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研究生: 高悅慈
Kao, Yueh-Tzu
論文名稱: 探討三氟敏 (Trifloxystrobin) 對斑馬魚胚胎離子調節、循環系統和感覺行為之影響
Effects of trifloxystrobin on ion regulation, circulatory system and sensory behavior of zebrafish embryos
指導教授: 林豊益
Lin, Li-Yih
口試委員: 林豊益
Lin, Li-Yih
洪君琳
Horng, Jiun-Lin
周銘翊
Chou, Ming-Yi
口試日期: 2024/06/18
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2024
畢業學年度: 112
語文別: 中文
論文頁數: 77
中文關鍵詞: 斑馬魚三氟敏基因組學感覺運動反應
英文關鍵詞: Zebrafish, Trifloxystrobin, Transcriptomics, Sensorimotor responses
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202400768
論文種類: 學術論文
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  • 三氟敏是一種被廣泛使用的甲氧基丙烯酸酯類除真菌劑。在三氟敏的過度使用下,已有研究證明其在環境中的殘留及對水生生物的風險。為了全面了解三氟敏對非目標生物的毒性作用及機制,本研究選擇斑馬魚作為實驗動物,探討其對胚胎之致死性、離子調節功能、感覺系統、循環系統及基因表達的影響。實驗將受精後4小時的胚胎暴露於0、100、200和300 ppb的三氟敏溶液中,持續96或120小時。結果顯示,濃度高於200 ppb會降低胚胎存活率和孵化率。同時,離子細胞數量及胚胎體內的Na+、K+和Ca2+水平顯著減少,表明其對離子調節功能產生了負面影響。在循環系統,除了圍心腔水腫、和心率降低,還造成了心室收縮力增加、心室血液逆流和血流流速變慢等心血管損傷。此外,本研究還揭示了三氟敏的感覺系統毒性,其不僅導致側線毛細胞數量減少,眼睛、耳石和耳石囊變小,通過行為反應實驗,還證實了側線感覺、聽覺、視覺和嗅覺功能亦受到損害。綜上所述,本研究闡明了三氟敏對斑馬魚胚胎的多種毒性作用,並透過RNA定序,從基因層面對其可能機制進行探討。

    Trifloxystrobin is a widely used strobilurin fungicide. Due to overuse, residues in the environment and risks to aquatic organisms have been identified. In order to comprehensively understand the toxic effects and mechanisms of trifloxystrobin on non-target organisms, zebrafish were selected as experimental animals to investigate basic toxicity, ion regulation, sensory system, circulatory system, and gene expression in this study. In the experiment, embryos were initially exposed at 4 hours post-fertilization to trifloxystrobin solutions at 0, 100, 200, and 300 ppb for 96 or 120 hours. The results showed that concentrations above 200 ppb decreased survival and hatchability. It also reduced the number of ionocytes and the levels of Na+, K+, and Ca2+ in the embryos, indicating a negative effect on ion regulatory function. In the sensory system, a reduction in lateral line hair cells and in the size of eyes, otoliths and otic vesicles was observed. Subsequent behavioral tests confirmed the impairment of lateral line sensory, auditory, visual and olfactory functions by trifloxystrobin. Furthermore, in addition to pericardial edema and decreased heart rate, it also causes cardiovascular damage such as increased ventricular contractility, ventricular blood reflux, and slowed blood flow velocity. In conclusion, this study clarified the various toxic effects of trifloxystrobin on zebrafish embryos and explored its possible mechanisms at the genetic level through RNA sequencing.

    摘要 iv Abstract vi 研究背景 1 三氟敏 (Trifloxystrobin, TFS) 介紹 1 三氟敏對環境與生物的風險 1 模式動物斑馬魚與三氟敏對其之毒性影響 2 斑馬魚的心血管循環系統 4 斑馬魚的離子調節 5 斑馬魚的側線與毛細胞 5 斑馬魚的驚嚇逃跑反應 6 斑馬魚的聽覺 7 斑馬魚的視覺 8 斑馬魚的嗅覺 9 研究目的 10 實驗架構 11 實驗設計 12 第一部分:三氟敏長時間暴露對斑馬魚胚胎之基本毒性 12 實驗一:斑馬魚胚胎長時間暴露於三氟敏之LC50、死亡率、孵化率 12 實驗二:斑馬魚胚胎暴露於三氟敏96小時之形態發育觀察 12 第二部分:三氟敏處理對斑馬魚胚胎循環系統之影響 12 實驗一:三氟敏暴露後之心臟指標 12 實驗二:三氟敏暴露後之血管指標 13 第三部分:三氟敏處理對斑馬魚胚胎離子調節功能之影響 13 實驗一:三氟敏暴露後之離子細胞密度與面積變化 13 實驗二:三氟敏暴露後之各離子含量變化 14 第四部分:三氟敏處理對斑馬魚胚胎感覺系統之影響 14 實驗一:三氟敏暴露後之側線感覺功能 14 實驗二:三氟敏暴露後之聲音驚嚇逃跑反應測試 15 實驗三:三氟敏暴露後之視運動反應測試 15 實驗四:三氟敏暴露後之鹽味迴避嗅覺測試 15 第五部分:三氟敏處理對斑馬魚胚胎基因層面之影響 16 實驗一:三氟敏暴露後之RNA定序 16 研究材料與方法 17 一、實驗動物 17 二、三氟敏溶液製備 17 三、死亡率、孵化率、發育形態與心率 17 四、循環系統影像錄製與分析 18 五、螢光染色 19 六、免疫細胞化學染色 (Whole mount Immunocytochemistry, ICC) 19 七、掃描式電子顯微鏡 (Scanning Electron Microscope, SEM) 20 八、電感耦合電漿體質普法 (Inductively coupled plasma mass spectrometry, ICP-MS) 21 九、感覺系統行為反應實驗 21 十、RNA定序 (RNA sequencing) 23 十一、統計方法 25 結果 26 第一部分:三氟敏長時間暴露對斑馬魚胚胎之基本毒性 26 實驗一:斑馬魚胚胎長時間暴露於三氟敏之死亡率、孵化率、LC50 26 實驗二:斑馬魚胚胎暴露於三氟敏96小時之形態發育觀察 26 第二部分:三氟敏處理對斑馬魚胚胎循環系統之影響 27 實驗一:三氟敏暴露後之心臟指標 27 實驗二:三氟敏暴露後之血管指標 27 第三部分:三氟敏處理對斑馬魚胚胎離子調節功能之影響 28 實驗一:三氟敏暴露後之離子細胞密度與面積變化 28 實驗二:三氟敏暴露後之各離子含量變化 28 第四部分:三氟敏處理對斑馬魚胚胎感覺系統之影響 29 實驗一:三氟敏暴露後之側線感覺功能 29 實驗二:三氟敏暴露後之聲音驚嚇逃跑反應測試 30 實驗三:三氟敏暴露後之視運動反應測試 30 實驗四:三氟敏暴露後之鹽味迴避嗅覺測試 30 第五部分:三氟敏處理對斑馬魚胚胎基因層面之影響 31 實驗一:三氟敏暴露後之RNA定序 31 討論 33 結論 46 參考資料 47

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