臺灣博碩士論文加值系統

虱目魚 (Chanos chanos) 是台灣重要的經濟魚種，具廣鹽性，可馴養在淡水及海水魚塭，但虱目魚不耐低溫的特性經常在冬季時造成漁民重大的損失。已知虱目魚馴養在海水中對於低溫的耐受性較優於馴養於淡水的魚隻。鰓是魚體進行離子調節的重要器官，並具有耗能的特性。因此，在魚體面臨溫度及鹽度逆境時，必須有足夠的能量透過有氧代謝與無氧代謝途徑供應給鰓以維持其生理功能。本研究藉由鰓的能量代謝相關產物，探討虱目魚面臨環境中不同的鹽度 (海水、淡水)及溫度 (28 oC、18 oC) 時，其鰓的能量供應變化。葡萄糖為脊椎動物 (包括魚類) 的重要能量來源， 經由葡萄糖轉運蛋白 (glucose transporter; GLUT) 將葡萄糖運輸進出細胞。其中，葡萄糖轉運蛋白1 (GLUT1) 為一種廣泛分布於身體各組織的蛋白質，其功能是將葡萄糖運送至目標細胞供其運用。因此，本研究首先分析GLUT1作為不同鹽度環境中虱目魚面臨低溫逆境時，其鰓的有氧代謝指標。免疫轉漬結果顯示，低溫逆境下的虱目魚鰓GLUT1蛋白表現量在淡水組顯著上升，海水組亦有上升趨勢但無顯著差異；而在常溫和低溫下，海水組蛋白表現量皆高於淡水組。以免疫螢光染色則觀察到GLUT1不論在淡水或海水虱目魚鰓，皆表現在代表離子細胞 (ionocytes)的NKA 免疫反應細胞 (NKA immunoreactive cells)旁邊的細胞上。其次，在有氧代謝方面，經由分析葡萄糖與肝醣含量，以及糖解作用的速率決定酵素,己糖激酶 (hexokinase)，之活性變化，探討GLUT1的蛋白表現量是否會影響鰓的能量來源。結果顯示，葡萄糖含量在海水低溫組的鰓顯著高於常溫組，而淡水組別無顯著差異。鰓肝醣的含量，在海水常溫組顯著高於低溫組，而淡水組無顯著差異。己糖激酶活性在常溫或低溫下，海水組皆顯著高於淡水組; 而在海水中，常溫組又顯著高於低溫組; 在淡水中常溫與低溫組的活性則無顯著差異。另一方面，本研究在無氧代謝方面則以乳酸與廣泛分佈型的乳酸運輸蛋白Monocarboxylate transporter 1(MCT1)作為指標。海水組或淡水組虱目魚鰓的乳酸含量皆是在常溫馴養的個體顯著高於低溫馴養的魚隻; 而mct1基因表現，海水組在常溫馴養的個體顯著高於低溫馴養的魚隻。在淡水組雖然表現平均值在低溫中降低，但是統計上無顯著差異。綜上所述，GLUT1蛋白表現確實會影響到葡萄糖在虱目魚鰓的含量，且虱目魚在常溫下，鰓行無氧代謝的狀況較低溫下更為旺盛。海水虱目魚在面臨低溫逆境時，鰓會使用暫存的肝醣作為能量利用的來源，可能是海水虱目魚較淡水虱目魚更能耐受低溫逆境的原因之一。

