【作者】 李二超；

【导师】 陈立侨； 曾嶒； Alain VAN WORMHOUDT；

【作者基本信息】 华东师范大学 ， 动物学， 2008， 博士

【摘要】 本项目结合水生动物养殖学、动物营养学、生物化学、分子生物学及组织学等研究手段，制定有限目标，较为系统和深入地研究不同养殖盐度下凡纳滨对虾基础营养生理学差异，并在此基础上从营养素（蛋白质和维生素）着手，研究了营养素对凡纳滨对虾生长速度和成活率提高方面的影响，探讨了营养素对凡纳滨对虾渗透调节的作用，初步阐明营养素对凡纳滨对虾渗透调节的调控机制，有针对性的对与凡纳滨对虾渗透调节相关的关键酶基因进性了定性和定量研究，研究成果不但可以为进一步了解甲壳动物渗透调节机理提供了一定的理论依据，也为进一步从分子生物学水平进行相关的研究建立了相关的检测平台，而且相关的营养学研究成果也为凡纳滨对虾的淡化养殖及相关的人工全价配饵提供更为准确的参考依据，具有十分重要的现实意义。1．盐度对凡纳滨对虾生长、体生化成份、呼吸及氨氮耐受性的影响在室内养殖条件下进行了凡纳滨对虾在高、中和低盐度下（分别为3．0、17．0和32．0‰）生长性能、成活率和体生化成分的研究，试验为期50天，期间投喂商用饲料，每个盐度设4个平行。结果表明，中盐度凡纳滨对虾增长率显著高于低盐度组，且低盐度组对虾成活率仅为70％左右，显著低于其它两处理组；然而，盐度对凡纳滨对虾肝体指数和肥满度却无显著影响。各盐度组凡纳滨对虾的体粗蛋白质和灰分含量无显著性差异，而对虾体水分含量却随盐度升高而升高，且高盐度组对虾的体粗脂肪含量显著低于其它两试验组。对不同盐度下凡纳滨对虾的呼吸率的研究发现，低盐度下对虾的耗氧率和吸收商显著高于中盐度和高盐度两试验组，而各盐度组对虾二氧化碳呼出率无显著性差异。此外，试验还研究了不同盐度下凡纳滨对虾对环境中氨氮耐受的差异，计算了低盐度下氨氮对凡纳滨对虾的半致死浓度，并与其它相关研究进行了比较。结果发现，低盐度下氨氮对凡纳滨对虾的半致死浓度（95％置信区间）为9．33（8．39-10．37）mg．l^-1，显著低于其它报道的半致死浓度。另外，本试验也发现，凡纳滨对虾对环境中氨氮的耐受力随盐度的升高而增强。综合以上述研究结果，可以看出，虽然凡纳滨对虾属广盐性虾类，对环境盐度范围非常广，但在低盐度下该对虾的养殖尚存在着生长速度低、成活率低和抗逆性低的“三低”现象，因此，在凡纳滨对虾的低盐度养殖过程中除了满足凡纳滨对虾生长和渗透调节所需能量和营养物质之外，还应时刻监测对虾养殖环境中对对虾有害物质的动态变化。2．不同盐度下凡纳滨对虾抗氧化和消化酶活力、血蓝蛋白含量和肝胰腺组织学结构的比较在前期研究结果的基础上，进一步比较了三个不同盐度（分别为3．0、17．0和32．0‰）下驯养50d后的凡纳滨对虾消化酶和抗氧化相关酶活力、血蓝蛋白含量及肝腺胰组织学结构的差异，探讨了凡纳滨对虾对不同盐度的生理适应性。结果发现，盐度3．0‰对虾组胰蛋白酶活力显著高于其它两个盐度实验组，盐度17．0‰对虾组总的淀粉酶活力却显著低于盐度3．0‰对虾组。各实验组脂肪酶和纤维素酶活力虽无显著性差异，但相比盐度17．0‰对虾组组，两者在高、低盐度下会有一定程度的升高趋势。低盐度还导致对虾氧合血蓝蛋白含量及氧合血蓝蛋白／总蛋白比值显著升高，提示凡纳滨对虾可通过提高对自身消化酶活力、代谢率和携氧能力，尽量满足低盐度下的高能量需要。