南美白對蝦(Litopenaeus vannamei)，又常簡稱為白蝦，在1994年被引進至台灣，成為了繼台灣草蝦養殖崩盤後的熱門養殖物種。能夠成為熱門養殖物種的原因有以下幾點：（1）成長快速，從苗到上市的養成期間只需要3 – 4個月；（2）養殖成本低，南美白對蝦有著廣鹽的特性，能夠適應1 - 2‰的低鹽度環境到40‰的高鹽度環境，業者可以利用此特性將白蝦苗淡化，以減少佔養殖成本中第二高位的海水成本。由於上述原因，台灣各地使用不同的鹽度環境來飼養白蝦，反觀墨西哥、印度等國家，卻因為水份蒸發率高，養殖的鹽度時常超過正常的海水鹽度。環境、病原、宿主，為養殖的三大重點，三者環環相扣，雖然白蝦能夠適應大範圍的鹽度，但其體內的滲透壓為718 mOsm/kg，相當於鹽度為25‰的環境，極端的鹽度環境對於白蝦而言，可能會帶來生理上的壓力，間接影響到體內的免疫能力。因此，本篇研究將白蝦長時間(2週)飼養在不同鹽度環境(2.5‰、5‰、15‰、35‰、45‰)下，觀察先天性免疫反應機制中的原酚氧化酵素系統(prophenoloxidase activation system, proPO system)，相關基因prophenoloxidase 1 (proPO1)、prophenoloxidase 2 (proPO2)、prophenoloxidase activating factor (PPAF)、serine proteinase (SP)及酵素層面的變化，並計算總血球細胞數量(total hemocyte count, THC)，藉此評估先天性免疫反應的變化。實驗結果顯示，在15‰的鹽度環境下，總血球數及血球之酚氧化酵素活性顯著性高於2.5‰與5‰的鹽度環境，但在原酚氧化酵素系統之相關基因表現量上，SP在35‰的鹽度環境下顯著高於其他鹽度。根據以上結果，低鹽度環境，會造成酚氧化酵素活性及總血球數的下降，推測因為白蝦需要花費較多的能量在調節體內的滲透壓，而沒有足夠的能量來維持體內的免疫反應。本研究的最終目的，是希望能夠提供養殖業者在選擇鹽度環境時的參考，考量鹽度對白蝦免疫力的影響，提高其養殖物種的獲利率。

White shrimp (Litopenaeus vannamei) was introduced to Taiwan in 1994 and immediately became a popular farming species after the market crash of tiger shrimp (Penaeus monodon). White shrimp is popular for the following reasons: (1) High growth rate. It takes only 3 - 4 months of cultivation before selling in the market. (2) Low cost of water. Water is the second highest cost in aquaculture. Because white shrimp is euryhaline, it can adapt to a wide range of salinities from 1 - 2 ‰ hypotonic to 40 ‰ hypertonic. Aquaculturists take advantage of this feature and desalinate seedlings, if their aquatic farms are far away from the source of seawater. Aquaculture of white shrimp in Taiwan is practiced in a wide range of salinities. In Mexico and India, the evaporation rate is very high, so the salinity of farming environment often exceeds the value of normal seawater. Environment, pathogen and host are the three main aspects in aquaculture. Although white shrimp can adapt to a wide range of salinities, the osmotic pressure of white shrimp is 718 mOsm/kg, which is equivalent to the salinity of 25 ‰. An extreme salinity environment may bring physiological stress and affect their immunity. In this study, we cultivated white shrimp in different salinity environment (2.5‰, 5‰, 15‰, 35‰ and 45‰) for two weeks and observe their innate immune responses. We estimated the total hemocyte count (THC), expression pattern of gene in prophenoloxidase activation system (proPO system): prophenoloxidase 1 (proPO1), prophenoloxidase 2 (proPO2), prophenoloxidase activating factor (PPAF), serine proteinase (SP), and activity level of phenoloxidase activity (PO activity). Our results showed that in the salinity of 15 ‰, PO activity and THC were significantly higher than in 2.5 ‰ and 5 ‰. However, the expression level of SP was significantly higher in 35‰. We suggested that more energy is required to adjust osmotic pressure in hypotonic environment, therefore organisms have insufficient to maintain immunity. The ultimate good of this study is to provide guidelines for white shrimp aquaculturists while choosing salinity environment for better immunity and higher harvest rate.

