摘要
【目的】探究高温和亚硝酸盐复合胁迫对凡纳滨对虾(Litopenaeus vannamei)渗透压调节相关指标的影响,为凡纳滨对虾健康养殖提供理论依据。【方法】试验设2个温度处理:常温组(25℃)和高温组(34℃),并在每个温度条件下设6个亚硝酸盐浓度处理:0.05(对照)、0.2、0.8、3.2、6.4、12.8 mg/L。水温通过加热棒调节并维持稳定,亚硝酸盐用于调控水体亚硝酸盐浓度。分别于0、6、72和168 h进行采样,于冰上解剖后取鳃组织,血淋巴利用1.0 mL无菌注射器经围心腔抽取,与等体积抗凝液混合。使用紫外分光光度计测定340 nm处的吸光度,并计算血淋巴中血蓝蛋白浓度。利用渗透压测定仪测定凡纳滨对虾血淋巴渗透压。采用Na^(+)/K^(+)-ATP和Ca^(2+)/Mg^(2+)-ATP酶活力测定试剂盒测定Na^(+)/K^(+)-ATP和Ca^(2+)/Mg^(2+)-ATP酶活力。实时荧光定量PCR检测凡纳滨对虾鳃组织中渗透压调节相关基因相对表达量。【结果】血蓝蛋白浓度检测结果显示,在25℃水温下,亚硝酸盐胁迫6、72和168 h,3.2、6.4和12.8 mg/L亚硝酸盐组血蓝蛋白浓度均显著低于0.05、0.2和0.8 mg/L亚硝酸盐组(P<0.05,下同),在25和34℃水温下,亚硝酸盐胁迫168 h,12.8 mg/L亚硝酸盐组血蓝蛋白浓度最低。渗透压测定结果显示,在25和34℃水温下,6.4和12.8 mg/L亚硝酸盐组凡纳滨对虾血淋巴渗透压均随胁迫时间延长而降低。酶活力检测结果显示,在34℃水温下,亚硝酸盐胁迫6 h,6.4 mg/L亚硝酸盐组凡纳滨对虾鳃组织中Ca^(2+)/Mg^(2+)-ATP酶活力最低,在25和34℃水温下,亚硝酸盐胁迫72和168 h,12.8 mg/L亚硝酸盐组凡纳滨对虾鳃组织中Na^(+)/K^(+)-ATP酶活力最低。实时荧光定量PCR检测结果显示,在25℃水温下,亚硝酸盐胁迫6 h,CLC基因相对表达量随亚硝酸盐浓度升高逐渐升高,12.8 mg/L亚硝酸盐组CCP基因相对表达量最高。【结论】高温和亚硝酸盐复合胁迫影响凡纳滨对虾血蓝蛋白浓度和血淋巴渗透压,导致凡纳滨对虾鳃组织中渗透压调节相关酶活力和基因相对表达量发生变化。亚硝酸盐胁迫能造成对凡纳滨对虾鳃组织生理紊乱,且高温会加重亚硝酸盐毒性,影响凡纳滨对虾鳃组织的生理功能。
【Objective】The aim of this study was to investigate the effects of combined stress of high temperature and nitrite on osmoregulation related indexes of Litopenaeus vannamei,which could provide theoretical basis for the healthy culture of Litopenaeus vannamei.【Method】Two temperature treatments were set up in the experiment:normal tempera‐ture group(25℃)and high temperature group(34℃),and six nitrite concentration treatments were set up under each temperature condition:0.05(control),0.2,0.8,3.2,6.4,and 12.8 mg/L.The water temperature was adjusted and main‐tained stable by a heating rod,and nitrite was used to regulate the nitrite concentration in the water.Samples were taken at 0,6,72 and 168 h,and gill tissues were dissected on ice.Hemolymph was extracted from the pericardial cavity with a 1.0 mL sterile syringe and mixed with anticoagulant.The absorbance at 340 nm was measured using an ultraviolet spectro‐photometer,and the hemocyanin concentration in the hemolymph was calculated.The hemolymph osmotic pressure of Litopenaeus vannamei was measured using an osmometer.Na^(+)/K^(+)-ATP and Ca^(2+)/Mg^(2+)-ATPase activity assay kits were used to determine the activity of Na^(+)/K^(+)-ATP and Ca^(2+)/Mg^(2+)-ATPase.Real-time fluorescence quantitative PCR was used to detect the relative expression of osmotic pressure regulation related genes in the gill tissues of Litopenaeus vannamei.【Re‐sult】The results of hemocyanin concentration test showed that at 25℃water temperature,the hemocyanin concentrations of 3.2,6.4,and 12.8 mg/L nitrite groups were significantly lower than those of 0.05,0.2,and 0.8 mg/L nitrite groups af‐ter nitrite stress for 6,72 and 168 h(P<0.05,the same below).At 25 and 34℃water temperatures,the hemocyanin con‐centration of 12.8 mg/L nitrite group was the lowest after nitrite stress for 168 h.The results of osmotic pressure measure‐ment showed that at 25 and 34℃water temperatures,the hemolymph osmotic pressure of the 6.4 and 12.8 mg/L nitrite groups decreased with the increase of stress time.The results of enzyme activity detection showed that at 34℃water tem‐perature,after nitrite stress for 6 h,the Ca^(2+)/Mg^(2+)-ATPase activity in the gill tissue of the 6.4 mg/L nitrite group was the lowest,and at 25 and 34℃water temperatures,after nitrite stress for 72 and 168 h,the Na^(+)/K^(+)-ATPase activity in the gill tissue of the 12.8 mg/L nitrite group was the lowest.The results of real-time fluorescence quantitative PCR showed that at 25℃,the relative expression of CLC gene increased gradually with the increase of nitrite concentration after ni‐trite stress for 6 h,and the relative expression of CCP gene was the highest in the 12.8 mg/L nitrite concentration group.【Conclusion】The combined stress of high temperature and nitrite affects the hemocyanin concentration and hemolymph osmotic pressure of Litopenaeus vannamei,leading to changes in the activity of osmoregulation related enzymes and rela‐tive expression of genes in the gill tissue of Litopenaeus vannamei.Nitrite stress can cause physiological disorders in the gill tissue of Litopenaeus vannamei,and high temperature can aggravate the toxicity of nitrite and affect the physiological function of the gill tissue of Litopenaeus vannamei.
作者
李嘉益
程曦
李雪男
王林薇
戴习林
LI Jia-yi;CHENG Xi;LI Xue-nan;WANG Lin-wei;DAI Xi-lin(National Demonstration Center for Experimental Fisheries Science Education(Shanghai Ocean University),Shanghai 201306,China;Shanghai Collaborative Innovation Center for Aquatic Animal Genetics and Breeding(Shanghai Ocean University),Shanghai 201306,China)
出处
《南方农业学报》
北大核心
2025年第6期1869-1878,共10页
Journal of Southern Agriculture
基金
上海市现代农业产业技术体系项目(沪农科产字[2022]第5号)。
关键词
凡纳滨对虾
高温
亚硝酸盐
渗透压
Litopenaeus vannamei
high temperature
nitrite
osmotic pressure
