Physiological Effects Of Ambient Salinity On Litopenaeus Vannamei And Nutrient Modulation

Pacific white shrimp,Litopenaeus vannamei,is a tropical species that has beenwidely cultured in extensive,intensive,and semi-intensive systems,and it has becomean attractive cultivar for inland saline water farming in many parts of the world including the United States,Thailand and China in these year.Therefore,research tounderstand the physiology and nutrition of this species in different ambient salinitiesis of very important role in the future inland low salinity culture.In the present study,the physiological statuses of L.vannamei at different ambient salinities were evaluatedsystematically,and the important roles of dietary protein and vitamin B₆ levels on growth and survival rate were also investigated.Besides,molecular biologytechniques were used to study qualitatively and quantitatively the som osmoreglationsrelated gene in L.vannamei.1.Growth,body composition,respiration and ambient ammonia nitrogentolerance of the juvenile white shrimp,Litopenaeus vannamei,at differentsalinitiesTrials were conducted in laboratory to investigate the growth performance,bodycomposition,respiration and ammonia-N tolerance of the white shrimp,Litopenaeusvannamei,at3.0,17.0 and 32.0‰,respectively.In the growth trial,40 juvenileshrimps were stocked into each tank with four replicates at each salinity,and were fedwith a commercial diet for 50 d.Shrimp weight gain at 17.0‰was the highest,andsignificantly higher than that of shrimps at 3‰.Shrimps survival rate at 3.0‰wassignificantly lower than that of other two groups.However,hepatosomatic index andcondition factor were not significantly affected by the ambient salinity.Shrimp bodyprotein and ash content were not affected significantly by salinity,while bodymoisture increased at high salinity,and crude lipid in shrimps was lowest at 32.0‰.After exposed to the above three salinities for 30 d prior to the test,shrimp oxygenconsumption and respiratory quotient of the shrimps at 3‰was significantly higherthan those of shrimps at medium and high salinities,while salinity did notsignificantly affect CO₂ production.When juvenile L.vannamei were exposed toseven ammonia-N concentrations(0,4.00,6.67,9.33,12.00,14.67,and 17.33 mg.l^-1)at the three above salinities to which shrimps had been separately acclimated for 10 d at pH 8.30 and 29±0.5℃,shrimps at 3‰were the most susceptible to ambientammonia-N,and the 96 h LC50 with 95% confidence limit to ambient ammonia-Nwas 9.33(8.39-10.37)mg.l^-1.This study suggests that L.vannamei could adapt to awide range of salinity,but the animals would be more susceptible to ammonia toxicityand spend more energy to compensate the cost for osmoregulation at low salinity.2. Comparison of digestive and antioxidant enzymes activities,haemolymphoxyhemocyanin contents and hepatopancreas histology of white shrimp,Litopenaeus vannamei,at various salinitiesAssessments on adaptation of Litopenaeus vannamei to three ambient salinities(3.0,17.0 and 32.0‰)were carried out with four replicates for 50 days Shrimpwere then sampled to measure digestive enzymes,superoxide dismutase(SOD),catalase(CAT)activities,haemolymph oxyhemocyanin content,and the histologicalstructure of hepatopancreas at each salinity.Trypsin activity at 3.0‰wassignificantly higher than that at 17.0 and 32.0‰,while total amylase activity at 17.0‰was lower than at 3.0‰.Cellulase and lipase activities were not significantly different across various salinities,though slight increases were observed at both 3.0and 32.0‰compared with 17.0‰.At 3.0‰,both haemolymph oxyhemocyanincontent and the ratio of oxyhemocyanin to heamolymph protein were enhanced.Inaddition,the SOD and CAT activities in the muscle and hepatopancreas at 3.0‰werehigher than those at 17.0‰.These results indicated that L.vannamei fed at a highlevel of nutrition,maintained metabolism and oxygen-carrying capacity to meet theirenergy requirement at low salinity.And low salinity had stimulated the production ofradicals for scavenging,and the activities increase of SOD and CAT for scavengingradicals insured the healthy status of L.vannamei in a certain degree.Besides,shrimpat 3.0‰produced more B cells in hepatopancreas tubules than at 17.0‰,while thevolume of B cells tended to increase at 32.0‰.The histological changes of thehepatopancreas tubules in concomitant with digestive and antioxidant enzymesprovide evidence on the mechanism of how L.vannamei can cope with salinitychange.3. Protein accumulation,amino acid profile and amino transferase activities ofthe white shrimp,Litopeneaus vannamei,at different salinitiesExperiments were conducted to investigate the protein accumulation of different tissues,muscle amino acid profile,Glatamic oxalacetic transferase(GOT)andGlutamat Pyruvat