The Nutritional And Physiological Functions Of Arginine In Chinese Mitten Crab Eriocheir Sinensis At Juvenile And Gonad Maturation Stage

It is urgent that to cultivate healthy and disease-resistant seed through nutrition manipulation and develop efficient fattening feed which can replace frozen fish feed in Chinese mitten crab(Eriocheir sinensis)industry.The key point to solve these problems is to thoroughly study the physiological functions of nutrients on growth regulation,stress tolerance and gonadal development of crabs.However,the studies on amino acids,which are play a crucial role in growth regulation,disease resistance,stress resistance and gonadal development in aquatic animals,is seriously insufficient,especially in the fattening period.Therefore,in this thesis,arginine(Arg),a key limiting amino acid for shrimp and crabs,was selected to investigate its role on protein synthesis,disease resistance,stress resistance,gonadal development and application strategies,using the methods of nutrition,molecular biology,immunology and transcriptomics.The main results and conclusions are as follows:1 Effects of arginine on protein synthesis in juvenile Chinese mitten crabThe mechanism of Arg regulating protein synthesis has been elucidated in mammals,however,the signal pathway of Arg regulating protein synthesis has not been reported in shrimp and crabs.Therefore,this thesis studied the ways of Arg regulating protein synthesis in juvenile Chinese mitten crab.Three diets containing Arg levels at1.72%(control),2.73% and 3.72% were formulated and fed to juvenile crab(0.22 ±0.03 g)for eight weeks.The results showed that Arg could regulate the ecdysterone and growth hormone in the serum,the expressions of PI3 K,Akt,mTOR,S6K1 and 4EPB1 genes in the hepatopancreas,which indicted that Arg promotes protein synthesis through the PI3K-Akt-mTOR signaling pathway in juvenile crab.2 Effects of arginine on immunity and stress resistance in juvenile Chinese mitten crabThe pathway of Arg regulating immunity in mammals and fish has been clearly reported.However,studies on the functions of Arg on the immune regulation and stress resistance in crustaceans are relatively lag.Moreover,the immune system of crustaceans and mammals are quite different,therefore,the way of Arg regulating immune function in crustaceans is not clear.In this study,the effects of Arg on immunity of Chinese mitten crab were further investigated by injection of lipopolysaccharide,Poly(I:C)and Aeromonas hydrophila.The results showed that Arg enhanced the immunity and resistance of A.hydrophilus by up-regulating the expressions of pro PO,cryptocyanin,and antimicrobial peptide(crustin,lysozyme)in Chinese mitten crab.The oxidative stress would occur when animals encounter environmental stress.Antioxidant capacity is an important indicator for evaluating the ability to cope with stress.This study found that Arg increased the antioxidant capacity of crab by increasing the activities of SOD and CAT.In order to verify whether Arg can truly improve the stress resistance of crab,high pH,the most common environmental stress factor in crab farming,was selected as the stress factor to test the role of Arg in stress resistance of Chinese mitten crab.Three diets containing 2.37%(control)3.14% and4.01% Arg were formulated and fed to crabs(3.37 ± 0.01 g)at high pH(pH = 9.5)for eight weeks.The results showed that Arg supplementation improved the antioxidant capacity of crabs at high pH by increasing the activities of SOD,CAT and GSH content in the hepatopancreas.In addition,Arg supplementation significantly downregulated the inflammatory and apoptotic genes including LPS-induced TNF-? factor,p38mitogen-activated protein kinase,a disintegrin and metalloprotease 17 under high pH stress.Meanwhile,Arg improved the growth and immunity of crab.These results show that Arg could alleviate the high pH stress by improving the antioxidant capacity and reducing the inflammatory response.In summary,Arg can enhance the immunity and stress resistance of Chinese mitten crab by up-regulating the activity of immune genes and antioxidant enzymes and down-regulating the expression of inflammatory genes.3 Application study of N-carbamylglutamate(NCG)in juvenile Chinese mitten crabThis study investigated the effects of NCG,a feed additive commonly used in mammals to promote endogenous synthesis of Arg,on endogenous synthesis of Arg in crab.These results showed that dietary supplementation with 0.04% and 0.08% NCG could improve the growth performance,antioxidant capacity,anti-inflammatory capacity and immunity of crab under high pH stress by promoting the endogenous synthesis of Arg.Therefore,NCG is a feed additive that can effectively promote the endogenous synthesis of Arg and improve the stress