誌謝 i
摘要 ii
Abstract iii
壹、前言 1
1.台灣南美白對蝦的養殖起源 1
2.白蝦主要的疾病 2
3.台灣白蝦養殖近況 3
4.廣鹽性(Euryhaline) 4
5.白蝦的免疫反應(Immunity of white shrimp) 5
6.原酚氧化酵素系統 6
Prophenoloxidase activation system (proPO system) 6
貳、材料與方法 9
2.1物種及飼養條件(Experimental animals and environments) 9
2.3總血球數量計數(Total hemocyte count, THC) 10
2.4原酚氧化酵素相關基因表現量 10
(Gene expression of prophenoloxidase activation system) 10
2.4.1萃取血球中的Total RNA 10
2.4.2反轉錄RNA 11
2.4.3即時聚合酶鏈式反應 11
2.5 酚氧化酵素活性(Phenoloxidase activity, PO activity) 12
2.5.1血球細胞 12
2.5.2血淋巴液 12
2.5.3總蛋白質 12
2.6 統計方法 13
參、結果 14
3.1長期暴露在不同鹽度的南美白對蝦其總血球數(Total hemocyte count, THC)的變化 14
3.2長期暴露在不同鹽度的南美白對蝦其原酚氧化酵素系統(Prophenoloxidase activation system, proPO system)相關基因的表現量 14
3.3長期暴露在不同鹽度的南美白對蝦其血球細胞及血淋巴液之酚氧化酵素活性(Phenoloxidase activity, PO activity)的變化 14
肆、討論 16
參考文獻 20
表次 27
圖次 37

Amparyup, P., Charoensapsri, W., & Tassanakajon, A. (2009). Two prophenoloxidases are important for the survival of Vibrio harveyi challenged shrimp Penaeus monodon. Developmental & Comparative Immunology, 33(2), 247-256.

Ayaz, M., Sumara, K., Vadher, K. H., & Ishakani, A. H. (2015). Effect of higher salinities on growth and survival of pacific white shrimp, Litopenaeus vannamei (Boone, 1931). Journal of Applied and Natural Science, 7(2), 885-888.

Battistella, S., Bonivento, P., & Amirante, G. A. (1996). Hemocytes and immunological reactions in crustaceans. Italian Journal of Zoology, 63(4), 337-343.

Castille, F. L., & Lawrence, A. L. (1981). The effect of salinity on the osmotic, sodium and chloride concentrations in the hemolymph of euryhaline shrimp of the genus Penaeus. Comparative Biochemistry and Physiology Part A: Physiology, 68(1), 75-80.

Chang, Z. W., Chiang, P. C., Cheng, W., & Chang, C. C. (2015). Impact of ammonia exposure on coagulation in white shrimp, Litopenaeus vannamei. Ecotoxicology and environmental safety, 118, 98-102.

Charoensapsri, W., Amparyup, P., Suriyachan, C., & Tassanakajon, A. (2014). Melanization reaction products of shrimp display antimicrobial properties against their major bacterial and fungal pathogens. Developmental & Comparative Immunology, 47(1), 150-159.

Chen, Y. Y., Chen, J. C., Tseng, K. C., Lin, Y. C., & Huang, C. L. (2015). Activation of immunity, immune response, antioxidant ability, and resistance against Vibrio alginolyticus in white shrimp Litopenaeus vannamei decrease under long-term culture at low pH. Fish & shellfish immunology, 46(2), 192-199.

Cheng, W., & Chen, J. C. (2000). Effects of pH, temperature and salinity on immune parameters of the freshwater prawn Macrobrachium rosenbergii. Fish & Shellfish Immunology, 10(4), 387-391.

