氨氮胁迫与恢复对罗氏沼虾幼虾非特异性免疫的影响
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摘要
在温度为(25±1)℃、p H为8. 0±0. 5条件下采用生物毒性实验方法研究了氨氮对罗氏沼虾(Macrobrachium rosenbergii)幼虾半致死浓度,并设置0、0. 43、0. 64、0. 85、1. 06 mg·L~(-1)5个浓度研究氨氮胁迫48 h及恢复48 h对罗氏沼虾非特异性免疫的影响。结果显示:非离子氨对罗氏沼虾幼虾24、48、72、96 h半致死浓度分别为4. 25、2. 35、1. 86、1. 54 mg·L~(-1),安全浓度为0. 154 mg·L~(-1)。氨氮胁迫48 h,罗氏沼虾SOD活性显著升高(P <0. 05),而CAT和GSH-Px活性变化不显著(P> 0. 05); 0. 43、0. 64、1. 06 mg·L~(-1)氨氮胁迫组罗氏沼虾MDA含量显著高于对照组(P <0. 05)。氨氮胁迫后恢复48 h,各组SOD活性均较胁迫时显著下降(P <0. 05),但除0. 85 mg·L~(-1)组外的其它试验组仍显著高于对照组(P <0. 05); 0. 85、1. 06 mg·L~(-1)组CAT活性较胁迫状态显著下降(P <0. 05);试验组与对照组MDA含量无显著差异(P> 0. 05)。结果表明,低浓度氨氮短期胁迫对罗氏沼虾造成的氧化损伤在解除胁迫后可修复,但肌肉仍处于应激状态。
Current study explored the influence of ammonia-N stress and post-exposure recovery on the nonspecific immunity of M. rosenbergii. The prawns were exposed to different concentrations of ammonia( 0,0. 43,0. 64,0. 85,1. 06 mg·L~(-1)) for 48 h,and then post-exposure recovery for 48 h was analyzed. The activities of superoxide dismutase( SOD), catalase( CAT), glutathione peroxidase( GSH-Px) and malondialdehyde( MDA) content were assessed in the muscle of M. rosenbergii under ammonia stress for 48 h and post-exposure recovery for 48 h. The results showed that the half lethal concentrations of non-ionic ammonia of 24 h,48 h,72 h and 96 h were 4. 25,2. 35,1. 86,1. 54 mg·L~(-1),respectively. And the safe concentration was 0. 154 mg·L~(-1) under the condition of( 25 ± 1) ℃,pH 8. 0 ± 0. 5. The SOD activity in the muscle of M. rosenbergii was significantly raised by ammonia nitrogen at 48 h. However,compared to the control,the activities of CAT and GSH-Px in all ammonia treatments didn't change significantly at 48 h( P >0. 05). The MDA content of 0. 43,0. 64,1. 06 mg·L~(-1) ammonia treatments were significantly higher than that of the control at 48 h( P < 0. 05). After 48 h post-exposure recovery,compared to the ammonia exposure,SOD activity reduced significantly with the same ammonia treatment( P < 0. 05),but they were still significantly higher than that of the control group except for 0. 85 mg·L~(-1)( P < 0. 05). CAT activities in0. 85,1. 06 mg · L~(-1) treatments reduced significantly at the same ammonia treatments after 48 h postexposure recovery( P < 0. 05),and MDA content of all treatments returned to the level of the control. The results indicate that oxidative damage of M. rosenbergii by ammonia-N recovers after 48 h post-exposure recovery,but oxidative stress still exists in the muscle.
