| The pH of the water body can affect various physiological functions of fish.mandarin fish(Siniperca chuatsi)is one of the famous and high-quality freshwater fishes in our country,and has high water quality requirements.Understanding the environmental adaptability of mandarin fish to acid-base stress is an important basis for its green and healthy breeding.In the thesis,mandarin fish was the research object,and the mandarin fish was directly transferred from fresh water to different acid-base gradient groups(p H = 4.0,5.5,7.0,8.5,10.0)for 72 h acute stress experiment.The changes of serum p H and oxygen consumption rate under different acid-base stresses were measured by physiological methods;the tissue morphological changes caused by acid-base stress on gills and liver were observed by optical microscopy;the liver and kidney were analyzed by real-time fluorescence quantitative method.Real-time fluorescence quantitative method was used to analyze the expression differences of cytochrome P450(CYP450),flavin monooxygenase(FMO),uridine diphosphate glucuronyl transferase(UGT)and glutathione S-transferase(GST)detoxification related genes in liver,kidney,brain,and gill tissues.;the activity of the above-mentioned detoxification enzymes in gills and liver was measured by enzyme-linked immunosorbent assay.It hopes to provide a theoretical basis for exploring the influence of acid-base stress on mandarin fish and its corresponding adaptation mechanism.The main findings are as follows:(1)When the mandarin fish enters the acid-base water environment,the initial stress response is strong,the operculum opens and closes violently,and the body color becomes lighter.In the later stage,the eyes are raised and cannot maintain balance.There was no dead fish in each stress group(p H = 4.0 ～ 10)within 72 hours of acid-base stress.The serum p H of fish in the p H = 7 group was relatively stable and maintained between 7.31 ～ 7.43,decreased continuously in the p H = 4 group.After 3 hours of stress,the serum p H of the other groups changed significantly.(P<0.05).The serum p H of the p H = 5.5 group showed a trend of first decreasing,then increasing and then decreasing.After 72 h,the serum p H was significantly lower than0 h.The serum p H of the p H = 8.5 group and the p H = 10 group increased first,then decreased and then increased,reaching the maximum value(7.79 and 7.84)after 72 hours.With the increase of stress time,the oxygen consumption rate of the other experimental groups showed a trend of first increasing and then decreasing after 12 hours,except that the oxygen consumption rate of the p H = 7 group was relatively stable.Among them,The oxygen consumption rate of the p H = 4 group was the lowest after 12 hours compared with the different p H stress groups at the same time period,and the oxygen consumption rate of each group was lower than the p H = 7 group after72 hours of stress.The results showed that the acidic environment with p H = 4.0 is more toxic to mandarin fish in the water environment range of p H 4.0 to 10.0;mandarin fish can adapt to acid-base stress by increasing the oxygen consumption rate between p H = 5.5-10.0.(2)After 72 hours of acid-base stress,observing the microstructure of the gill tissue showed that the gill lamellae were relatively normal in the p H = 7 group,and the morphology of various cells was relatively complete,arranged regularly and evenly distributed.The gill lamellae under acidic or alkaline stress become thicker or shrink,and epithelial cells swell and fall off.Observing the microstructure of the liver tissue,it can be seen that the hepatocytes in the p H = 7 group were mostly regular oval-shaped,evenly distributed,and the nucleus was normal.Under acidic or alkaline stress environment,hepatocytes are enlarged or significantly reduced in number,hepatic cord relaxation is disordered,cell nucleus is deformed,hepatic sinusoids are expanded and even fusion occurs.The tissue morphology changes of gills and liver did not produce obvious differences between acid and alkali stress,and the effects caused by other environmental stresses were similar.(3)The mRNA expression levels of CYP450,FMO,UGT,GST in the liver,kidney,brain and gills showed an obvious trend of first increasing and then decreasing except the p H = 7 group,.After 3 hours of acid-base stress,UGT and GST genes in the liver began to increase significantly,and after 12 hours of stress,CYP450 and FMO genes began to increase significantly(P<0.05);CYP450 and FMO genes in the kidney were both reached a peak after 24 h stress and then showed a downward trend.The m RNA expression of CYP450,FMO and GST genes in the brain reached the highest value after 48 hours,and it decreased after 72 hours of stress;UGT and GST genes were all reached a peak value after 24 hours stress and then showed a downward trend in the gills;the relative expression of CYP450 gene and FMO gene both reached a peak value at 48 h after the stress,and then showed a declining trend.GST enzyme activity increased along with prolonged stress time in liver tissue,and the activity and m RNA expression of CYP450,FMO,UGT enzymes were basically same,showing a obvious trend that increasing first and then decreasing with time.There was no significant change in the activities of the detoxification enzymes in the gill and liver in the p H = 7 groups(P>0.05).In the acidity and alkalinity groups,the activities of UGT and GST were significantly increased at 3h after stress in the gill(P<0.05),and the activities of CYP450 and FMO both reached their peaks at 24 h after stress.The CYP450,FMO and GST enzyme activities in the liver were significantly higher than the p H = 7 group after 3 hours of stress(P<0.05).Although the UGT enzyme activity fluctuated during the 72 hours of stress,it first increased and then decreased as a whole trend.The activity of CYP450 enzyme reached the peak at 24 h after the stress and then start decline,the activity was higher than that of the p H = 7 group after 72 hours of stress;the activity of FMO enzyme reached the peak at 48 h and then start decline,and the activity was higher than the p H = 7 group after the stress for 72h;UGT Enzyme activity fluctuated in each stress group.UGT enzyme activity in p H = 7 group was higher than other stress groups after 48h;GST enzyme activity was significantly higher than p H = 7 group after 72 h stress(P<0.05).The results show that CYP450,FMO,UGT and GST participate in the response process of mandarin fish under acid-base stress.Mandarin fish can mobilize the chemical defense mechanism by increasing the activity of detoxification enzymes,strengthen the excretion of acid-base toxicity,and maintain its own survival. |
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