急性氨氮胁迫对虎斑乌贼肝脏、鳃和脑组织结构的影响
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摘要
为了探讨氨氮对虎斑乌贼器官组织结构的影响,以体质量为(13.80±0.65)g的幼虎斑乌贼为对象,研究了氨氮胁迫对其肝脏、鳃和脑组织结构的影响。根据96 h的LC50实验结果设计5个梯度(0、1、3、6和12 mg/L)进行96 h的氨氮胁迫后,利用光学显微镜和透射电子显微镜观察其肝脏、鳃和脑组织结构。结果显示,在相同浓度的氨氮胁迫下,虎斑乌贼不同组织器官之间损伤程度存在差异,其中肝脏损伤的程度最大,对氨氮胁迫表现最敏感,其次是鳃组织和脑组织,组织器官损伤程度与氨氮浓度呈现正相关。氨氮胁迫浓度越大或胁迫时间越长,肉眼观察发现肝脏颜色越鲜红,肿胀和易糜烂程度越明显,可通过肉眼观察肝脏颜色和肿胀程度初步判断机体氨中毒程度;氨氮胁迫后,对肝脏和鳃组织造成较为严重的损伤,可能是虎斑乌贼氨中毒致死的原因。当氨氮高于或等于6 mg/L时胁迫96 h后,通过显微观察发现肝小叶轮廓模糊不完整、排列不紧密和胞浆疏松透明,大量的细胞核溶解,细胞出现空泡化,肝血窦扩张;通过电镜观察发现细胞核皱缩变形、核仁消失、线粒体嵴紊乱、线粒体空泡化、线粒体外室肿胀,高尔基体数量减少;显微观察发现鳃组织的泌氯细胞和上皮细胞核溶解,细胞出现空泡化、排列紊乱,鳃丝肿胀淤血、轮廓模糊不完整,并出现坏死脱落和缺损;通过透射电镜观察,发现细胞核皱缩、核膜破损和细胞核裂解,线粒体出现了皱缩变形、空泡化和不完整破损现象;观察脑组织的神经团和视叶,未发现脑组织的细胞有显著损伤。
The aim of this study is to investigate the toxic effects of ammonia-N stress on the histopathological changes of liver, gill and brain in juvenile cuttlefish(Sepia pharaonis). The S. pharaonis, whose initial average weight was(13.80±0.65) g, were first exposed to ammonia-N for 96 hours and then the 96 hours median lethal concentration was obtained. In this study, we established a control group(0 mg/L) and four different ammonia nitrogen concentrations of 1, 3, 6, and 12 mg/L for the acute ammonia toxicity experiment according to preliminary experimental results, and each ammonia-N level was respectively sampled at 96 h. The liver, gill and brain tissues were observed by optical microscope and transmission electron microscope. The results showed that the severity of lesions clearly differed among organs with the liver showing the most extensive damages, followed in order by the gill and brain. And there was no damage to the brain tissue. The damages of liver and gill increased with increasing exposure concentration and time. It is found that the liver is bright red, swollen, and easily eroding from the 96 h after exposure to no less than 6 mg/L ammonia. Microscopical observation showed that the liver displayed hypertrophy of hepatocytes, nuclear hypertrophy, cellular peripheral nucleus, hepato cellular vacuolation, hydropic degeneration, necrosis, dilatation in sinusoids, cellular outline indistinguishable and cytolysis. The transmission electron microscope observation showed the hepatocyte nuclear crinkle deformation, the nucleolus disappearance, the mitochondrial crista disorder, the vacuolation and the outer chamber swelling, and the number of Golgi bodies decreased. Microscopical observation showed shedding epithelial cells from gill lamella, irregular array and some necrosis phenomenon, vessel of gill filaments was pycnosis or even became cavum, and branchial swelling and congestion. The transmission electron microscope observation showed gill epithelium and chloride nuclear membrane breakage, and nuclear cleavage, mitochondrion has crinkle deformation, vacuolization and incomplete breakage. While the brain tissue and the optic lobes were observed, there was no cell damage in the brain tissue.