Chong-Robles, J., Charmantier, G., Boulo, V., Lizárraga-Valdéz, J., Enríquez-Paredes, L. M., & Giffard-Mena, I. (2014). Osmoregulation pattern and salinity tolerance of the white shrimp Litopenaeus vannamei (Boone, 1931) during post-embryonic development. Aquaculture, 422, 261-267.

Decker, H., & Tuczek, F. (2000). Tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism. Trends in biochemical sciences, 25(8), 392-397.

Gao, W., Tan, B., Mai, K., Chi, S., Liu, H., Dong, X., & Yang, Q. (2012). Profiling of differentially expressed genes in hepatopancreas of white shrimp (Litopenaeus vannamei) exposed to long-term low salinity stress. Aquaculture, 364, 186-191.

Gao, W., Tian, L., Huang, T., Yao, M., Hu, W., & Xu, Q. (2016). Effect of salinity on the growth performance, osmolarity and metabolism-related gene expression in white shrimp Litopenaeus vannamei. Aquaculture Reports, 4, 125-129.

González Santoyo, I., & Córdoba Aguilar, A. (2012). Phenoloxidase: a key component of the insect immune system. Entomologia Experimentalis et Applicata, 142(1), 1-16.

Huang, H. H., Liu, X. L., Xiang, J. H., & Wang, P. (2013). Immune response of Litopenaeus vannamei after infection with Vibrio harveyi. Aquaculture, 406, 115-120.

Jasmani, S., Jayasankar, V., & Wilder, M. (2010). Na/K-ATPase activity and osmo-ionic regulation in adult whiteleg shrimp Litopenaeus vannamei exposed to low salinities. Aquaculture, 304(1), 88-94.

Ji, P. F., Yao, C. L., & Wang, Z. Y. (2009). Immune response and gene expression in shrimp (Litopenaeus vannamei) hemocytes and hepatopancreas against some pathogen-associated molecular patterns. Fish & shellfish immunology, 27(4), 563-570.

Johansson, M. W., & Söderhäll, K. (1985). Exocytosis of the prophenoloxidase activating system from crayfish haemocytes. Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, 156(2), 175-181.

Lai, C. Y., Cheng, W., & Kuo, C. M. (2005). Molecular cloning and characterisation of prophenoloxidase from haemocytes of the white shrimp, Litopenaeus vannamei. Fish & shellfish immunology, 18(5), 417-430.

Lavine, M. D., & Strand, M. R. (2002). Insect hemocytes and their role in immunity. Insect biochemistry and molecular biology, 32(10), 1295-1309.


Li, C. C., & Chen, J. C. (2008). The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under low and high pH stress. Fish & shellfish immunology, 25(6), 701-709.

Li, C. C., Yeh, S. T., & Chen, J. C. (2010). Innate immunity of the white shrimp Litopenaeus vannamei weakened by the combination of a Vibrio alginolyticus injection and low-salinity stress. Fish & shellfish immunology, 28(1), 121-127.

Li, E., Chen, L., Zeng, C., Chen, X., Yu, N., Lai, Q., & Qin, J. G. (2007). Growth, body composition, respiration and ambient ammonia nitrogen tolerance of the juvenile white shrimp, Litopenaeus vannamei, at different salinities. Aquaculture, 265(1), 385-390.

Lin, Y. C., & Chen, J. C. (2001). Acute toxicity of ammonia on Litopenaeus vannamei Boone juveniles at different salinity levels. Journal of Experimental Marine Biology and Ecology, 259(1), 109-119.

Lin, Y. C., & Chen, J. C. (2003). Acute toxicity of nitrite on Litopenaeus vannamei (Boone) juveniles at different salinity levels. Aquaculture, 224(1), 193-201.

Lin, Y. C., Chen, J. C., Chen, Y. Y., Yeh, S. T., Chen, L. L., Huang, C. L., ... & Li, C. C. (2015). Crowding of white shrimp Litopenaeus vananmei depresses their immunity to and resistance against Vibrio alginolyticus and white spot syndrome virus. Fish & shellfish immunology, 45(1), 104-111.

