Ammonia Metabolism Pathway And Ammonia Transport Of Rh Proteins Of Oreochromis Niloticus Under The Stress Of Carbonate Alkalinity

Nile tilapia（Oreochromis niloticus）is an important aquaculture fish in China.It does not only grow fast,but also show a strong salinity and alkalinity tolerance,which can be used as a candidate to breed saline-alkaline tolerance varieties.This thesis assesses salinity-alkalinity tolerance of juvenile fish of the 3^rd selected generation of Nile tilapia and growth performance of the adult.Also,the timing of the ammonia metabolism pathways（glutamine metabolism pathway and urea metabolism pathway）and the Rh protein ammonia transport function in gills under the stress of carbonate alkali stress were investigated.The main results are as follows:（1）In order to assess salinity-alkalinity tolerance of juvenile fish of the 3^rd selected generation of Nile tilapia and growth performance of the adult,chronic salinity-alkalinity lethal test was carried out for the 3rd selected generation of tilapia fingerling and the control group in tanks of0.6 m×0.4 m×0.5 m.And 60 days growth trail were carried out in cement tanks of each 6.0 m×5.0 m×1.6 m for the 3rd selected generation and the control 5 group reared in fresh water（S0A0 group）,salinity group（10 g/L,S10A0 group）and different salinity-alkalinity waters（10,2）g/L,S10A2group;（10,4）g/L,S10A4 group;and（10,6）g/L,S10A6 group.It was found that the median lethal salinity was 39.63±2.12 g/L for the third selected generation,and 31.42±2.42 g/L for the control group,and that the median lethal alkalinity was 13.94±0.25 g/L for the third selected generation group and 11.26±0.15 g/L for the control group.The average growth rate of the 3rd generation and control group was decreased in S10A0,S10A2,S10A4 and S10A6 groups with the elevation of alkalinity.In the same salinity-alkalinity condition,the 3rd selected generation group was significantly higher than the control group（P<0.05）.（2）This study was conducted to comprehend the mechanism of ammonia metabolism during the adaptation of Nile tilapia to alkaline environment.In this study,tilapia was subjected to acute stress in（2,4,6g/L）NaHCO3 alkaline environment.This study analyses the following changes of tilapia under acute stress in 72 h:the concentration of serum ammonia;the concentrations of urea in liver,kidney,gill,water,urine and blood;the concentration of glutamine（Gln）in liver,brain and gill;the activities of glutamine synthetase（GS）in liver,brain and the activities of carbamyl phosphate synthetase（CPS）in liver;the changes in gene expression of synthetase,carbamyl phosphate synthetase,glutaminase（GLS）in different tissues.The results showed that the blood ammonia concentration of Nile tilapia increased under acute stress,reaching its peak at 12 h.With the increase of blood ammonia,the concentration of urea in each tissue substantially increased from 0 to 6 h.There was a dramatic growth of the activity of carbamyl phosphate synthase from 0 to 2 h.The expression quantity of carbamyl phosphate synthase went up from 0 to 24 h,which indicating that the urea metabolization pathway was activated within0-6 h.The concentration of glutamine in the liver grew rapidly from 0 to 6h and reached its highest point.The activity of glutamine synthase in the liver soared from 0 to 6 h and from 12 to 24 h.The expression of glutamine synthase and glutaminase in each tissue was on a rise at 0 to 24 h.It illustrated that glutamine metabolism was also activated within 0-6 h.The results showed that Nile tilapia activated urea metabolism pathway and glutamine metabolism pathway simultaneously to adjust blood ammonia concentration in the early stage of stress under alkali stress conditions.（3）In this paper,the ammonia transport pathway of euryhaline fishes in carbonate alkaline environment was studied.The Nile tilapia was simultaneously subjected to acute alkalinity stress（2,4,6 g/L）and chronic alkalinity stress.The blood ammonia concentration within 120 h stress was detected,and the mRNA expression of Rhag,Rhbg,Rhcg1 and Rhcg2 in the gill was analyzed,respectively.Immunohistochemistry was used to detect the positive reaction of Rh glycoprotein in gill.The results showed that under acute alkalinity stress,blood ammonia increased rapidly to peak at 12 h,and the expression of four kinds of Rh genes increased,and reached the peak at 24 h.Due to the increasing alkalinity of the chronic alkali stress group,the concentration of blood ammonia was fluctuated,and the expression of the four Rh genes was maintained at a high level of expression.It is suggested that four kinds of Rh proteins may participate in the regulation of blood ammonia concentration.The expression of Rhcg1gene was significantly higher than that of the other three genes at 24 h.The immunohistochemical results showed that Rhag,Rhbg and Rhcg in the acute and chronic stress groups were found to be positive in the gill,and the positive response increased with the increase of alkalinity.The results of this study indicated that,in the context of alkali stress,Nile tilapia could increase the expression of Rh gene and protein in the process of transamination.