Current study explored the influence of ammonia-N stress and post-exposure recovery on the nonspecific immunity of M. rosenbergii. The prawns were exposed to different concentrations of ammonia( 0,0. 43,0. 64,0. 85,1. 06 mg·L~(-1)) for 48 h,and then post-exposure recovery for 48 h was analyzed. The activities of superoxide dismutase( SOD), catalase( CAT), glutathione peroxidase( GSH-Px) and malondialdehyde( MDA) content were assessed in the muscle of M. rosenbergii under ammonia stress for 48 h and post-exposure recovery for 48 h. The results showed that the half lethal concentrations of non-ionic ammonia of 24 h,48 h,72 h and 96 h were 4. 25,2. 35,1. 86,1. 54 mg·L~(-1),respectively. And the safe concentration was 0. 154 mg·L~(-1) under the condition of( 25 ± 1) ℃,pH 8. 0 ± 0. 5. The SOD activity in the muscle of M. rosenbergii was significantly raised by ammonia nitrogen at 48 h. However,compared to the control,the activities of CAT and GSH-Px in all ammonia treatments didn't change significantly at 48 h( P >0. 05). The MDA content of 0. 43,0. 64,1. 06 mg·L~(-1) ammonia treatments were significantly higher than that of the control at 48 h( P < 0. 05). After 48 h post-exposure recovery,compared to the ammonia exposure,SOD activity reduced significantly with the same ammonia treatment( P < 0. 05),but they were still significantly higher than that of the control group except for 0. 85 mg·L~(-1)( P < 0. 05). CAT activities in0. 85,1. 06 mg · L~(-1) treatments reduced significantly at the same ammonia treatments after 48 h postexposure recovery( P < 0. 05),and MDA content of all treatments returned to the level of the control. The results indicate that oxidative damage of M. rosenbergii by ammonia-N recovers after 48 h post-exposure recovery,but oxidative stress still exists in the muscle.
引文
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[6] CHENG W,CHEN S M,WANG F I,et al. Effects of temperature,p H,salinity and ammonia on the phagocytic activity and clearance efficiency of giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae[J]. Aquaculture,2003,219(1-4):111-121.
[7]贾旭颖.淡水养殖凡纳滨对虾(Litopenaeus vannamei)对环境胁迫的生理响应[D].青岛:中国海洋大学. 2014.JIA X Y. The ecophysiological responses of Litopenaeus vannamei under freshwater condition to environmental stress[D]. Qingdao:Ocean University of China. 2014.
[8]方金龙,王元,李新苍,等.氨氮和亚硝基氨共同胁迫对凡纳滨对虾感染WSSV的影响[J].海洋渔业,2017,39(1):85-91.FANG J L. WANG Y,LI X C,et al. Effects of WSSV on cultured Litopenaeus vannamei under both ammonia and nitrite stress[J]. Marine Fisheries,2017,39(1):85-91.
[9]李永,杨其彬,苏天凤,等.氨氮对斑节对虾的毒性及免疫指标的影响[J].上海海洋大学学报,2012,21(3):358-362.LI Y,YANG Q B,SU T F,et al. The toxicity of ammonia-N on Penaeus monodon and immune parameters[J]. Journal of Shanghai Ocean University,2012,21(3):358-362.
[10]王芸. p H、氨氮胁迫对中国对虾细胞凋亡和抗氧化系统影响机理的研究[D].上海:上海海洋大学,2011.WANG Y. Effects of p H, Ammonia Stress on Apoptosis and antioxidant system of Chinese shrimp,Fenneropenaeus chinensis[D]. Shanghai:Shanghai Ocean University,2011.
[11]李波,樊启学,张磊,等.不同溶氧水平下氨氮和亚硝酸盐对黄颡鱼的急性毒性研究[J].淡水渔业,2009,39(3):31-35.LI B,FAN Q X,ZHANG L,et al. Acute toxic effect of ammonia and nitrite on yellow catfish(Pelteobagrus fulvidraco)at different dissolved oxygen levels[J]. Freshwater Fisheries,2009,39(3):31-35.
[12]王龙郝志敏王晶.两种溶氧条件下亚硝酸盐和氨氮对罗氏沼虾毒性比较的研究[J].饲料与畜牧,2011(8):12-16WANG L,HAO Z M,WANG J. Acute toxic effect of ammonia and nitrite on Macrobrachium rosenbergii two dissolved oxygen levels[J]. Feed and Husbandry. 2011(8):12-16.
[13]王娟,曲克明,刘海英,等.不同溶氧条件下亚硝酸盐和氨氮对中国对虾的急性毒性效应[J].海洋水产研究,2007,28(6):1-6.WANG J,QU K M,LIU H Y,et al. Acute toxic effects of nitrite and non-ion ammonia on Fenneropenaeus chinensis at different dissolved oxygen levels[J]. Marine Fisheries Research,2007,28(6):1-6.
