| Today,in the feed-dependent aquaculture system,approximately 75% of the exogenous nitrogen is not utilized by farmed aquatic animals.Instead,unnecessary nitrogen uses excreta as a medium to enter the aquaculture water that produced a large amount of toxic ammonia under the action of ammonization of environmental microorganisms.It leads to the frequent occurrence of ammonia poisoning in fish.During evolution,various physiological detoxification strategies in response to ammonia have emerged in fishes.The vast majority of fish are mainly dependent on glutamine synthesis and urea cycle.Previous successful experience of mammalian studies has shown that deficiency of the key enzyme of ammonia detoxification is the critical factor causing the difference of ammonia tolerance in the organism,which is also the key target of accurate nutrition regulation.Dolly varden trout(Salvelinus malma)is a small and medium-sized cold water rare fish with poor ammonia tolerance.With the continuous expansion of intensive farming scale,the normalization of ammonia exposure has a dramatic impact on the growth,reproduction and survival of dolly varden trout,which has seriously affected the yield and quality of the fish.In present study,a series of experiments was conducted to investigate the reasons of low ammonia tolerance of dolly varden trout aiming at providing a new opportunity for genetic breeding and the development of artificial compound feed.In this study,the toxicity of ammonia exposure to dolly varden trout was investigated firstly.Two treatment groups were set up: control group [0.01 mg/L total ammonia nitrogen(TA-N),non-ionic ammonia nitrogen(UIA-N)< 0.001 mg/L] and ammonia stress group(1/2 96 h LC50: 12.33 mg/L TA-N,0.19 mg/L UIA-N),the chronic ammonia stress experiment was conducted for 28 d.The result shows that the contents of ammonia in brain had increased 15-fold when it reached the maximum value(1 day)compared with control group,but the time of reach the maximum value was later than that in the liver(12 h);The maximum concentration of glutamine in the brain was 35.33 ?mol/g which is much higher than the lethal concentration(1 ?mol/g)of mammals.The activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPx)and glutathione reductase(GST)in liver showed a gradually decreasing trend and reached a stable level after 1 day.The activities of lysozyme(LZM),acid phosphatase(ACP)and alkaline phosphatase(AKP)in liver and respiratory burst(RB)of macrophages in head kidney were significantly decreased after 1 day.In addition,the concentrations of ammonia and glutamine in brain and liver of experimental fish in ammonia stress group were significantly higher than those in control group,while the activities of SOD,CAT,GPx,GST,LZM,AKP,ACP and RB in liver of experimental fish in ammonia stress group were significantly lower than those in control group after 1 h.These results suggest that ammonia exposure can induce accumulation of ammonia and glutamine in the liver and brain of dolly varden trout as well as leading to oxidative damage and immunosuppression.However,the accumulation of glutamine in the liver and brain of dolly varden trout is not the immediate reason of ammonia poisoning death.On the contrary,glutamine synthesis is likely to be a significant pathway of ammonia detoxification in dolly varden trout.In order to understand the role of glutamine synthesis pathway in ammonia-nitrogen detoxification of dolly varden trout,this study used the physiological method of intraperitoneal injection of methionine sulfoximide(MSO,glutamine synthase inhibitor)to set up four treatment groups: fish in Na Cl group were intraperitoneally injected with 0.9% Na Cl;Fish in the NH3 group were intraperitoneally injected with half lethal concentration of ammonium acetate(2.5 ?g / g fish);Fish in the NH3+MSO group were intraperitoneally injected with half lethal concentration of ammonium acetate and methionine sulfoximide(2.0 ?g /g fish);Fish in the MSO group was intraperitoneally injected with methionine sulfone imide(2.0 ?g/g fish),and the acute toxicity test lasted for 96 h.These results showed that the white blood cell count,the content of malondialdehyde and the m RNA expression of TNF,IL 1 and IL 8 genes in liver of fish in NH3 groupwere increased;While the serum glucose content,total antioxidant capacity(T-AOC),SOD,CAT,GPx,LZM activities,complement,immunoglobulin G(Ig G)content,phagocytic index(PI),and m RNA expressions of SOD,CAT and GPX genes in liver were decreased.The activities of glutamine synthetase and glutaminase in liver of fish in NH3 group were significantly higher than those of fish in NH3+MSO group and MSO group.The survival rate in NH3+MSO group was the lowest.The results suggested that ammonia poisoning could induce blood deterioration,oxidative stress,immunosuppression and inflammation of dolly varden trout;The ammonia detoxification of dolly varden trout depends on glutamine synthesis pathway and the urea cycle does not play a crucial role in the ammonia detoxification process.The purpose of this study was to investigate the reason why urea cycle did not participate in ammonia detoxification of dolly varden trout;Three experimental treatment groups were set up in this experiment:The control group was intraperitoneally injected with 0.9% sodium chloride;The low ammonia group was intraperitoneally injected with 0.5 m L ammonium acetate(1.25 ?g/g fish,1/2 96 h LC50);Fish in the high ammonia group were intraperitoneally injected with 0.5 m L ammonium