Carbohydrate Metabolism And Regulation In Litopenaeus Vannamei At Low Salinity

The white shrimp,Litopenaeus vannamei,is the most important shrimp species,representing more than 70%of the world farmed shrimp production.With the development of inland low salinity farms,L.vannamei has become one of the most popular shrimp species for aquaculture in America and Asia.Even high profite can be obtained from the inland culture of L.vannamei,many problems related to low salinity stress still exsists.As carbohydrate plays important role in the osmoregulation process.This study explored the carbohydrate metabolism and osmoreguation mechanism in L.vannamei at low salinity using comprehensive experiment technology of nutrition,biochemistry,physiology and molecular biology.It is consisted of several parts:the effects of dietary carbohydrate nutrion on the growth,ammonia tolerance and hepatopancreas histology of L.vannamei at low salinity;the protein sparing effect of carbohydrate and the molecular response of carbohydrate metabolism in L.vannamei at low salinity.Results obtained from this study would provide a practical solution,through dietary manipulation,to improve growth performance and to reduce environmental stress for inland white shrimp farming at low salinities.1.Molecular pathway and gene responses of the white shrimp L.vannamei to acute low salinity stressTo understand the underlying mechanism of the white shrimp L.vannamei responding to acute salinity stress,RNA-seq was used to determine the transcriptome response of shrimp muscle and gill after ambient salinity changed from salinity of 20(control)to 3 in 24 h.A total of 281.4 million reads were obtained and assembled into 105,153 contigs with an average length of 984 bp.Comparison of gene expression between shrimp exposed to salinity of 3 and the salinity control revealed that 991 and 3709 genes were differently expressed in the gill and muscle,respectively.Both in muscle and gill,the changes of pathway can be categorized into oxidative pathways,signal transduction pathways,and metabolism pathways.More pathways significantly responded in gill than in muscle in metabolism and signal transduction.The significant change of pathways revealed that under acute low salinity stress,the increase of energy derived from carbohydrate,amino acid or lipid in gill could satisfy the extra energy requirement of shrimp under salinity stress,but also lead to an over-production of reactive oxygen species.For the maintenance of homeostasis,protein ubiquitination and relevant pathways were activated to remove the excessive reactive oxygen species and metabolite waste.2.Molecular pathway and protien responses of the white shrimp L.vanntamei to long-term low salinity stressIn this study,reverse phase high-performance liquid chromatography-MS/MS with isobaric tags for relative and absolute quantification(iTRAQ)method is applied to identify and quantify the differentially expressed proteins between the L.vannamei under long-term(8 weeks)low salinity stress at 3 practical salinity unit(psu)and the shrimp at 25 psu serving as the control.533 proteins(unique peptide?1)were identified by PEAKS8.0 studio program and 60 proteins were detected to be regulated for more than 1.2-fold change after long-term low salinity stress,including 34 down-regulated and 26 up-regulated proteins.These proteins participated in 41 different patyways,e.g.metabolic pathway,wnt signaling pathway,Phagosome,Ribosome biogenesis,Ubiquitin mecdiated proteolysis and Ribosome biogenesis.Among these 60 proteins,6 proteins were involved in pathways related to carbohydrate metabolism.3.Growth,body composition,and ammonia tolerance of juvenile white shrimp L.vannamei fed diets containing different carbohydrate levels at low salinityThe effects of dietary carbohydrate levels on growth,body composition and ammonia tolerance of juvenile white shrimp L.vannamei at a low salinity of 3.0psu were evaluated.Six iso-nitrogenous and iso-lipid experimental diets containing different dietary carbohydrate(CBH)levels were formulated and fed to juvenile L.vannamei for 42 days in triplicate for each treatment.Weight gain and survival rate of shrimp fed 20%CBH were the highest and differed from those fed 5%and 30%CBH.Shrimp body crude protein of the 20%CBH group was significantly higher than that of other treatments.Whole body crude lipid increased with the increase of dietary CBH and was significantly higher in 20%,25%and 30%CBH groups than the control.Hepatosomatic index,condition factor,whole body moisture and ash content were not affected by dietary CBH.Hepatopancreas soluble proteins peaked in shrimp fed 20%CBH,and were significantly higher than in other groups.Hepatopancreas and muscle glycogen showed similar tendency,but peaked in shrimp fed 15%CBH.Malate dehydrogenase,pyravate kinase and glucose-6-phosphate dehydrogenase activities were not affected significantly by dietary carbohydrate levels.Ammonia challenge of 96-h showed that the shrimp in the 20%CBH group had the highest survival rate,though no significant differences were observed between treatments.This study indicates that 15%-20%dietary carbohydrate is optimal for growth,and can improve the anti-stress ability of L.vannamei at low salinity.4.Growth,ammonia tolerance and hepatopancreas histology of white shrimp L.vannamei fed diets containing different carbohydrate sources at low salinityFive diets with glucose,sucrose,wheat starch,corn starch or potato starch as the carbohydrate(CBH)source were fed to juvenile L.vannamei in triplicate at a low salinity of 3.0psu for 50 days to explore the effect of dietary carbohydrate