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研究生:藍雅媂
研究生(外文):Atiek Rahmawaty
論文名稱:探討有機磷殺蟲劑地特松對淡水長臂大蝦的免疫因子及免疫相關基因之影響
論文名稱(外文):The Effects of an Organophosphorus Insecticide, Trichlorfon on the Immunological Responses and Immune Related Genes of Giant Freshwater Prawn, Macrobrachium rosenbergii
指導教授:張欽泉Eddy Suprayitno
指導教授(外文):Chin-Chyuan ChangEddy Suprayitno
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:水產養殖系所
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
論文頁數:70
中文關鍵詞:淡水長臂大蝦三氯松乙醯膽鹼酯酶免疫反應基因表現
外文關鍵詞:Macrobrachium rosenbergiitrichlorfonacetylcholinesteraseimmunological responsesgenes expressions
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三氯松為有機磷之藥劑,其被應用於農業作為殺蟲劑之用途,以及應用於人類醫學作為防制體內之寄生蟲之效用。其亦應用於畜牧業及水產養殖業作為殺蟲劑之用途,並在蝦類養殖業上造成環境汙染之問題。因此,本研究目的為評估淡水長臂大蝦(Macrobrachium rosenbergii)於不同時間長度下(0, 3, 6, 12 及24 小時)接觸不同濃度之三氯松(分別為0, 0.2, 0.4 mg L-1)對其免疫反應、相關免疫基因表現及乙醯膽鹼酯酶(AChE)活性之影響。總血球數(THC)、 不同血球數(DHC)、吞噬細胞活性、酚氧化酵素活性(PO)、超氧化歧化酵素活性(SOD)、 麩胱甘肽過氧化酵素活性(GPx) 、轉麩胺醯胺酵素活性(TGase) 、及血細胞之凝血時間的作為評估免疫反應之指標。實驗結果顯示淡水長臂大蝦暴露於三氯松(濃度0.2及 0.4 mg L-1)12及24小時後,其總血球數顯著增加。不同血球數結果顯示顆粒細胞增加而透明細胞減少。淡水長臂大蝦暴露在三氯松(濃度0.2 mg L-1)時在6及12小時其酚氧化酵素活性顯著提升。超氧化歧化酵素活性於三氯松之濃度0.2 mg L-1時,在3, 12 及24 小時顯著提高。超氧化歧化酵素活性於三氯松之濃度0.4 mg L-1時,在6及12 小時顯著提高。不同濃度實驗組之胱甘肽過氧化酵素活性相較下低於控制組。不同濃度實驗組之呼吸爆在12及24小時高於控制組。轉麩胺醯胺酵素活性降低及血細胞之凝血時間增加,則顯示凝血受到抑制。以有機磷藥劑作為潛在之生物標記,淡水長臂大蝦之乙醯膽鹼酯酶在六小時後顯著降低,且有濃度依賴之趨勢。免疫相關基因之表現,以蝦類之PO、LGBP、黏附蛋白、巨球蛋白、轉麩胺醯胺酵素、Cu,Zn-SOD及麩胱甘肽過氧化酵素作為評估指標。在暴露於三氯松之濃度0.4 mg L-1於24小時,所有免疫相關基因之表現皆顯著降低。蝦類短期暴露於三氯松,不僅降低血細胞之循環,且誘發氧化壓力,從而導致免疫基因DNA之損害。本研究之結論為淡水長臂大蝦曝露在濃度0.4 mg L-1之三氯松在24小時對其免疫反應、免疫基因表現之影響、免疫力之降低及後續對病原感染之敏感性,相較於其他濃度及時間更為顯著。
Trichlorfon is an organophosphorous pesticide and has been used in agriculture as a pesticide and as a human medicine to combat internal parasites. It is also use as an ectoparasiticide in the livestock and aquaculture industries, which was pollutant to prawn industry. Thus, the aim of this study is to assessed the effects of trichlorfon during short-term exposure in immunological responses, immune related genes expressions and acetylcholinesterase (AChE) enzyme activity of the giant freshwater prawn, Macrobrachium rosenbergii at 0, 0.2, 0.4 mg L-1 for 0, 3, 6, 12 and 24 hrs, respectively. Total hemocyte count (THC), differential hemocyte count (DHC), respiratory burst activity, phenoloxidase (PO) activity, superoxide dismutase (SOD) activity, glutathion peroxidase (GPx) activity, transglutaminase (TGase) activity, and clotting time of hemocytes were conducted to assessed the immunological responses. The results showed that THC increased after 12 and 24 hrs exposed of trichlorfon at both concentration. DHC showed that Granular cells value increased and Hyaline cells reduced whereas PO activity increased at 6 and 12 hrs under 0.2 mg L-1 TRF. SOD activity increased at 3, 12, and 24 hrs for 0.2 mg L-1 TRF treatment and at 6 and 12 hrs for 0.4 mg L-1 TRF whereas, GPx activity decreased in under exposed of time-dependent in both TRF concentration compared with control. Production of respiratory burst increased generally after 12-24 hrs exposed trichlorfon at both concentration compared to the control. TGase activity and clotting showed TGase activity reduced whereas clotting time of hemocytes increased were indicates that coagulation system inhibited. The