，達到 500 隻/mL 。實驗證明 Strombidium parasulcatum 實際上是以附著在生米上的 Erwinia 與 Kluyvera 等細菌為食物。而進一步的實驗也證實除了上述兩種細菌之外，大腸桿菌以及病原弧菌等也是 Strombidium parasulcatum 可攝食的對象。我們也以 EGFP 螢光轉殖大腸桿菌測試，發現單隻 Strombidium parasulcatum 移除細菌的平均速率約為 241 cells/hr。而為了未來實務上的應用，我們也嘗試使用其他培養條件，提升 Strombidium parasulcatum 的養殖密度。例如我們改用餵食 Erwinia 菌所製備的培養基，可將纖毛蟲的飽和濃度提高 20 倍，達到約 10000 隻/mL。由上述的初步實驗顯示，以 Strombidium parasulcatum 作為養殖病原菌的生物防治是可行的。我們將繼續進行動物實驗及田野實驗，期望能以更環保的方式解決水產養殖的問題。

Disease control is an important issue in aquaculture. Because of bad management, traditional treatments, such as drugs, may have residues and resistance problems that will cause impact on food security and environment. On the other hand, biological control can relieve these problems. However, current biological control methods are passive and inefficient. Here, we used Strombidium parasulcatum, an oligotrich ciliate, to ingest pathogenic bacteria and evaluates the feasibility of biological control with ciliates in aquaculture. Using the raw rice-seawater medium, Strombidium parasulcatum could ingest bacteria, such as Erwinia and Kluyvera, grown on the raw rice and increased its cell number 500-fold (from 1 cell/ mL to 500 cells/ mL) in 18 days. Experiments showed that bacteria, Erwinia and Kluyvera, attached to the raw rice are food of Strombidium parasulcatum Further, pathogenic Vibrio and E.coli were also used to culture Strombidium parasulcatum By using EGFP-expressing E. coli, the forage rate of Strombidium parasulcatum was estimated as 241 E.coli / hr. Adjustments of culture medium to Erwinia may further increase the maximum cell density of Strombidium parasulcatum to 20 folds (10000 cells/mL) that is enough for practical applications. Although the animal tests and field experiments are still ongoing, our preliminary experiments showed that Strombidium parasulcatum is a high potential biological control agent for aquaculture disease. We will continue to conduct animal experiments and field experiments and to solve the problem of aquaculture disease in an environmentally friendly way.

目錄
謝誌 II
Abstract VII
摘要 VIII
1.1 水產養殖的發展價值與重要性 1
1.2 水產養殖面臨的疾病問題 2
1.2.1疾病對水產養殖的影響 2
1.2.2 水產養殖的細菌性疾病 3
1.2.3 水產養殖上重要的細菌性病原菌: 弧菌 (Vibrio) 4
1.2.4 水產養殖對細菌性疾病的防治方法與問題 6
1.3 生物防治的應用 7
1.3.1 生物防治用於水產養殖 7
1.3.2 纖毛蟲用於生物防治的可行性 8
1.4 海洋微型浮遊動物: 寡毛綱纖毛蟲 (Oligotrichea) 9
1.4.1 寡毛綱纖毛蟲在海洋的生態地位 9
1.4.2 寡毛綱 (Oligotrich) 纖毛蟲的培養 10
1.5 研究方法與動機 11
貳、材料與方法 12
2.1 寡毛綱纖毛蟲 Strombidium parasulcatum 實驗方法 12
2.1.1 纖毛蟲品種 12
2.1.2 Strombidium parasulcatum 培養與保存 12
2.1.2.1 人工海水製備 12
2.1.2.2 無菌人工海水製備 13
2.1.2.3 毛細管吸蟲法 14
2.1.2.4 米粒培養基培養法 15
2.1.2.5 3% NaCl TSB 製備 15
2.1.2.6 3% NaCl TSB 平板製備 16
2.1.2.7 餌料菌分離與鑑定 17
2.1.2.8 菌種保存 17
2.1.2.9 冷凍菌餌料製備 18
2.1.3 Strombidium parasulcatum 觀察與計數 18
2.1.3.1 Lugol’s 固定液製備 18
2.1.3.2 Strombidium parasulcatum 低濃度計數法 19
2.1.3.3 Strombidium parasulcatum 高濃度計數法 19
2.1.3.4 Strombidium parasulcatum 纖毛蟲清洗法 20
2.1.3.6 Strombidium parasulcatum 之免疫螢光染色 20
2.1.4 Strombidium parasulcatum 攝食細菌實驗 23
2.1.4.1 eGFP-E.coli 攝食測試 23
2.1.4.2 病源弧菌攝食測試 23
2.1.5 Strombidium parasulcatum 魚病測試實驗 24
2.1.5.1 弧菌攻毒實驗 (浸泡法) 24
2.1.5.1 弧菌攻毒實驗 (含Strombidium parasulcatum) 24
2.2 分子生物學方法 - eGFP-E.coli 製備 25
2.2.1 聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 25
2.2.2 DNA 洋菜膠體電泳 26
2.2.3 DNA電泳膠純化 26
2.2.4 限制酶剪切 27
2.2.5 質體接合重組 27
2.2.6 洋菜培養基製備法 28
2.2.7 勝任細胞製備與轉型 28
2.2.8 小量質體抽取 29
參、研究結果 30
3.1 Strombidium parasulcatum 培養方式的改良 30
3.1.1 Strombidium parasulcatum 米粒培養基培養法 30
3.1.2 Strombidium parasulcatum 餌料菌的選擇與分離 30
3.1.3 Erwinia 餌料菌的培養測試 31
3.1.4 餌料菌的保存 31
3.2 Strombidium parasulcatum 攝食細菌的情形與消化率 32
3.2.1 Strombidium parasulcatum 攝食不同細菌對其生長的影響 32
3.2.2 以 eGFP-E.coli 來觀察細菌在纖毛蟲體內的消化情形 32
3.3 Strombidium parasulcatum 防治弧菌感染的效果 33
3.3.1 Strombidium parasulcatum 攝食弧菌 33
3.3.2生物防治測試 34
3.3.3 Strombidium parasulcatum 治療弧菌感染測試 34
肆、討論及未來方向 49
4.1 Strombidium parasulcatum 培養方式的改良 49
4.2 Strombidium parasulcatum 應用於弧菌生物防治的可行性 49
4.3 Strombidium parasulcatum 作為海水仔魚餌料生物的潛力 49
伍、參考文獻 51

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