Study On The Triglycerol Catabolism,dietary Lipid And ?-lipoic Acid On Chinese Mitten Crab Eriocheir Sinensis

Lipid is one of the most important energy sources in diet of aquatic animals.The first key step in lipid catabolism is hydrolysis of triglycerol which is the main composition of lipid in most organisms.Glycerol and fatty acid can be released by triglyceride hydrolysis.In this study,three triglyceride lipases were identified and cloned in Chinese mitten crab(Eriocheir sinensis)and named es_TGL1,es_TGL2 and es_IL,respectively.mRNA expressions of these three lipases were analyzed by qPCR in hepatopanaeus during starvation.After the resrarch of optimum addition and metabonomics mechanism of ?-lipoic acid,it was added to diet with different lipid percentage and lipid sources.Growth performance,proximate composition,antioxidant capacity,triglyceride catabolism and hepatopancreas fatty acid composition were analyzed in E.sinensis after cultivation for 8 weeks.Results in the study not only filled the gap of triglycerol catabolism in E.sinensis,but also can provide us a new insight to the efficient use of lipid in diet of aquatic animal.1.Triglyceride lipase identified,clone and expression in E.sinensisAs an important energy source in aquatic diet,lipid can both supply a large quantity energy and sturctural substrate for organism.The first key step of lipid utilize is the hydrolysis of triglyceride which containing both dietary triglyceride and intracellular triglyceride.Therefore,in this section,three riglyceride lipases were identified and cloned from E.sinensis which named es_TGL1,es_TGL2 and es_IL,respectively.Through the constract of conversed domain by NCBI,es_TGL1 and es_TGL2 are digestive gland lipase which responsing for dietary triglyceride and es_IL is intracellular lipase which responsing for intracellular triglyceride.The full-length cDNAs of the es_TGL1,es_TGL2 and es_IL were 2129,1423 and 1939 bp long,respectively,with open reading frames of 1797,1266 and 1611 bp encoding for 599,422 and 537 amino acids,respectively.es_TGL1,es_TGL2 and es_IL containing 82,51 and 132 bp 5'-untranslated region(UTR),respectively,and 268,103 and 193 bp 3'-UTR,respectively.Three lipases were secretory proteins which all containing signal peptide.NCBI accession number are MF182403,MF182404 and MF182405,respectively.es_TGL1 showed higher mRNA expressions in gill,stomach and hepatopancreas and showed lowest mRNA expression in cardiocoelom.es_TGL2 have highest mRNA expression in muscle and lowest expressions in cardiocoelom,eyestalk and intestinal.es_IL showed highest mRNA expression in hepatopancreas and showed lower expressions in muscle and stomach.mRNA expressions of es_IL were lowest in the other four tissues.After feeding diet(13% lipid)for 4 weeks,crabs were starvation for 2 weeks.During starvation,TG in serum were increased significantly with the starvation.Glycerol contents in serum showed significantly increase in 72 h and 2 week than other time during starvation.Free fatty acid in serum increased before 24 h and dicreased after 48 h.In hepatopancreas,TG contents were decreased gradually during starvation and no significant difference was found in free fatty acid during starvation.Contents of glycerol in hepatopancreas showed fluctuating change before 48 h and recovered to control level after 72 h.mRNA expressions of es_TGL1 showed significantlt up-regulation in 6 and 12 h and decreased gradually afer 24 h.Similarly,mRNA expressions of es_TGL2 increased in 6,12 and 24 h and down-regulation after 48 h.es_IL showed significant high mRNA expressions in 6,12 and 72 h and downregulation after 96 h during starvation.Therefore,in this study,three triglyceride lipases were cloned and analyzed in E.sinensis.es_TGL1 and es_TGL2 have similar mRNA expression tendency during starvation.2.Optimum supplement of ?-lipoic acid in E.sinensis dietary and metabonomics mechanism analysisAs a new feed additive,?-lipoic acid is an important coenzyme in tricarboxylic acid cycle which involved in energy metabolism in organism.It has received extensive attention in the field of medicine and animal husbandry due to oral efficiently.In aquaculture,?-lipoic acid has attracted more and more attention.Therefore,this section was to determine the dietary ?-lipoic acid optimum addition of juvenile E.sinensis.?-lipoic acid was added to diets with seven levels(0,300,600,1200,2400,4800 and 9600 mg/kg diet).Each diet was fed to E.sinensis in four replicates for 8 weeks.Results showed that the optimun supplement of ?-lipoic acid in diet for E.sinensis is 1339.13 and 1574.09 mg/kg diet according to weight gain and specific growth rate,respectively,by line regression equation.No significant difference was found in proximate composition among treatments.SOD and GHS-Px activities in hepatopancreas were highest in crab fed diet with 600 mg/kg ?