potential biomarker of organophosphorous pesticide, AChE revealed significant decreases after exposing for six hours, and showed a concentration-dependent tendency. Immune related genes expressions including PO, LGBP, peroxinectin, α2-macroglubulin, transglutaminase, Cu,Zn-SOD, and GPx of the prawn were also carried in the evaluation. All the immune genes expression decreased significantly when the prawn exposed to 0.4 mg L-1 for 24 hours. The prawn exposed to trichlorfon for short-term not only decrease the circulating hemocyte, but also induced the oxidative stress, which caused the subsequent damage to DNA formation of the immune genes. These concluded that the immunological responses and immune genes expressions of the prawn exposed to trichlorfon at concentration 0.4 mg L-1 for 24 hour were perturbed and then caused the deficiency of immunity and the increase of susceptibility to pathogens infection subsequently.
CONTENTS
ABSTRACT i
CONTENTS v
LIST OF TABLES viii
LIST OF FIGURES ix
1. INTRODUCTION 1
1.1. Background 1
1.2. Objective 7
2. LITERATURE REVIEW 8
2.1. Palaemonidae Shrimp Macrobrachium rosenbergii 8
2.1.1 Distribution 8
2.1.2 Taxonomy and life history 9
2.1.3 Morphology 10
2.2. Crustacean Immune System 11
2.2.1 Hemocytes and hematopoiesis 11
2.2.2 Immune pattern recognition receptors (PRR) 12
2.2.3 Phagocitosis activity 14
2.2.4 Coagulation system 16
2.2.5 Prophenoloxidase (ProPO) system 17
2.3. Trichlorfon 18
2.4. Acetilcholinesterase System 21
3. MATERIALS AND METHODS 23
3.1. Research Flow Chart 23
3.2. Experimental Animal 24
3.3. Hemolymph Sample Collection 25
3.4. THC (Total hemolymph count) 25
3.5. Total RNA Isolation and Reverse Transcription 25
3.6. Quantitative real-time PCR Analysis of Gene Expression 26
3.7. Phenoloxidase Activity 28
3.8. Respiratory Burst 29
3.9. Acetylcholinesterase (AChE) Activity 29
3.10. Superoxide dismutase, Gluthation peroxidase and Transgluta-
minase activity 30
3.11. Haemolymph Coagulation Time Measurement 31
3.12. Statistical Analysis 32
4. RESULTS 33
4.1 Effect of Trichlorfon on the Achetilcholinesterase Activity of
Prawn Hemocytes 33
4.2 Immunological Responses 34
4.2.1 Total hemocyte count (THC) and differential
hemocyte count (DHC) of prawn hemocytes
exposed to trichlorfon 29
4.2.2 Antioxidant and oxidative stress statues of prawn
hemocytes exposed to trichlorfon 38
4.2.3 Phenoloxidase activity, transglutaminase activity
and clotting activity of prawn hemocytes exposed
to trichlorfon 41
4.3 Immune-related Gene Expressions 44
4.3.1 Antioxidant genes expressions of prawn hemocytes
exposed to trichlorfon 44
4.3.2 Immune-related molecules genes expressions of prawn hemocytes exposed to trichlorfon 46
5. DISCUSSIONS 51
5.1 Effect of Trichlorfon on the Acetylcholinesterase Activity
of Prawn Hemocytes 51
5.2 Immunological Responses 52
5.2.1 Total hemocytes count (THC) and differential hemocyte count (DHC) of prawn hemocytes exposed to trichlor-
fon 52
5.2.3 Oxidative stress statues of prawn hemocytes exposed to trichlorfon 53
5.2.4 Phenoloxidase activity, transglutaminase activity
and clotting activity of prawn hemocytes exposed
to trichlorfon 55
5.3 Immune-related Gene Expressions 57
6. CONCLUSIONS 61
REFERENCES 62
APPENDIX 59


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