The euryhaline milkfish (Chanos chanos) is one of the economically important aquaculture species in Taiwan. However, the cold-intolerant characteristic of milkfish usually caused huge economic loss of the fishermen in winter. The seawater-acclimated milkfish has better cold tolerance than the fresh water-acclimated individuals. The gill is an energy-consuming organ mainly for ionoregulation. When fish faced changes in environmental temperatures or salinities, sufficient energy was required through regulating aerobic and anaerobic metabolic pathways to maintain physiological functions in gills. This study compared the mechanisms of energy metabolism, including aerobic and anaerobic pathways, in gills between seawater (SW) and freshwater (FW) milkfish under hypothermal stress (18oC vs. 28oC) to reveal the changes in energy supply. The glucose is an essential energy resource for vertebrates including fish and is transported through facilitative glucose transporters (GLUTs) in cells. Among the GLUTs, the glucose transporter 1 (GLUT1) is widely distributed in various tissues, responsible for transporting glucose into target cells to be utilized. Hence, relative protein abundance and localization of GLUT1 was first studied as the indicator of aerobic metabolism. Upon hypothermal challenge, branchial GLUT1 protein expression of FW milkfish was up-regulated. At normal or hypothermal temperature, relative protein abundance of GLUT1 was higher in gills of SW milkfish than FW ones. In addition, localization of GLUT1 was found in gill epithelial cells adjacent to NKA-immunoreactive (MR) cells (ionocytes). Subsequently, the other indicators of the aerobic metabolism pathway including glucose contents, glycogen contents, and the activities of hexokinase (HK), the rate-limiting enzyme for glycolysis that catalyzed glucose, were compared. Glucose contents were significantly lower in the SW/18oC group than the SW/28oC group, while there was no significant change between the FW/18oC and FW/28oC group. Glycogen contents were not changed in the FW/18oC group. Glycogen contents were down-regulated in the SW/18oC group. In the FW group, HK activities were not changed under hypothermal stress. However, in SW, HK activities were down-regulated in the hypothermal group. On the other hand, lactate and monocarboxylate transporter 1 (MCT1), responsible for lactate uptake in cells, were assayed as the anaerobic metabolism indicators. Under hypothermal environments, lactate contents and mct1 expression were both down-regulated in SW and FW milkfish. Taken together, GLUT1 protein expression was found to influence glucose contents in the SW groups. Anaerobic metabolism was down-regulated in both hypothermal SW and FW milkfish. Under hypothermal stress, SW-acclimated milkfish could use glycogen as an energy resource that might make SW milkfish more tolerant to hypothermal stress.

