探討吳郭魚面臨亞硝酸曝露時的生理反應機制

Investigation of physiological responses to nitrite exposure in tilapia (Oreochromis mossambicus x O. niloticus)

謝易璋(Yi-Jang Hsieh)

指導教授 ： 胡紹揚 ； 湯政豪

國立屏東科技大學/農學院/生物科技研究所/碩士(2014年)

https://doi.org/10.6346/NPUST.2014.00253

摘要

亞硝酸為水生環境中常見的含氮廢物，特別是在高密度或循環水養殖系統當中。一旦亞硝酸累積則會對水生動物造成生理層次上的傷害。然而，當魚類面臨非致死濃度的亞硝酸時，所誘發的分子生理反應機制的探討仍相當缺乏。因此，本篇報告運用全球養殖魚種，吳郭魚(Oreochromis mossambicus #westeur024# O. niloticus)，搭配整合性的實驗方法，探討曝露於兩種亞硝酸濃度 (0.15 mM, 0.3 mM)為期一週時，其形質因子、逆境反應、離子調節與能量代謝與生長等生理反應。結果顯示，吳郭魚其血糖濃度、肝指數以及肥滿度在亞硝酸曝露下並無顯著變化。另一方面，鰓上的熱休克蛋白(Heat shock proteins, HSPs; HSP70 and HSP90)與泛素蛋白(Ubiquitin-conjugated proteins)的結果顯示熱休克蛋白70在0.15與0.3 mM亞硝酸曝露下表現量皆有上升的趨勢，顯著性上升在熱休克蛋白90與泛素蛋白則是在0.3 mM。進一步本研究的實驗發現血液中滲透壓與氯離子濃度皆會受到亞硝酸的曝露而顯著性的下降，而更重要的是在驅動離子運輸系統的Na+/K+-ATPase (NKA)其表現量與活性也有上升的趨勢。接著鰓上氯離子運輸相關通道的結果，顯示氯離子吸收通道的Na+/Cl- cotransporter (NCC)與chloride channel 3 (ClC-3)的mRNA表現量受到亞硝酸曝露後呈現上調控的趨勢，然而排氯方面的cystic fibrosis transmembrane conductance regulator (CFTR)與則是具有下降的趨勢。此外，能量代謝與肌肉成長方面，在0.3 mM亞硝酸曝露下，鰓與肝臟的肝糖含量顯著下降，然而在肌肉則沒有變化。進一步的實驗結果證實，與肌肉成長調控相關的myosin其表現量在0.3 mM亞硝酸曝露下具有顯著性的上升，而負向調控肌肉成長的基因，myostatin mRNA則是在兩個濃度的亞硝酸暴露下皆有顯著提高表現量的變化。綜合以上結果推測吳郭魚藉由活化抗逆境相關的蛋白質品質恆定機轉以進行細胞防衛機制，並且調整其離子調節反應以應付亞硝酸逆境的刺激。然而，伴隨著影響整體能量儲存與分配，其個體生長調控也可能受到影響。本研究釐清了亞致死濃度的亞硝酸逆境，對魚類生理各方面的威脅；同時提供了相關的研究資料，以了解並建立可能評估方式，來偵測亞硝酸對硬骨魚類的影響。

關鍵字

亞硝酸 ； 吳郭魚 ； 蛋白質恆定機轉 ； 離子調節 ； 能量代謝 ； 肌肉成長調節

並列摘要

Nitrite is a common pollutant in aquatic environment, especially in intensive or recirculating aquaculture system. In this study, the integrated approaches were used to clarify the nitrite-induced physiological responses in tilapia (Oreochromis mossambicus #westeur024# O. niloticus), a worldwide aquaculture species. Tilapia was exposed to two sublethal concentrations of nitrite (0.15 and 0.3 mM) for exploration of the impacts of nitrite on morphometric indices, stress responses, ionoregulation, metabolic responses and muscle growth regulation. The results showed no evident changes of hepatosomatic index, condition factor and plasma glucose were observed. In the responses of protein quality control (PQC) mechanism, branchial protein abundance of heat shock protein 70 (HSP70) was significantly induced at two studied concentrations of nitrite while the levels of HSP90 and ubiquitin-conjugated proteins elevated at 0.3 mM nitrite exposure. Accordingly, up-regulation of gill HSPs and ubiquitinated proteins was essential for preventing the accumulation of protein aggregations in branchial cells of tilapia under nitrite stress. Sublethal nitrite exposure also led to evidently decreasing plasma chloride concentration and osmolality. Then elevation of gill Na+/K+-ATPase (NKA) expression and transcript levels of Na+/Cl- cotransporter (NCC) and chloride channel 3 (ClC-3) in combination with down-regulation of cystic fibrosis transmembrane conductance regulator (CFTR) were found as the compensatory responses to adjust the ionic conditions within certain physiological ranges. In metabolic responses, hepatic and branchial glycogen contents significantly decreased in 0.3 mM nitrite exposed fish, no significant difference was found in muscle glycogen content though. It was suggested that the energy storage and allocation were affected by sublethal nitrite exposure. In addition, to assess whether sublethal nitrite stress affects muscle growth, mRNA expression of muscle growth related genes, myosin and myostatin was examined. Up-regulation of muscle myosin and myostatin mRNA implied that nitrite exposure might interfere in muscle growth regulation. Taken together, our findings revealed that tilapia could activate gill PQC mechanism and ionoregulatory responses to maintain cell viability and ionic balance, respectively, for coping with the challenge of nitrite exposure. Subsequently, alteration of energy storage and allocation was found due to the energy-consuming processes was activated and then it potentially affected the muscle growth. The present study clarified the threats of sublethal nitrite stress to fish physiology at various aspects to provide the important insights into waterborne nitrite stress as well as the potential ways to monitor the nitrite effects on finfish.

並列關鍵字

nitrite ； tilapia ； protein quality control ； ionoregulation ； energy metabolism ； muscle growth regulation

參考文獻

Acosta, J., Carpio, Y., Borroto, I., Gonz#westeur034#lez, O., Estrada, M.P., 2005. Myostatin gene silenced by RNAi show a zebrafish giant phenotype. Journal of Biotechnology 119, 324-331.

Afonso, L., Basu, N., Nakano, K., Devlin, R., Iwama, G., 2003. Sex-related differences in the organismal and cellular stress response in juvenile salmon exposed to treated bleached kraft mill effluent. Fish Physiology and Biochemistry 29, 173-179.

Aggergaard, S., Jensen, F.B., 2001. Cardiovascular changes and physiological response during nitrite exposure in rainbow trout. Journal of Fish Biology 59, 13-27.

Alonso, A., Camargo, J.A., 2006. Toxicity of nitrite to three species of freshwater invertebrates. Environmental Toxicology 21, 90-94.

Alves, R.N., Cordeiro, O., Silva, T.S., Richard, N., de Vareilles, M., Marino, G., Di Marco, P., Rodrigues, P.M., Concei#westeur040##westeur036#o, L.E., 2010. Metabolic molecular indicators of chronic stress in gilthead seabream (Sparus aurata) using comparative proteomics. Aquaculture 299, 57-66.