Transferase(GPT)activities of the white shrimp,Litopeneaus vannamei,reared at three ambient salinities which were 3.0‰,17.0‰and 32.0‰respectively for 50 days.The results showed that shrimps at 3.0‰had significantlyhigher soluble protein content of hepatopancreases and heamolymph than those atboth 17.0‰and 32.0‰,while no significant differences were observed in the shrimptissue of muscle among three treatments.Low salinity 3.0‰and high salinity 32‰led to the increasing of both GOT and GPT activities in muscle though withoutsignificant differences comparing with 17.0‰treatment.Shrimps at 3.0‰and 32.0‰had significant higher levels of total amino acids and total essential amino acids inmuscle than these at 17.0‰,and non essential amino acids in shrimps at 3.0‰and 17.0‰showed no obvious differences.However,the total muscle methionine,serine,cysteine and proline contents of shrimps at 3.0‰were significantly lower than theseat 17.0‰.Most muscle free amino acids were not different significantly among all thetreatments.Besides,as one of the five osmolyte amino acids,alanine of shrimps at3.0‰showed similar tendency as proline though no significant difference wasobserved when compared with shrimps at 17.0‰.All these results indicated thatunder stress of higher and lower salinity,the white shrimp ensured itself to havesufficient amino acids for energy supply in the osmoregulation by accumulatingprotein in the hepatopancreas and heamolymphs and increasing GOT and GPTactivities in muscle,and proline and alanine might be the main two among theseamino acids which play important roles in osmoregulation.4.Effects of dietary protein level on growth,survival,body composition andhepatopancreas histological structure of the white shrimp,Litopenaeus vannamei,at different ambient salinitiesAn 8-week trial was conducted to investigate the effects of dietary protein level whichwere 20.61,30.52,40.43 and 50.34%(named CP20,CP30,CP40 and CP50 respectively)on the growth,survival,body composition of the white shrimp,Litopenaeus vannamei,at three ambient salinities which were 2‰,22‰and 32‰respectively.The results showed that ambient salinity had significant effects onshrimp growth,survival,conditional factor(CF)and body ash,while no significantdifferences were observed in the hepatosomatic index(HSI),body crude protein,lipid and moisture.Shrimps at 22‰were the highest in all the parameters followed by these at 32‰and 2‰.Weight gain,specific growth rate for both weight and bodylength of shrimps at 32‰and 22‰were significantly higher than those at 2‰,while no differences were found in shrimps at 32‰and 22‰.Dietary protein level onlysignificantly affected the growth performance,body crude protein,CF and HSI of theshrimps.Weight gain and the body crude protein content of shrimps increased withthe increasing of dietary protein level,and shrimps fed with CP20 showed significantlower growth performance and body crude protein.CF and HSI increased firstly withthe dietary protein level increasing to CP40,and then decreased slightly when fedwith CP50.Dietary protein level had no effect on the survival shrimp at all theambient salinities.Ambient salinity and dietary protein level only had significantinteraction on the body ash content,while no significant interaction effects wereobserved in other parameters.Tubules of shrimp fed with CP30 and CP40 had thenormal structure,though CP40 increased the number of R cells.Tubules of shrimpsfed with CP20 had relatively fewer R cells and arranged incompactly.And the basalmembranes of partial tubules were in disrepair.While tubules of shrimps fed withCP50 compactly arranged with much ambiguous materials in B cells.The resultsindicated that though the growth performance and conditional factor could beincreased by increasing dietary protein level,low survival rate at low salinity did notenhanced.And improper content of dietary protein,especially extremely lower dietaryprotein,would results in pathological changes of the hepatopancreans' histologicalstructure.5.Factorial effect of salinity and dietary animal to plant protein on growth andsurvival in juvenile white shrimp,Litopenaeus vannameiThe factorial effects of ambient salinity(3.0 and 22.0 ppt)and plant to animalprotein ratio(0/38,7/30,14/23,21/16,29/8 and 36/0 approximately)on growth andsurvival of juvenile white shrimp,Litopeneaus vannamei were investigated.Theresults showed that dietary animal to plant protein had significant effects on theshrimp weight gain,hepatosomatic index,conditional factor,survival rate and thehepatopancreas soluble protein.Weight gain increased with the dietary animal to plantprotein ratio increased,while for other index measured,they exhibited the tendency ofincreased first with the dietary animal to plant protein ratio increasing,and thendecreased.Increasing salinity