臺灣博碩士論文加值系統

清白招潮蟹是一種廣泛分佈於海岸潮間帶的半陸生性螃蟹，由於每日潮汐的漲退，生存環境鹽度的變化相當的大。雖然過去文獻中指出Na+, K+ -ATPase及carbonic anhydrase（CA）同為參與離子調節的重要酵素，但在蟹類相關研究中並未發現它們被同時探討的文獻。從前人的報告中發現，在不同鹽度處理下，螃蟹鰓上Na+, K+ -ATPase活性在低鹽度處理下表現較高，且後半部鰓比前半部鰓高的趨勢。在CA部分，在不同鹽度處理下，各對鰓間CA活性表現趨勢有種別性而呈現不一致的情形。在本實驗中，希望討論清白招潮蟹不同對鰓間活性表現的趨勢。實驗設計首先進行5、25、45 ‰鹽度馴養試驗，測量滲透壓、鰓間Na+, K+ -ATPase與CA活性的變化，並使用western blotting方法，比較鰓間Na+, K+ -ATPase與CA含量的變化。由體液滲透壓變化的結果指出，清白招潮蟹屬於hyper-hypo osmoregulator。在不同鹽度處理下，清白招潮蟹後半部鰓的Na+, K+ -ATPase及CA活性表現較前鰓高，且低鹽度處理顯著比高鹽度處理表現高。Western blotting結果同樣顯示在不同鹽度馴養下後鰓表現比前鰓高。由CA活性在45 ‰處理下，各對鰓活性表現顯著比25 ‰處理組高的現象，可能暗示在高鹽環境下CA所扮演離子調節的角色比Na+, K+ -ATPase更為重要。

Uca lactea is a semi-terrestrial crab and widely distributes along the west coast of Taiwan. It lives in intertidal areas and is subjected to large daily and seasonal changes in salinity. Previous literature indicated that not only Na+, K+-ATPase but also carbonic anhydrase (CA) are important in osmoregulation. However, these two enzymes have never been studied simultaneously for the osmoregulation of brachyuran crabs. From previous studies, the CA activity in different pairs of gills was species-specific. No significant difference among gill CA activity was found in one crab species with high salinity treatment while a significantly higher CA activity in the posterior gills in a Uca species was reported in another report. In my study, I examined the variation of these two enzymes in Uca lactea. The experiment started with salinity acclimation of 5, 25, 45 ‰ and osmolality, Na+, K+-ATPase and CA activity and western blotting were examined. From osmolality results, Uca lactea is a hyper-hypo osmoregulator. Both Na+, K+-ATPase and CA activity were higher in diluted seawater and in posterior gills. The results from western blotting had a similar pattern in that Na+, K+-ATPase and CA were higher in posterior gills. The CA activity in Uca lactea was higher in 45 ‰ than in 25 ‰, suggesting that the CA may be more important than Na+, K+-ATPase in osmoregulation in concentrated seawater.

目錄
中文摘要…………………………………………………………..1
Abstract……………………………………………………………2
前言………………………………………………………………..3
（一）蟹類滲透壓調節的方式………………………………….3
（二）蟹類滲透壓調節的器官………………………………….4
（三）Na+, K+-ATPase和carbonic anhydrase活性研究…….5
（四）實驗目的…....………………………………………….6
材料與方法………………………………………………………..7
（一）實驗動物………………………………………………….7
（二）研究方法………………………………………………….7
一.蓄養水槽的設置…...................................7
二.體液的測量.........................................8
三.水質測定...........................................8
四.蛋白質粹取液的準備…...............................8
五.Na+, K+-ATPase反應產生Pi濃度的測定與計算............9
六.Carbonic anhydrase活性測定與計算...................10
七.西方墨點法（Western blotting）.....................11
八.藥品的配置.........................................12
九.實驗設計及步驟.....................................15
十.統計...............................................15
實驗結果..............................................16
鹽度轉移試驗..........................................16
一.體液滲透壓.........................................16
二.體液離子濃度.......................................17
三.Na+, K+-ATPase.....................................18
四.Carbonic anhydrase.................................20
討論..................................................22
（一）體液滲透壓與環境的處理..........................22
（二）Na+, K+-ATPase專一活性..........................23
（三）Carbonic anhydrase活性..........................26
結語..................................................29
參考文獻..............................................30
圖說..................................................36

蘇詠超。 1998。 四種招潮蟹（Ocypodidae : Uca）滲透壓調節方式的比較。 私立東海大學生物系碩士論文。
Ahearn, G. A., J. M. Duerr, Z. Zhuang, R. J. Brown, A. Aslamkhan and D. A. Killebrew. 1999. Ion transport processes of crustacean epithelial cells. Physiol. Biochem. Zool. 72(1): 1-18.
Boaden, P. J. S. and R. Seed. 1985. An introduction to coastal ecology. Pp.80. Blackie Academic & Professional, New York.
