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研究生:莊淑君
研究生(外文):Shu-Chun Chuang
論文名稱:蚯蚓照射紫外線A或紫外線B(UV-AorUV-B)後對爬行行為、呼吸及體表氧化壓力之研究
論文名稱(外文):The study of crawling behavior, oxygen consumption and oxidative stress of skin in earthworms after UV-A or UV-B exposure
指導教授:陳俊宏陳俊宏引用關係
指導教授(外文):Jung-Hong Chen
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:動物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:192
中文關鍵詞:蚯蚓紫外線行為呼吸氧化壓力
外文關鍵詞:earthwormultravioletbehavioroxygen consumptionoxidative stress
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本文主要探討兩個主題:蚯蚓為何在大雨過後的夜晚到清晨爬出地表的原因及日出後為何不爬回土壤而死於地表。以往的學者常將”為何爬出地表”及”爬出地表後會死亡”視為一件事,但在本文中發現爬出地表的原因與耗氧有關,而紫外線的照射是蚯蚓致死的重要原因。
從呼吸生理的角度,綜合實驗結果及野外觀察,有一類蚯蚓包含了A. gracilis,會在下過大雨後的夜晚爬出地表,其耗氧量在夜晚(從晚上到清晨)較白天高,因此在大雨後,土壤飽含雨水,會將孔隙封住,使得土壤中氧氣降低,由於這類蚯蚓清晨時的活動力及耗氧量皆最高,因此當蚯蚓將土壤及雨水中氧氣用盡時,就會被迫此時必須爬出地表呼吸;但另一類包括P. corethrurus的蚯蚓,耗氧量較低,對無氧環境的耐受力高,因此不需在下過大雨後的清晨爬出地表,以求獲得更多的氧氣。
蚯蚓在爬出地表後會死亡的原因與紫外線有很大的關係,以UV-A及UV-B進行實驗發現,一般而言,UV-A所產生的傷害遠低於UV-B,不同蚯蚓對紫外線的耐受性也不同,P. corethrurus耐受性最佳,M. posthuma次之,而A. gracilis則最差。根據本文的研究發現這些耐受性與多種生理反應有關,如:呼吸調節,行為收縮,體表組織的氧化壓力等等有關。UV-B會影響Amynthas gracilis會的爬行能力並造成死亡率的增加。由照射時立即的行為反應及之後發生的體表病變,可分為急性及慢性反應,急性為蚯蚓照射時會立即出現肌肉強烈收縮,有不正常S型爬行行為及跳動;慢性反應則會造成表皮和肌肉細胞的破損及個體的死亡。在測量蚯蚓照射UV-B後耗氧會降低,但泡過水再加以UVB照射,其耗氧會明顯降的更低,推測是由於蚯蚓氧氣的取得是藉由濕潤的體表讓氧氣滲透,再經由肌肉的收縮使血液中的氧氣帶至身體各處,因此當紫外線照射後,體表的傷害會阻擋部分的氧氣進入血管中,且紫外線使肌肉無法正常運動,造成蚯蚓血液循環變緩,最後造成缺氧(耗氧降低)而死。在缺氧情況下,容易造成細胞的氧化壓力增加,再者,紫外線也可能直接影響體表組織的氧化壓力,在測量與氧化壓力有關的物質及酵素發現,蚯蚓體表組織在照射UV-B後,脂質過氧化現象會增加,抗氧化酵素(catalase及GPx)的活性會降低,並且直接測量其中的H2O2發現含量會增加,顯示氧化壓力亦是造成蚯蚓體表傷害及死亡的原因之一。
因此UV-B對A. gracilis的影響,可能開始於神經傳導的異常,造成肌肉的收縮不正常,爬行能力降低,並且出現體表的病變,這些變化導致蚯蚓的耗氧降低,所需氧氣無法獲得,加上體表細胞的氧化壓力增加,大量細胞死亡,最後造成蚯蚓的死亡。而P. corethrurus或M. posthuma沒有如此嚴重的行為及死亡現象,可能與在耗氧上的適應,體表氧化壓力的耐受性較佳有關,甚至可能身體具有一些物質可以有效保護他們免於紫外線的傷害,但此部分有待進一步實驗證實。
The main purpose of this thesis is to investigate two topics: 1. why do earthworms crawl out of soil at night after a heavy rain. 2. why earthworm do not crawl back into soil after sunrise and die on the ground? In the past, researchers considered them as the same. In this study, I found oxygen consumption was one of the major reasons for earthworms crawled out the soil and ultraviolet exposure is the lethal event for earthworms.
