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研究生: 劉昌奇
Liu, Chang-Chi
論文名稱: 酪胺對淡水長臂大蝦生理及免疫反應之影響
Effects of tyramine on the physiological and immunological responses of giant freshwater prawn, Macrobrachium rosenbergii
指導教授: 鄭文騰
Cheng, Winton
學位類別: 碩士
Master
系所名稱: 農學院 - 水產養殖系所
Department of Aquaculture
畢業學年度: 108
語文別: 中文
論文頁數: 120
中文關鍵詞: 淡水長臂大蝦酪胺免疫能力乳酸鏈球菌
外文關鍵詞: Macrobrachium rosenbergii, Tyramine, Immunity, Lactococcus garvieae
DOI URL: http://doi.org/10.6346/NPUST202000342
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  • 酪胺 (tyramine)為一種生物單胺類,於無脊椎動物中參與多種重要之生理作用,包括免疫調節。本研究目的為評估酪胺對淡水長臂大蝦免疫能力之影響。試驗分為兩部分包括酪胺注射與餵食試驗, (1)注射酪胺濃度為 0、1、10 nmol prawn-1,於0、0.5、1、2、4、8小時採樣, (2)飼料添加酪胺濃度為0、1、10 mg (kg diet)-1,餵食試驗持續7天,並於0、1、3、7天採樣,檢測其總血球數 (THC)、不同血球數 (DHC)、酚氧化酵素活性 (PO)、超氧陰離子活性 (O2-)與溶菌酶活性之免疫反應變化;血淋巴葡萄糖、乳酸、多巴胺 (DA)與正腎上腺素 (NE)之生理變化;對乳酸鏈球菌 (Lactococcus garvieae)之吞噬作用、清除效率以及攻擊試驗對病原體之抵抗性;測定免疫相關基因表現量。注射試驗結果顯示,酪胺注射1 nmol prawn-1試驗組於注射後0.5小時其總血球數、透明血球、半顆粒血球、酚氧化酵素活性及其注射後1小時對乳酸鏈球菌之吞噬活性、清除效率顯著增加。酪胺注射10 nmol prawn-1試驗組於注射後0.5小時其溶菌酶活性、吞噬活性、清除效率及血淋巴葡萄糖、多巴胺含量顯著降低。注射酪胺濃度為1、10 nmol prawn-1試驗組於0.5小時其呼吸爆與血淋巴乳酸顯著高於控制組,此外,酪胺注射10 nmol prawn-1試驗組於注射後0.5小時抑制多巴胺釋放且具有濃度關係。酪胺注射後2小時,所有免疫指標均恢復至生理值。酪胺注射1小時後,進行乳酸鏈球菌攻擊試驗,酪胺注射1 nmol prawn-1試驗組之活存率顯著提升;餵食試驗結果顯示,餵飼添加酪胺1、10 mg (kg diet)-1試驗組於第3天其總血球數、顆粒血球、半顆粒血球、酚氧化酵素活性及超氧陰離子活性顯著高於控制組。餵飼添加酪胺10 mg (kg diet)-1試驗組於第7天其超氧陰離子活性顯著高於控制組。餵飼添加酪胺1、10 mg (kg diet)-1試驗組於第3天其吞噬作用及清除效率顯著高於控制組。所有免疫指標皆於餵飼7天後恢復至生理值與控制組無顯著差異。淡水長臂大蝦餵飼含酪胺飼料亦顯著提升經乳酸鏈球菌攻擊後之活存率。此外,攻擊後持續餵食添加酪胺10 mg (kg diet)-1試驗組其存活率於168小時能夠維持90%。餵飼添加酪胺1 mg (kg diet)-1試驗組於第3天其proPO系統相關基因表現量顯著高於控制組。以上結果證明神經傳遞物質酪胺不論以注射或餵食方式,於短期間內皆可能促進免疫調節,且增強淡水長臂大蝦對乳酸鏈球菌之抵抗力。

    Tyramine (TA), a biogenic monoamine, plays various important physiological roles and immunological regulation in invertebrates. The purpose of this study was to evaluate the effects of tyramine on the immune responses of giant freshwater prawn, Macrobrachium rosenbergii. Two experiments TA injection and feeding were conducted to examine the effects on physiological responses and immunological regulation. (1) In injection group, M. rosenbergii (9.2  0.5 g) received TA at 0, 1, 10 nmol prawn-1, and were sampled at 0, 0.5, 1, 2, 4, 8 hrs. (2) In feeding group, three experiment diets, including the control diet (without of tyramine), dietary supplement of TA 1 and 10 mg (kg diet)-1 were carried out. During 7 days of culture, M. rosenbergii (10.3  0.2 g) was sampled at 0, 1, 3, 7 days. The total hemocyte count (THC), different hemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (release of superoxide anion), lysozyme activity, Hemolymph glucose, lactate, dopamine and norepinephrine, phagocytic activity, clearance efficiency, challenge test to the