曾萬年臺灣大學：漁業科學研究所楊竣菘Yang, Chun-SungChun-SungYang2007-11-282018-07-062007-11-282018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/59355大眼海鰱 (Megalops cyprinoides) 與鰻鱺目魚類相同，都具有特殊柳葉魚(Leptocephalus) 發育階段的生活史。研究指出，柳葉魚仔魚轉換到稚魚的變態過程(Metamorphosis)，須經歷行為、棲地轉換與身體組織重組等個體發生(Ontogenetic) 的劇烈變化；在此過程將觀察到耳石日週輪明顯變寬、耳石Sr/Ca比因GAGs降解而急速下降等現象。另外，影響耳石微化學變化的眾多環境因子中，以水體元素濃度的影響最為明顯，其次為鹽度及溫度。為了探討變態時期耳石微化學受生理效應和外在水體元素濃度的影響程度，本研究設計一室內養殖控制實驗，來比較大眼海鰱不同發育階段耳石元素比值的變化情形。 實驗用的大眼海鰱柳葉魚標本，於2005年8月到9月間，採自於淡水公司田溪，其發育階段已進入變態期 (StageⅡ) 的第一天，隨即帶回實驗室以河口半淡鹹水馴化一天後，用四環黴素 (Tetracycline) 螢光標示耳石當作實驗開始的第一天。個別添加 SrCl2 ˙ 6H2O和Ba(NO3)2 兩種藥品，將飼育水依2×、4×、8×的比例調配成含控制組的四個濃度加兩重複添加實驗，飼育螢光標示後的柳葉魚。根據耳石日週輪的變化與發育階段的關係，得知變態期大約14天，以此將耳石劃分為柳葉魚期、變態期和稚魚期三個發育階段。水樣分析在添加Sr、Ba後的倍率變化為Sr/Ca比1×、5.3×、13.4×和29.0×，Ba/Ca比1.00×、0.99×、1.15×和1.53×非等倍率的變化關係。利用EPMA及LA-ICPMS測定結果，發現變態時期耳石Sr/Ca劇烈下降的現象只出現在Sr1×，而在Sr的四個濃度下，耳石Sr/Ca比呈現變態期1.0×、2.5×、7.4×和16.0×，稚魚期1.0×、6.5×、20.0×和47.1×的倍率關係，表示Sr/Ca比從水體進入到耳石後，具有放大的效果；同樣地，耳石Ba/Ca比在Ba的四個濃度下，倍率關係則呈現變態期1.00×、1.19×、1.15×和1.16×，稚魚期1.00×、1.37×、1.60×和1.81×，相較於Sr/Ca比，Ba/Ca比的放大效果則較不明顯，但兩者皆反應了水樣中Sr/Ca、Ba/Ca比非等倍率增加的關係。耳石Sr/Ca比在兩發育階段皆隨水體Sr濃度增加而上升，並呈現高度的正相關 (r=0.99)，且斜率檢定為 p>0.05，表示不論在變態期或稚魚期，耳石元素吸收的狀況皆是環境元素濃度的影響程度遠大於生理的效應；但Ba/Ca比在變態時期與水體Ba元素濃度之間的關係卻不顯著，而稚魚期耳石則同樣地受飼育水元素濃度增加而有正相關關係 (r=0.7)；由以上結果顯示，耳石對於水中元素的吸收除具有選擇性差異外，亦會受不同發育階段關鍵性的影響。此外，魚體長、體重及存活率也受Ba元素濃度而變化較鍶實驗組來得大。 本研究重要的發現為大眼海鰱柳葉魚在變態過程中耳石元素比值與飼育水之間的關係，水體元素濃度的效應超過過去所認為很重要的變態期生理因子的調控，且其關係在不同元素間又有很大的差異。並由分配係數得知，耳石吸收鍶的效率約為鋇的20倍，鍶鋇雖同為鹼土族元素 (Alkali metals)，在控制組(1×)和稚魚期也有著相似的變化趨勢，但進入耳石內的比例卻有很大的差別。經由本研究耳石對元素利用的發現，將有助於耳石微化學組成在魚群洄游環境及生態研究上之應用。The Pacific tarpon Megalops cyprinoides are similar with Anguilliformes which has a willow-leaf-shaped larva called leptocephalus. Previous research indicated a drastic ontogenetic change in behavior, habitat, body shape, and recombination of tissue during metamorphosing from leptocephalus to juvenile stages. A maximum otolith increment width (OIW) and sharp decreasing in otolith Sr/Ca ratios resulting from glycosaminoglycan (GAGs) broken–down were observed during metamorphosis. In addition to such physiological factor, otolith microchemistry was affected mostly by ambient concentration rather than salinity and temperature. In order to compare with the affected degree of physiological factors and water chemistry during metamorphosis, we designed a controlled experiment with adding strontium (Sr) and barium (Ba) in rearing water to discuss the relationships between otolith chemistry and life stages in Pacific tarpon. Leptocephali (entering StageⅡ) were collected in the estuary of Gong-shy-tyan Creek, a tributary of the Tanshui river in August to September 2005. After acclimating 1 day, the leptocephali were immersed in a tetracycline solution to mark the otoliths with a fluorescent band as the onset of the experiment (Day 1) and then reared in the treatments spiked with different concentrations (2×、4×、8×) of Sr and Ba. According to the changes of otolith daily growth increments (DGIs), the leptocephalus completed metamorphosis is approximately 14d during rearing conditions. The results of Sr:Ca and Ba:Ca ratios in the rearing water were measured to be 1×、5.3×、13.4×、29.0× and 1.00×、0.99×、1.15×、1.53× respectively after spiking of Sr and Ba. The Sr:Ca ratios of the otoliths were analysed with EPMA and the period of metamorphosis (M-zone) increased times in:1.0×、2.5×、7.4×、16.0× and juvenile (J-zone) in:1.0×、6.5×、20.0×、47.1×, and the phenomenon of Sr:Ca ratios rapid decreasing only presented in control treatment (Sr1×). Other Ba:Ca ratios in the otoliths were analysed with LA-ICPMS and the concentration ratios at M-zone were: 1.00×、1.19×、1.15×、1.16× and J-zone: 1.00×、1.37×、1.60×、1.81×. Although Ba:Ca ratios has less magnification effects than Sr:Ca in the otoliths, both