Effects Of Salinity On Growth And Physiology Of Oriental River Prawn,Macrobrachium Nipponense

Salinity is an important environmental factor affecting the regulation of osmotic pressure in aquatic animals,and changes in salinity also can cause effects on growth,development,immunity and other physiological activities.The research animal of this paper is Macrobrachium nipponense,also called oriental river prawn,which is a traditional freshwater aquaculture species in China.Because of its delicious taste and rich nutrition,this prawn has become one of the favorite aquatic products for consumers.With the increasing demand for aquatic products,it has become the research direction of some countries and regions to domesticate the seawater species with low salinity and cultivate the freshwater species with saline water.In this study,the effects of salinity acclimation on the osmotic physiology of M.nipponense and the effects of salinity culture on the growth of M.nipponense were studied.The effects of salinity on the growth physiology of M.nipponense were discussed for the first time by integrating the theories and methods of enzymology,molecular biology and transcriptomics.The main results and conclusions are as follows:1 Effects of salinity acclimation on osmotic pressure and ion concentration of M.nipponenseBefore salinity culture,it is necessary to acclimate M.nipponense to salinity by gradually increasing salinity.The process of acclimation is to adjust osmotic pressure to adapt to salinity.In this study,four salinity gradients were set:fresh water group(control group),salinity 8,14 and 22.The salinity was increased by 2 every day.After one week of adaptation to the correspond salinity,the serum osmotic pressure and water environment osmotic pressure were measured,and the concentration of ions in serum was measured.According to the formula,the isoosmotic point of M.nipponense is 490m Osm/kg H₂O,and the corresponding salinity is about 14.4.The concentrations of Na⁺and Cl^-in serum showed similar trends,both of which increased with the increase of salinity.However,the concentrations of Na⁺and Cl^-in serum decreased significantly in high salinity.There was no significant difference in the effects of salinity on the concentrations of K⁺in serum.The results showed that the osmotic pressure of M.nipponense increased with the increase of salinity.Na and Cl ions play an important role in the osmotic pressure regulation.2 Cloning and expression of osmoregulation related genesNa⁺/K⁺-ATPase(NKA)and carbonic anhydrase(CA)are ion transporters that play important roles in osmotic pressure balance in crustaceans.In this study,the full length of cDNA of Na⁺/K⁺-ATPase(NKA)and carbonic anhydrase(CA)were cloned for the first time,and named Mn?NKA and MnCA.GenBank numbers were MH378774 and MH827971,respectively.Bioinformatic analysis showed that Mn?NKA and MnCA showed high homology and conservation with other crustaceans,90%and 76%respectively and contain the representative features of NKAs and CAs in other crustaceans.Quantitative real-time PCR revealed that Mn?-NKA and MnCA were expressed in various tissues,with highest expression in posterior gills and hepatopancreas.Both Mn?-NKA and MnCA were up-regulated in response to salinity acclimation,but expression patterns differed.And the localization of Mn?-NKA and MnCA was studied by immunohistochemistry and in situ hybridization,respectively.It is widely distributed in gill tissue.Our results provide a theoretical basis for the mechanism regulating salinity acclimation in M.nipponense.3 Effects of salinity on growth and non-specific immunity of M.nipponenseAfter M.nipponense has adapted to the salinity through osmotic pressure regulation,what kind of impact will it have on its growth if it continues to be cultured in salinity?Will it cause oxidative damage and affect its immune function?In order to verify,we studied the growth and non-specific immunity of M.nipponense after cultured at different salinities(0,8,14,22)for 6 weeks.The results showed that M.nipponense in salinity 14 group had higher survival rate,weight gain rate and hepatopancreatic index.Salinity can cause effect on the contents of water,crude protein,crude fat and ash.Besides,trypsin,amylase,lipase and pepsin were affected by salinity in different degrees,and the highest activity was found in salinity 14 group.Suitable salinity could stimulate the expression of EcR,CDA1 and CHS,indicating that a certain salinity could promote the growth of M.nipponense,and was in a good growth state at salinity 14.Meanwhile,we detected the changes of non-specific immune enzymes(AKP and ACP),antioxidant related enzymes(SOD,CAT,GPX,GST),GSH and MDA contents in hepatopancreas of M.nipponense,and detected the effects of antioxidant enzyme related genes(SOD,CAT,GPX,GST)and key genes in immune pathway.We found that appropriate salinity can improve the antioxidant capacity and non-specific immunity of M.nipponense.However,high salinity can cause oxidative damage and even apoptosis in hepatopancreas.4 Transcriptomics of M.nipponense under isoosmotic salinityIsotonic salinity is beneficial to the growth of M.nipponense,it is interested to study which specific pathways are affected.Therefore,we performed the transcriptome of M.nipponense under isotonic salinity(14).A total of 35991 unigenes larger than 300bp were obtained.A total of 733 genes were significantly different between control group and isotonic group in M.nipponense,including 416 up regulated genes and 317down regulated genes.A total of 170 differential genes were enriched in the biological process,cell composition and molecular function of GO database.Blast to KEGG database,only 91 differentially expressed genes were successfully compared with KEGG database.Most of the enrichment pathways with significant differences were metabolic pathways.Among the metabolic pathways,glycerophospholipids and glyceride metabolic signaling pathways had the most abundant genes in lipid metabolism pathway,which indicated that isotonic salinity had a significant effect on lipid metabolism.It may be related to the development of hepatopancreas and gonad,it provides a theoretical basis for the study of salinity adaptability of M.nipponense.5 Effects of salinity on metabolism related enzymes of M.nipponense and cloning and expression of AKBased on the transcriptomic data,salinity had significantly effects on metabolic pathway.Therefore,we determined the key enzymes in the process of lipid metabolism and glucose metabolism.The results showed that salinity culture had a certain effect on glucose metabolism in hepatopancreas and muscle tissue of M.nipponense.Under the condition of isotonic salinity,so it does not need to use the liver glycogen and muscle glycogen to produce more energy to adjust the osmotic pressure.Salinity did not affect the triglyceride content in muscle,but had a great impact on the triglyceride content in hepatopancreas,which may be due to the fact that under isotonic condition,M.nipponense does not need to active osmotic pressure exchange,so it can store more energy for growth and development.Besides,we cloned the full length of two subtypes of arginine kinase,the key genes of energy metabolism in crustacean,and named MnAK2 and MnAK3.GenBank numbers were MN149533 and MN149534.1,respectively.Both MnAK2 and MnAK3 contain the conserved domains of arginine kinase and multiple sequence alignment analysis showed that AKs of M.nipponense had high similarity with other crustaceans.q RT-PCR results showed that MnAK2 and MnAK3 were expressed in all tissues of M.nipponense,but the highest expression was found in hepatopancreas and muscle.With the increase of salinity,the expression of vincreased gradually,while MnAK3 increased significantly in all salinity groups.WB results showed that the expression of AK protein was up-regulated in salinity 14 and 22groups,suggesting that AK may play an important role in the regulation of energy metabolism in M.nipponense.Based on the above experiments,this study found that M.nipponense could adapt to salinity acclimation through osmotic pressure regulation genes and ion concentration changes.After long-term salinity culture,it was found that low salinity could promot the growth of M.nipponense,and M.nipponense had a wide salinity tolerance,which laid a certain foundation for the realization of salinity culture of M.nipponense.