【作者】 梁俊平；

【导师】 赵法箴； 李健；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2013， 博士

【摘要】 本研究首次解决了脊尾白虾全人工繁育技术，提出了人工控制亲虾同步性成熟的方法，以及受精卵人工孵化及幼体选优的方法，明确了幼体孵化、培育的适宜温度和盐度范围。同时借助分子生物学手段，克隆了脊尾白虾卵黄蛋白原(Vg)、卵黄蛋白原受体(VgR)和蜕皮激素受体(EcR)基因，并分析了Vg、VgR和EcR基因在脊尾白虾繁殖过程中的表达特征。主要结果如下：一、脊尾白虾亲虾人工培育1、脊尾白虾卵巢解剖结构与组织学特征取卵巢发育不同阶段的脊尾白虾雌虾卵巢，研究了卵巢结构、组织学特征以及卵黄蛋白积累。结果显示，脊尾白虾卵巢一对，左右对称，中间分离，前后端愈合，成熟的卵巢外形呈“⊥”型或“橄榄球”型。随着卵巢发育，性腺指数逐渐增大，卵巢成熟时(IV期)最大，排卵后(V期)又降到最小；组织学特征显示，在卵细胞增殖期，细胞核清晰可见，一般能观察到1-2个沿核膜内侧分布的核仁，同时随着卵细胞逐渐成熟，卵原细胞周围的滤泡细胞逐渐增多，卵原细胞的卵黄蛋白逐渐增多。卵细胞成熟时，并未观察到类似对虾的皮质棒，明显的标志是细胞核消失，滤泡细胞数量减少。排卵后的恢复期，细胞核重新出现，卵原细胞又开始增殖，卵黄蛋白重新开始积累。2、脊尾白虾亲虾人工培育及其同步性成熟调控方法的建立提出了一种全人工培育脊尾白虾亲虾的方法，在24°C、26°C温度下，分别经过120天和110天的培育，80%雌虾性腺可达到成熟。此方法可切断其病原传播途径，在人工控制的养殖环境下实现脊尾白虾性腺同步发育成熟，为培育出健康优质脊尾白虾苗种打下良好的基础。二、脊尾白虾受精卵人工孵化1、不同温度和盐度对脊尾白虾胚胎发育速度的影响选用实验室内人工控制交尾的脊尾白虾，研究了不同温度和盐度对脊尾白虾胚胎发育的影响。结果显示，5-30盐度范围内，盐度20时的孵化时间最短，但与5、10、15、25、30盐度组无显著差异(P>0.05)。脊尾白虾胚胎发育的生物学零度为12.18°C，有效积温为3828.27°C h。在15-28°C范围内，温度对脊尾白虾胚胎发育影响显著，胚胎发育时间随着温度升高而呈双曲线性缩短，而胚胎发育速度随着温度的升高而呈直线性加快，但当温度超过30°C时，胚胎无法正常完成发育。因此在脊尾白虾育苗中，幼体孵化温度不应低于12°C，最高不超过28°C，盐度控制在5-30范围内即可。2、脊尾白虾受精卵人工孵化及幼体选优方法的建立提出了一种脊尾白虾人工室内孵化及幼体选优的方法。主要包括以下特点：通过调节水温，可调控受精卵发育速度；可以将活力强幼体与活力差幼体、死亡幼体很好地分离，幼体变态为仔虾的存活率在85%以上；每尾抱卵虾单独培育，幼体孵化后亲缘关系明确，可用于家系建立及选择育种。三、脊尾白虾幼体人工培育1、不同温度对脊尾白虾幼体变态存活的影响选用实验室内人工控制交尾的脊尾白虾，研究了不同温度对脊尾白幼体变态、存活的影响。结果显示，在盐度为31的条件下，脊尾白虾幼体变态发育速度随着温度的升高而加快，18°C、20°C、22°C、24°C、26°C和28°C各实验组开始出现仔虾的时间依次为17、14、11、9、8和8d，各组90%以上幼体变态为仔虾的时间依次为21、18、15、14、11和11d。各实验组在幼体变态过程中存活率都呈明显的阶梯式下降趋势，且28°C组的存活率下降最快，但当存活幼体全部变为仔虾时，各实验组间的存活率并无显著性差异(P>0.05)。