此外，结果还发现低盐度下凡纳滨对虾肌肉和肝胰腺组织的超氧化物歧化酶和过氧化氢酶活力显著高于盐度17．0‰对虾组，表明对虾机体抗氧化系统在长期处理低盐度应激下，已经开始启动用于最大程度地清除机体产生的自由基。相比盐度17．0‰对虾组，低盐度下凡纳滨对虾肝胰腺肝小体中分泌细胞（B细胞）数量增多，而高盐度下却表现出B细胞体积增大的趋势，在一定程度上佐证了对虾消化酶和抗氧化相关酶活力升高的现象，从组织学角度进一步证实了凡纳滨对虾对不同盐度的适应情况。3．盐度对凡纳滨对虾体组织蛋白质积累、氨基酸组成和转氨酶活性的影响研究了低、中和高三个盐度水平（分别为3‰、17‰和32‰）对凡纳滨对虾各组织蛋白质的积累、肌肉谷草转氨酶和谷丙转氨酶活力、肌肉总氨基酸和游离氨基酸组成和含量的影响。结果显示，经过50d不同盐度水平的试验，低盐度组对虾的肝胰腺和血淋巴中可溶性蛋白质含量显著高于中、高盐度组（P（0．05），而肌肉中可溶性蛋白质含量在各处理组间无显著性差异；低、高盐度均导致肌肉中谷丙转氨酶和谷草转氨酶活力升高，但是各处理间的差异不显著；低、高盐度组凡纳滨对虾肌肉总氨基酸和总必需氨基酸含量均显著高于中盐度组（P<0．05），中、低盐度处理组非必需氨基酸含量差异不显著，而低盐度组对虾肌肉中蛋氨酸、丝氨酸、半胱氨酸和脯氨酸含量均显著低于中盐度组（P<0．05），其中脯氨酸为常见的5种主要渗透调节氨基酸之一；低、高盐度组对虾肌肉总游离氨基酸含量显著高于中盐度组（P<0．05），而盐度对机体绝大部分肌肉游离氨基酸含量的影响不显著（P>0．05）。结果显示，当环境盐度偏离凡纳滨对虾最适生长盐度时，机体可通过在肝胰腺和血淋巴中积累蛋白质，及提高自身转氨酶活力，来获得机体在渗透调节供能时所需的氨基酸，而这些氨基酸以脯氨酸为主。4．不同盐度下饵料蛋白质含量对凡纳滨对虾生长、体成份和肝胰腺组织结构的影响设计了蛋白质含量为20．61、30．52、40．43和50．34％的四种饵料（分别为CP20、CP30、CP40和CP50），研究了饵料蛋白质含量对不同盐度下（分别为低盐度LS 2‰、中盐度MS22‰和高盐度HS32‰）凡纳滨对虾（0．0144±0．0047g）生长、成活及体成份的影响，测定了不同处理组对虾的肝胰腺指数（HIS）和肥满度（CF），试验为期8周。结果显示：（1）盐度对凡纳滨对虾的生长、成活、肥满度和灰分含量均有显著影响，而对肝体指数、体粗蛋白、体粗脂肪和水分无显著影响，中盐度组对虾各指标均最高，其次为高盐度组，低盐度组最低；中、高盐度组对虾的增重率、特殊体重（长）增长率均显著高于低盐度组对虾，而中、高盐度组对虾的各生长指标无显著差异；（2）饵料蛋白质含量对凡纳滨对虾的各生长指标和体粗蛋白含量影响显著，对其它各指标影响不显著。各盐度下，对虾生长和体粗蛋白含量均随饵料蛋白质含量升高而升高，投喂CP20的对虾组显著低于其它各处理组；肥满度和肝体指数均先随饵料蛋白质含量升高至40．43％而升高，然后稍有下降；饵料蛋白质含量对各盐度下对虾成活率影响均不显著。（3）双因素方差分析结果显示，盐度和饵料蛋白质含量，除对体灰分含量存着着显著的交互作用外，对其它体生化成份含量、生长及体形态指标的交互作用均不显著。