慢性氨氮胁迫对凡纳滨对虾生理生化指标及血蓝蛋白基因表达的影响

【目的】探究慢性氨氮胁迫对凡纳滨对虾（Litopenaeus vannamei）生理生化和血蓝蛋白基因表达的影响，明确氨氮胁迫的毒性作用，为养殖户在对虾中后期健康养殖管理上提供技术参考，同时丰富凡纳滨对虾的毒理研究资料。【方法】挑选540尾平均初始湿体重9.92±0.24 g/尾的健康凡纳滨对虾，随机分成6个组，每组设3个重复，暴露于不同氨氮浓度［0（对照）、4、6、8、12和16 mg/L］的曝气自来水中，分别于胁迫16、17、18、19和20 d时取样测定凡纳滨对虾的血淋巴氨氮、尿素氮及血蓝蛋白含量，胁迫结束后统计凡纳滨对虾的体长增长率、体重增长率和存活率，同时取鳃组织和肝胰腺制作石蜡切片。【结果】慢性氨氮胁迫16～20 d，各胁迫处理组凡纳滨对虾的存活率、体重增长率和体长增长率均随氨氮浓度的增加而依次降低，且显著低于对照组凡纳滨对虾（P<0.05，下同）。随着氨氮胁迫时间的延长，各胁迫处理组凡纳滨对虾血淋巴氨氮和尿素氮含量均明显高于对照组凡纳滨对虾，且二者的变化趋势基本一致；各胁迫处理组凡纳滨对虾血蓝蛋白含量均显著低于对照组凡纳滨对虾，且血蓝蛋白含量的变化趋势总体上与水体氨氮浓度呈负相关，血蓝蛋白基因的表达水平与血蓝蛋白含量变化趋势基本相符，总体上表现为氨氮胁迫时间越长，凡纳滨对虾血蓝蛋白基因的相对表达量越低。经慢性氨氮胁迫后凡纳滨对虾鳃组织和肝胰腺严重受损，鳃丝肿胀，核固缩，血淋巴细胞增多，局部空泡化、结构不完整，鳃呼吸上皮细胞大面积脱落；胰腺管肿大，空泡化严重，肝小管排列紊乱，管腔扩大，边界模糊，肝小管间隙和管腔中可观察到破碎的细胞组织。【结论】慢性氨氮胁迫对凡纳滨对虾的生长及生存有明显影响，造成血淋巴氨氮和尿素氮含量增加，并下调血蓝蛋白基因表达及阻碍血蓝蛋白合成，凡纳滨对虾的鳃组织和肝胰腺严重受损。即慢性氨氮胁迫引起的呼吸功能障碍及肝胰腺代谢功能紊乱，是导致凡纳滨对虾应激死亡的主要原因。

Effects of chronic ammonia nitrogen stress on physiology and biochemistry indexes and hemocyanin gene expression of Litopenaeus vannamei

【Objective】To investigate the effects of chronic ammonia nitrogen stress on the physiology, biochemistry and expression of hemocyanin gene of Litopenaeus vannamei, clarify the toxic effect of ammonia nitrogen stress, and provide reference for farmers in the middle and late stage of healthy culture management of shrimp, and enrich the toxicological research data of L. vannamei at the same time.【Method】540 healthy L. vannamei with an average initial wet body weight of 9.92±0.24 g per shrimp were randomly divided into 6 groups, each group was set with 3 replicates, each of which was exposed to aerated tap water at different ammonia nitrogen concentrations(0, 4, 6, 8, 12 and 16 mg/L). Samples were taken at 16, 17, 18, 19 and 20 d to determine the contents of ammonia nitrogen, urea nitrogen and hemocyanin of L. vannamei, respectively. After the stress, the growth rate of body length, the rate of weight gain and the survival rate were counted. At the same time, the gill tissue and hepatopancreas were taken for paraffin section.【Result】From 16 to 20 d of chronic ammonia nitrogen stress, the survival rate, the growth rate of weight gain and body length of L. vannamei in each stress group decreased in turn with the increase of ammonia nitrogen concentration, and significantly lower than that of the control group(P<0.05, the same below). With the prolongation of ammonia nitrogen stress time, ammonia nitrogen and urea nitrogen in hemolymph of each stress group were higher than those of the control group, and the change trend of the two was basically consistent;the content of hemocyanin in each stress group was significantly lower than that of the control group, and the variation trend of hemocyanin content was negatively correlated with the concentration of ammonia nitrogen in water, the expression of hemocyanin gene was basically consistent with the change trend of hemocyanin content, which generally showed that the longer ammonia-nitrogen stress time was, the lower the relative expression level of hemocyanin gene was. After chronic ammonia nitrogen stress, the gill tissue and hepatopancreas of L. vannamei were seriously damaged, including gill filament swelling, nuclear pyknosis, increased blood lymphocytes, local vacuolation and incomplete structure, and large area of gill respiratory epithelial cells were exfoliated. Pancreatic ducts were enlarged with severe vacuolization, hepatic tubules were arranged disorderly, lumen was enlarged and the boundary was blurred, and broken cells and tissues could be observed in the space between the hepatic tubules and lumen.【Conclusion】Chronic ammonia nitrogen stress has a great inhibition on the growth and survival rate of L. vanname. It will increase the content of ammonia nitrogen and urea nitrogen in hemolymph, decrease the relative expression level of hemocyanin mRNA, block the synthesis of hemocyanin, and cause serious damage to gill and hepatopancreas. Respiratory dysfunction and hepatopancreas metabolic dysfunction caused by chronic ammo nianitrogen stress are the main causes of stress death of L. vannamei.