-lipoic acid and decreased gradually with the increasing of ?-lipoic acid in diets.T-AOC was increased and showed highest value in crab fed diet with 4800 mg/kg ?-lipoic acid.MDA contents in crab fed diet with 4800 and 9600 mg/kg ?-lipoic acid were significantly higher than that in crab fed other diets.There was no significant difference in GSH contents among seven treatments.Total lipid in hepatopancreas of crab fed diet with 300,600 and 1200 mg/kg ?-lipoic acid were higher than crab fed other diets,though there was no significant different among these treatments.There were significantly increase TG contents in hepatopancreas with the incerase ?-lipoic acid in diets.es_TGL1,es_TGL2 and es_IL all showed highest mRNA expressions in hepatopancreas of crab fed diet with 2400 mg/kg ?-lipoic acid.mRNA expressions of es_TGL1 were increased gradually with the increasing ?-lipoic acid supplement in diets within 2400 mg/kg dose.es_DGL showed highest mRNA expression in carb fed diet with 1200 mg/kg ?-lipoic acid which has no significant difference with mRNA expressions in crab fed diet with 300 and 2400 mg/kg ?-lipoic acid.Crab fed diet with 300 mg/kg ?-lipoic acid showed significantly higher CPT-1 expression than crabs fed other diets.Metabonomics contrast result of serum between crab fed diet with 0 and 2400 mg/kg ?-lipoic acid indicated there were 45 significantly changed metabolites.Among these metabolites,22 metabolites were significantly increased and 23 metabolites were significantly decreased in crab fed diet with 2400 mg/kg ?-lipoic acid contrast to control group.Main metabolish patyways of these significantly changed metabolites involved in fatty acid metabolish,amino acid metabolism,acyl carnitine metabolism,autophagy,antioxidant substrate etc.In conclusion,the optimun addition of ?-lipoic acid in diet for E.sinensis is 1339.13-1574.09 mg/kg diet.Metabonomics results indicated that ?-lipoic acid can signifiantly affect fatty acid metabolism and amino acid metabolism in E.sinensis and can also significantly increased the contents of antioxidant substrate.3.The effect of ?-lipoic acid on growth performance and physiology in E.sinensis under different dietary lipid percentageLipid is one of the important energy sources in diet of aquatic animals which can provide twice energy than protein and carbohydrate of units.Therefore,lipid efficient utilization can provide a new method for sparing diet protein and mitigating environmental pollution.In this section,effects of dietary lipid percentage(7% and 13%)on growth performance,body composition,antioxidantive status and hepatopancreas lipid catabolism of E.sinensis.Each lipid diet was supplemented with three concentrations of ?-lipoic acid at 0,700 and 1400 mg/kg diet,and fed to E.sinensis juveniles for eight weeks.The weight gain and specific growth rate of crabs fed the diets supplemented with ?-lipoic acid were significantly higher than those fed the control diet without ?-lipoic acid,regardless of dietary lipid levels.The ?-lipoic acid significantly increased lipid contents in the whole body and hepatopancreas in a dose-dependent manner.E.sinensis fed 13% lipid showed a significantly higher hepatosomatic index than those fed 7% lipid.The mRNA expressions of es_TGL2 and es_IL increased with the increase of ?-lipoic acid supplement in crabs fed diet with 7% lipid.No significant difference was found in the CPT-1 mRNA expression among all treatments.The ?-lipoic acid addition at 1400 mg/kg diet significantly improved oxidative stress due to lipid accumulation in the hepatopancreas of crab fed 7% lipid as indicated by the high activity of SOD and GSH-Px and low content of MDA.The diet with 13% lipid level increased the lipid content in the hepatopancreas but suppressed the activity of GSH-Px.Meanwhile,the T-AOC increased with the increase of ?-lipoic acid concentration in crabs fed 13% lipid percentage.This section indicates that ?-lipoic acid supplementation can improve growth performance and accelerate lipid accumulation in the hepatopancreas by increasing lipid utilization efficiency.Furthermore,?-lipoic acid can relieve hepatopancreas oxidative damage induced by lipid accumulation and improve the health of E.sinensis.4.Reciprocal effects of ?-lipoic acid and lipid sources on E.sinensis and lipidomics analysis of hepatopancreas in crabs fed different lipid sourcesDiet lipid nutrition depend on lipid quality which hinge on fatty acid composition and proportion.Therefore,in this section,effects of four dietary lipid sources(palm oil,olive oil,safflower oil and purple perilla seed oil)on growth performance,body composition,antioxidant capacity,hepatopancreas lipid catabolism and hepatopancreas fatty acid composition of E.sinensis were evaluated.Each lipid source diet was supplemented with two concentrations of ?