Effects Of Ammonia Nitrogen Stress On Detoxification Metabolism And Functional Verification Of Related Genes In Cyclina Sinensis

Ammonia nitrogen is a common stress factor in aquaculture environment,which can affect the growth and development,physiological metabolism and behavioral response of aquatic animals,and may even directly cause large-scale death of aquatic animals.Studies have shown that some freshwater fish,crustaceans and aerobic marine fish can reduce ammonia nitrogen toxicity by regulating detoxification metabolic pathways and changing excretion behavior.However,there is little research on how the tidal flat shellfish reduce the toxicity of ammonia nitrogen and the adaptation mechanism of ammonia nitrogen tolerance.The Cyclina sinensis is an important aquaculture species in China,and the adaptation mechanism of the C.sinensis to tolerate ammonia nitrogen is lacking in-depth research.Therefore,this paper took the C.sinensis as the research object.And firstly using the ecotoxicological method to find out the semi-lethal concentration and safe concentration of ammonia nitrogen to C.sinensis.At the tissue level,the toxicity mechanism and detoxification metabolism mechanism of C.sinensis under ammonia nitrogen stress were explored to reveal the adaptive tolerance mechanism of C.sinensis to ammonia nitrogen.This study can provide theoretical guidance for the regulation of water quality in the process of shellfish culture and the breeding of new varieties of Clams tolerant to ammonia nitrogen.The results are as follows:1.Effects of acute ammonia nitrogen stress on the antioxidant and detoxification metabolism in C.sinensisIn this study,the 96-h ammonia nitrogen median lethal concentration(LC₅₀-96 h)and safe concentration（SC）were first evaluated in the C.sinensis.The results showed that the LC₅₀-96 h and SC of ammonia nitrogen to the C.sinensis were 66.79 mg/L and 6.58 mg/L,respectively.where the LC₅₀-96 h and SC of molecular ammonia（NH₃）were 1.70 mg/L and 0.17 mg/L,respectively.On this basis,the activities of superoxide dismutase（SOD）,catalase（CAT）,glutamine synthase（GS）,and glutamine（Gln）in the hepatopancreas of the C.sinensis under different ammonia nitrogen concentrations were studied.and urea,the results showed that both SOD and CAT activities were dose-and time-dependent with TAN concentration.GS activity,Gln content and urea content were significantly increased.The above results showed that the combined effect of enhancing the antioxidant capacity of the hepatopancreas and converting ammonia into Gln and urea could reduce the toxicity of ammonia nitrogen to Clams.2.Cloning,sequence characterization and analysis of genes related to detoxification metabolism in C.sinensisIn this study,glutathione sulfur transferase gene（Cs GST）and ammonia transporter gene（Cs Rhbg）,the key genes in the detoxification metabolic pathway in C.sinensis,were screened for cloning and functional analysis.The results show that the full-length sequence of Cs GST is1141 bp,and the open reading frame has 609 bp,encoding 202 amino acids.Sequence alignment showed that it belonged to the cytoplasmic sigma-like GST.The protein includes two conserved domains:the conserved domain GST＿N contains the GSH binding site（G site）;the conserved domain GST＿C contains the substrate binding pocket site（H site）.The results of the phylogenetic tree showed that the C.sinensis has 76.35%homology with the M.mercenaria.Tissue expression studies showed that Cs GST was expressed in all tissues of C.sinensis.Cs Rhbg has 1777 bp,including 355 bp in the 5’non-coding region and 1422 bp in the ORF region,encoding 473 amino acids.The protein contains a typical conserved domain:the ammonia transporter family domain.The results of the phylogenetic tree showed that the C.sinensis has91.33%homology with the M.mercenaria.Tissue expression studies showed that Cs Rhbg was expressed in all tissues of the C.sinensis.3.Effects of chronic ammonia nitrogen stress on the transport pathway in C.sinensisTo investigate the effect of chronic ammonia stress on the transport pathway of ammonia in C.sinensis,the effects of ammonia stress on the expression of the ammonia transport protein gene（Cs Rhbg）,the activities of glutamine synthetase（GS）and glutamate dehydrogenase（GDH）in gill and hepatopancreas tissues and histological features of gill tissue were investigated.The results showed that:the GS activity of the gills of the experimental group increased significantly within 12 hours（P<0.05）;the GDH activity of the experimental group of the gills increased significantly within 3 hours（P<0.05）and reached the maximum value,and returned to 6 hours later.The level of control group;the expression level of Cs Rhbg m RNA in gills increased significantly within 3 hours（P<0.05）,and reached the maximum value at 96 hours.The GS activity of the hepatopancreas in the experimental group increased significantly and reached the maximum within 12 h,then increased first and then decreased;the GDH activity of the hepatopancreas in the experimental group reached the maximum at 12 h,and recovered to the level of the control group from 24 h to 768 h;The expression level of Cs Rhbg m RNA in hepatopancreas reached the maximum at 24 h.Under ammonia nitrogen stress for 768 h,the gill cilia were shed,the interlamellar space was increased,the nucleus was pyknosis and the gill filaments were adhered.The above research results show that the GS activity and GDH activity in the Gln synthesis pathway of gill and hepatopancreas significantly increased under ammonia nitrogen stress,and the expression level of Cs Rhbg m RNA was significantly increased,indicating that the C.sinensis was mainly transformed in the gill and hepatopancreas tissue.Glutamine storage and utilization.The Gln content of gills and hepatopancreas can be used as an evaluation index for the detoxification and metabolism of C.sinensis under ammonia nitrogen stress.4.Effects of chronic ammonia nitrogen stress on metabolic pathways in C.sinensisIn order to study the effect of chronic ammonia stress on the metabolic pathways in C.sinensis,the effects of ammonia stress on the m RNA expression of glutathione sulfotransferase gene（Cs GST）and the activity of glutathione sulfotransferase（GST）and the concentration of urea nitrogen（BUN）in gill and hepatopancreas tissues and histological features of hepatopancreas tissue were investigated.The results showed that the BUN content in the gills of the experimental group reached the maximum value at 96 h,and then showed a downward trend;the GST of the gills of the experimental group showed a fluctuating trend,reached the maximum value at 24 h,and then showed a downward trend,and was still significantly higher than that of the control after 192 h.group level（P<0.05）;the expression of gill Cs GST m RNA in the experimental group reached the maximum value at 24 h,then showed a downward trend,but increased again at 768 h,and was higher than that in the control group（P<0.05）.The hepatopancreas BUN content of the experimental group reached the maximum value at 192 h and showed a downward trend;the hepatopancreatic GST of the experimental group reached the maximum value at 24 h and showed a downward trend;the hepatopancreatic Cs GST m RNA expression of the experimental group reached the maximum value at 24 h,showing a downward trend.Under ammonia nitrogen stress for 768 h,the hepatopancreatic ducts of the C.sinensis were enlarged,the lumen narrowed,blood cells infiltrated,lipofuscin accumulated,digested cells atrophied,and epithelial cells were shed.Injury intensifies.The above research results showed that urea was mainly synthesized in the hepatopancreas under ammonia nitrogen stress,and urea could be excreted through excretory organs.