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

Acute effects of ammonia exposure on selected immunological and metabolic parameters in juvenile red claw crayfish(Cherax quadricarinatus)and the post-exposure recovery

应用实时荧光定量PCR技术,结合生物酶和代谢产物测定,研究了氨氮急性胁迫对红螯光壳螯虾幼虾代谢及免疫系统的毒性影响及其毒后恢复情况。实验首先进行3 d的氨氮胁迫,取样后剩余虾移入曝气自来水进行7 d的毒后恢复实验。结果表明,3 d氨氮胁迫后,肌肉ACP、AKP、SOD活性表达均受到显著影响,随着氨氮浓度的升高酶活性分别降低,最高浓度组(16 mg/L)降低为对照组的76%、68%和62%。线粒体MnSOD、胞外Cu/ZnSOD的mRNA表达量也随着氨氮浓度增加而下降,最高浓度组降低至对照组的69%和68%。CAT、GPX活性以及GPX和GST的mRNA表达量变化不显著。肝胰腺中可溶性蛋白和甘油三酯含量随着氨氮浓度升高而降低,最高浓度组分别降低至对照组的72%和59%,AST活性在12 mg/L浓度组显著升高至对照组的134%。7 d恢复期过后,ACP和AKP活性以及各代谢指标恢复到正常水平;而SOD和GPX活性高于对照组。各抗氧化基因的表达量都不同程度高于对照组。实验表明,高浓度氨氮胁迫能抑制部分免疫相关酶的活性及基因表达,对免疫系统造成损害。氨氮胁迫下,红螯光壳螯虾动员蛋白质和脂肪来供能应对胁迫。7 d的恢复时间不足以让红螯光壳螯虾从胁迫中完全恢复,其肌肉仍处于轻度氧化应激状态。

The acute effects of ammonia exposure on selected immunological and metabolic parameters in juvenile red claw crayfish, Cherax quadricarinatus as well as the post-exposure recovery were studied by using Real-time PCR technique and biochemical assays. The crayfish were first exposed to ammonia for 3 d, then half of them were sampled and the other half were moved into aerated tap water for a 7 d post-exposure recovery. The results showed that a 3 d ammonia exposure had significant impact on the activities of ACP, AKP and SOD in the muscle of juvenile C. quadricarinatus. As ammonia concentration increased, the activities of these enzymes significantly decreased. At the highest concentration （16 mg/L）, they decreased to 76% ,68% and 62% of the control, respectively. The mRNA expression of mitochondrial manganese superoxide dismutase （ mMnSOD ） , cytosolic MnSOD （ cMnSOD ）, extracellular copper/zinc SOD （ exCu/ ZnSOD） also showed a decreasing trend following increased ammonia concentration. In contrast, the mRNA expression of catalase （ CAT）, glutathione peroxidase （ GPX ） and glutathione transferase （GST） in muscle remained unchanged compared to the control. Dissolved protein and triglycerides in hepatopancreas significantly decreased as ammonia concentration increased. At the highest concentration, they decreased to 72% and 59% of the control. AST showed a noble elevation which was 134% of the control in 12 mg/L group. After a 7 d post-exposure recovery, ACP and AKP activities returned to the normal level in ammonia treatments. On the contrary, the activities of SOD and GPX in all ammonia treatments were significantly higher than the control. The expression of antioxidant genes was higher than the control in all ammonia treatments. Metabolic parameters showed no differences between all groups. The results indicate that high levels of ammonia might suppress activities and gene expression patterns of immune-related enzymes, leading to a loss of defence mechanism. The crayfish which had been exposed to ammonia for 3 d could not fully recover after a 7 d post-exposure recovery and oxidative stress still existed in the muscle in C. quadricarinatus.