The aim of this study is to investigate the toxic effects of ammonia-N stress on the histopathological changes of liver, gill and brain in juvenile cuttlefish(Sepia pharaonis). The S. pharaonis, whose initial average weight was(13.80±0.65) g, were first exposed to ammonia-N for 96 hours and then the 96 hours median lethal concentration was obtained. In this study, we established a control group(0 mg/L) and four different ammonia nitrogen concentrations of 1, 3, 6, and 12 mg/L for the acute ammonia toxicity experiment according to preliminary experimental results, and each ammonia-N level was respectively sampled at 96 h. The liver, gill and brain tissues were observed by optical microscope and transmission electron microscope. The results showed that the severity of lesions clearly differed among organs with the liver showing the most extensive damages, followed in order by the gill and brain. And there was no damage to the brain tissue. The damages of liver and gill increased with increasing exposure concentration and time. It is found that the liver is bright red, swollen, and easily eroding from the 96 h after exposure to no less than 6 mg/L ammonia. Microscopical observation showed that the liver displayed hypertrophy of hepatocytes, nuclear hypertrophy, cellular peripheral nucleus, hepato cellular vacuolation, hydropic degeneration, necrosis, dilatation in sinusoids, cellular outline indistinguishable and cytolysis. The transmission electron microscope observation showed the hepatocyte nuclear crinkle deformation, the nucleolus disappearance, the mitochondrial crista disorder, the vacuolation and the outer chamber swelling, and the number of Golgi bodies decreased. Microscopical observation showed shedding epithelial cells from gill lamella, irregular array and some necrosis phenomenon, vessel of gill filaments was pycnosis or even became cavum, and branchial swelling and congestion. The transmission electron microscope observation showed gill epithelium and chloride nuclear membrane breakage, and nuclear cleavage, mitochondrion has crinkle deformation, vacuolization and incomplete breakage. While the brain tissue and the optic lobes were observed, there was no cell damage in the brain tissue.
引文
[1]彭瑞冰,乐可鑫,蒋霞敏,等.虎斑乌贼受精卵卵黄营养成分分析[J].水产学报,2015,39(7):1034-1042.Peng R B,Le K X,Jiang X M,et al.Analysis of nutritive composition of yolk of fertilized egg of Sepia pharaonis[J].Journal of Fisheries of China,2015,39(7):1034-1042(in Chinese).
[2]阮鹏,蒋霞敏,韩庆喜,等.社会等级因素对虎斑乌贼生长、存活及相关酶活的影响[J].水产学报,2016,40(12):1897-1905.Ruan P,Jiang X M,Han Q X,et al.Effects of social hierarchy on the growth,survival and related enzyme activities of Sepia pharaonis[J].Journal of Fisheries of China,2016,40(12):1897-1905(in Chinese).
[3]Peng R B,Le K X,Wang P S,et al.Detoxification Pathways in response to environmental ammonia exposure of the cuttlefish,Sepia pharaonis:glutamine and urea formation[J].Journal of the World Aquaculture Society,2017,48(2):342-352.
[4]Peng R B,Wang P S,Le K X,et al.Acute and chronic effects of ammonia on juvenile cuttlefish,Sepia pharaonis[J].Journal of the World Aquaculture Society,2017,48(4):602-610.
[5]Sinha A K,Rasoloniriana R,Dasan A F,et al.Interactive effect of high environmental ammonia and nutritional status on ecophysiological performance of European sea bass(Dicentrarchus labrax)acclimated to reduced seawater salinities[J].Aquatic Toxicology,2015,160:39-56.
[6]许星鸿,张雁秋,阎斌伦,等.氨氮胁迫对日本蟳免疫生理指标及器官结构的影响[J].生态学报,2014,34(14):3885-3894.Xu X H,Zhang Y Q,Yan B L,et al.Effects of ammonia-N stress on immunity-related indicators and histological structure of some organs of the marine crab,Charybdis japonica[J].Acta Ecologica Sinica,2014,34(14):3885-3894(in Chinese).
[7]Benli A?K,K?ksal G,?zkul A.Sublethal ammonia exposure of Nile tilapia(Oreochromis niloticus L.):Effects on gill,liver and kidney histology[J].Chemosphere,2008,72(9):1355-1358.
[8]Chen J C,Cheng S Y,Chen C T.Changes of haemocyanin,protein and free amino acid levels in the haemolymph of Penaeus japonicus exposed to ambient ammonia[J].Comparative Biochemistry and Physiology Part A:Physiology,1994,109(2):339-347.
