Study On Tolerance Of Chinese Razor Clam(Sinonovacula Constricta)to Important Water Environmental Factors In Inland Waters

China is the largest aquatic country in world.The pressure of ecological environment on aquaculture is also increasing by day up.Bivalve is one of the important species to solve the eutrophication of inland cultured water.Carrying out research on bivalve breeding in inland waters is conducive to the development of aquaculture,adjusting the agricultural economic structure,improving the breeding ecological environment,and increasing farmers'production and income.The research on aquaculture marine bivalve to inland waters started late,with few reports.S.constricta is an important marine economic bivalve in China,and has strong resistance to stress.In this paper,the S.constricta was used to study its tolerance to inland water conditions,Long-term breeding experiments were carried out in conditions,and the preliminary mechanism of its osmotic regulation was discussed.1. In this study,we studied the tolerance,growth and physiological response of S.constricta under low salinity stress.Experiment 1:Acute low salinity stress.Acute low salinity stress method was used in here,and the survival rate,Na⁺/K⁺-ATPase activity and phagocytosis of hemocytes of S.constricta was measured.The results showed when the salinity was set from 8 to 2 the survival rate of three different sizes S.constricta were almost 100%.However,when salinity was set from 2 to 0 the survival rate of three different sizes S.constricta decreased with the decrease of salinity,and the survival rate of adult was higher than that the sub-adult and juvenile all the time.Meanwhile,the Na⁺/K⁺-ATPase activity was correspondingly increased to maintain the osmolality balance inside and outside of the body.The low salinity LC₅₀ of juveniles,sub-adults and adult were 1.45,1.25 and 0.75,respectively.Experiment 2:Long-term low salinity stress.In this study,we investigated the possibility of rearing and breeding the razor clam in inland low salinity water or freshwater.Long-term low salinity?LS?rearing was performed for 3 months to determine the effects of LS on the survival rate,growth rate,and the activities of critical enzymes in juvenile S.constricta?JSC?.The survival rate in the LS group was only?15%at the end of the LS rearing test.In the first month,the survival rate in the LS group was significantly lower than that in the control group?P<0.001?.The growth rate?shell length growth rate and weight gain rate?was significantly lower in the LS group than the control group in the first month?P<0.001 for length and weight?.However,the growth rates in the two groups differed little during the second and third months.The oxygen consumption and ammonia excretion rates by JSCs were significantly higher in the LS group than the control group during the first month,but they decreased gradually during the following 2 months.The Na⁺/K⁺-ATPase and superoxide dismutase activities were significantly higher in the LS group than the control group during the first month,but they then decreased gradually until there were no significant differences between the groups.However,the aspartate aminotransferase activity was higher in the LS group than the control group during all 3 months.2. In this study,we studied the tolerance,growth and physiological response of S.constricta under high carbonate alkalinity and p H stress.Experiment 1:The effect of acute high p H and carbonate alkalinity?C_A?stress on three kinds of body sizes?JSC,small-size SSc and large-size LSc?of S.constricta.The results showed that when the C_A concentration was 2.5 mmol/L and the p H was 7.5-9.5,the survival rates of the three size were close to 100%.The p H value was greater than 9.5,the concentration of C_Awas 0-44.58 mmol/L and the p H value was 9.0-10.0,the survival rate of clams decreased following with the increase of p H and C_A concentration,respectively.Under the condition of p H 9.5,The Na⁺/K⁺-ATPase activity of LSc gill tissue increased with the increase of C_A concentration,and the phagocytic capalacity of LSc haemolymph decreased with the increase of C_A concentration.Experiment 2:long-term stress test.After 3 months of stress,for the JSCs in various groups,the survival rate shell length growth rate and weight gain rate were significantly lower in the C_A and p H co-stress?CA-p HS?group than the other groups?p<0.05 for shell length;p<0.001 for weight gain?in the first month.However,the difference in the growth rate among groups decreased in the next two months.For the JSCs in the CA-p HS group,the oxygen consumption,ammonia-N excretion,Na⁺/K⁺-ATPase,aspartate aminotransferase,and superoxide dismutase levels were significantly higher than those in the other groups during the first month,but there were no significant differences between the groups subsequently.The acetylcholinesterase and lysozyme levels did not