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研究生:林淵智
研究生(外文):Yuan Chih Lin
論文名稱:大鼻孔叫姑魚(Johniusmacrorhynus)生物音學特性及發音肌蛋白質體學研究
論文名稱(外文):Bioacoustics and sonic-muscle proteomics of big-snout croaker (Johnius macrorhynus)
指導教授:黃寶貴莫顯蕎莫顯蕎引用關係李明安李明安引用關係
指導教授(外文):Bao Quey HuangHin Kiu MokMing Anne Lee
學位類別:博士
校院名稱:國立臺灣海洋大學
系所名稱:環境生物與漁業科學學系
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:2008
畢業學年度:96
語文別:中文
論文頁數:125
中文關鍵詞:大鼻孔叫姑魚石首魚發音肌生物聲學被動聲納蛋白質體學季節性膨大萎縮
外文關鍵詞:big-snout croakerJohnius macrorhynussciaenidssonic musclebioacousticspassive acousticproteomicsseasonal hypertrophy and atrophy
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本研究首先比較大鼻孔叫姑魚 (Johnius macrorhynus)在三種情形(受壓迫手抓情況、養殖缸內及野外環境)的鳴音特徵變化,受壓迫手抓著的狀態下只會發出purr類型聲音,而養殖缸內及野外環境都可收錄到”purr”及”dual-knocks”兩種類型聲音,purr鳴音的主要特徵為第一脈衝間距(first inter-pulse interval)大約為主要脈衝間距(main inter-pulse interval)的6-9倍;聲音頻率可達5 kHz,具有兩個頻率高峰(frequency peak)分別在1 kHz及2 kHz,前者為主頻(dominant frequency)。此類型聲音特徵在三種情形下並沒有顯著差異,顯示受壓迫手抓情況下所產生的聲音可用來推測野外環境下產生鳴音的魚種。第一脈衝間距、主要脈衝間距、脈衝重複率(repetition rate of pulse)及平均單一脈衝時間(pulse duration)可做為鑑別魚種鳴音的特徵。
研究中亦包括大鼻孔叫姑魚鳴音在台灣沿岸海域的季節分佈情形,發現聲音都分佈於水深40公尺以內的西岸海域,春到秋季都可發現鳴音,其中以夏季鳴音較多。鳴音特徵在三個季節具有變化,夏季聲音具有較高的鳴音長度(call duration)、鳴音脈衝數(number of pulse per call)、平均單一脈衝時間及頻率高峰;但其第一脈衝間距及主要脈衝間距卻以夏季較小。利用雌雄魚之生殖腺指數(gonadosomatic index)及雌魚的卵巢卵細胞成熟階段(ovarian maturity stages)研究可判定大鼻孔叫姑魚的生殖時間大約為3到10月,高峰值則為6到9月。發音肌只在雄魚發現,雌魚並不具有此構造,在生殖季時發音肌之重量及厚度會比非生殖季分別多1.6及1.9倍,而發音肌的長度及寬度並沒有季節性的變化。發音肌的組織切片研究上亦發現發音肌肌纖維橫切面面積(fiber cross-sectional areas)、中間核心構造(central core)面積、收縮肌原纖維(contractile myofibrillar)區域面積及周圍環狀肌漿(peripheral ring of sarcoplasm)區域都會在生殖季節變大。
石首魚鳴音是經由發音肌的快速收縮及放鬆以震動泳鰾而產生,因此,本研究利用二維電泳(two-dimensional electrophoresos, 2DE)技術將蛋白質進行分離以比較大鼻孔叫姑魚之發音肌與兩種體側肌(紅肌及白肌),以了解發音肌肉快速收縮對其結構或生理上的適應。發音肌與體側肌蛋白質最主要的差異位於等電點5.0到5.7及分子量60到130 kDa之區域,其他區域的蛋白質點比較亦於文中描述。之後利用基質輔助雷射脫附游離-飛行式質譜儀(mass spectrometry, matrix assisted laser desorption ionization time of flight, MALDI-TOF)及胜?質量指紋(peptide mass fingerprinting, PMF)技術進行蛋白質種類鑑定,共分辨出32個點位,包括以下蛋白質種類:六種肌肉收縮相關蛋白(fast muscle myosin heavy chain, skeletal alpha actin, alpha actin cardiac, tropomyosin, myosin light chain 2, and myosin light chain 3)、四種能量代謝相關蛋白(enolase, acyl-CoA synthetase, isocitrate dehydrogenase, and creatine kinase)、混合蛋白(cytochrome P450 monooxygenase and DEAD box protein mix to fast skeletal myosin heavy chain)及三種其他蛋白(DEAD box protein, voltage-dependent calcium channel isoform, and cyclin H)。
利用二維電泳及1D-SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)技術比較發音肌季節性膨大(hypertrophy)及萎縮(atrophy)之蛋白質,發現以下五種蛋白質為發音肌膨大時表現量較多,包括:cytochrome P450 monooxygenase、fast muscle myosin heavy chain、DEAD box proteins、isocitrate dehydrogenase及creatine kinase。另外三種蛋白質則為發音肌萎縮時表現量較多,包括:alpha actin cardiac、myosin light 2及myosin light 3。
The sounds of male big-snout croaker, Johnius macrorhynus, produced under hand-held and voluntary conditions (in large aquarium and in the field) were compared. Voluntary calls included “purr” and “dual-knocks”; only purrs were produced when the fish was hand-held. The purr is composed of pulses in which the first inter-pulse interval was 6-9 times longer than the other inter-pulse intervals. Purrs emitted under these conditions did not differ significantly, suggesting that the hand-held sound can be employed to match the sound in the field. First inter-pulse interval, main inter-pulse interval, repetition rate of pulse and pulse duration may serve as the diagnostic characters for the species-specific sound (i.e. purrs).