pathogen Lactococcus garvieae and immune-related genes expression were measured. TA-injection results showed that THC, hyaline cells, semigranular cells, and PO activity at 0.5 hr as well as phagocytic activity and clearance efficiency to L. garvieae at 1 hr of prawn injected with TA at 1 nmol prawn-1 significantly increased, but the significantly decreased plasma lysozyme activity, phagocytic activity, clearance efficiency, and Hemolymph glucose and dopamine were observed in prawn injected with TA at 10 nmol prawn-1 for 0.5 hr. Respiratory bursts and Hemolymph lactate in two TA-injection treatments at 0.5 hr, were significantly higher than those of the saline control, and in addition, TA depressed dopamine release in a dose-dependent manner after 0.5 hr of TA 10 nmol prawn-1-injection. All the examined parameters returned to control levels after prawn injected with TA for 2 hrs. For prawn received TA for 1 hr then challenged with L. garvieae, the survival ratio of TA 1 nmol prawn-1-injected prawn significantly increased. Feeding results showed that THC, granular cells, semigranular cells, PO activity, respiratory bursts of prawn fed with dietary supplement of TA 1 and 10 mg (kg diet)-1 significantly increased at 3 days. In prawns fed with dietary supplement of TA 10 mg (kg diet)-1, respiratory bursts had increased at 7 days. Phagocytic activity and clearance efficiency of prawn fed with dietary supplement of TA 1 and 10 mg (kg diet)-1 significantly increased at 3 days. All of the immune parameters had returned to control values by 7 days after prawn fed. Dietary supplement of TA 10 mg (kg diet)-1 significantly increased the survival ratio (80%) of M. rosenbergii challenged with the pathogen L. garvieae after 72 hrs. However, continuous feeding dietary supplement of TA 1 and 10 mg (kg diet)-1 also significantly increased the survival ratio (73% and 90%) of M. rosenbergii challenged with L. garvieae after 168 hrs. proPO system-related genes expression of prawn fed with dietary supplement of TA 1 mg (kg diet)-1 significantly increased at 3 days. These results suggest that neurotransmitters TA to dietary supplement promotes the immune responses, which in turn enhance the resistance against to L. garvieae of M. rosenbergii.

    目錄
    摘要………………………………………………………………………
    I
    Abstract……………………………………………………………………
    III
    謝誌………………………………………………………………………
    V
    目錄………………………………………………………………………
    VI
    表目錄……………………………………………………………………
    VIII
    圖目錄……………………………………………………………………
    IX
    1.
    前言…………………………………………………………………
    1
    2.
    文獻回顧……………………………………………………………
    6
    2.1.
    淡水長臂大蝦之介紹…………………………………………
    6
    2.2.
    甲殼類動物之免疫機制………………………………………
    7
    2.3.