Two types of earthworms were investigated. Amynthas gracilis, may crawl out of the soil after a rain. However, the secondary types of earthworms like Pontoscolex corethrurus, never disperses after a rain, which suggest that they could stay in oxygen-deprived water for a couple days. A. gracilis has a diurnal rhythm of oxygen consumption which consumes more oxygen at night. This could explain why A. gracilis disperses on the soil surface at night after a heavy rain. When a rain is heavy enough to fill up the soil, the water in the soil content less oxygen for earthworm respiration. A. gracilis showed higher activity and consumed more oxygen at night, they were forced to surface at night after a heavy raining day to consume more oxygen.
After the earthworms surface the soil, they might die in few hours after sunrise. According to the results for this study, ultraviolet exposure is the cause of death. In general, UV-B has stronger damage effect than UV-A. Different species of earthworms have different tolerance to UV exposure. In this study, P. corethrurus showed the highest tolerance and A. gracilis is most sensitive to UV. The damage of ultraviolet radiation on earthworms can induce two responses: acute and chronic response. The acute response is charactering by the appearance of abnormally strong muscle contraction generate S-shape movement and jumping behavior. The phenomenon might be caused by bad coordination between circular muscle and longitudinal muscle in the earthworms. The chronic response showed damage of the skin and muscle cells and generate high mortality rate. The oxygen consumption of A. gracilis was significantly decreased after UV-B exposure. And the effect was more significant in the drown earthworms. Since the circulation of earthworms is mediated by muscle contraction, and the oxygen has to diffuse through their moisture skin, it is reasonable too explain abnormal muscle contraction and damaged epithelium could cause the stifle of earthworm. In addition, UV also increased oxidative stress on earthworm tissue. It was found the lipid peroxidation and the activity of antioxidant enzymes (such aaaaas catalase, GPx or SOD) were increased in the UV-B exposure earthworms. Therefore, I suggest that oxidative stress is the other reason that caused earthworms dead after UV-B exposure.
The adaptation of low oxygen consumption and high toleration of oxidative stress may explain why P. corethrurus and M. posthuma could alive under such ultraviolet exposure. They maybe have other specialized mechanisms to prevent the damage of ultraviolet exposure. However, it need more study to prove that in the future.
總目錄
中文摘要……………………………………………………………………………15
英文摘要……………………………………………………………………………18
第一章 緒論………………………………………………………………………20
第一節紫外線的介紹………………………………………………………20
第二節 紫外線對生物的影響………………………………………………22
不同紫外線對生物的影響程度不同………………………………23
紫外線對水生生物的影響………………………………………..25
紫外線對陸生動物的影響…………………………………………26
紫外線對細胞產生的影響………………………………………….28
紫外線對動物的間接影響………………………………………….29
第三節 蚯蚓漫遊地表的行為與紫外線的關係…………………………31
第四節 研究動機及目的…………………………………………………...33
第二章 蚯蚓在雨後清晨爬出地表與耗氧關連性之探討………………………...34
第一節 前言……………………………………………………………...…34