摘要 i

Abstract iii

前言 1

研究目的 6

材料方法 7

結果 17

討論.22

結論.26

表.27

圖.28

Supplementary data.38

參考文獻.48

附錄.54

Anni, I. S. A., Bianchini, A., Barcarolli, I. F., Junior, A. S. V., Robaldo, R. B., Tesser, M. B., & Sampaio, L. A. (2016). Salinity influence on growth, osmoregulation and energy turnover in juvenile pompano Trachinotus marginatus Cuvier 1832. Aquaculture, 455, 63-72.
Bagarinao, T.U. (1991). Biology of milkfish (Chanos chanos Forsskal). SEAFDEC Aquaculture Department, Iloilo, Philippines, pp. 12-64.
Baker, S. K., McCullagh, K. J., & Bonen, A. (1998). Training intensity-dependent and tissue-specific increases in lactate uptake and MCT-1 in heart and muscle. Journal of Applied Physiology, 84(3), 987-994.
Balmaceda-Aguilera, C., Martos-Sitcha, J. A., Mancera, J. M., & Martínez‐Rodríguez, G. (2012). Cloning and expression pattern of facilitative glucose transporter 1 (GLUT1) in gilthead sea bream Sparus aurata in response to salinity acclimation. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 163(1), 38-46.
Barton, B. A. (2002). Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids. Integrative and Comparative Biology, 42(3), 517-525.
Barton, B. A., & Iwama, G. K. (1991). Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids. Annual Review of Fish Diseases, 1, 3-26.
Beitinger, T. L., & Bennett, W. A. (2000). Quantification of the role of acclimation temperature in temperature tolerance of fishes. Environmental Biology of Fishes, 58(3), 277-288.
Bennett, W. A., & Beitinger, T. L. (1997). Temperature tolerance of the sheepshead minnow, Cyprinodon variegatus. Copeia, 77-87.
Bergersen, L. H. (2007). Is lactate food for neurons? Comparison of monocarboxylate transporter subtypes in brain and muscle. Neuroscience, 145(1), 11-19.
Blázquez, M. A., Lagunas, R., Gancedo, C., & Gancedo, J. M. (1993). Trehalose‐6‐phosphate, a new regulator of yeast glycolysis that inhibits hexokinases. FEBS letters, 329(1-2), 51-54
Boeuf, G., & Payan, P. (2001). How should salinity influence fish growth?. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 130(4), 411-423.
Chang, C. H., Huang, J. J., Yeh, C. Y., Tang, C. H., Hwang, L. Y., & Lee, T. H. (2018). Salinity effects on strategies of glycogen utilization in livers of euryhaline milkfish (Chanos chanos) under hypothermal stress. Frontiers in Physiology, 9, 81.
Chang, C. H., Tang, C. H., Kang, C. K., Lo, W. Y., & Lee, T. H. (2016). Comparison of integrated responses to nonlethal and lethal hypothermal stress in milkfish (Chanos chanos): a proteomics study. PloS one, 11(9), e0163538.
Chang, J. C. H., Wu, S. M., Tseng, Y. C., Lee, Y. C., Baba, O., & Hwang, P. P. (2007). Regulation of glycogen metabolism in gills and liver of the euryhaline tilapia (Oreochromis mossambicus) during acclimation to seawater. Journal of Experimental Biology, 210(19), 3494-3504.
Cheng, S. Y., Chen, C. S., & Chen, J. C. (2013). Salinity and temperature tolerance of brown-marbled grouper Epinephelus fuscoguttatus. Fish Physiology and Biochemistry, 39(2), 277-286.
Clow, K. A., Short, C. E., Hall, J. R., Gendron, R. L., Paradis, H., Ralhan, A., & Driedzic, W. R. (2016). Journal of Experimental Biology, 219(17), 2763-2773.
Deng, D., Xu, C., Sun, P., Wu, J., Yan, C., Hu, M., & Yan, N. (2014). Crystal structure of the human glucose transporter GLUT1. Nature, 510(7503), 121.
Donaldson, M. R., Cooke, S. J., Patterson, D. A., & Macdonald, J. S. (2008). Cold shock and fish. Journal of Fish Biology, 73(7), 1491-1530.
Dunn, J. F. (1988). Low-temperature adaptation of oxidative energy production in cold-water fishes. Canadian Journal of Zoology, 66(5), 1098-1104.
Ern, R., Huong, D. T. T., Cong, N. V., Bayley, M., & Wang, T. (2014). Effect of salinity on oxygen consumption in fishes: a review. Journal of Fish Biology, 84(4), 1210-1220.
Evans, D. H., Piermarini, P. M., & Choe, K. P. (2005). The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiological Reviews, 85(1), 97-177.
Ferraris, R. P., Almendras, J. M., & Jazul, A. P. (1988). Changes in plasma osmolality and chloride concentration during abrupt transfer of milkfish (Chanos chanos) from seawater to different test salinities. Aquaculture, 70(1-2), 145-157.
Fiess, J. C., Kunkel-Patterson, A., Mathias, L., Riley, L. G., Yancey, P. H., Hirano, T., & Grau, E. G. (2007). Effects of environmental salinity and temperature on osmoregulatory ability, organic osmolytes, and plasma hormone profiles in the Mozambique tilapia (Oreochromis mossambicus). Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 146(2), 252-264.
Hall, J. R., MacCormack, T. J., Barry, C. A., & Driedzic, W. R. (2004). Sequence and expression of a constitutive, facilitated glucose transporter (GLUT1) in Atlantic cod Gadus morhua. Journal of Experimental Biology, 207(26), 4697-4706.