    甲殼類緊迫與神經內分泌機制………………………………
    15
    3.
    材料與方法…………………………………………………………
    20
    3.1.
    試驗設計………………………………………………………
    20
    3.2.
    試驗飼料製備…………………………………………………
    21
    3.3.
    淡水長臂大蝦免疫因子影響之分析…………………………
    21
    3.4.
    淡水長臂大蝦抗病能力影響之分析…………………………
    23
    3.5.
    淡水長臂大蝦生理因子影響之分析…………………………
    25
    3.6.
    餵食酪胺對淡水長臂大蝦免疫相關基因表現量之分析……
    27
    3.7.
    統計分析………………………………………………………
    30
    4.
    結果…………………………………………………………………
    33
    4.1.
    注射酪胺對淡水長臂大蝦免疫因子之影響…………………
    33
    4.2.
    注射酪胺對淡水長臂大蝦抗病能力之影響…………………
    43
    4.3.
    注射酪胺對淡水長臂大蝦生理因子之影響…………………
    49
    4.4.
    餵食酪胺對淡水長臂大蝦免疫因子之影響…………………
    53
    4.5.
    餵食酪胺對淡水長臂大蝦抗病能力之影響…………………
    67
    4.6.
    餵食酪胺對淡水長臂大蝦生理因子之影響…………………
    75
    4.7.
    餵食酪胺對淡水長臂大蝦免疫相關基因表現之影響………
    83
    5.
    討論…………………………………………………………………
    85
    5.1.
    酪胺對淡水長臂大蝦免疫反應之影響………………………
    85
    5.2.
    酪胺對淡水長臂大蝦抗病能力之影響………………………
    88
    5.3.
    酪胺對淡水長臂大蝦生理因子之影響………………………
    89
    5.4.
    餵食酪胺對淡水長臂大蝦免疫相關基因表現之影響………
    91
    6.
    結論…………………………………………………………………
    93
    參考文獻…………………………………………………………………
    94
    作者簡介…………………………………………………………………
    120
    表目錄
    Table 1.
    Composition of the basal diet (g kg-1) for Macrobrachium
    rosenbergii………………………………………………
    31
    Table 2.
    Specific primers for real-time PCR used in the
    experiment………………………………………………
    32
    Table 3.
    Effect of tyramine on the survival ratio of M. rosenbergii
    challenged with L. garvieae………………………………
    48
    Table 4.
    Dietary supplement of tyramine on the survival ratio of M.
    rosenbergii challenged with L. garvieae……………………
    72
    Table 5.
    Continuous feeding dietary supplement of tyramine on the
    survival ratio of M. rosenbergii challenged with L. garvieae
    74
    圖目錄
    Figure 1.
    蝦類生物胺合成路徑……………………………………
    4
    Figure 2.
    本研究之試驗架構…………………………………………
    5
    Figure 3.
    Total hemocyte count of M. rosenbergii received saline or
    tyramine at 1 or 10 nmol prawn−1…………………………
    34
    Figure 4.
    Hyaline cells count of M. rosenbergii received saline or
    tyramine at 1 or 10 nmol prawn−1…………………………
    35
    Figure 5.
    Semigranular cells count of M. rosenbergii received saline
    or tyramine at 1 or 10 nmol prawn−1……………………
    36
    Figure 6.
    Granular cells count of M. rosenbergii received saline or
    tyramine at 1 or 10 nmol prawn−1………………………
    37
    Figure 7.
    Phenoloxidase (PO) activity in hemocytes per 50 μL of
    Hemolymph (A) and in per 107 granulocytes (the sum of
    granular and semigranular cells) (B) of M. rosenbergii
    received saline or tyramine at 1 or 10 nmol prawn−1………
    39
    Figure 8.
    Respiratory bursts in hemocytes per 10 μL of Hemolymph
    (A) and in per 107 hemocytes (B) of M. rosenbergii
    received saline or tyramine at 1 or 10 nmol prawn−1………
    41
    Figure 9.