resistance of Chinese mitten crab.4 Effects of arginine on growth and ovarian maturation in adult female crabThere is a quite physiological difference between gonadal development stage and juvenile stage,whether Arg is a key limiting amino acid in gonadal development stage is still unknown.Therefore,the effects of Arg on growth and ovarian maturation were studied to determine the physiological function of Arg in female crab during gonadal development stage.Female crabs with an average weight of 60.05 ± 4.33 g were fed with diets containing Arg levels at 1.83%,2.52%,3.26%,3.96%,4.81% and 5.52%,respectively.After eleven weeks,the optimal Arg levels in diets(2.52%-3.26%)can improve the weight gain and ovarian index of female crab,indicating that Arg is an essential amino acid in ovarian maturation of female crab.Subsequently,the optimal requirement of Arg during the fattening period was further studied,and it was found that the Arg requirement for ovarian development was higher,up to 4.09% during the fattening period.In order to investigate the ways that Arg regulates gonadal development,the further study on the effects of Arg on vitellogenin synthesis,transport and deposition in female was performed.The results show that Arg can promote the expression of vitellogenin gene in the hepatopancreas through regulating the second messenger cGMP/cAMP and the downstream PKG/PKA,which were affected by the vitellogenesis inhibitory hormone(VIH)in the hemolymph.In addition,Arg not only promotes the synthesis of vitellogenin,it can also improve the expression of the gene of vitellogenin receptor in the ovary,so as to promote the deposition of vitellogenin in ovary.In summary,Arg promotes ovarian development by promoting the synthesis,transport and accumulation of vitellogenin,and Arg is an essential amino acid in ovarian development.5 Effects of arginine on growth and protein synthesis in adult male Chinese mitten crabIn adult stage,male crabs with an average weight of 78.19 ± 8.34 g were fed with diets containing Arg levels at 1.83%,2.52%,3.26%,3.96%,4.81% and 5.52%,respectively.After eleven weeks,the optimal Arg levels in diets(3.26%-3.96%)significantly increased the weight gain,hepatopancreas index,accessory gland index and muscle percentage of male parents,indicating that Arg plays an important physiological function in the growth and development of adult male crab.Subsequently,the optimal requirement of Arg during the fattening period was further studied,and it was found that the optimal Arg level for the development of the accessory gland is 3.76%during the fattening period.Based on the above results,the further study was performed to explore the effects of Arg on protein synthesis.The results show that crabs fed the 3.26% Arg diet obtained the highest muscle percentage,and in which group,mTOR,S6K1 4EBP1 and other related genes were up-regulated in the hepatopancreas and muscle.However,there were no significant differences in genes of PI3 K and Akt.The genes of GATOR2 family were significantly up-regulated,and the genes of GATOR1 family were significantly downregulated,meanwhile,RagA,RagC,Rheb and Raptor were all significantly upregulated in crabs fed the 3.26% Arg diet.These results indicated that Arg did not affect mTOR through PI3K-Akt pathway,but through GATOR2,RagA and RagC to regulate mTOR,thereby promoting the synthesis and deposition of proteins in the hepatopancreas and muscle.6 Effects of arginine on the synthesis of accessory gonad protein in adult male crabIn male crab,the mature accessory gland accounts for more than 70% of the whole male reproductive system.It is the place where the main nutrients of the male sex gland are deposited.The effects of amino acids on the development of accessory gland have not been reported.Therefore,this study aims to estimate the physiological function of Arg in the life stage of gonad development by studying the effect of Arg on the accessory gland index and transcriptome analysis.The results show that 3.26% Arg significantly promoted the accessory gland development,transcriptome results showed that the Arg promoted the anabolism of glycoproteins,then promote the accessory gland development by upregulating the genes including oligosaccharide transferase,galactosyl transferase,Fucosyl transferase,Acetyl galactosyl transferase.Therefore,Arg is an essential amino acid in accessory gland development stage.In order to further determine the synthetic sites of accessory gland protein,comparative transcriptome analysis was performed in this thesis.And the results show that the accessory gland is the only site for the synthesis of accessory gland glycoprotein by comparing transcriptome and analyzing the distribution of target genes regulating the synthesis of accessory gland protein.