differ significantly among groups during stress for 3 months.These results indicate that water C_A and p H together affect the survival,growth,and physiological activity of JSCs.3. In this study,we investigated the response of the S.constricta under the Na⁺/K⁺ratio in inland saline water.Acute and long-term?30 days?stresses were studied to better delineate the process of the effect.At the end of the test,only the groups of Na⁺/K⁺ratio from 21.28 to 159.95 contained surviving Juvenile S.constricta,and the Na⁺/K⁺ratio 31.91 and 47.27 groups showed significantly higher survival rates?P<0.001?.In test groups of Na⁺/K⁺ratio,the growth rate?shell length growth rate and weight gain rate?was significantly lower under than that the control group?P<0.05 and P<0.001 for length and weight?.The Na⁺/K⁺-ATPase activities in the test Na⁺/K⁺ratio groups were significantly higher than that in the control group during 30 days?P<0.05?.The superoxide dismutase activity in the higher Na⁺/K⁺ratio groups was significantly higher than that in the control group during 30 days.In the test groups,the aspartate aminotransferase,acetylcholinesterase,and lysozyme activities were lower than that control group during 30 days.Under ultrahigh and ultralow Na⁺/K⁺ratio acute stress,the oxygen consumption rate,phagocytosis rate,and metabolic activity fluctuated significantly.Unbalances of Na⁺/K⁺ratio may affect ion regulation,metabolism,immunity,and neuromodulation.Under long-term stress,JSC can tolerate a wide range of Na⁺/K⁺ratio in water environment.4. In this study,we evaluated the tolerance of JSC to Ca²⁺and Mg²⁺concentrations,and determined the effects of these ions on JSC growth and physiological parameters.After 30 days stress,the tolerable ranges of JSC to Ca²⁺and Mg²⁺were determined to be 0.19 mmol/L-19.46 mmol/L and 0 mmol/L-29.54 mmol/L,respectively.The concentrations of Ca²⁺?less than 0.65 mmol/L or more than 3.24 mmol/L?and Mg²⁺?less than 0.37 mmol/L or more than 14.17 mmol/L?significantly inhibit JSC growth.Physiological enzyme activity no significant response when the concentrations range ofrespectively.Under acute stress with extremely high or low Ca²⁺and Mg²⁺concentrations,the oxygen consumption rate,phagocytosis rate and metabolic activity were significantly increased.The imbalance of Ca²⁺and Mg²⁺concentration may affect the ion balance,metabolism,immunity,and nerve regulation.Under long-term stress,JSC can tolerate a wide range of Ca²⁺and Mg²⁺concentrations of in water environment.5. In this study,we investigated aquaculture JSC in target inland alkaline blackish water?TBW?.The results showed that shell length and weight were significantly inhibited in the TBW group?p<0.001?.Although the mortality rate was 81.479±7.028?p<0.001?at 15 days,it dropped sharply to 10.637±2.116?p<0.001?at 45 days.The enzyme activities of Na⁺/K⁺-ATPase,aspartate aminotransferase and superoxide dismutase peaked at 15 days,and JSC engaged in partial anaerobic metabolism,which may explain the high mortality rate at this time.Phagocytic ability of haemocytes was inhibited?p<0.001?,but the metabolic activity was enhanced?p<0.001?.Comparing the previous results,indicate that the effects of main ionic components from TBW on JSC are relatively independent,or there is a weak joint effect between ions,and the Na⁺/K⁺ratio may be the most important limiting factor.Approximately 7%of animals adapted successfully to TBW after 45 days.In summary,S.constricta has a great potential in further anti-TBW conditions selective breeding research.6. In this study,the liver,gill and mantle transcriptomes of razor clam target inland blackish water and low salinity water stress were analyzed.From the results,it can be seen that the target inland blackish water and low salinity water stress have a lot in common.The metabolism of taurine and hypotaurine was significantly up-regulated in all tissue types involved in the experiment.The two most significant genes are cysteine sulfinic acid decarboxylase?CSAD?and glutamate decarboxylase 1?GAD?.Significant changes in lysosomal enzymes occur in liver tissues,as cathepsins B,C,and L,V-type H⁺-transporting ATPase subunit a?ATPe V?,AP-1 complex subunit gamma-1?AP-1?and AP-3 complex subunit delta?AP-3,which guide exocytosis of lysosomes?,were significantly inhibited.This leads to inefficient protein digestion and puts pressure on the energy supply.The metabolism of amino acids in gills and mantle tissues was significantly up-regulated.The most significant amino acid metabolism pathways were arginine and proline metabolism,histidine metabolism,and alanine-aspartic acid-glutamic acid metabolism.Systemic energy metabolism was observed to be up-regulated,the most significant differential gene was phosphoenolpyruvate carboxykinase?PEPCK?.Gill tissue is probably the most important osmotic pressure regulating tissue.Liver tissues may play an important role as a source of energy.