Effects Of Short-term Low Salinity Stress On The Physiology,Metabolism And Non-volatile Flavor Substances Of Portunus Trituberculatus

Portunus trituberculatus is one of the main cultured economic crabs in coastal areas of China.Salinity as an important physical and chemical index of seawater,has great impact on the growth and survival rate of P.trituberculatus.In order to adapt to salinity alterations,P.trituberculatus can adjust some metabolites by adjusting its osmotic pressure in hemolymph to maintain stability.Therefore,this study first investigated the effects of short-term low salinity stress on the physiological and biochemical characteristics of P.trituberculatus;then based on GC-MS technology,metabolites differences in the key organs of osmoregulation-gill under different salinities were detected,the metabolic mechanism of P.trituberculatus in response to low salt stress was preliminarily revealed.Finally,alterations in the contents of non-volatile flavor substances under low salinity stress were also analyzed,revealing the effects of different salinity levels on their nutritional components and flavor.This study focused on a more comprehensive explanation of its regulatory mechanism under low salinity stress,which may provide technical guidance and theoretical support for the selection and cultivation of new varieties.The main results in this research were presented as follows:1.Effects of short-term low salinity stress on physiological and biochemical characteristics of P.trituberculatus were studied.Results showed that low salt stress had significant impact on survival rate,osmoregulation,enzyme activity and gill filament morphology of P.trituberculatus.Under sudden short-time low salt stress,several crabs in the salinity 8 experimental group began to die at24 h,and the survival rate was 77%at 48 h,while the survival rate of crabs in the gradual short-time low salinity and recovery stress experiment was still 93%when the salinity dropped to 8;secondly,low salinity stress changed the structure and morphology of gill tissue,damaging the normal physiological functions of gill tissue,while short-time low salinity stress had greater impact on the survival rate and gill tissue structure of P.trituberculatus than gradual low salinity stress.In addition,the osmotic pressure of hemolymph decreased significantly with the decrease of salinity,and then increased significantly with the recovery of salinity.The osmotic pressure of serum was always higher than that of seawater;changes in the activities of osmotic regulation-related enzymes Na~+-K~+-ATPase、Carbonic anhydrase ATPase and ATPase indicated that salinity 8 might be an adaptive low salinity for P.trituberculatus within a short period of time.The results confirmed that P.trituberculatus belongs to osmotic adjustable species that can survive in a certain range of low salinity.Furthermore,it was adaptive under salinity 12,while it can still maintain a relatively high survival rate under salinity 8.With the recovery of salinity,its physiological function tends to be stable,but the osmotic pressure after recovery was lower than the initial osmotic pressure level,and the enzyme activities after recovery were higher than the initial enzyme activity levels as well.2.Based on GC-MS technology,metabolic mechanism about how to adapt to the gradual low salinity stress was analyzed.A total of 342 different metabolites were identified in this study,and a total of 18 differential metabolites were screened in the S24 and S16 groups;a total of 36 differential metabolites were screened in the S24 and S12 groups;a total of 46 different metabolites were screened in the S24 and S8 groups.In addition,9 mutual metabolites,including six mutual up-regulated metabolites and 3 mutual down-regulated metabolites,were screened out through the joint analysis of the three groups of P.trituberculatus.Then differential metabolites were analyzed by KEGG enrichment,and 62 metabolic pathways were obtained,20 of which were significantly different(P<0.05),including metabolic pathways such as TCA cycle,Glyoxylate and dicarboxylate metabolism,and Galactose metabolism.Those metabolic pathways mainly belong to amino acid metabolism,carbohydrate metabolism,lipid metabolism,metabolism of cofactors and vitamins,signal transduction,nucleotide metabolism,etc.Results indicated that organic acids and carbohydrates play important roles in the adapting process of P.trituberculatus under gradual low salinity stress;while energy metabolism offer basic energy supplement during the above process.3.The contents of free amino acids,nucleotides and lactic acid in both muscle and hepatopancreas tissues of P.trituberculatus were determined by high performance liquid chromatography(HPLC).Results showed that short-time low salinity stress reduced the contents of free amino acids(FAA)and essential amino acids(EAA)in the above two tissues of P.trituberculatus;muscle tissue contained a higher content of sweet free amino acids(SFA),however low salinity stress can greatly increase the content of bitter free amino acid(BFA),leading muscle tissue a bitter feature under extremely low salinity;hepatopancreas tissue contained a higher content of umami free amino acids(UFA)and a higher MSG equivalent(EUC)index,and the EUC index increased significantly with the decrease of salinity level,the index of umami feature reached the highest when salinity dropped to 8.Therefore,low salinity level resulted in a better umami taste to a certain extent in hepatopancreas tissue of P.trituberculatus,while stimulated bitter feature in muscle tissue.Results above revealed that different salinities levels influenced the contents of non-volatile flavor in both the muscle and hepatopancreas tissues of P.trituberculatus,which provided a guidance for improvement of low salinity varieties cultivation from the perspectives of nutrition and flavor.