[9]郝小凤,刘洋,凌去非.氨氮对泥鳅的急性毒性及对其肝、鳃组织超微结构的影响[J].水生态学杂志,2012,33(5):101-107.Hao X F,Liu Y,Ling Q F.Acute toxicity test of ammonia nitrogen and effects of ammonia-N stress on the ultrastructure of gill and liver of Misgurnus anguillicaudatus[J].Journal of Hydroecology,2012,33(5):101-107(in Chinese).
[10]Hegazi M M,Attia Z I,Ashour O A.Oxidative stress and antioxidant enzymes in liver and white muscle of Nile tilapia juveniles in chronic ammonia exposure[J].Aquatic Toxicology,2010,99(2):118-125.
[11]张武肖,孙盛明,戈贤平,等.急性氨氮胁迫及毒后恢复对团头鲂幼鱼鳃、肝和肾组织结构的影响[J].水产学报,2015,39(2):233-244.Zhang W X,Sun S M,Ge X P,et al.Acute effects of ammonia exposure on histopathology of gill,liver and kidney in juvenile Megalobrama amblycephala and the post-exposure recovery[J].Journal of Fisheries of China,2015,39(2):233-244(in Chinese).
[12]洪美玲,陈立侨,顾顺樟,等.氨氮胁迫对中华绒螯蟹免疫指标及肝胰腺组织结构的影响[J].中国水产科学,2007,14(3):412-418.Hong M L,Chen L Q,Gu S Z,et al.Effects of ammonia exposure on immunity indicators of haemolymph and histological structure of hepatopancreas in Chinese mitten crab(Eriocheir sinensis)[J].Journal of Fishery Sciences of China,2007,14(3):412-418(in Chinese).
[13]王贞杰,陈四清,曹栋正,等.急性氨氮胁迫对圆斑星鲽(Verasper variegatus)幼鱼鳃和肝组织结构及相关酶活性的影响[J].渔业科学进展,2017,38(2):59-69.Wang Z J,Chen S Q,Cao D Z,et al.Effects of acute ammonia nitrogen stress on histopathology of gill andliver and enzyme activities of juvenile Verasper variegatus[J].Progress in Fishery Sciences,2017,38(2):59-69(in Chinese).
[14]Wong C K C,Wong M H.Morphological and biochemical changes in the gills of tilapia(Oreochromis mossambicus)to ambient cadmium exposure[J].Aquatic Toxicology,2000,48(4):517-527.
[15]Shiogiri N S,Paulino M G,Carraschi S P,et al.Acute exposure of a glyphosate-based herbicide affects the gills and liver of the Neotropical fish,Piaractus mesopotamicus[J].Environmental Toxicology and Pharmacology,2012,34(2):388-396.
[16]Velmurugan B,Selvanayagam M,Cengiz E I,et al.Histopathology of lambda-cyhalothrin on tissues(gill,Kidney,liver and intestine)of Cirrhinus mrigala[J].Environmental Toxicology and Pharmacology,2007,24(3):286-291.
[17]胡毅,黄云,钟蕾,等.氨氮胁迫对青鱼幼鱼鳃丝Na+/K+-ATP酶、组织结构及血清部分生理生化指标的影响[J].水产学报,2012,36(4):538-545.Hu Y,Huang Y,Zhong L,et al.Effects of ammonia stress on the gill Na+/K+-ATPase,microstructure and some serum physiological-biochemical indices of juvenile black carp(Mylopharyngodon piceus)[J].Journal of Fisheries of China,2012,36(4):538-545(in Chinese).
[18]Suzuki H,Yanaka A,Shibahara T,et al.Ammonia-induced apoptosis is accelerated at higher p H in gastric surface mucous cells[J].Gastrointestinal and Liver Physiology,2002,283(4):G986-G995.
[19]Smart G R.Investigations of the toxic mechanisms of ammonia to fish-gas exchange in rainbow trout(Salmo gairdneri)exposed to acutely lethal concentrations[J].Journal of Fish Biology,1978,12(1):93-104.
[20]Wicks B J,Randall D J.The effect of sub-lethal ammonia exposure on fed and unfed rainbow trout:the role of glutamine in regulation of ammonia[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2002,132(2):275-285.