[14]曲克明,徐勇,马绍赛,等.不同溶解氧条件下亚硝酸盐和非离子氨对大菱鲆的急性毒性效应[J].海洋水产研究,2007,28(4):83-85.QU K M,XU Y,MA S S,et al. Acute toxic effects of nitrite and non-ion ammonia on turbot(Scophthalmus maximus)at different DO levels[J].Marine Fisheries Research,2007,28(4):83-85.
[15]潘训彬,张秀霞,鲁耀鹏,等.氨氮和亚硝酸盐对红螯螯虾幼虾和亚成虾的急性毒力[J].生物安全学报,2017,26(4):316-322.PAN X B,ZHANG X X,LU Y P,et al. Acute toxicity of ammonia and nitrite on juvenile and subadult redclaw crayfish(Cherax quadricarinatus)[J]. Journal of Biosafety,2017,26(4):316-322.
[16]钟君伟,朱永安,孟庆磊,等.氨氮对2种规格克氏原螯虾的急性毒性研究[J].长江大学学报(自然科学版),2013,10(23):55-59.ZHONG J W,ZHU Y A,MENG Q L,et al. Acute toxicity of Ammonia-N to two specifications of Procambarus clarkii adult[J]. Journal of Yangtze University(Natural Science Edition), 2013,10(23):55-59.
[17]罗静波,曹志华,蔡太锐,等.氨氮对克氏原螯虾幼虾的急性毒性研究[J].长江大学学报(自然科学版),2006,3(4):183-185.LUO J B,CAO Z H,CAI T R,et al. Acute toxic research of ammonia nitrogen on Procambarus clarkia juvenile[J]. Journal of Yangtze University(Natural Science Edition),2006,3(4):183-185.
[18] CHEN J C,LIN C Y. Lethal effects of ammonia on Penaeus chinensis Osbeck juveniles at different salinity levels[J] Journal of Experimental Marine Biology and Ecology,1992,156(1):139-148.
[19] ZHANG Y,YE C,WANG A,et al. Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn(Macrobrachium rosenbergii):Effects on the oxidative stress,antioxidant enzymatic activities and apoptosis in haemocytes[J].Ecotoxicology,2015(24):1601-1610.
[20] LEPAGE G,MNUOZ G,CHAMPAGNE J,et al.Preparative steps necessary for the accurate measurement of malondialdehyde by highperformance liquid chromatography[J]. Analytical Biochemistry,1991,197(2):277-283.
[21]任海,李健,李吉涛,等.急性氨氮胁迫对脊尾白虾(Exopalaemon carinicauda)抗氧化系统酶活力及GPx基因表达的影响[J].农业环境科学学报,2014,33(4):647-655.REN H, LI J, LI J T, et al. Effects of acute ammonia stresses on antioxidant enzyme activities and GPx gene expression in Exopalaemon carinicauda[J]. Journal of Agro-Environment Science,2014,33(4):647-655.
[22]包杰,姜宏波,程慧,等.氨氮对中华小长臂虾的急性毒性及非特异性免疫指标的影响[J].水生生物学报,2017,41(3):516-522.BAO J,JIANG H B,CHENG H,et al. Effect of ammonia nitrogen on acute toxicity and nonspecific immune parameters of Palaemonetes sinensis[J].Acta Hydrobiologica Sinica,2017,41(3):516-522.
[23] YILDIRIM O, BUYUKBINGOL Z. Effects of supplementation with a combination of cobalt and ascorbic acid on antioxidant enzymes and lipid peroxidation levels in streptozocin-diabetic rat liver[J]. Biological Trace Element Research,2002,90(1-3):143-154.
[24]蒋琦辰,顾曙余,张文逸,等.氨氮急性胁迫及其毒后恢复对红螯光壳螯虾幼虾相关免疫和代谢指标的影响[J].水产学报,2013,37(7):1066-1072.JIANG Q C,GU S Y,ZHANG W Y,et al. Acute effects of ammonia exposure on selected immunological and metabolic parameters in juvenile red claw crayfish(Cherax quadricarinatus)and the post-exposure recovery[J]. Journal of Fisheries of China,2013,37(7):1066-1072.
[25]曾媛媛,蒋云霞,艾春香.氨氮胁迫对拟穴青蟹组织器官中SOD及GPX活性的影响[J].台湾海峡,2011,30(2):210-215.ZENG Y Y, JIANG Y X, AI C X. Effects of ammonia-N stress on the activities of superoxide dismutase and glutathione peroxidase in different tissues and organs of Scylla paramamosain[J].Journal of Oceanography in Taiwan Strait,2011,30(2):210-215.