第二節 材料及方法……………………………………………………..….37
實驗動物…………………………………………………………….37
測量蚯蚓在水中存活的時間……………………………………….37
蚯蚓耗氧的測量…………………………………………………….38
耗氧速率的計算…………………………………………………….38
數據分析…………………………………………………………….39
第三節 結果…………………………………………………………….…40
各實驗組蚯蚓的體重……………………………………………….40
蚯蚓在水中的存活時間及最後的水中溶氧量…………………….40
在不同溫度下蚯蚓耗氧的日週期變化…………………………….40
溫度對蚯蚓耗氧的影響………………………………………….....41
兩種蚯蚓的耗氧比較……………………………………………….42
第四節 討論……………………………………………………………....43
附表……………………………………………………………………….50
附圖…………………………………………………………………….…56
第三章 紫外線A或B對蚯蚓爬行行為及死亡率的影響………………………..52
第一節前言……………………………………………………………..…52
第二節 材料及方法……………………………………………………...65
實驗動物………………………………………………………….…65
紫外線的照射………………………………………………………66
照射UV-A及UV-B時發生的蚯蚓行為…………………………..66
照射UV-A及UV-B後蚯蚓的爬行活力………………………..…66
蚯蚓在照射UV-A及UV-B後的死亡率…………………………..67
蚯蚓UV-A或UV-B在照射後,體表組織出現的傷害……………67
數據統計…………………………………………………………….68
第三節 結果………………………………………………………………69
蚯蚓在照射UV-A及UV-B時的行為…………………………..…69
UV-A或UV-B對蚯蚓爬行能力的影響……………………………69
UV-A或UV-B照射蚯蚓後的死亡率………………………………70
UV-A及UV-B照射下體表組織的傷害……………………………71
第四節 討論……………………………………………………………....72
附表……………………………………………………………………….77
附圖…………………………………………………………………….…79
第四章 紫外線對蚯蚓耗氧的影響……………………………………………..….89
第一節 前言………………………………………………………………89
第二節 材料及方法…………………………………………………..….92
實驗動物…………………………………………………………….92
紫外線的照射……………………………………………………….92
蚯蚓耗氧的測量………………………………………………….…93
耗氧速率的計算…………………………………………………….94
處理後蚯蚓之存活率及存活時間測定…………………………….94
數據分析…………………………………………………………….94
第三節 結果………………………………………………………………96
蚯蚓各種處理的體重……………………………………………….96
蚯蚓在照射UV-B時不同處理下的耗氧量…………………….…96
蚯蚓在照射UV-A時不同處理下的耗氧量……………………….97
蚯蚓在照射UV-B或UB-A後的死亡率及存活時間………….…97
第四節 討論………………………………………………………………99
附表………………………………………………………………………104
附圖……………………………………………………………………104
第五章 蚯蚓照射紫外線後的氧化壓力之研究………………………………….123
第一節 前言…………………………………………………………..…124
第二節 材料與方法…………………………………………………..…132
實驗動物…………………………………………………………132
蚯蚓照射紫外線…………………………………………………...132
蚯蚓組織均質液的製備…………………………………………133
脂質過氧化的測定………………………………………………133
GSH的含量測定………………………………………………….134
Catalase的活性測定…………………………………………..134
Glutathione peroxidase (GPx) 的活性測定…………………….135
H2O2的含量測定…………………………………………………..136
總蛋白質含量的測定……………………………………………136
細胞程式死亡判定………………………………………………136
數據統計…………………………………………………………137
第三節 實驗結果………………………………………………………..138
總蛋白質含量與組織重量的關係………………………………138
蚯蚓組織中TBARS含量…………………………………………138
組織中GSH的含量………………………………………………138
蚯蚓組織中catalase的活性……………………………………..138
蚯蚓組織中Glutathione peroxidase(GPx)的活性………….…140
蚯蚓組織中H2O2的含量………………………………………….141
外加抗氧化酵素對於UVB引起脂質過氧化現象影響…………..141
蚯蚓體表組織在照射UV-B後產生apoptosis的現象………….142
第四節 討論……………………………………………………………..143
附圖……………………………………………………………………...151
第六章 總結……………………………………………………………………….156
附圖…………………………………………………………………..….176
參考文獻…………………………………………………………………………...177
表目錄
Table 2-1 蚯蚓在不同處理下的體重…………………………………………..…50
Table 2-2 A. gracilis在溫度、時段及各時間點的耗氧量以摺疊設計進行變方分析………………………………………………………………………51
Table2-3. Table 2-3. 摺疊設計中,蚯蚓Amynthas gracilis 在不同溫度及不同時段下的耗氧量以最小顯著差異測驗法(LSD)之統計結果………………………………………………………………….…......52
Table2-4. 摺疊設計中,蚯蚓P. corethtutus 在不同溫度及不同時段下的耗氧量以最小顯著差異測驗法(LSD)之統計結果……………………………53
Table 2-5. Amynthas gracilis 及 Pontoscolex corethrurus耗氧量(oxygen consumption (Mo2))的Q10值…………………………………….……54
Table 2-6. A. gracils and P. corethrurus在25℃下不同時段的耗氧比較…..…55
Table 3-1先冰敷再照射UV-B 1,500 J/m2後蚯蚓死亡率……………………….77