（4）饲料蛋白质含量明显影响了凡纳滨对虾肝胰腺的组织结构，投喂CP30和CP40饵料的对虾肝小体基膜完整，投喂CP40对虾的肝小体中还出现了大量的存储细胞（R细胞）；而投喂CP20饵料的对虾肝小体分布松散，R细胞数量较小，并且部分肝小体基膜破损；而投喂CP50饵料的对虾的肝小体排列紧密，且B细胞内出现大量内容物质。结果提示，提高饵料蛋白质含量虽然在一定程度上加快对虾的生长速度和增加肥满度，但是并不能提高低盐底下凡纳滨对虾的成活率。饲料中不适宜的蛋白质含量，尤其是含量过低，会导致对虾肝胰腺的结构发生变化，甚至发生不同程度的病理变化。5．盐度和饵料动植物蛋白比对凡纳滨对虾生长、成活和肝胰腺可溶性蛋白质含量的交互作用试验以鱼粉和大豆浓缩蛋白为蛋白源，配制了6种不同饵料动植物蛋白比的饵料，研究了饵料不同动植物蛋白比对凡纳滨对虾生长、成活和肝胰腺可溶性蛋白质含量的影响，试验为期40天。结果显示：1）饵料动植物蛋白比可显著影响凡纳滨对虾增重率、成活率、肝体指数、肥满度和肝胰腺可溶性蛋白质含量。增重率随饵料动植物蛋白升高而升高，但当饵料中动物性蛋白比至29／8时，增重速率不再明显升高，其它指标均先随饵料动植物蛋白升高至一定程度后，稍有下降；2)220‰盐度组对虾的增重率、成活率和肥满度显著高于3‰盐度组对虾，而肝体指数却显著低于3‰盐度组，且盐度对凡纳滨对虾肝胰腺可溶性蛋白含量的影响不显著；3)双因素方差分析结果显示，盐度和饵料动植物蛋白比对凡纳滨对虾增重率、成活率和肝体指数存在交互作用；4)Broken-Line模型分析结果显示，3‰盐度下凡纳滨对虾最适饵料蛋白比为分别29．12／7．79-30．29／6．71，盐度为22‰时为26．05／10．95-29．03／7．97。结果提示，由于不同蛋白源的氨基酸组成和含量不同，配饵中适当的动植物蛋白可以满足凡纳滨对各种氨基酸的适宜需求，且不同盐度下凡纳滨对虾对饵料中动植物蛋白比要求不同。因此，在养殖过程中，一定要结合实际的养殖环境和饵料蛋白源种类，来确定适合自身的饵料配方，才能达到低本高效的养殖目的。6．低盐度下凡纳滨对虾对饵料维生素B₆的营养需求研究了凡纳滨对虾对饵料中维生素B₆的最适需求含量。试验根据凡纳滨对虾的营养需求，以不含维生素的酪蛋白比为蛋白源配制成试验用基础饵料，在基础饵料中分别以0、35、70、105、140和200mg／kg饵料的比例添加维生素B₆配制成6种不同维生素B₆含量的试验用饵料，饵料中维生素B₆含量的实际测量值分别为2．17、32．43、65．79、96．97、137．13和189．56mg／kg饵料。用试验用饵料投喂体重为0．014±0．005g凡纳滨对虾幼虾，每一饵料组设3个平行，试验为期30天。结果表明，饵料中维生素B6含量可显著影响凡纳滨对虾的增重率、特殊体重和体长增长率、成活率和肥满度，而对于对虾的肝体指数却无显著影响。对虾增重率、特殊体重和体长增长率及肥满度均随饵料中维生素B₆含量升高而升高，但当饵料中维生素B₆含量升高到137．13mg／kg饵料后，各指标虽继续随饵料中维生B₆含量升高而提高，但各组之间无显著性差异。对虾成活率总体上是先随饵料中维生素B₆含量升高而升高，饵料中维生素B₆含量为137．13mg／kg时，成活率达到最大值，而当饵料中维生素B₆含量升高至189．56mg／kg饵料时，对虾成活率却稍有下降，但137．13和189．56rag／kg饵料两试验组的成活无显著差异。