Hazel, J. R., & Prosser, C. L. (1974). Molecular mechanisms of temperature compensation in poikilotherms. Physiological Reviews, 54(3), 620-677.
Hirose, S., Kaneko, T., Naito, N., & Takei, Y. (2003). Molecular biology of major components of chloride cells. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 136(4), 593-620.
Hrytsenko, O., Pohajdak, B., Xu, B. Y., Morrison, C., & Wright Jr, J. R. (2010). Cloning and molecular characterization of the glucose transporter 1 in tilapia (Oreochromis niloticus). General and Comparative Endocrinology, 165(2), 293-303.
Hwang, D. Y., & Ismail-Beigi, F. (2001). Stimulation of GLUT-1 glucose transporter expression in response to hyperosmolarity. American Journal of Physiology-Cell Physiology, 281(4), C1365-C1372.
Hwang, P. P., & Lee, T. H. (2007). New insights into fish ion regulation and mitochondrion-rich cells. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 148(3), 479-497.
Jana, S. N., Garg, S. K., & Patra, B. C. (2006). Effect of inland water salinity on growth performance and nutritional physiology in growing milkfish, Chanos chanos (Forsskal): field and laboratory studies. Journal of Applied Ichthyology, 22(1), 25-34.
Johnston, I. A., & Maitland, B. (1980). Temperature acclimation in crucian carp, Carassius carassius L., morphometric analyses of muscle fibre ultrastructure. Journal of Fish Biology, 17(1), 113-125.
Juel, C. (1997). Lactate-proton cotransport in skeletal muscle. Physiological Reviews, 77(2), 321-358.
Kang, C. K., Chen, Y. C., Chang, C. H., Tsai, S. C., & Lee, T. H. (2015). Seawater-acclimation abates cold effects on Na+, K+-ATPase activity in gills of the juvenile milkfish, Chanos chanos. Aquaculture, 446, 67-73.
Kang, C. K., Tsai, S. C., Lee, T. H., & Hwang, P. P. (2008). Differential expression of branchial Na+/K+-ATPase of two medaka species, Oryzias latipes and Oryzias dancena, with different salinity tolerances acclimated to fresh water, brackish water and seawater. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 151(4), 566-575.
Kültz, D. (2015). Physiological mechanisms used by fish to cope with salinity stress. Journal of Experimental Biology, 218(12), 1907-1914.
Lin, Y. M., Chen, C. N., & Lee, T. H. (2003). The expression of gill Na, K-ATPase in milkfish, Chanos chanos, acclimated to seawater, brackish water and fresh water. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 135(3), 489-497.
Lin, Y. M., Chen, C. N., Yoshinaga, T., Tsai, S. C., Shen, I. D., & Lee, T. H. (2006). Short-term effects of hyposmotic shock on Na+/K+-ATPase expression in gills of the euryhaline milkfish, Chanos chanos. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 143(3), 406-415.
Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 25(4), 402-408.
Luz, R. K., Martínez-Álvarez, R. M., De Pedro, N., & Delgado, M. J. (2008). Growth, food intake regulation and metabolic adaptations in goldfish (Carassius auratus) exposed to different salinities. Aquaculture, 276(1-4), 171-178.
McClelland, G. B., Craig, P. M., Dhekney, K., & Dipardo, S. (2006). Temperature‐and exercise‐induced gene expression and metabolic enzyme changes in skeletal muscle of adult zebrafish (Danio rerio). The Journal of Physiology, 577(2), 739-751.
Menoyo, D., Diez, A., Lopez-Bote, C. J., Casado, S., Obach, A., & Bautista, J. M. (2006). Dietary fat type affects lipid metabolism in Atlantic salmon (Salmo salar L.) and differentially regulates glucose transporter GLUT4 expression in muscle. Aquaculture, 261(1), 294-304.
Metz, J. R., Van Den Burg, E. H., Bonga, S. E. W., & Flik, G. (2003). Regulation of branchial Na+/K+-ATPase in common carp Cyprinus carpio L. acclimated to different temperatures. Journal of Experimental Biology, 206(13), 2273-2280.
Mommsen, T.P.( 1984). Metabolism of the fish gill. Fish Physiol. Vol. 10, Academic Press, pp. 203–238.
Orczewska, J. I., Hartleben, G., & O'Brien, K. M. (2010). The molecular basis of aerobic metabolic remodeling differs between oxidative muscle and liver of threespine sticklebacks in response to cold acclimation. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 299(1), R352-R364.
Polakof, S., Arjona, F. J., Sangiao-Alvarellos, S., Del Río, M. P. M., Mancera, J. M., & Soengas, J. L. (2006). Food deprivation alters osmoregulatory and metabolic responses to salinity acclimation in gilthead sea bream Sparus auratus. Journal of Comparative Physiology B, 176(5), 441-452.
Polakof, S., Panserat, S., Soengas, J. L., & Moon, T. W. (2012). Glucose metabolism in fish: a review. Journal of Comparative Physiology B, 182(8), 1015-1045.
Pörtner, H. O. (2010). Oxygen-and capacity-limitation of thermal tolerance: a matrix for integrating climate-related stressor effects in marine ecosystems. Journal of Experimental Biology, 213(6), 881-893.