Bottcher, K. and D. Siebers. 1993. Biochemistry, localization, and physiology of carbonic anhydrase in the gills of euryhaline crabs. J. Exp. Zool. 265: 397-409.
Bottcher, K., D. Siebers and W. Becker. 1990. Carbonic anhydrase in branchial tissues of osmoregulating shore crabs, Carcinus maenas. J. Exp. Zool. 255: 251-261.
Bottcher, K., A., D. Siebers and S. Sender. 1995. Carbonic anhydrase, a respiratory enzyme in the gills of the shore crab Carcinus maenas. Helgolander Meeresunters. 49: 737-745.
Bottcher, K., A. Waheed and W. S. Sly. 1994. Membrane-associated carbonic anhydrase from the crab gill: purification, characterization, and comparison with mammalian CAs. Arch. Biochem. Biophys. 312(2): 429-435.
Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Brion, L. P., J. H. Schwartz, B. J. Zavilowitz and G. J. Schwartz. 1988. Micro-method for the measurement of carbonic anhydrase activity in cellular homogenates. Anal. Biochem. 175: 289-297.
Castilho, P. C., I. A. Martins and A. Bianchini. 2001. Gill Na+,K+-ATPase and osmoregulation in the estuarine crab, Chasmagnathus granulata Dana, 1851 (Decapoda, Grapsidae). J. Exp. Mar. Biol. Ecol. 256: 215-227.
Chu, K. H. 1986. Sodium transport across the perfused midgut and hindgut of the blue crab, Callinectes sapidus: the possible role of the gut in crustacean osmoregulation. Comp. Biochem. Physiol. 87A(1): 21-26.
Corotto, S. F. and C. W. Holliday. 1996. Branchial Na, K-ATPase and osmoregulation in the purple shore crab, Hemigrapsus nudus (Dana). Comp. Biochem. Physiol. 113A(4): 361-368.
Daures, M. C., G. Charmantier, K. P. C. Janssen, D. E. Aiken and F. V. Herp. 1994. Involvement of eyestalk factors in the neuroendocrine control of osmoregulation in adult american lobster Homarus americanus. Gen. Comp. Endocrinol. 94:281-293.
Gerencser, G. A., G. A. Ahearn, F. Robbins. and M. A. Cattey. 2000. Chloride transport by lobster hepatopancreas is facilitated by several anion antiport mechanisms. Comp. Biochem. Physiol. 125A(4): 223-228.
Henry, R. P. 1988a. Subcellular distribution of carbonic anhydrase activity in the gills of the blue crab, Callinectes sapidus. J. Exp. Zool. 245: 1-8.
Henry, R. P. 1988b. Multiple functions of carbonic anhydrase in the crustacean gill. J. Exp. Zool. 248: 19-24.
Henry, R. P. 1991. Branchial and Branchiostegite carbonic anhydrase in decapoda crustaceans: the aquatic to terrestrial transition. J. Exp. Zool. 259: 294-303.
Henry, R. P. 2001. Enviromentally mediated carbonic anhydrase induction in the gills of euryhaline crustaceans. J. Exp. Biol. 204: 991-1002.
Henry, R. P. and J. N. Cameron. 1982. The distribution and partial characterization of carbonic anhydrase in selected aquatic and terrestrial decapod crustacean. J. Exp. Zool. 221: 309-321.
Henry, R. P. and E. R. Swenson. 2000. The distribution and physiological significance of carbonic anhydrase in vertebrate gas exchange organs. Resp. Physiol. 121:1-12.
Holliday, C. W. 1985. Salinity-induced changes in gill Na, K-ATPase activity in the mud fiddler crab, Uca pugnax. J. Exp. Zool. 233: 199-208.
Hwang, P. P., M. J. Fang, J. C. Tsai, C. J. Huang and S. T. Chen. 1998. Expression of mRNA and protein of Na, K-ATPase α subunit in gills of tilapia (Oreochromis mossambicus). Fish Physiol. Biochem. 18: 363-373.
Kultz, D., R. Bastrop, K. Jurss and D. Siebers. 1992. Mitochondria-rich(MR) cells and the activities of the Na+/K+-ATPase and carbonic anhydrase in the gill and opercular epithelium of Oreochromis Mossambicus adapted to various salinities. Comp. Biochem. Physiol. 102B(2): 293-301.
Lima, A. G., J. C. McNamara and W. R. Terra. 1997. Regulation of hemolymph osmolytes and gill Na+/K+-ATPase activities during acclimation to saline media in the freshwater shrimp Macrobrachium olfersii (Wiegmann, 1836) (Decapoda, Palaemonidae). J. Exp. Mar. Biol. Ecol. 215: 81-91.
Lucu, C. and M. Devescovi. 1999. Osmoregulation and branchial Na+,K+-ATPase in the lobster Homarus gammarus acclimated to dilute seawater. J. Exp. Mar. Biol. Ecol. 234: 291-304.