Lin, Y. C., Chen, J. C., Li, C. C., Morni, W. Z. W., Suhaili, A. S. N., Kuo, Y. H., ... & Huang, C. L. (2012). Modulation of the innate immune system in white shrimp Litopenaeus vannamei following long-term low salinity exposure. Fish & shellfish immunology, 33(2), 324-331.

Liu, C. H., & Chen, J. C. (2004). Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus. Fish & Shellfish Immunology, 16(3), 321-334.

Liu, C. H., Yeh, S. T., Cheng, S. Y., & Chen, J. C. (2004). The immune response of the white shrimp Litopenaeus vannamei and its susceptibility to Vibrio infection in relation with the moult cycle. Fish & shellfish immunology, 16(2), 151-161.

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.

Ma, T. H., Benzie, J. A., He, J. G., Sun, C. B., & Chan, S. F. (2014). PmPPAF is a pro-phenoloxidase activating factor involved in innate immunity response of the shrimp Penaeus monodon. Developmental & Comparative Immunology, 44(1), 163-172.

Menz, A., & Blake, B. F. (1980). Experiments on the growth of Penaeus vannamei Boone. Journal of Experimental Marine Biology and Ecology, 48(2), 99-111.

Perazzolo, L. M., Gargioni, R., Ogliari, P., & Barracco, M. A. (2002). Evaluation of some hemato-immunological parameters in the shrimp Farfantepenaeus paulensis submitted to environmental and physiological stress. Aquaculture, 214(1), 19-33.

Perez Farfante, I. S. A. B. E. L., & Kensley, B. (1997). Penaeoid and sergestoid shrimps and prawns of the world. Keys and diagnoses for the families and genera. Editions du Muséum National d'Histoire Naturelle.

Qiao, G., Xu, D. H., Wang, Z., Jang, I. K., Qi, Z., Zhang, M., & Kim, S. K. (2015). Comparison of immune response of Pacific white shrimp, Litopenaeus vannamei, after multiple and single infections with WSSV and Vibrio anguillarum. Fish & shellfish immunology, 44(1), 257-264.

Söderhäll, K. (1992). Biochemical and molecular aspects of cellular communication in arthropods. Italian Journal of Zoology, 59(2), 141-151.

Söderhäll, K., & Cerenius, L. (1998). Role of the prophenoloxidase-activating system in invertebrate immunity. Current opinion in immunology, 10(1), 23-28.

Song, Y. L., Yu, C. I., Lien, T. W., Huang, C. C., & Lin, M. N. (2003). Haemolymph parameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taura syndrome virus. Fish & shellfish immunology, 14(4), 317-331.

Sun, H., Zhang, L., Ren, C., Chen, C., Fan, S., Xia, J. J., ... & Hu, C. (2011). The expression of Na, K-ATPase in Litopenaeus vannamei under salinity stress. Marine Biology Research, 7(6), 623-628.

Yeh, M. S., Lai, C. Y., Liu, C. H., Kuo, C. M., & Cheng, W. (2009). A second proPO present in white shrimp Litopenaeus vannamei and expression of the proPOs during a Vibrio alginolyticus injection, molt stage, and oral sodium alginate ingestion. Fish & shellfish immunology, 26(1), 49-55.

Yu, Z., Li, C., & Guan, Y. (2003). Effect of salinity on the immune responses and outbreak of white spot syndrome in the shrimp Marsupenaeus japonicus. Ophelia, 57(2), 99-106.

丁雲源 (2005)，白蝦養殖產業發展與技術創新-白蝦養殖發展史。水產試驗所特刊, 第6號: 3 - 4。

董明澄 (2005)，白蝦養殖產業發展與技術創新-白蝦疾病診斷與防治。水產試驗所特刊,第6號: 101 - 115, 2005。

王瑜琦 (2007)， 白蝦免疫相關基因之表現及其受口服葡聚多醣的影響 。國立中山大學海洋生物研究所。博士論文。