Sampaio, L. A., & Bianchini, A. (2002). Salinity effects on osmoregulation and growth of the euryhaline flounder Paralichthys orbignyanus. Journal of Experimental Marine Biology and Ecology, 269(2), 187-196.
Sangiao-Alvarellos, S., Arjona, F. J., del Río, M. P. M., Míguez, J. M., Mancera, J. M., & Soengas, J. L. (2005). Time course of osmoregulatory and metabolic changes during osmotic acclimation in Sparus auratus. Journal of Experimental Biology, 208(22), 4291-4304.
Sangiao-Alvarellos, S., Laiz-Carrión, R., Guzmán, J. M., Martín del Río, M. P., Miguez, J. M., Mancera, J. M., & Soengas, J. L. (2003). Acclimation of S. aurata to various salinities alters energy metabolism of osmoregulatory and nonosmoregulatory organs. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 285(4), R897-R907.
Sardella, B. A., Kültz, D., Cech, J. J., & Brauner, C. J. (2008). Salinity-dependent changes in Na+/K+-ATPase content of mitochondria-rich cells contribute to differences in thermal tolerance of Mozambique tilapia. Journal of Comparative Physiology B, 178(3), 249-256.
Somero, G. N. (2010). Comparative physiology: a “crystal ball” for predicting consequences of global change. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 301(1), R1-R14.
Somero, G. N., & DeVries, A. L. (1967). Temperature tolerance of some Antarctic fishes. Science, 156(3772), 257-258.
Stickney, R. R. (1986). Tilapia tolerance of saline waters: a review. The Progressive Fish‐Culturist, 48(3), 161-167.
Tang, C. H., Tzeng, C. S., Hwang, L. Y., & Lee, T. H. (2009). Constant muscle water content and renal HSP90 expression reflect osmotic homeostasis in euryhaline teleosts acclimated to different environmental salinities. Zoological Studies, 48(4), 435-441.
Teerijoki, H., Krasnov, A., Gorodilov, Y., Krishna, S., & Mölsä, H. (2001). Rainbow trout glucose transporter (OnmyGLUT1): functional assessment in Xenopus laevis oocytes and expression in fish embryos. Journal of Experimental Biology, 204(15), 2667-2673.
Tsai, H. J., & Wilson, J. E. (1997). Functional organization of mammalian hexokinases: characterization of the rat type III isozyme and its chimeric forms, constructed with the N-and C-terminal halves of the type I and type II isozymes. Archives of Biochemistry and Biophysics, 338(2), 183-192.
Tseng, Y. C., & Hwang, P. P. (2008). Some insights into energy metabolism for osmoregulation in fish. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 148(4), 419-429.
Tseng, Y. C., Chen, R. D., Lee, J. R., Liu, S. T., Lee, S. J., & Hwang, P. P. (2009). American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 297(2), R275-R290.
Tseng, Y. C., Huang, C. J., Chang, J. C. H., Teng, W. Y., Baba, O., Fann, M. J., & Hwang, P. P. (2007). Glycogen phosphorylase in glycogen-rich cells is involved in the energy supply for ion regulation in fish gill epithelia. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 293(1), R482-R491.
van den Thillart, G., & Smit, H. (1984). Carbohydrate metabolism of goldfish (Carassius auratus L.). Journal of Comparative Physiology B, 154(5), 477-486.
Vargas-Chacoff, L., Arjona, F. J., Polakof, S., del Río, M. P. M., Soengas, J. L., & Mancera, J. M. (2009). Interactive effects of environmental salinity and temperature on metabolic responses of gilthead sea bream Sparus aurata. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 154(3), 417-424.
Verbost, P.M., Schoenmakers, T.J., Flik, G., Wendelaar Bonga, S.E. (1994). Kinetics of ATP- and Na+-gradient driven Ca2+ transport in basolateral membranes from gills of freshwater- and seawater-adapted tilapia. Journal of Experimental Biology, 186: 95-108.
Virani, N. A., & Rees, B. B. (2000). Oxygen consumption, blood lactate and inter-individual variation in the gulf killifish, Fundulus grandis, during hypoxia and recovery. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 126(3), 397-405.
Wang, X. D., Li, E. C., Chen, K., Wang, S. F., Li, T. Y., Xu, C., ... & Chen, L. Q. (2017). Response of facilitative glucose transporter 1 to salinity stress and dietary carbohydrate nutrition in white shrimp Litopenaeus vannamei. Aquaculture Nutrition, 23(1), 90-100.
Woo, N. Y., & Kelly, S. P. (1995). Effects of salinity and nutritional status on growth and metabolism of Spams sarba in a closed seawater system. Aquaculture, 135(1-3), 229-238.
Wood, I. S., & Trayhurn, P. (2003). Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. British Journal of Nutrition, 89(1), 3-9.
Wurts, W. A. (1998). Why can some fish live in freshwater, some in salt water, and some in both. World Aquaculture, 29(1), 65.
劉宗政, 2016。低溫逆境引發淡水與海水馴化之虱目魚肝臟有氧代謝變化。國立中興大學生命科學系碩士論文，台中，台灣。
周修緯, 2015。環境鹽度對於虱目魚在面臨低溫時其乳酸脫氫酶在肝臟與肌肉表現變化之影響。國立中興大學生命科學系碩士論文，台中，台灣。