饵料中维生素B₆含量对凡纳滨对虾肌肉中谷草转氨酶和谷丙转氨酶活力影响显著，两转氨酶活力变化呈现相同的变化趋势，即先随饵料中维生素B₆含量升高而缓慢升高，而当饵料中维生素B₆含量从65．79mg／kg升高至96．97mg／kg时，对虾肌肉中转氨酶活力急速升高至最大值。之后，凡纳滨对虾肌肉中转氨酶活力却随着饵料中维生素B₆含量升高呈稍下降的趋势，但于96．97、137．13和189．56mg／kg三试验组之间无显著性差异，且均显著高于另外三试验组。采用Broken-line模型对凡纳滨对虾饵料中最适维生素B₆需求的分析结果范围为106．96-151．92 mg／kg饵料，平均值±标准差为129．94±18．86mg／kg饵料。7．低盐度下饵料中蛋白质和维生素B₆对凡纳滨对虾生长、成活和转氨酶活力交互作用研究了低盐度3．0‰下饵料中维生素B₆和蛋白质对凡纳滨对虾生长成活、体形态学参数及肌肉中转氨酶活力的交互作用。根据凡纳滨对虾的营养需求，以不含维生素的酪蛋白为蛋白源配制成蛋白质含量分别为25％和40％的两种等能试验用基础饵料，在基础饵料中分别以0和200mg／kg饵料的比例添加维生素B₆配制成4种不同试验用饵料。用试验用饵料投喂体重为0．014±0．005g凡纳滨对虾幼虾，每一饵料组设3个平行，试验为期30天。结果表明，饵料中添加维生素B₆可以显著提高凡纳滨对虾的增重率、成活率、肥满度及谷草和谷丙两转氨酶活力，但该饵料组对虾的肝体指数却显著低于不添加维生素B₆饵料组对虾；饵料蛋白质对凡纳滨对虾增重率、体形态学参数和转氨酶活力均不显著影响，但40％饵料组对虾成活率显著高于25％组对虾组；双因素方差分析结果显示饵料中维生素Be和蛋白质对低盐度下凡纳滨对虾各测定指标均无显著交互作用。结果提示，维生素B₆和蛋白质对低盐度下凡纳滨对虾具有不同的营养作用，配制饵料时应同时满足凡纳滨对虾对两者的营养需求量，只通过满足或者提高两者之一而节约另外一种营养素的方式，来获得凡纳滨对虾的最佳生长和成活率是不可行的。8．凡纳滨对虾谷氨酶脱氢基因的定性研究谷氨酸脱氢酶在甲壳动物游离氨基酸中，尤其是在脯氨酸和丙氨酸的合成代谢中扮演着十分重要的角色。由于脯氨酸和丙氨酸是甲壳动物常见的起渗透调节作用的游离氨基酸，因此谷氨酸脱氢酶在甲壳动物渗透调节中的作用应受到重视。本研究对凡纳滨对虾谷氨酸脱氢酶基因进行了定性的研究，结果发现凡纳滨对虾中存在两种谷氨酸脱氢酶基因，其编码的氨基酸的长度分别为474和552个氨基酸，两氨基酸序列中前461个氨基酸完全相同，从第462个氨基酸两者不同。与其它物种谷氨酸脱氢酶编码的氨基酸序列进行比较发现，凡纳滨对虾谷氨酸脱氢酶是一种相对保守的蛋白质，两种凡纳滨对虾谷氨酸脱氢酶氨基酸序列均与果蝇表现出较大的同源性。以不同物种cDNA序列建立的系统树，也可以得到较理想的系统进化物，在所有的选择的物种中，凡纳滨对虾与果蝇表现出较近的亲缘关系。通过比较凡纳滨对虾谷氨酸脱氢酶cDNA序列和基因全序列比较，发现在凡纳滨对虾两谷氨酸脱氢酶基因中均存在三个内含子，长别分别为202、333和256bp，且相位判断结果发现三个内含子均为0相位内含子。而与其它物种相比较，只有长度为256bp的第三个内含子在各物中均存在，提示内含子在研究系统进化中可能具有重要的作用。本文研究结果为以后凡纳滨对虾该基因的进一步定性和定量研究提供了基础资料。9．