Sound distribution of the big-snout croaker around Taiwan was reported. The sounds were major found on the western coast at sites where depths are shallower than 40 m and were heard in spring, summer and autumn, with a peak in summer. In summer, the call duration, number of pulse per call, pulse duration, energy frequency peak were higher, but the interpulse interval (first and main interpulse interval) of the sounds was shorter. Spawning season lasts from March to October with peaks appearing from June to September. Mass and thickness of the sonic muscles, which are present only in males, in the spawning season became nearly 1.6- and 1.9- folds larger than that in the non-spawning season, respectively. However, there were no significant seasonal differences in the sonic-muscle length, and width. Cross-sectional areas of the sonic-muscle fiber, central core, contractile myofibrillar region, and peripheral ring of sarcoplasm region were also larger in the spawning season.
Sciaenids produce sound through the contraction and relaxation of the sonic muscles vibration movements of the swim bladder. The protein profiles of the sonic muscle, red muscle and white muscle shown by two-dimensional electrophoresis (2-DE) were compared to reveal differential protein expressions of the big-snout croaker’s sonic muscle that can be accounted for certain structural and physiological adaptations. The major differences of the expressed proteins (about 80 up-regulated protein spots in the sonic muscle) were in the regions of isoelectric point range from 5.0 to 5.7, and molecular weight range from 60 to 130 kDa. Other protein spots expressed differentially in these three muscles were also described. The matrix assisted laser desorption ionization time of flight (MALDI-TOF) and peptide mass fingerprinting (PMF) were used for protein identification. A total of 32 spots related to six contractile proteins (fast muscle myosin heavy chain, skeletal alpha actin, alpha actin cardiac, tropomyosin, myosin light chain 2, and myosin light chain 3), four energy metabolic enzymes (enolase, acyl-CoA synthetase, isocitrate dehydrogenase, and creatine kinase), mixture proteins (cytochrome P450 monooxygenase and DEAD box protein mix to fast skeletal myosin heavy chain), and three miscellaneous proteins (DEAD box protein, voltage-dependent calcium channel isoform, and cyclin H) were identified.
The 2-DE and sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS PAGE) were used to decipher the protein content and to compare sonic muscles in hypertrophic and atrophic conditions. The contents of muscle proteins were significantly higher in hypertrophic muscle than atrophic muscle. The levels of cytochrome P450 monooxygenase, fast muscle myosin heavy chain, DEAD box proteins, isocitrate dehydrogenase, and creatine kinase were up-regulated in hypertrophy muscle, but the levels of alpha actin cardiac, myosin light 2, and myosin light 3 were lower than the atrophy muscle.
Abstract............................................ 1
摘要................................................. 3
Chapter 1: Background................................ 5
1.1. Sound production in fish........................ 5
1.2. Passive acoustics survey........................ 5
1.3. Sounds production mechanisms.................... 7
1.4. Function of sound production.................... 7
1.5. Sonic muscle.................................... 8
1.6. Single sonic muscle twitch (SSMT) model of sciaenid 10
1.7. Proteomics...................................... 11
1.8. Motive of this study............................ 12
1.9. Plan of the dissertation........................ 14