source on growth,body composition and ammonia tolerance.Weight gain and survival of shrimp fed glucose were highest and significantly differed from those fed wheat starch and potato starch.Shrimp fed com starch had the lowest weight gain.Whole shrimp body crude protein and lipid of the com starch group were highest and significantly differed from the shrimp fed wheat starch,but whole body moisture and ash contents,hepatosomatic index,condition factor were not affected by dietary carbohydrate sources.Shrimp fed glucose had the highest hepatopancrease soluble protein and differed from those fed sucrose or wheat starch.Shrimp fed potato starch had higher hepatopancreas and muscle glycogen than in other dietary groups.Shrimp fed sucrose had higher glucose-6-phosphate dehydrogenase and lower pyruvate kinase activities than in other dietary groups.Besides,shrimp fed starch produced more B cells in hepatopancreas tubules than those fed glucose or sucrose.Furthermore,shrimp fed different sources of carbohydrate differed in the number of R cells in the hepatopancreas tubules.After ammonia challenge for 96 h,the shrimp fed glucose,followed by those fed wheat starch,corm starch and sucrose group,had the highest survival rate,though no significant differences were observed between various treatments.This study indicates that glucose can be used directly to meet the energy demand of L.vannamei under low salinity stress,leading to enhanced growth and improved anti-stress ability of L.vannamei.We recommend that wheat starch be a suitable carbohydrate for L.vannamei farming in low salinity.5.Protein sparing effect of carbohydrate in the diet of white shrimp L.vannamei at low salinityJuvenile L.vannamei were fed for 8 weeks with diets containing four ratios of protein to carbohydrate(CBH)at P260:CBH300,P300:CBH250,P340:CBH190 and P380:CBH140,respectively at salinity of 3.Shrimp weight gain of P340:CBH 190 group was the highest and differed from the shrimp fed the P260:CBH300 or P300:CBH250 diet.Shrimp fed the P340:CBH190 diet contained the highest body protein and lipid,which were significantly higher than those fed the P380:CBH140 diet.While shrimp fed the P260:CBH300 diet obtained higher survival than those fed other diets.The P300:CBH250 diet group had the highest hemolymph glucose content,which were significantly higher than those fed the P260:CBH300 or P380:CBH140 diet.Hepatopancreas B cell number of shrimp fed the P260:CBH300 diet was lower than those fed other diets and the R cell number was the highest in shrimp of P300:CBH250 group.This study indicates that the protein sparing effect by CBH occurred in the P300:CBH250 and P340:CBH 190 groups because these protein to CBH ratios can support normal growth.Within the range of basic energy demand,the high dietary CBH to protein ratio can improve L.vannamei survival at low salinity6.Response of facilitative glucose transporter 1 in response to salinity stress and dietary carbohydrate nutrition in white shrimp L.vannameiFacilitative glucose transporter 1(GLUT1)is a transporter protein for glucose transport via the plasma membrane of the cells to provide energy through carbohydrate metabolism.GLUT1 cDNA from L.vannamei was obtained and analyzed in this study.Full-length GLUT1 cDNA is 2,062 bp long,containing a 1,506 bp ORF encoding a 502 amino acid protein,a 270 bp 5'UTR and a 284 bp 3'UTR.When shrimp were under acute low salinity stress,the expression in hepatopancreas,muscle,gill and eyestalk was all up-regulated at 12 h(P<0.05)and 96 h(P<0.05),while the expression in the four tissues was all down-regulated at 6 h(P<0.05)and 48 h.The expression in the muscle of shrimp at water salinity of 3 was lower than that at 30 independent of dietary carbohydrate levels.While expression in hepatopancreas,gill and eyestalk were up-regulated at 200 and 300 g kg-1 carbohydrate levels.The expression in all tissues fed glucose was up-regulated when compared to the expression in shrimp held at a water salinity of 30.This study suggests that GLUT1 is a conserved protein in L.vannamei,and changes in expression due to environmental salinity and dietary carbohydrate level and source.7.Molecular response of carbohydrate metabolism to dietary carbohydrate and acute low salinity stress in white shrimp L.vannameiMolecular response of carbohydrate metabolism in L.vannamei was evaluated at transcriptional level through quantitative real-time PCR analysis.The mRNA expressions of six genes hexose-6-phosphotransferase,pyruvate kinase,phosphoenolpyravate carboxykinase,crustacean hyperglycemic hormone,glucose transporter and insulin-like growth factors binding protein in the hepatopancreas,muscle,gill and eyestalk were analyzed in shrimp fed different levels of carbohydrate and challenged with acute salinity stress.Among these six genes,the relative expression of GLUT1,CHH and IGF-BP in the muscle was lower or nearly the same as the control group regardless of the dietary carbohydrate level.The relative expressions of HK and PK were much higher in the hepatopancreas,muscle,gill and eyestalk at 12 h after salinity stress.The relative expression of PEPCK showed a down-up-down tendency and the expression was much higher in different tissues(except in eyestalk)than in the control from 12 to 24 h after stress.This study indicates that there was no significant dietary carbohydrate regulation in muscle.The glycogen and amino acid in muscle were the main source energy for osmoregulation in L.vannamei when the animals were challenged with acute salinity stress.The muscle and gills were the main osmoregulation organ in shrimp under hypo-saline stress.