of them reflected the relationships of non-equal times of the element spiked in rearing water. Whether in metamorphasis or juvenile stages, the Sr:Ca ratios in the otoliths were highly related to water chemistry (r=0.99) which indicated that otolith Sr:Ca ratios were mostly influenced by ambient elemental concentrations. Besides, the relationships only appeared within juvenile (r=0.70) in Ba treatments. Sr and Ba belong to alkali metals, but mean partition coefficients DMe in Sr:Ca ratios are around 20 times more than Ba:Ca ratios. In this study the great discovery is the effects of water chemistry are more important than physical factor in metamorphosis, and selective absorption rate from ambient element to otoliths is also existed especially in different life stages.摘要……………………………………………………………………...………………3 Abstract.............................................................................................................................5 壹、前言……………………………………………………...................………………7 1. 大眼海鰱的分類地位………………………………………………………….7 2. 大眼海鰱的特殊生活史………………………………………….....................7 2.1 柳葉魚發育階段的劃分…….…………………………………………….8 2.2 柳葉魚的發育生理學……………………………………………………..9 2.3 變態與激素之關係………………………………………………………10 3. 耳石學………………………………………………………………………...11 3.1 耳石形態與功能………………………………………………………....11 3.2 耳石形成的機制………………………………………………………....12 3.3 耳石微化學的研究………………………………………………………13 4. 研究目的……………………………………………………………………..15 貳、材料與方法…………………………………………………….............…………17 1. 實驗魚體的來源及標定……………………………………………………..17 1.1 採樣………………………………………………………………………17 1.2 耳石螢光標示……………………………………………………………17 2. 實驗設計……………………………………………………………………..17 3. 水樣的採集與分析…………………………………………………………..18 3.1 採集………………………………………………………………………18 3.2 分析………………………………………………………………………19 4. 耳石製備與判讀……………………………………………………………..19 4.1 製備………………………………………………………………………19 4.2 螢光照相…………………………………………………………………20 4.3 耳石日週輪的判讀及測量………………………………………………20 5. 微化學分析…………………………………………………………………..20 5.1 電子微探儀 (EPMA)................................................................................20 5.2 雷射感應耦合電漿質譜儀 (LA-ICPMS)………………………….........21 6. 資料分析……………………………………………………………………..22 6.1 耳石直徑與不同發育階段耳石距離之測量…………………………....22 6.2 不同發育階段耳石元素測量值之統計………………………………....22 6.3 統計方法………………………………………………………………....22 參、結果……………………………………………….................………………........23 1. 添加鍶、鋇元素後飼育水中Sr/Ca比和Ba/Ca比之變化……...................23 2. 飼育水的鍶、鋇濃度對體成長之影響…………………………………......24 3. 耳石微細構造隨發育階段的變化…………………………………………..24 3.1 變態期的長短…………………………………………........................…24 3.2 耳石構造的變化與發育階段之關係………………................................25 4. 飼育水的鍶、鋇濃度對耳石成長及耳石Sr/Ca比和Ba/Ca比之影響…...25 4.1 飼育水的元素濃度對耳石成長之影響…………………………………25 4.2 飼育水的元素濃度對耳石Sr/Ca比和Ba/Ca比之影響……….............26 5. 飼育水與耳石之間元素比之關係……………………………………..........27 6. 飼育水與耳石之間的Sr/Ca比和Ba/Ca比分配係數之關係.......................28 肆、討論………………….........................……………………………………………29 1. 飼育水中Sr/Ca比與Ba/Ca比倍率變化的差異性…………………….......29 2. 不同元素對體成長的影響…………………………………………………..29 3. 變態時間的長短及其影響因子……………………………………………..30 4. 耳石微細構造的變化與發育階段之關係…………………………………..31 4.1 耳石上的發育階段之劃分………………................................................31 4.2 耳石同分異構物對於元素吸收的影響………………………………....32 5. 耳石微化學…………………………………………………....……………..33 5.1 耳石Sr/Ca比與Ba/Ca比和發育階段之關係………………………….33 5.2 元素進入耳石內所需要的反應時間………………................................33 6. 飼育水的元素濃度對耳石微化學之影響……………………………..........34 6.1 水體中Sr的影響….........................………..............................................34 6.2 水體中Ba的影響………………………………………………………..35 6.3 環境中複雜因子間的交互作用對於魚體吸收元素比例之影響………35 7. 結論…………………………………………………………………………..36 8. 未來的工作…………………………………………………………………..37 伍、附錄…………………………………………….....................................…………38 參考文獻…........……………………………….....................…………………………40 表……………………………………………………………………………………….51 圖……………………………………………………………………………………….58en-US大眼海鰱耳石變態柳葉魚鍶鋇Megalops cyprinoidesotolithmetamorphosisleptocephalusstrontiumbariumother