Chapter 2: Sound characteristics of big-snout croaker, Johnius macrorhynus.................................. 18
2.1. Introduction.................................... 18
2.2. Materials and Methods........................... 20
2.2.1. Sound recording............................... 20
2.2.2. Sound analysis................................ 22
2.2.3. Statistical analysis.......................... 22
2.3. Results......................................... 23
2.3.1. Characteristics of fish sounds................ 23
2.3.2. Voluntary vocal activity under captivity...... 25
2.4. Discussion...................................... 26

Chapter 3: Seasonal variations of sounds and sonic muscle of the big-snout croaker, Johnius macrorhynus (Sciaenidae)......................................... 38
3.1. Introduction.................................... 38
3.2. Materials and Methods........................... 39
3.2.1. Surveys of underwater ambient biological noises............................................... 39
3.2.2. Sound recording, analysis and sound parameters........................................... 40
3.2.3. Monthly variation of sonic muscle parameters.. 41
3.2.4. Reproductive condition........................ 42
3.2.5. Statistical analysis.......................... 43
3.3. Results......................................... 43
3.3.1. Big-snout croaker sounds...................... 43
3.3.2. Regional and temporal distributions of the big-snout croaker’s sounds in the wild........................ 44
3.3.3. Seasonal variation in the parameters of purr sounds............................................... 44
3.3.4. Monthly variation of the sonic muscle parameters 45
3.3.5. Monthly change in reproductive condition....... 46
3.4. Discussion....................................... 47
Supplementary data – The spatial distributions of big-snout croaker’s sounds............................... 65

Chapter 4: Proteomic analysis of the sonic muscle in big-snout croaker, Johnius macrorhynus.................... 73
4.1. Introduction..................................... 73
4.2. Materials and Methods............................ 75
4.2.1. Sample collection.............................. 75
4.2.2. Preparation of protein extraction.............. 75
4.2.3. Two-dimensional electrophoresis (2-DE)......... 76
4.2.3.1. First dimension.............................. 76
4.2.3.2. Second dimension............................. 76
4.2.4. Protein staining and image analysis............ 76
4.2.5. Protein identification......................... 77
4.2.6. Statistical analysis........................... 78
4.3. Results.......................................... 78
4.3.1. 2-DE gel protein spots......................... 78
4.3.2. Protein identification......................... 79
4.4. Discussion....................................... 80

Chapter 5: Differential expression profiling of the proteomes in the hypertrophy and atrophy sonic muscle in big-snout croaker (Johnius macrorhynus).............. 102
5.1. Introduction ................................... 102
5.2. Materials and Methods .......................... 103
5.2.1. Sample collection............................. 103
5.2.2. Protein concentration analyses................ 103
5.2.3. Two-dimensional electrophoresis (2-DE) and protein identification....................................... 103
5.2.4. Myosin heavy chain (MHC) and actin (1D-SDS PAGE)................................................ 104
5.2.5. Statistical analysis.......................... 104
5.3. Results......................................... 104
5.3.1. Protein concentration......................... 104
5.3.2. Two-dimensional electrophoresis (2-DE)........ 105
5.3.3. Higher expression proteins in hypertrophy sonic muscle............................................... 105
5.3.4. Higher expression proteins in atrophy sonic muscle............................................... 106
5.4. Discussion...................................... 106

Chapter 6: Summary and conclusions................... 115

References........................................... 117
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