Table 3-2 照射UV-B1,500J/m2後,以冰浴方式處理蚯蚓後的死亡時間…..…78
Table 4-1. UV-A處理中,蚯蚓A. gracilis及M. posthuma的重量……………….106
Table 4-2. UV-B處理中,蚯蚓A. gracilis及M. posthuma的重量…………….107
Table 4-3. M. posthuma 在UV-B照射後,各種處理間耗氧的差異…………...108
Table 4-4. A. gracilis 在UV-B照射後,各種處理間耗氧的差異………………109
Table 4-5. M.posthuma 在UV-A照射後,各種處理間的差異………………...110
Table 4-6. A. gracilis 在UV-A照射後,各種處理間的差異……………………111
Table 4-7. 蚯蚓A. gracilis及M. posthuma在UV-B 照射下,各種處理的死亡率及平均死亡時間………………………………………………………112
Table 4-8. 蚯蚓A. gracilis及M. posthuma在UV-A 照射下,各種處理的死亡率及平均死亡時間……………………………………………………..103
圖目錄
Fig. 1-1紅斑作用光譜、日光作用光譜及太陽紅斑有效光譜………………….24
Fig. 2-1 測量蚯蚓耗氧儀器………………………………………………………55
Fig. 2-2 蚯蚓的存活時間………………………………………………………….57
Fig. 2-3 蚯蚓存活後的人工礦泉水在25 ℃所剩的溶氧量……………………58
Fig. 2-4 Amynthas gracilis 在不同溫度及不同時段下的累積耗氧量…………..59
Fig. 2-5 P. corethrurus 在不同溫度及不同時段下的累積耗氧量………………60
Fig. 2-6 兩種蚯蚓在不同溫度下的總耗氧量比較……………………………….61
Fig. 3-1 蚯蚓A. gracilis在照射 UV-A 或 UV-B後出現S形爬行…………...79
Fig. 3-2 在UV-A 或 UV-B 照射下,A. gracilis出現跳動的個體百分比….…79
Fig. 3-3 蚯蚓在照射UV-A or UV-B後的延遲爬行活力值………………………81
Fig. 3-4 A. gracilis及M. posthuma在照射UV-B後的死亡率…………………83
Fig. 3-5 A. gracilis 照射 UV-B 前後的個體外觀圖…………………………….85
Fig 3-6 A. gracilis 在照射UV-B 1500 J/m2 後的體表變化………………….…86
Fig. 3-7. A. gracilis的體表組織及個體傷害量化圖……………………………..87
Fig. 3-8 M. posthuma在照射UV-B 1500 J/m2 後的體表變化…………………88
Fig. 4-1 未照射UV-B時,兩種正常蚯蚓的耗氧量……………………………..104
Fig. 4-2 M.posthuma 在UV-B的各種處理下20分鐘內的總耗氧量及累積耗氧量……………………………………………………………………..…115
Fig. 4-3 A. gracilis 在UV-B的各種處理下20分鐘內的總耗氧量及累積耗氧量………………………………………………………………………..116
Fig. 4-4未照射UV-A時,兩種正常蚯蚓的耗氧量………………………………118
Fig. 4-5 M .posthuma 在UV-A的各種處理下30分鐘內的總耗氧量及累積耗氧量……………………………………………………………….….…119
Fig. 4-6 A. gracilis 在UV-A的各種處理下30分鐘內的總耗氧量……………120
Fig. 4-7 紫外線照射造成蚯蚓A. gracilis耗氧降低引發相關效應示意圖….…122
Fig. 5-1 紫外線造成光氧化反應圖………………………………………………124
Fig. 5-2 光氧化反應後的脂質過氧化物型態…………………………………...125
Fig. 5-3組織重量與總蛋白質含量吸光值……………………………………….151
Fig. 5-4 A. gracilis 與P. corethrurus在UV-B照射後脂質過氧化現象………152
Fig. 5-5 以A. gracilis組織粹取液照射UV-B後脂質過氧化現象…………….154
Fig. 5-6 將蚯蚓照射UV-B後GSH含量變化………………………………….155
Fig. 5-7以A. gracilis組織粹取液照射UV-B後的GSH含量…………………157
Fig. 5-8蚯蚓A. gracilis及P. corethrurus在不同處理下的catalase活性…..158
Fig. 5-9以A. gracilis去粒線體上清液照射不同輻射量UV-B後的變化…….160
Fig. 5-10將純化的catalase 照射不同紫外線輻射量後的活性變化…………161
Fig. 5-11 A. gracilis組織中GPx在照射UV-B後的活性變化…………………162
Fig. 5-12以A. gracilis去粒線組織粹取液照射UV-B後的GPx的活性變化…163
Fig. 5-13 蚯蚓A. gracilis及P. corethrurus在照射不同輻射量UV-B後的H2O2的變化……………………………………………………………………164
Fig. 5-14以去粒線體組織粹取液照射UVB後,H2O2的含量…………………166
Fig. 5-15外加抗氧化酵素對於UVB引起脂質過氧化現象影響……………….167
Fig. 5-16在A. gracilis照射1,500J/m2會出現DNA片段化的現象…………..168
Fig. 5-17在A. gracilis照射3,000J/m2會出現DNA片段化的現象……….…169
Fig. 5-18 蚯蚓照射UV-B後,產生光氧化反應的途徑………………………..174
Fig. 6-1 紫外線照射對蚯蚓A. gracilis產生影響示意圖………………………175
參考文獻
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Albro PW, Bilski P, Corbett JT, Schroeder JL and Chignell CF. 1997. Photochemical reactions and phototoxicity of sterols: novel self-perpetuating mechanism for lipid photooxidation. Photochem Photobiol 66: 316-325.
Albro P W, Schroeder JL and Corbett JT. 1992. Lipids of the earthworm Lumbricius terrestris. Lipid 27(2):136-143.
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