金錢魚 (Scatophagus argus)常作為與文蛤混養之工作魚種，以清除池中大型藻類。近年來，與文蛤混養之工作魚種每逢冬季寒流來襲之際常大量死亡，同時造成養殖業嚴重損失。本研究針對金錢魚幼魚於長期低溫馴養三十天後，面臨寒流低溫之刺激下，是否提升其低溫耐受能力，並與虱目魚低溫耐受能力相比較。熱休克蛋白 (HSP70) 主要功能在維持細胞正常生理機能，幫助受到環境迫害、失去功能的蛋白質重新正常摺疊。因此，利用極限耐受溫度實驗 (Critical thermal minima) 測定金錢魚於三個馴養溫度組別 (17.0、22.5和28.0°C) 之最低耐受溫度，並比較將經過馴養的金錢魚回復到正常水溫三十天後之最低耐受溫度。另外，利用西方墨點法 (Western Blotting) 探討不同水溫馴養後金錢魚體內熱休克蛋白質 (Heat shock protein) 表現量之改變情形。結果顯示，虱目魚最低耐受溫度達15.0±0.5°C，且死亡率達70%。而金錢魚幼魚馴養於不同水溫一個月後，水溫28°C組最低耐受溫度達14.0±0.5°C；水溫22.5°C 組最低耐受溫度可達8±0.5°C；水溫17.0°C組最低耐受溫度可達6.0±0.5°C。此外，相比虱目魚高死亡率，金錢於三個溫度組達最低耐受溫度後皆無死亡率情形發生。在HSP70表現方面，經過一個月馴養於不同水溫後，水溫22.5°C組HSP70表現量與水溫28°C組並無顯著差異；而水溫17.0°C組HSP70表現量顯著大於水溫28.0°C組。另外，將馴養於不同溫度三十天後的金錢魚，回復到水溫28.0°C繼續馴養三十天後，再測定其最低耐受溫度。結果顯示，水溫28.0°C組最低耐受溫度依舊達14.0±0.5°C；水溫22.5°C 組最低耐受溫度達12.0±0.5°C；水溫17.0°C組最低耐受溫度可達11.0±0.5°C。經過三十天馴養後，將三個溫度組之金錢魚曝露至水溫15.0°C並測定其肝組織中HSP70表現量改變情形。結果顯示，水溫17.0°C組經過馴養後，暴露於水溫15.0°C中HSP70並無顯著改變情形；而水溫28.0°C和22.5°C組之HSP70表現量皆顯著提升。本研究結果顯示，長期低溫馴養能提升金錢魚最低耐受溫度。且經過回復到水溫28.0°C三十天後之金錢魚，其最低耐受溫度雖上升，卻依舊低於未經過低溫馴養之個體。本研究對於金錢魚是否能於冬天與文蛤混養提供基礎的生理學建議。

The spotted scat is usually farmed for controlling macro-algae density in poly-aquaculture practice in Taiwan. Nowadays, massive mortality of economic aquaculture fish poly-cultured with hard clam, such as milkfish, often occurs during dramatic temperature drop in winter. The present study intended to estimate whether the capability of cold tolerance of the spotted scat (Scatophagus argus) would be altered after 30 days acclimation to different water temperatures. Moreover, heat shock protein 70 (HSP70) plays a significant role in cytoprotection for maintaining cellular homeostasis to refold and repair the proteins which are damaged by the environment stressors. Hence, the correlation between protein expression of hepatic HSP70 and low temperature tolerance ability of spotted scat were investigated. This study revealed that critical temperature minima (CTMin) after 30 days acclimation were observed at 14±0.5, 8±0.5 and 6±0.5°C for S. argus acclimated at 28.0±0.5, 22.5±0.5 and 17.0±0.5°C, respectively. Individuals of the acclimation experiment were then transferred back to 28°C for additional 30 days and their CTMin was examined again. The CTMin values for S. argus previously acclimated at 28.0±0.5, 22.5±0.5 and 17.0±0.5°C were 14±0.5, 12±0.5 and 11±0.5°C, respectively. HSP70 expression level of liver in S. argus acclimated for 30 days at different temperatures was highest at 17.0°C and the lowest at 22.5°C. These results indicated that the cold tolerance capacity of S. argus may be enhanced by acclimating to low temperature. Despite transferring back to 28°C for 30 days, fish which acclimated to low temperature had better cold tolerance capacity than untreated fish. Besides, acclimation to 22.5°C for S. argus may be a practical way to enhanced cold tolerance during winter to below 10°C since fish acclimated to 17°C had high HSP70 expression level and lower locomotion.

學位論文審定書 i
謝辭 ii
摘要 iii
Abstract iv
Content vi
1. Introduction 1
1.1 Temperature acclimation 1
1.2 Heat shock proteins 70 (HSP70) 3
1.3 The current situation of poly-cultured species with hard clam 7
2. Materials and methods 10
2.1 Stablization conditions 10
2.2 Low temperature acclimation 10
2.3 Effect of additional acclimation to 28°C after low temperature acclimation 12
2.4 Critical thermal minima (CTMin) determination 12
2.5 Cold exposure experiment 14
2.6 Sampling liver tissue and protein extraction 15
2.7 Western blot analysis 16
2.8 Statistical analysis 17
3. Results 19
3.1 Critical thermal minima (CTMin) after 30 days acclimation 19
3.2 Critical thermal minima (CTMin) after additional 30 days acclimation at 28°C 20
3.3 Heat shock protein70 (HSP70) expression level after 30 days acclimation 21
3.4 HSP70 expression level during cold exposure trial 22
3.5 Feed intake during 30 days acclimation 23
4. Discussions 24
4.1 Low temperature acclimation triggers cold tolerance 24
4.2 Tolerance capability after additional 30 days recovery period 25
4.3 30 days acclimation induces HSP70 expression in response to cold acclimation 26
4.4 HSP70 expression level during cold exposure trial at 15°C 29
4.5 Aquaculture application 30
5. Conclusions 32
Reference 33
Figures 44

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