[26]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.
[2] YANG W,XIANG F H,LIANG L G,et al. Toxicity of ammonia and its effects on oxidative stress mechanisms of juvenile crucian carp(Carassius auratus)[J]. Journal of Freshwater Ecology,2010,25(2):297-302.
[3] ROMANO N,ZENG C. Toxic effects of ammonia,nitrite,and nitrate to decapod crustaceans:A review on factors influencing their toxicity, physiological consequences, and coping mechanisms[J].Reviews in Fisheries Science,2013,21(1):1-21.
[4] CHEN J C,KOU Y Z. Effects of ammonia on growth and molting on Penaeus japonicus juveniles[J].Aquaculture,1992,104(3-4):249-260.
[5] CHENG W,CHEN J C. The virulence of Enterococcus to freshwater prawn Macrobrachium rosenbergii and its immune resistance under ammonia stress[J].Fish&Shellfish Immunology,2002,12(2):97-109.
[6] CHENG W,CHEN S M,WANG F I,et al. Effects of temperature,p H,salinity and ammonia on the phagocytic activity and clearance efficiency of giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae[J]. Aquaculture,2003,219(1-4):111-121.
[7]贾旭颖.淡水养殖凡纳滨对虾(Litopenaeus vannamei)对环境胁迫的生理响应[D].青岛:中国海洋大学. 2014.JIA X Y. The ecophysiological responses of Litopenaeus vannamei under freshwater condition to environmental stress[D]. Qingdao:Ocean University of China. 2014.
[8]方金龙,王元,李新苍,等.氨氮和亚硝基氨共同胁迫对凡纳滨对虾感染WSSV的影响[J].海洋渔业,2017,39(1):85-91.FANG J L. WANG Y,LI X C,et al. Effects of WSSV on cultured Litopenaeus vannamei under both ammonia and nitrite stress[J]. Marine Fisheries,2017,39(1):85-91.
[9]李永,杨其彬,苏天凤,等.氨氮对斑节对虾的毒性及免疫指标的影响[J].上海海洋大学学报,2012,21(3):358-362.LI Y,YANG Q B,SU T F,et al. The toxicity of ammonia-N on Penaeus monodon and immune parameters[J]. Journal of Shanghai Ocean University,2012,21(3):358-362.
[10]王芸. p H、氨氮胁迫对中国对虾细胞凋亡和抗氧化系统影响机理的研究[D].上海:上海海洋大学,2011.WANG Y. Effects of p H, Ammonia Stress on Apoptosis and antioxidant system of Chinese shrimp,Fenneropenaeus chinensis[D]. Shanghai:Shanghai Ocean University,2011.
[11]李波,樊启学,张磊,等.不同溶氧水平下氨氮和亚硝酸盐对黄颡鱼的急性毒性研究[J].淡水渔业,2009,39(3):31-35.LI B,FAN Q X,ZHANG L,et al. Acute toxic effect of ammonia and nitrite on yellow catfish(Pelteobagrus fulvidraco)at different dissolved oxygen levels[J]. Freshwater Fisheries,2009,39(3):31-35.
[12]王龙郝志敏王晶.两种溶氧条件下亚硝酸盐和氨氮对罗氏沼虾毒性比较的研究[J].饲料与畜牧,2011(8):12-16WANG L,HAO Z M,WANG J. Acute toxic effect of ammonia and nitrite on Macrobrachium rosenbergii two dissolved oxygen levels[J]. Feed and Husbandry. 2011(8):12-16.
[13]王娟,曲克明,刘海英,等.不同溶氧条件下亚硝酸盐和氨氮对中国对虾的急性毒性效应[J].海洋水产研究,2007,28(6):1-6.WANG J,QU K M,LIU H Y,et al. Acute toxic effects of nitrite and non-ion ammonia on Fenneropenaeus chinensis at different dissolved oxygen levels[J]. Marine Fisheries Research,2007,28(6):1-6.
[14]曲克明,徐勇,马绍赛,等.不同溶解氧条件下亚硝酸盐和非离子氨对大菱鲆的急性毒性效应[J].海洋水产研究,2007,28(4):83-85.QU K M,XU Y,MA S S,et al. Acute toxic effects of nitrite and non-ion ammonia on turbot(Scophthalmus maximus)at different DO levels[J].Marine Fisheries Research,2007,28(4):83-85.