谷氨酸脱氢酶和Na⁺-K⁺ ATPase酶基因在凡纳滨对虾不同组织中的特异表达试验设计了用于定量研究凡纳滨对虾渗透调节两种主要相关酶，即谷氨酸脱氢酶和Na⁺-K⁺ ATPase酶的引物和实时定量PCR的反应程序，研究了两种基因在凡纳滨对虾不同组织中的特异性表达。结果表明：1)采用本试验设计的所有引物和反应程度均可成功的扩增出目的基因片段，且Na⁺-K⁺ ATPase酶和谷氨酸脱氢酶基因的表达均表现出组织特异性：2)Na⁺-K⁺ ATPase酶基因在凡纳滨对虾鳃组织中表达量显著高于其它4个组织的表达量，而该基因在对虾肌肉和上皮组织中的表达量次之，但显著高于肝胰腺和眼柄两组织中的表达量，该酶基因在肝胰腺和眼柄两组织中的表达量最低；3)谷氨酸脱氢酶基因A和基因B在凡纳滨对虾不同组织中的表达量稍有不同，但两者均在对虾肌肉组织中的表达量最高，且在肝胰腺组织中的表达量均最低，在其它3组织的表达量介于肌肉和肝胰腺两者之间；4)谷氨酸脱氢酶基因B与基因A两者表达量比值在凡纳滨对虾各组织之间虽然不显著性差异，但基因B的表达量却显著高于基因A在各组织的表达量，比值介于在眼柄中的32．04和肌肉中的64．52之间。结果提示，研究凡纳滨对虾渗透调节主要相关酶谷氨酸脱氢酶和Na⁺-k⁺ ATPase酶基因表达时，对虾肌肉和鳃是比较理想的研究靶点，肝胰腺与其它各组织相比则不适合作为研究该基因表达的靶组织。10．谷氨酸脱氢酶、Na⁺-K⁺ ATPase和淀粉酶基因在凡纳滨对虾不同发育阶段中的定量表达采用实时定量PCR技术研究了谷氨酸脱氢酶、Na⁺-K⁺ ATPase和淀粉酶基因在凡纳滨对虾不同发育阶段中的定量表达情况。结果发现，凡纳滨对虾两谷氨酸脱氢酶基因表达量均随着对虾的发育而呈现缓慢升高的趋势，但直到凡纳滨对虾发育到后期幼体后，两谷氨酸脱氢酶基因表达量才表现出显著的升高趋势。谷氨酸脱氢酶B与A基因表达量的比值在对虾卵中最高，但至无节幼体各阶段，两基因表达量的比值一直呈降低的趋势，而待对虾发育至潘状幼体时，比值开始稍有升高到一定水平，并在糠虾中保持较稳定的状态，但待对虾发育至后期幼体2时，比值稍有下降，而后又呈稍上升。Na⁺-K⁺ ATPase基因表达随着凡纳滨对虾的发育，一直保持在一个稳定的水平，直到对虾发育到后期幼体16，其表达量才急速升高。淀粉酶基因在凡纳滨对虾发育过程中的卵和无节幼体的表达量非常低，且虽然在溞状幼体中表达量稍有升高，但是表达量却依然保持在很低的水平，直到对虾发育到糠虾幼体，淀粉酶基因表达量才开始骤然升高。由于谷氨酸脱氢酶和Na⁺-K⁺ATPase均在凡纳滨对虾渗透调节过程中起着重要的作用，但两者基因表达量均在对虾发育阶段后期幼体的后期才有大幅度地升高，说明凡纳滨对虾只有发育到后期幼体后的某一个阶段，才开始具有较强的渗透调节能力。所以，在这一阶段之前的幼体培养过程中应该保持对虾幼体最适的盐度环境。而淀粉酶基因表达的变化规律说明凡纳滨对虾只有发育到糠虾阶段时，对外源食物的消化吸收能力才开始加强。因为结合两方面的试验结果，可以初步认为，如果想要通过营养强化的方式来提高对虾幼体的渗透调节能力，以期提高对虾在养成过程中的成活率，要待对虾发育至糠虾幼体阶段再开始进行营养调节为宜。更多还原

【Abstract】 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.更多还原