[21]Randall D J,Tsui T K N.Ammonia toxicity in fish[J].Marine Pollution Bulletin,2002,45(1-12):17-23.
[22]Van der Linden A,Verhoye M,Nilsson G E.Does anoxia induce cell swelling in carp brains?In vivo MRI measurements in crucian carp and common carp[J].Journal of Neurophysiology,2001,85(1):125-131.
[23]Ip Y K,Tay A S L,Lee K H,et al.Strategies adopted by the swamp eel Monopterus albus to survive in high concentrations of environmental ammonia[J].Physiological and Biochemical Zoology,2004,77:390-405.
[24]黎庆,龚诗雁,黎明.慢性氨氮暴露诱发黄颡鱼幼鱼谷氨酰胺积累、氧化损伤及免疫抑制的研究[J].水产学报,2015,39(5):728-734.Li Q,Gong S Y,Li M.Chronic ammonia toxicity induces glutamine accumulation,oxidative damage and immunosuppression of juvenile yellow catfish Pelteobagrus fulvidraco[J].Journal of Fisheries of China,2015,39 (5):728-734(in Chinese).
[2]阮鹏,蒋霞敏,韩庆喜,等.社会等级因素对虎斑乌贼生长、存活及相关酶活的影响[J].水产学报,2016,40(12):1897-1905.Ruan P,Jiang X M,Han Q X,et al.Effects of social hierarchy on the growth,survival and related enzyme activities of Sepia pharaonis[J].Journal of Fisheries of China,2016,40(12):1897-1905(in Chinese).
[3]Peng R B,Le K X,Wang P S,et al.Detoxification Pathways in response to environmental ammonia exposure of the cuttlefish,Sepia pharaonis:glutamine and urea formation[J].Journal of the World Aquaculture Society,2017,48(2):342-352.
[4]Peng R B,Wang P S,Le K X,et al.Acute and chronic effects of ammonia on juvenile cuttlefish,Sepia pharaonis[J].Journal of the World Aquaculture Society,2017,48(4):602-610.
[5]Sinha A K,Rasoloniriana R,Dasan A F,et al.Interactive effect of high environmental ammonia and nutritional status on ecophysiological performance of European sea bass(Dicentrarchus labrax)acclimated to reduced seawater salinities[J].Aquatic Toxicology,2015,160:39-56.
[6]许星鸿,张雁秋,阎斌伦,等.氨氮胁迫对日本蟳免疫生理指标及器官结构的影响[J].生态学报,2014,34(14):3885-3894.Xu X H,Zhang Y Q,Yan B L,et al.Effects of ammonia-N stress on immunity-related indicators and histological structure of some organs of the marine crab,Charybdis japonica[J].Acta Ecologica Sinica,2014,34(14):3885-3894(in Chinese).
[7]Benli A?K,K?ksal G,?zkul A.Sublethal ammonia exposure of Nile tilapia(Oreochromis niloticus L.):Effects on gill,liver and kidney histology[J].Chemosphere,2008,72(9):1355-1358.
[8]Chen J C,Cheng S Y,Chen C T.Changes of haemocyanin,protein and free amino acid levels in the haemolymph of Penaeus japonicus exposed to ambient ammonia[J].Comparative Biochemistry and Physiology Part A:Physiology,1994,109(2):339-347.
[9]郝小凤,刘洋,凌去非.氨氮对泥鳅的急性毒性及对其肝、鳃组织超微结构的影响[J].水生态学杂志,2012,33(5):101-107.Hao X F,Liu Y,Ling Q F.Acute toxicity test of ammonia nitrogen and effects of ammonia-N stress on the ultrastructure of gill and liver of Misgurnus anguillicaudatus[J].Journal of Hydroecology,2012,33(5):101-107(in Chinese).
[10]Hegazi M M,Attia Z I,Ashour O A.Oxidative stress and antioxidant enzymes in liver and white muscle of Nile tilapia juveniles in chronic ammonia exposure[J].Aquatic Toxicology,2010,99(2):118-125.
[11]张武肖,孙盛明,戈贤平,等.急性氨氮胁迫及毒后恢复对团头鲂幼鱼鳃、肝和肾组织结构的影响[J].水产学报,2015,39(2):233-244.Zhang W X,Sun S M,Ge X P,et al.Acute effects of ammonia exposure on histopathology of gill,liver and kidney in juvenile Megalobrama amblycephala and the post-exposure recovery[J].Journal of Fisheries of China,2015,39(2):233-244(in Chinese).