[15]潘训彬,张秀霞,鲁耀鹏,等.氨氮和亚硝酸盐对红螯螯虾幼虾和亚成虾的急性毒力[J].生物安全学报,2017,26(4):316-322.PAN X B,ZHANG X X,LU Y P,et al. Acute toxicity of ammonia and nitrite on juvenile and subadult redclaw crayfish(Cherax quadricarinatus)[J]. Journal of Biosafety,2017,26(4):316-322.
[16]钟君伟,朱永安,孟庆磊,等.氨氮对2种规格克氏原螯虾的急性毒性研究[J].长江大学学报(自然科学版),2013,10(23):55-59.ZHONG J W,ZHU Y A,MENG Q L,et al. Acute toxicity of Ammonia-N to two specifications of Procambarus clarkii adult[J]. Journal of Yangtze University(Natural Science Edition), 2013,10(23):55-59.
[17]罗静波,曹志华,蔡太锐,等.氨氮对克氏原螯虾幼虾的急性毒性研究[J].长江大学学报(自然科学版),2006,3(4):183-185.LUO J B,CAO Z H,CAI T R,et al. Acute toxic research of ammonia nitrogen on Procambarus clarkia juvenile[J]. Journal of Yangtze University(Natural Science Edition),2006,3(4):183-185.
[18] CHEN J C,LIN C Y. Lethal effects of ammonia on Penaeus chinensis Osbeck juveniles at different salinity levels[J] Journal of Experimental Marine Biology and Ecology,1992,156(1):139-148.
[19] ZHANG Y,YE C,WANG A,et al. Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn(Macrobrachium rosenbergii):Effects on the oxidative stress,antioxidant enzymatic activities and apoptosis in haemocytes[J].Ecotoxicology,2015(24):1601-1610.
[20] LEPAGE G,MNUOZ G,CHAMPAGNE J,et al.Preparative steps necessary for the accurate measurement of malondialdehyde by highperformance liquid chromatography[J]. Analytical Biochemistry,1991,197(2):277-283.
[21]任海,李健,李吉涛,等.急性氨氮胁迫对脊尾白虾(Exopalaemon carinicauda)抗氧化系统酶活力及GPx基因表达的影响[J].农业环境科学学报,2014,33(4):647-655.REN H, LI J, LI J T, et al. Effects of acute ammonia stresses on antioxidant enzyme activities and GPx gene expression in Exopalaemon carinicauda[J]. Journal of Agro-Environment Science,2014,33(4):647-655.
[22]包杰,姜宏波,程慧,等.氨氮对中华小长臂虾的急性毒性及非特异性免疫指标的影响[J].水生生物学报,2017,41(3):516-522.BAO J,JIANG H B,CHENG H,et al. Effect of ammonia nitrogen on acute toxicity and nonspecific immune parameters of Palaemonetes sinensis[J].Acta Hydrobiologica Sinica,2017,41(3):516-522.
[23] YILDIRIM O, BUYUKBINGOL Z. Effects of supplementation with a combination of cobalt and ascorbic acid on antioxidant enzymes and lipid peroxidation levels in streptozocin-diabetic rat liver[J]. Biological Trace Element Research,2002,90(1-3):143-154.
[24]蒋琦辰,顾曙余,张文逸,等.氨氮急性胁迫及其毒后恢复对红螯光壳螯虾幼虾相关免疫和代谢指标的影响[J].水产学报,2013,37(7):1066-1072.JIANG Q C,GU S Y,ZHANG W Y,et al. Acute effects of ammonia exposure on selected immunological and metabolic parameters in juvenile red claw crayfish(Cherax quadricarinatus)and the post-exposure recovery[J]. Journal of Fisheries of China,2013,37(7):1066-1072.
[25]曾媛媛,蒋云霞,艾春香.氨氮胁迫对拟穴青蟹组织器官中SOD及GPX活性的影响[J].台湾海峡,2011,30(2):210-215.ZENG Y Y, JIANG Y X, AI C X. Effects of ammonia-N stress on the activities of superoxide dismutase and glutathione peroxidase in different tissues and organs of Scylla paramamosain[J].Journal of Oceanography in Taiwan Strait,2011,30(2):210-215.
[26]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.