Studies On The Pathway Of Detoxication And Evaluation Of Physical Health On The Exposure Of Ammonia In Sea Cucumber Apostichopus Japonicus

Ammonia is one of the most important stress factors in aquaculture. This textresearched the expression of related gene in detoxification metabolism and thevariation of enzymatic activity, the content of nitrogen compounds, the indicators ofimmunity and antioxidant. Thus, we explored the mechanisms in ammoniadetoxification and physical health level on the exposure of ammonia in the seacucumber Apostichopus japonicus. The article mainly includes three parts:(1) Geneclone of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) andsequences analysis.(2) The study of ammonia transport and metabolism on theexposure of ammonia in A. japonicus.(3) The influence on immunity and antioxidantindicators on the exposure of ammonia in A. japonicus. The results are listed below:1. Ammonia detoxification metabolism related genes clone and sequence analysis.In this test, we used respiratory tree of A. jaopnicus as template and cloned twogene segments through the website of COEDHOP and RT-PCR. GDH gene segmentreached a length of693bp, coding231amino acids. It has the identity as high as78%compared with Riptortus pedestris. GS gene segment reached a length of636bp,coding212amino acids. It has the identity as high as78%compared withParacentrotus lividus.2. The study of ammonia transport and metabolism on the exposure of ammonia in A.japonicus.In this study, we set up three different ammonia concentrations of0(control),0.5and1.25mg L1for ammonia exposure, and sampled three tissues of coelomic fluid,respiratory tree and body wall at the time of0,2,6,12,24,48h. GDH activity, GSactivity, Glutamine (Gln) content, Xanthine oxidoreductase (XOR) activity, uric acid (UA) content, Arginase (Arg) activity, Urea content and the key gene expression weremeasured.The results indicated that ammonia exposure had significant influence onammonia content of coelomic fluid, GDH activity, GS activity, Gln content, XORactivity, Arg activity, Urea content of coelomic fluid, respiratory tree and body wall,and gene relative expression of GDH, GS, XOR, Arg of respiratory tree. The contentof UA in all tissues and XOR activity in coelomic fluid could not be measured duringthe test. Ammonia concentration in coelomic fluid increased significantly and peakedat6h in0.5and1.25mg L-1ammonia treatment groups. Afterwards, they alldecreased gradually and remained stable after24h, but had significant differencecompared with the control.GDH activity, GDH relative mRNA expression and GSActivity, GS relative mRNA expression Gln content in respiratory tree of treatmentgroups increased within12and24h, respectively, and then tended to be stable. GDHactivity in body wall increased to the maximum at12h, and then decreased to thecontrol level at48h. GS activity, Gln content in body wall increased during the first12h, and then remained stable. GDH activity in coelomic fluid increased significantlyand tended to be stable, while there was no significant change in GS activity and Glncontent of coelomic fluid during the investigation time. Arg activity, Arg relativemRNA expression and Urea content in respiratory treeincreased significantly during12h, and then remained stable. XOR, Arg activity and Urea content in body wallpeaked at6h,12h,12h respectively, and recovered to the control level at48h. XORactivity, XOR relative mRNA expression in respiratory tree and Arg activity, Ureacontent in coelomic fluid had no significant difference compared with control. Thisimplies that ammonia detoxification strategies existed in A. japonicus, which couldconvert ammonia to Gln and Urea to reduce the poison.3. The influence of immunity and antioxidant on the exposure of ammonia in A.japonicus.In this study, we set up three different ammonia concentrations of0(control),0.5and1.25mg L1for ammonia exposure, and sampled the tissues of coelomic fluid andbody wall at the time of0,2,6,12,24,48h. Total coelomocyte counts, agglutinating activity, lysozyme activity (lsz), antimicrobial activity in coelomic fluid and totalantioxidant ability (T-AOC), superoxide dismutase (SOD), glutathione (GSH) contentand GSH/GSSG in coelomic fluid and body wall. The results showed that ammoniaexposure had significant influence on Total coelomocyte counts, agglutinating activity,lsz activity, antimicrobial activity in coelomic fluid and T-AOC, SOD, GSH contentand GSH/GSSG in coelomic fluid and body wall. Total coelomocyte counts in0.5and1.25mg L-1ammonia treatment groups decreased during12h, and then recovered tothe control level at48h. Lsz activity increased significantly within6h, and thenrecovered to the control level. Antimicrobial activity and agglutinating activitydecreased during48h. T-AOC, SOD activity, GSH content decreased significantly andremained stable after12h. T-AOC and SOD activity in body wall increased to themaximum at6h, and then decreased and significantly lower the control level at48h.GSH content and GSH/GSSG increased to the maximum at6h, and then recovered tothe control level. The results indicated that ammonia exposure had significantinfluence on immunity and antioxidant system in A. japonicus, and can lead to adecreased level of immunity and antioxidant.