-lipoic acid at 0 and 1400 mg/kg,and fed to E.sinensis juveiles for eight weeks.Results indicated that weight gain,specific growth rate,hepatosomatic index and survival were not significantly effected by dietary lipid sources and ?-lipoic acid.Similarly,moisture,crude protein and ash contents of E.sinensis were neither significantly influenced by dietary lipid sources and ?-lipoic acid.Crude lipid contents were significantly decreased by ?-lipoic acid in crabs fed lipid sources of olive oil and purple perilla seed oil.Crude lipid content of E.sinensis in palm oil was significantly higher than crabs fed the other three lipid sources with ?-lipoic acid spplement.In crabs fed with purple perilla seed oil,?-lipoic acid supplement can significantly improve antioxidant capacity with significantly increase activities of SOD,GSH-Px,T-AOC and content of GSH in hepatopancreas compared to control group.However,MDA content in hepatopancreas of crabs fed diet with ?-lipoic acid was higher than control group.No significant influence on antioxidant status was found in crabs fed with the other three lipid sources by ?-lipoic acid supplement in diets.In four lipid sources without ?-lipoic acid supplement,mRNA expressions of es_TGL2,es_IL,es_DGL and CPT-1 in hepatopancreas were all significantly lower than crabs fed the other three lipid sources.es_IL and CPT-1 showed highest mRNA expressions in hepatopancreas of crabs fed with olive oil.?-lipoic acid supplement can significantly increase es_TGL1 mRNA expression and decrease es_DGL mRNA expression in hepatopancreas of crabs fed with palm oil.?-lipoic acid supplement increased mRNA expressions of es_TGL2 and es_IL significantly in crabs fed with olive oil.In crabs fed with purple perilla seed oil,?-lipoic acid can significantly increase the mRNA expressions of es_IL and CPT-1.Lipid sources in diets significantly effect the contents of C20:4 and DHA in hepatopanaeus and extremely significantly affect the contents of other detected fatty acid except EPA.?-lipoic acid supplement in diets influence the contents of C14:0,C16:0,C20:5n3 and C22:6n3 in hepatopancreas significantly and affect the contents of C18:0,C18:1n9 and C18:2n6 extremely significantly.Meanwhile,contents of ? PUFA,? SAFA,? MUFA and ? n-6 in hepatopancreas were significantly influence by dietary ?-lipoic acid.There was still no research on fatty acid composition and content in TG,PE and PC of crustacean when fed diets with different lipis sources till now.In this section,lipidomics which is a novel methodologies was used to analyze diverse lipids and fatty acids fabrication in hepatopancreas of E.sinensis.Results indicated that crabs fed OA showed lowest percentage composition of TG and crabs fed LNA showed highest TG content in hepatopancreas which is significantly higher than the contents of TG in E.sinensis fed the other two lipid sources.There was no significant difference of PC contents between crabs fed PA and OA which were significantly higher than crabs fed with LA and LNA.Contents of PE in hepatopancreas were significantly decreased with the increased unsaturated degree of main fatty acid in diets.In sn-1,3 and sn-2 of TG,reserved degree of fatty acid in hepatopancreas was consistant with the main fatty acid in diets.However,content of C18:2 in sn-2 was highest in crabs fed LNA.In sn-1,3 of TG,HUFA contents of crabs fed OA were lowest among four lipid sources in diets and the contents of EPA and DHA in crabs fed with LA was higher than crabs fed the other diets.There was no C18:3 in sn-1 and C18:0 in sn-2 of PC.Saturated fatty acids were mainly connected to sn-1 and unsaturated fatty acids were mainly connected to sn-2 of PC.There was no C20:4,C20:5 and C22:6 in sn-1 and C18:0 in sn-2 of PE.Meanwhile,C16:0 and C18:0 were mainly connected to sn-1 and C20:5 and C22:6 were mainly connected to sn-2 of PE.In sn-1 and sn-2 of PE,reserved degree of fatty acid was consistant with the main fatty acid in diets.In conclusion,lipid sources and ?-lipoic acid neither affect growth performance of E.sinensis in this study.However,?-lipoic acid can significantly improve antioxidant status in E.sinensis fed with purple perilla seed oil.?-lipoic acid showed distinguishing influence on triglycerol catabolism in crabs fed different dietary lipid sources.Dietary lipid sources and ?-lipoic acid both showed significant influence on fatty acid composition in hepatopancreas of E.sinensis.There was good retention of fatty aicd in TG and PE in hepatopancreas with diet main fatty acid in E.sinensis.There was selective connection of saturated and unsaturated fatty acids to sn-1 and sn-2 of PC and PE.The research of hepatopancreas lipidomics in E.sinensis enrich basic knowledge of fatty acids composition in various lipids and provide a new insight for lipid research in aquaculture.