[12]洪美玲,陈立侨,顾顺樟,等.氨氮胁迫对中华绒螯蟹免疫指标及肝胰腺组织结构的影响[J].中国水产科学,2007,14(3):412-418.Hong M L,Chen L Q,Gu S Z,et al.Effects of ammonia exposure on immunity indicators of haemolymph and histological structure of hepatopancreas in Chinese mitten crab(Eriocheir sinensis)[J].Journal of Fishery Sciences of China,2007,14(3):412-418(in Chinese).
[13]王贞杰,陈四清,曹栋正,等.急性氨氮胁迫对圆斑星鲽(Verasper variegatus)幼鱼鳃和肝组织结构及相关酶活性的影响[J].渔业科学进展,2017,38(2):59-69.Wang Z J,Chen S Q,Cao D Z,et al.Effects of acute ammonia nitrogen stress on histopathology of gill andliver and enzyme activities of juvenile Verasper variegatus[J].Progress in Fishery Sciences,2017,38(2):59-69(in Chinese).
[14]Wong C K C,Wong M H.Morphological and biochemical changes in the gills of tilapia(Oreochromis mossambicus)to ambient cadmium exposure[J].Aquatic Toxicology,2000,48(4):517-527.
[15]Shiogiri N S,Paulino M G,Carraschi S P,et al.Acute exposure of a glyphosate-based herbicide affects the gills and liver of the Neotropical fish,Piaractus mesopotamicus[J].Environmental Toxicology and Pharmacology,2012,34(2):388-396.
[16]Velmurugan B,Selvanayagam M,Cengiz E I,et al.Histopathology of lambda-cyhalothrin on tissues(gill,Kidney,liver and intestine)of Cirrhinus mrigala[J].Environmental Toxicology and Pharmacology,2007,24(3):286-291.
[17]胡毅,黄云,钟蕾,等.氨氮胁迫对青鱼幼鱼鳃丝Na+/K+-ATP酶、组织结构及血清部分生理生化指标的影响[J].水产学报,2012,36(4):538-545.Hu Y,Huang Y,Zhong L,et al.Effects of ammonia stress on the gill Na+/K+-ATPase,microstructure and some serum physiological-biochemical indices of juvenile black carp(Mylopharyngodon piceus)[J].Journal of Fisheries of China,2012,36(4):538-545(in Chinese).
[18]Suzuki H,Yanaka A,Shibahara T,et al.Ammonia-induced apoptosis is accelerated at higher p H in gastric surface mucous cells[J].Gastrointestinal and Liver Physiology,2002,283(4):G986-G995.
[19]Smart G R.Investigations of the toxic mechanisms of ammonia to fish-gas exchange in rainbow trout(Salmo gairdneri)exposed to acutely lethal concentrations[J].Journal of Fish Biology,1978,12(1):93-104.
[20]Wicks B J,Randall D J.The effect of sub-lethal ammonia exposure on fed and unfed rainbow trout:the role of glutamine in regulation of ammonia[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2002,132(2):275-285.
[21]Randall D J,Tsui T K N.Ammonia toxicity in fish[J].Marine Pollution Bulletin,2002,45(1-12):17-23.
[22]Van der Linden A,Verhoye M,Nilsson G E.Does anoxia induce cell swelling in carp brains?In vivo MRI measurements in crucian carp and common carp[J].Journal of Neurophysiology,2001,85(1):125-131.
[23]Ip Y K,Tay A S L,Lee K H,et al.Strategies adopted by the swamp eel Monopterus albus to survive in high concentrations of environmental ammonia[J].Physiological and Biochemical Zoology,2004,77:390-405.
[24]黎庆,龚诗雁,黎明.慢性氨氮暴露诱发黄颡鱼幼鱼谷氨酰胺积累、氧化损伤及免疫抑制的研究[J].水产学报,2015,39(5):728-734.Li Q,Gong S Y,Li M.Chronic ammonia toxicity induces glutamine accumulation,oxidative damage and immunosuppression of juvenile yellow catfish Pelteobagrus fulvidraco[J].Journal of Fisheries of China,2015,39 (5):728-734(in Chinese).