Lipid Nutrition Of The Pacific White Shrimp,Litopenaeus Vannamei Under Low Salinity

The Pacific white shrimp(Litopenaeus vannamei)is one of the most important shrimp species cultured worldwide.Nowadays,the rearing of L.vannamei in inland saline water has become an emerging and pillar industry due to its wide range of salinity tolerance.Although the relatively high economic profit with over 0.7 million tons production can be obtained from the culture of L.vannamei in inland low salinity water,there are some problems and potential risks of failure in commercial production,such as slow growth and low survival,low immune ability,and low stress resistance to some water born toxicants.However,these problems and potential risks were still damaging the quality and production of L.vannamei.Indeed,the L.vannamei is a typical euryhaline crustacean species,a hyper-hypo-osmoregulation process should be existed in L.vannamei to cope with the environmental salinity fluctuation,but a long-term low salinity stress still will cause many metabolism disorder and even death.Therefore,this adaptive mechanisms and biological functions of osmoregulation in euryhaline crustacean is the key to solve the problems.This studies aimed at investigating the mechanisms and function of lipid metabolism on osmoregulation of L.vannamei by using nutrient regulation including different dietary lipid sources and levels supplementation.1.Growth,lipid metabolism and transcriptomics analysis of the pacific white shrimp Litopenaeus vannamei at different salinitiesJuvenile white shrimp Litopenaeus vannamei(1.98±0.28 g)were fed a commercial diet for 8 week in triplicate to investigate growth and lipid metabolism at 3 salinities(3,17,and 30).Shrimp weight gain and survival at 3 were significantly less than that at 17 and 30.No differences were found in whole-body proximate composition.Linolenic acid(18:3n-3)and(n-3)long-chain unsaturated fatty acid levels in the hepatopancreas,and n-3 long-chain polyunsaturated fatty acid level,especially eicosapentaenoic acid(EPA;C20:5n-3)and docosahexaenoic acid(DHA;C22:6n-3)in muscle at 3 were significantly greater than at other salinities.Fatty acid synthase,hormone sensitive lipase,lipoprotein lipase,adipose triacylglycerol lipase,acyl-CoA,diacylglycerol acyltransferase 2,elongase of very long-chain fatty acid 6,and ?5 and A6 fatty acid desaturase activity was detected and showed a negative trend with an increase of salinity,and no significant differences were found among salinity groups.In transcriptomics results,26,034 genes were obtained from the hepatopancreas tissue of L.vannamei using the Illumina HiSeq 2000 system,and 855 genes showed significant changes in expression under salinity stress.Eighteen top Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were significantly involved in physiological responses,particularly in lipid metabolism,including fatty-acid biosynthesis,arachidonic acid metabolism and glycosphingolipid and glycosaminoglycan metabolism.Lipids or fatty acids can reduce osmotic stress in L.vannamei by providing additional energy or changing the membrane structure to allow osmoregulation in relevant organs,such as the gills.The results indicate that the low salinity will decrease the growth of L.vannamei,and the requirement of n-3 HUFA will increase due to the n-3 HUFA could improve osmoregulation and growth under such low salinity.Also,this study is the first report on the long-term adaptive transcriptomic response of L.vannamei to low salinity,and the results will further our understanding of the mechanisms underlying osmoregulation in euryhaline crustaceans.2.Effects of different lipid sources diets on growth performance and lipid metabolism of pacific white shrimp Litopenaeus vannamei at low salinity,and transcriptomics analysisLitopenaeus vanname(1.98±0.28 g)were fed diets containing soybean oil(SO),beef tallow(BT),fish oil(FO),linseed oil(LO),and an equal combination of SO +BT + FO(SBF)or SO + BT + LO(SBL)as dietary lipid source respectively for 8 weeks at low salinity of 3 psu.The shrimp fed the SBL diet had the highest weight gain and survival rate.The whole body fatty acid composition including the EPA and DHA of L.vannamei generally reflected the composition of dietary fatty acids with the highest DHA and EPA found in L.vannamei fed FO.The activities of fatty acid synthetase,acyl-CoA,diacylgycerol acyltransferase 2,elongase of long-chain fatty acids family member 6,?5 and ?6 fatty acid desaturases of shrimp fed SBL were significantly lower than those fed BT.There are 17,232(76.83%)?6298(28.08%)?3720(16.58%)?302(1.35%)predicted protein squences could be annotate in NR?string?KOG?COG ? NOG,respectively.A total of 9622 isogenes were annotated in 316 KEGG pathways and 39,42 and 32 pathways significantly changed in the paired comparisons of FO vs SBL,BT vs SBL,or FO vs BT,respectively.All the significantly changed pathways are associated with energy metabolism and cell membrane structure.The results indicate that lipids sources affect the adaptation of L.vannamei to low salinity by providing extra energy or specific fatty acids to change gill membrane structure and control iron balance.Appropriately increased n-3 HUFA diet can significantly improve L.vannamei osmoregulation under low salinity,but excess n-3 HUFA will casue harmful effects.Beside,L.vannamei may have abilities to biosynthesize n-3 HUFA.3.The effects of highly unsaturated fatty acids on osmoregulation and growth performance of Litopenaeus vannamei under different salinityThe effect of fish oil and coconut oil on osmoregulatory capacity of the L vannamei were investigated.The juvenile shrimp(1.89±0.16 g)were fed by three concentration purified fish diet(fish oil,coconut oil and equal mixed oil)and cultured at 3 and 30 psu salinities respectively(LFO,HFO,LCO,HCO,LMO and HMO respectively).The survival rate of each diet treatment had no differences under 30 psu salinity but significantly higher than 3 psu groups and showed a significantly decreasing tendency as the decrease of fish oil concentration in 3 psu salinity.The weight gain in two salinities was significantly increased as the dietary fish oil concentration increase,and the weight gain in high salinity was higher than that in low salinity.The results of fatty acids composition