significantly increased the weight gain,survival and theconditional factor,and significantly decreased the hepatosomatic index,while no significant differences of salinity were observed in the hepatosomatic index of theshrimps.Shrimp weight gain,survival and hepatosomatic index were significantlyeffected by the interaction between ambient salinity and dietary animal to plant protein ratio,while shrimp conditional factor and the hepatopancreas were not significantly affected by the interaction between ambient salinity and dietary animal to plant protein ratio.The broken-line model analysis showed that the optimal dietary animal to plant protein content in the diets for the white shrimps at 3.0 and 22.0 ppt ranged from 29.12/7.79 to 30.29/6.71 and from 26.05/10.95 to 29.03/7.97 approximately.6.Dietary vitamin B₆ requirement of Pacific white shrimp,Litopenaeusvannamei,at low salinityA feeding trial was conducted to determine the adequate level of dietary vitamin B₆(pyridoxine,PN)for juvenile Pacific white shrimp,Litopenaeus vannamei.Purifiedbasal diets were formulated using vitamin-free casein as the protein source.Gradedlevels(0,35,70,105,140 and 200 mg PN/kg diet)of PN were added to the basal dietwhich upon analyzed contained 2.17,32.43,65.79,96.97,137.13 and 189.56 mg/kgdiet,respectively.Each diet was fed to three replicate groups of shrimp(mean weight)for 30 days.Results showed that dietary vitamin B₆ significantly affected the weightgain,special growth rate,survival rate and conditional factor of L.vannamei),whileno significant differences were observed in the HIS of shrimp.Shrimp weight gainand specific growth rate and conditional factor increased significantly with theincrease of dietary vitamin B₆ content.But when the dietary vitamin B₆ contentincreased to 137.13 mg/kg diet,increase of dietary vitamin B₆ content slightlyincreased the growth rate,but no significant differences were observed.Overall,shrimp survival rate increased firstly with dietary vitamin B₆ increase to 137.13 mg/kgdiet,and then slightly decreased when dietary vitamin B₆ content increased to 189.56mg/kg diet,glatamic oxalacetic transferase(GOT)and glutamat Pyruvat Transferase(GPT)activities showed the same tendency which were increased first with theincrease of dietary vitamin B₆ content to 96.97 mg/kg diet,and then the activitiesdecreased slightly when dietary vitamin B₆ content continue increased,but nosignificant differences were observed in both GOT and GPT activeties of shrimp fedvitamin B₆ content of 96.97,137.13 and 189.56 mg/kg diets.Weight gain,specificgrowth rate for both weight and length,conditional factor and amino transferase activities of shrimp analyzed by broken-line regression indicated that the dietaryvitamin B₆ requirement of L.vannamei ranged from 106.96 to 151.92 mg/kg diet,andthe mean±standard deviation is 129.94±18.86mg/kg.7. Interaction of dietary vitamin B₆ and protein on growth,survival and aminotransferase activities of Pacific white shrimp,Litopenaeus vannantei,at lowsalinityA feeding trial was conducted to study of the interaction of dietary vitamin B₆(pyridoxine,PN)and protein on growth,survival,morphological parameters andamino transferase activities of juvenile Pacific white shrimp,Litopenaeus vannamei.Purified basal diets were formulated using vitamin-free casein as the protein source.Graded levels(0 and 200 mg PN/kg diet)of PN were added to the two basal dietswhich contain 25.50 and 40.80% dietary protein,respectively.Each diet was fed tothree replicate groups of shrimp(mean weight 0.014±0.005g)for 30 days.Resultsshowed that supplementation of vitamin B₆ in diets significantly increased weight gain,special growth rate,survival rate,conditional factor and both GOT and GPT activitiesof L.vannamei,while significantly decreased thee HIS of L.vannamei.Shrimp growth,morphological parameters and transferase activities of L.vannamei fed dietscontaining 25% and 40% dietary protein had no significantly differences,and onlysurvival rate was significantly by increasing dietary protein level from 25% to 40%.Significant interaction between dietary protein and vitamin B₆ was not observed in allparameters tested in L.vannamei in present study.Results in present study indicatedthat dietary vitamin B₆ and protein had different nutritional effect in L.vannamei,andwhen preparing diets for L.vannamei cultured at low salinity,dietary vitamin B-6 andprotein must meet the requirement of L.vannamei at the same time.And it is notfeasible to try to meet and increase one of these two nutrients to get the maximumgrowth and survival rate of L.vannamei with the aim to save the other nutrient.8. Characterization of the glutamate dehydrogenase gene in pacific whiteshrimp,Litopenaeus vannameiGlutamate dehydrogenase(GDH)plays a key role in the metabolism of freeamino