    Lysozyme activity of M. rosenbergii received saline or tyramine at 1 or 10 nmol prawn−1………………………
    42
    Figure 10.
    Phagocytic activity of M. rosenbergii received saline or
    tyramine at 1 or 10 nmol prawn−1……………………
    44
    Figure 11.
    Clearance efficiency of M. rosenbergii received saline or
    tyramine at 1 or 10 nmol prawn−1………………………
    46
    Figure 12.
    Hemolymph glucose of M. rosenbergii received saline or
    tyramine at 1 or 10 nmol prawn−1………………………
    49
    Figure 13.
    Hemolymph lactate of M. rosenbergii received saline or
    tyramine at 1 or 10 nmol prawn−1………………………
    50
    Figure 14.
    Dopamine concentration in Hemolymph of M. rosenbergii
    received saline or tyramine at 1 or 10 nmol prawn−1………
    51
    Figure 15.
    Norepinephrine concentration in Hemolymph of M.
    rosenbergii received saline or tyramine at 1 or 10 nmol
    prawn−1…………………………………………………
    52
    Figure 16.
    Total hemocyte count of M. rosenbergii fed with dietary
    supplement of tyramine 0, 1 and 10 mg (kg diet)-1…………
    54
    Figure 17.
    Hyaline cells count of M. rosenbergii fed with dietary
    supplement of tyramine 0, 1 and 10 mg (kg diet)-1…………
    56
    Figure 18.
    Semigranular cells count of M. rosenbergii fed with dietary supplement of tyramine 0, 1 and 10 mg (kg diet)-1…………
    58
    Figure 19.
    Granular cells count of M. rosenbergii fed with dietary
    supplement of tyramine 0, 1 and 10 mg (kg diet)-1…………
    60
    Figure 20.
    Phenoloxidase (PO) activity in hemocytes per 50 μL of
    Hemolymph (A) and in per 107 granulocytes (the sum of
    granular and semigranular cells) (B) of M. rosenbergii fed
    with dietary supplement of tyramine 0, 1 and 10 mg (kg
    diet)-1………………………………………………………
    62
    Figure 21.
    Respiratory bursts in hemocytes per 10 μL of Hemolymph
    (A) and in per 107 hemocytes (B) of M. rosenbergii fed
    with dietary supplement of tyramine 0, 1 and 10 mg (kg
    diet)-1……………………………………………………
    64
    Figure 22.
    Lysozyme activity of M. rosenbergii fed with dietary
    supplement of tyramine 0, 1 and 10 mg (kg diet)-1…………
    66
    Figure 23.
    Phagocytic activity of M. rosenbergii fed with dietary
    supplement of tyramine 0, 1 and 10 mg (kg diet)-1…………
    68
    Figure 24.
    Clearance efficiency of M. rosenbergii fed with dietary
    supplement of tyramine 0, 1 and 10 mg (kg diet)-1…………
    70
    Figure 25.
    Hemolymph glucose of M. rosenbergii fed with dietary supplement of tyramine 0, 1 and 10 mg (kg diet)-1…………
    76
    Figure 26.
    Hemolymph lactate of M. rosenbergii fed with dietary
    supplement of tyramine 0, 1 and 10 mg (kg diet)-1…………
    78
    Figure 27.
    Dopamine concentration in Hemolymph of M. rosenbergii
    fed with dietary supplement of tyramine 0, 1 and 10 mg (kg
    diet)-1………………………………………………………
    80
    Figure 28.
    Norepinephrine concentration in Hemolymph of M.
    rosenbergii fed with dietary supplement of tyramine 0, 1
    and 10 mg (kg diet)-1………………………………………
    82
    Figure 29.
    Immune-related gene expressions of M. rosenbergii fed with
    dietary supplement of tyramine 0, 1 and 10 mg (kg diet)-1…
    84

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