in gill and hepatopancreas showed that the highly unsaturated fatty acids increased with the rise of fish oil concentration.The enzyme activities of Na~+/K~+-ATPase and total ATPase declined as the decrease of fish oil concentration in 30 psu salinity but the activities in LMO were significantly lower than LCO and LFO.On the contrary,the relative expression level of Na~+/K~+-ATPase showed the opposite tendency as the enzyme activities.This results suggested that fish oil indeed has a positive influence indispensably on osmoregulation.But L.vannamei fed by a mixed oil contained with fish oil and coconut oil presented a higher osmoregulatory capacity and obtained a better weight gain in high salinity stress.It indicated that dietary lipid sources contained with saturated fatty acids could improve the osmoregulatory capacity in cooperation with highly unsaturated fatty acids,and it is more efficient than single highly unsaturated fatty acids.4.The influence of ?-linolenic acid on growth performance,osmoregulation and fatty acid biosynthesis of Litopenaeus vannamei under different salinity This study aimed at investigating the influence of a-linolenic acid on osmoregulation of L.vannamei under different salinities.The juvenile shrimp(1.89±0.16 g)were fed by three concentration purified perilla diet(perilla oil,coconut oil and equal mixed oil)and cultured at 3 and 30 psu salinities respectively.The survival rate and weight gain showed that of all the oil group under 30 psu salinity significantly higher than those under 3 psu salinity.But the survival rate no significantly difference between each oil group under 30 psu salinity,and the mixed oil and perilla oil group were significantly higher than coconut oil group under 3 psu salinity.However,there is an increasing tendency of weight gain with the increased dietary perilla oil concentration under 3 psu salinity.But only the mixed oil group was significantly higher than coconut oil group under 30 psu salinity.On the other hand,the highly unsaturated fatty acid content in gill showed an increasing tendency with the dietary increased perilla oil under both 30 psu and 3 psu salinity.Meanwhile,the a-linolenic acid content in hepatopancreas of mixed oil and perilla oil group under 3 psu salinity was significantly lower that those under 30 psu salinity.The activities of Na~+/K~+-ATPase also showed an increasing tendency with the dietary increased perilla oil under 30 psu salinity,but the mixed oil group was significantly lower than coconut oil and perilla oil group under 3 psu salinity.This results suggested that dietary perilla oil can significantly improve the osmoregulation capacity and also improve growth performance of L.vannamei under low salinity.In addition,L.vannamei may has the ability to convert the a-linolenic acid to highly unsaturated fatty acid,and this ability was greatly enhaced under low salinity stress.5.Effect of salinity and different dietary lipid sources on Pacific white shrimp(Litopenaeus vannamei)using GC-TOF/MS-Based metabolomicsNuclear magnetic resonance spectroscopy(NMR)was used to compare the metabolomics response of the effects of different lipid sources in L.vannamei plasma.Shrimp were fed by three diets with different lipid sources to investigate the function of different fatty acids profile in osmoregulation,including fish oil(FO),perilla oil(PO)and coconut oil(CO).We have pair-compared effects of lipid sources under low or high salinity,and we also compared the different metabolites in two salinities fed by the same lipid diet.The general classifications for the major significantly changed metabolites of interest are amino acids,carbohydrates and lipids.When shrimp cultured in low salinity(3 psu),the serine and tyrosine were detected highest in CO group and followed by PO group,the FO group is lowest.Meanwhile,the oleic acid and glucose were detected highest in PO group,and followed by CO group,the FO group is lowest.However,when shrimp cultured under high salinity(30 psu),the tyrosine and lysine were detected highest in PO group and followed by CO group,the FO group is lowest;and the palmitic acid,oleic acid,glycerol phosphate were detected highest in CO group and followed by PO group,the FO group is lowest.On the other hand,we also compared the different metabolites in two salinities fed by the same diets.In both CO and FO group,the significantly increased metabolites were Trans-4-hydroxy-l-proline and tyrosine,but the glycine was significantly decreased when shrimp cultured in low salinity compared with high salinity.But the tyrosine was significantly decreased in PO group when shrimp facing the low salinity environment.The results could suggest that fatty acids not only could modify the membrane permeability,fluidity and could supply sufficient energy via ?-oxidation,but also could modify the amino acid composition in plasma for osmoregulation.But the function of fatty acids and underlying mechanisms of lipid sources in osmoregulation are still need further studies.6.The effects of salinitiy and dietary lipids on the A6 fatty acyl desaturase-like gene of pacific white shrimp(Litopenaeus vannamei)In this study,a ?6 fatty acyl desaturase(Fad)like gene was identified and characterized with a full length of 1491 bp,including a 39 bp of 5'-UTR(untranslated regions),a 108 bp of 3'-UTR and a 1344 bp open reading frame encoding a 448 amino acid protein.The protein contained a cytochrome b5-like heme/steroid binding domain and a fatty acid desaturase domain.The A6 Fad-like protein of L.vannamei shared 70%to 77%sequence homology with that of Macrobrachium nipponense,Scylla paramamosain,Portunus trituberculatus and Eriocheir sinensis.When the salinity was reduced from 30 to 3 psu,expression of the ?6 Fad like gene increased significantly in gills,eyestalk and hepatopancreas.The highest expression was observed in hepatopancreas under 3 psu in the linseed oil group,and followed by the fish oil group.The results suggest that L.vannamei has the gene encoding a ?6 Fad like protein,whose expression increases with the decreased salinity,and also depends on the level of C18 fatty acids such as a-linolenic acid and linoleic acid in the diet.This study provides a deeper insight of the effects of sainity and dietary lipid on A6 Fad like gene of L.vannamei.