Lucu, C., M. Devescovi., B. Skaramuca and V. Kozul. 2000. Gill Na,K-ATPase in the spiny lobster Palinurus elephas and other marine osmoconformers adaptiveness of enzymes from osmoconformity to hyperregulation. J. Exp. Mar. Biol. Ecol. 246: 163-178.
Luquet, C. M. 1995. Gill morphology and terrestrial adaptation in the estuarine crab Uca uruguayensis (Decapoda, Brachyura). Crustaceana 68(7): 882-892.
Luquet, C. M., C. O. Cervino, M. Ansaldo, V. C. Pereyra, S. Kocmur, R. E. Dezi. 1998. Physiological response to emersion in the amphibious crab Chasmagnathus granulata Dana (Decapoda Grapsidae)：biochemical and ventilatory adaptations. Comp. Biochem. Physiol. 121A:385-393.
Luquet, C., G. Pellerano and G. Rosa. 1997. Salinity-induced changes in the fine structure of the gills of the semiterrestrial estuarian crab, Uca uruguayensis (Decapoda, Brachyura). Tissue & Cell 29(4): 495-501.
Luquet, C. M., G. A. Rosa, C. C. Ferrari, G. Genovese and G. N. Pellerano. 2000. Gill morphology of the intertidal estuarine crab Chasmagnathus granulatus Dana, 1851 (Decapoda, Grapsidae) in relation to habitat and respiratory habits. Crustaceana 73(1): 53-67.
Mananes, A. A. L., L. J. Magnoni and A. L. Goldemberg. 2000. Branchial carbonic anhydrase (CA) of gills of Chasmagnathus granulata (Crustacea Decapoda). Comp. Biochem. Physiol. 127B: 85-95.
Martinez, C. B. R., R. R. Harris and M. C. F. Santos. 1998. Transepithelial potential differences and sodium fluxes in isolated perfused gills of the mangrove crab Ucides cordatus. Comp. Biochem. Physiol. 120A: 227-236.
McNamara, J. C. and A. G. Lima. 1997. The route of ion and water movements across the gill epithelium of the freshwater shrimp Macrobrachium olfersii (Decapoda, Palaemonidae): evidence from ultrastructural changes induced by acclimation to saline media. Biol. Bull. 192: 321-331.
Morris, S. 2001. Neuroendocrine regulation of osmoregulation and the evolution of air-breathing in decapod crustaceans. J. Exp. Biol. 204: 979-989.
Morritt, D. and A. M. M. Richardson. 1999. Osmoregulation and evolutionary patterns in coastal terrestrial amphipods (Talitridae). J. Crust. Biol. 20(1): 67-74.
Pequex, A. 1995. Osmotic regulation in crustaceans. J. Crust. Biol. 15(1): 1-60.
Peterson, G. L. 1978. A simplified method for analysis of inorganic phosphate in the presence of interfering substances. Anal. Biochem. 84: 164-172.
Piller, S. C., R. P. Henry, J. E. Doeller and D. W. Kraus. 1995. A comparison of the gill physiology of two euryhaline crab species, Callinectes sapidus and Callinectes similes: energy production, transport-related enzymes and osmoregulation as a function of acclimation salinity. J. Exp. Biol. 198: 349-358.
Santos, M. C. F. and G. S. Moreira. 1999. Time course of osmoionic compensations to acute salinity exposure in the ghost crab Ocypode quadrata (Fabricius, 1787). J. Exp. Mar. Biol. Ecol. 235: 91-104.
Sender, S., K. Bottcher, Y. Cetin and G. Gros. 1999. Carbonic anhydrase in the gills of seawater- and freshwater-acclimated flounders Platichthys flesus : purification, characterization, and immunohistochemical location. J. Histochem. Cytochem. 47(1): 43-50.
Siebers, D., C. Lucu and A. Winkler. 1987. Active influx of ions across the gills of osmoregulating shore crabs Carcinus maenas. In “Biochemistry, localization, and physiology of carbonic anhydrase in the gills of euryhaline crabs” (Bottcher, K. and D. Siebers), Pp.404. J. Exp. Zool. 265: 397-409.
Towle, D. W. 1993. Ion transport systems in membrane vesicles isolated from crustacean tissues. J. Exp. Zool. 265: 387-396.
Towle, D. W. 1997. Molecular approaches to understanding salinity adaptation of estuarine animals. Amer. Zool. 37: 575-584.
Warner, G. F. 1977. The Biology of Crabs. Van Nostrand Reinhold, New York, USA.
Whiteley, N. M., J. L. Scott, S. J. Breeze and L. Mccann. 2001. Effect of water salinity on acid-base balance in decapod crustaceans. J. Exp. Biol. 204: 1003-1011.
Zanders, I. P. and W. E. Rojas. 1996a. Transbranchial potentials and ion fluxes across isolated, perfused gills of Uca rapax. Mar. Biol. 125: 307-314.
Zanders, I. P. and W. E. Rojas. 1996b. Osmotic and ionic regulation in the fiddler crab Uca rapax acclimated to dilute and hypersaline seawater. Mar. Biol. 125: 315-320.