acetate(96 h LC50),the acute toxicity ammonia stress lasted for 96 h.The results show that with the increase of ammonium acetate content,the contents of glutamine,glutamic acid,arginine and ornithine in plasma were significantly increased,while the contents of citrulline and aspartate were significantly decreased;The activities of argininase synthase,argininase lyase,glutamine synthase and glutaminase in liver of fish in high ammonia group had the highest value,while the activities of carbamyl phosphate synthase,ornithine aminotransferase and arginase had the lowest value;In particular,the activity of argininase was only 6% of the control group;After the liver cells were stained with acridine orange,irregular dense yellowish-green fluorescent signal was detected in the high ammonia group.These results suggest that there may be a urea circulation disorder indolly varden trout which is closely related to the absence of argininase activity.To investigate whether there is an argininase deficiency in dolly varden trout,different levels of arginine(activator)and lysine(inhibitor)were supplemented into the diet to filter out the arginase activation and inhibition models ofdolly varden trout.On this basis,the physiology of argininase deficiency was studied.The experiment consisted of two stages: in the first stage,seven experimental diets were prepared: control group,3 levels of arginine(1.00,2.00 and 3.00%)and 3 levels of lysine(1.00,2.00 and 3.00%),a 10 weeks nutritional regulation experiment was conducted.The results showed that the weight gain(WG)of fish in the 2.00% and 3.00% arginine groups was significantly higher than that in the control group.Similarly,the WG of fish in the 2.00% and 3.00% lysine groups was significantly higher than that in the control group.Supplemented with 3.00% lysine in the diet significantly increased the white blood cell count of fish;The activities of SOD,CAT,GPx,LZM and complement contents in serum of fish were significantly increased with the increase of dietary arginine content while significantly decreased with the increase of dietary lysine content.Comprehensive analysis based on the above indicators,the 2.00% arginine group was used as the arginase activation model(the activation group),while the 3.00% lysine group was used as the arginase inhibition model(the inhibition group).In the second stage,three groups were set up: control group,activation group and inhibition group,all three groups were intraperitoneally injected with ammonium acetate(96 h LC50,2.5 ?g/g fish),this acute experiment lasted for 96 h.The results showed that the cumulative mortality of the inhibition group was significantly lower than that of the activation group,but there was no significant difference between the inhibition group and the control group.The contents of arginase and carbamyl phosphate synthetase in liver of fish in activated group were significantly higher than those of fish in inhibited group,while the contents of ammonia and glutamine were opposite;The contents of glutamine synthase and glutaminase of fish in activated group were not significantly different from those in control group.These results suggest that the disturbance of urea cycle in ammonia detoxification process of dolly varden trout is related to the deficiency of argininase,however,the molecular mechanism is still unclear.In order to understand the molecular mechanism of ammonia detoxification of dolly varden trout,two treatment groups were set up using comparative transcriptome analyses: the control group was intraperitoneally injected with 0.9% Na Cl,and the ammonia group was intraperitoneally injected with 0.5 m L ammonium acetate(96 h LC50,2.50 ?g/g fish).The acute ammonia toxicity test lasted for 96 h.The results showed that 225.54 Gb of available data were obtained by sequencing,and 74 502 nonredundant genes were obtained by assembly.Functional annotation of non-redundant genes was performed,and 25 921 annotation results were obtained,of which 650 genes were differentially expressed,,which were mainly involved in protein/amino acid metabolism,TCA cycle and ammonia metabolism signaling pathway,etc..The activity of arginase in liver of fish in ammonia group was only 5.5% of that of control group.These results indicate that the physiological pathways involved in ammonia detoxification of dolly varden trout mainly including reduction of protein decomposition,partial amino acid decomposition to form alanine and glutamine synthesis were perturbed at the gene transcript level.The reason why the urea cycle does not play a role in ammonia detoxification is probably related to the argininase deficiency.In order to understand whether ammonia stress can cause argininase methylation in dolly varden trout,two treatment groups were set up using comparative transcriptome analyses: the control group was intraperitoneally injected with 0.9% Na Cl,and the ammonia group was intraperitoneally injected with 0.5 m L ammonium acetate(96 h LC50,2.50 ?g/g fish).The acute ammonia toxicity test lasted for 96 h.The results showed that the full length of ARG 1 gene was 1014 bp,encoding 338 amino acids.There were CpG islands in the core promoter region of ARG 1,containing 16 CG sites.Under ammonia stress,the methylation level of CpG islands in the core promoter region of ARG 1 was significantly higher than that in the control group.The results showed that ammonia stress could cause ARG 1 methylation in the liver of dolly varden char. |
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