acid in crustaceans.Glutamate synthesized by GDH via reductive amination isthe amino group donor for alanine synthesis and the precursor required for prolinesynthesis.Since both proline and alanine are important intracellular osmolytes in many marine invertebrates,GDH has been widely implicated as playing a central rolein response to hyperosmotic stress in these animals.In the current study,two GDH genes were obtained,and named GDH A and GDH B which encode 474 and 552 amino acids respectively.The alignment of the two GDH A amino acid sequenceswith other those of other species showed that the difference of the GDH A and Bstarted only at the 462 th amino acids,and both GDH A and B were all fairyconserved protein.Besides,three exact phase-0 introns were found in both GDH Aand B which were 202,333 and 256 bp respectively.And as a control,a phylogenetictree was constructed with the cDNA of the two GDHs of L.vannamei and otherspecies,and the results showed that the two L.vannamei GDHs fall into the expectedposition with the sequences of other arthropod based on organism phylogen,showedclosed evolutional relationship with D.melanogaster and B.mori of Arthropoda.Thisis the first time that two different GDH genes have been identified from a crustacean.Results of the present study would provide basic knowledge for future quantitativeand qualitative study of this gene in L.vannamei and other species of crustacean.9.Tissue expression of Na+-K+ ATPase and two glutamate dehydrogenasegenes in pacific white shrimp,Litopenaeus vannameiA semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)assay was developed to estimate the tissue specific expressions of Na⁺-K⁺ ATPase andtwo glutamate dehydrogenase(GDH)genes in five different tissues of pacific whiteshrimp,Litopenaeus vannamei.The results showed that primers designed and reactioncondition in the present study for Na⁺-K⁺ ATPase and GDH genes were feasible,andcould be used in the future study.The expressions of all test genes were tissue specific.Na⁺-K⁺ATPase was mainly expressed in shrimp gill,and both GDH A and B weremainly expressed in shrimp muscle,while expression of all test genes in shrimphepatopancreas were the lowest when compared with that in other tissues.Beside,though no significant difference was observed in the ratio of GDH B to Atranscription between all the test tissues,the ratio of GDH B to A transcription keptvery high which ranged 32.04 in eye stalk to 64.52 in muscle indicating that GDH Bwas the dominated gene for GDH activity in L.vannamei.All these results in thepresent study indicated that shrimp muscle and gill but not hepatopancreas were themost suitable site to study the gene expression of Na⁺-K⁺ ATPase and GDH inL.vannamei.The results in the present study provide baseline data of physiological expressions for Na⁺-K⁺ ATPase and GDH genes which relates to osmoregulation of L.vannamei and a guideline of tissue or organ sampling for effective gene expressionanalyses for future related studies.10.Ontogenetic changes in two glutamate dehydrogenase,Na+-K+ ATPase andamylase gene expression of thepacific white shrimp,Litopenaeus vannameiQuantitative polymerase chain reaction(qPCR)was used to study the geneexpressions of the two glutamate dehydrogenase,Na⁺-K⁺ ATPase and amylase ofL.vannamei at different developmental stages.The results showed that both GDH Aand GDH B transcripts relative toβ-actin transcripts tended to increase from stage ofegg to postlarvae steadily,and a sharp increase at the stage of postlarvae was observed.Ratio of GDH B to GDH A transcripts was first decreased from stage of egg to thestage of nauplii,and then increase at the stage of zoea,and kept relatively constant inmysis,and then decreased lightly to the stage of postlarvae 2,and then there wasanother increase with the development of L.vannamei.Na⁺-K⁺ ATPase geneexpression kept relatively constant in egg,nauplii,zoea,mysisi and even in thepostlarvae 10.But there was a sharp increase in postlarvae 16 at which the amount ofNa⁺-K⁺ ATPase relative toP-actin transcripts was several times higher than those atother developmental stages.Amylase gene expression of L.vannamei in egg,nauplii,and zoea were very low,and it began to increase sharply only at the stage of mysis.Results of GDH and Na+-K+ ATPase gene expression in this study indicated thatL.vannamei larvae had no strong osmoregulation capacity until it developed to thestage of later postlarvae,while the amylase gene expression change in this study thatL.vannamei larvae could only digest and utilize food efficiently when it developed tothe stage of mysis.Therefore,if we want to modulate the osmoregulation capacityfrom the L.vannamei larvae on by some nutritional methods,the best time fornutritional modulation is when the shrimp develop to the stage of mysis at least.