18°C组仔虾干质量明显高于其它各组(P<0.05)，28°C组仔虾干质量最低，但与20°C、22°C、24°C和26°C组无显著性差异(P>0.05)。因此在脊尾白虾育苗中，幼体培育温度，建议控制在22-26°C为最佳。2、不同盐度对不同日龄脊尾白虾生长发育的影响选用室内人工培养的卵巢发育到II期的脊尾白虾雌虾以及性成熟雄虾，经逐级淡化，盐度稳定在5、10、15、20、25、30。雌抱卵孵化后，研究了不同盐度对溞状幼体变态、生长及存活的影响，以及不同盐度对仔虾后生长、存活的影响。结果显示，不同盐度对脊尾白虾溞状幼体的变态率和存活率无显著性影响，但对仔虾的干质量影响显著，15和20盐度下仔虾的干质量显著高于其它盐度组(P<0.05)；不同盐度对20日龄脊尾白虾的生长影响显著(P<0.05)，其特定生长率随着盐度升高而逐渐增大，在盐度20时达到最大(P<0.05)，当盐度超过20时其特定生长率又开始降低；同时由60日龄脊尾白虾鳃Na+-K+-ATPase的相对表达量可以看出，在高盐或低盐时Na+-K+-ATPase的相对表达量均较高，在盐度20时鳃Na+-K+-ATPase的相对表达量最低(P<0.05)，经二次方程拟合计算，理论Na+-K+-ATPase的相对表达量最低时的盐度为17.53，此盐度可能为脊尾白虾成体的等渗点。因此，在脊尾白虾人工育苗及养殖过程中，建议适宜盐度控制在15-20。3、不同日龄脊尾白虾对氨氮的耐受性在水温24°C、盐度31、pH8.1的条件下，研究了氨氮对30日龄和120日龄脊尾白虾的毒性。结果表明，氨氮对30日龄脊尾白虾24、48、72、96h的半致死质量浓度分别为155.81、116.71、92.55、80.40mg/L，氨氮对120日龄脊尾白虾24、48、72、96h的半致死质量浓度分别为178.80、156.37、140.28、120.86mg/L。30日龄脊尾白虾总氨氮和非离子氨的安全质量浓度为8.04、0.26mg/L，120日龄脊尾白虾总氨氮和非离子氨的安全质量浓度为12.09、0.50mg/L。因此，在脊尾白虾养殖过程中，水体中的非离子氨浓度不应超过0.26mg/L。四、脊尾白虾卵黄蛋白原基因的克隆及其在卵巢发育过程中的表达分析首次克隆得到了脊尾白虾卵黄蛋白原基因(Vg)cDNA，全长7841bp，开放阅读框7632bp，编码2543个氨基酸。脊尾白虾Vg氨基酸与罗氏沼虾(Macrobrachium rosenbergii)一致性最高为75%，与高背长额虾(Pandalus hypsinotus)一致性为62%，与日本仿长额虾(Pandalopsis japonica)为61%，而与对虾科的对虾一致性却相对较低，为33-38%。该蛋白存在7个N-糖基化位点(N151，N159，N168，N614，N660，N936和N2306)，而在枝鳃亚目中几乎没有糖基化位点。荧光定量分析显示，脊尾白虾Vg主要在肝胰腺内表达，其次为卵巢，肌肉和鳃有微量的表达。卵巢发育期间，卵巢中Vg mRNA的相对表达量却是一直处于上升趋势，在第V期时达到最大；而肝胰腺中Vg mRNA的相对表达量在IV期时却最高，在V期的表达量下降至与I、II期水平。但在卵巢整个发育阶段，肝胰腺中Vg mRNA的相对表达量始终显著高于卵巢(P>0.05)，因此推测，在脊尾白虾卵巢整个发育过程中，Vg主要在肝胰腺内合成，只是在恢复期时卵巢中的Vg合成明显起到了协助作用，为脊尾白虾卵巢再次成熟奠定了基础。五、脊尾白虾卵黄蛋白原受体基因的克隆及其在卵巢发育过程中表达模式成功获得了脊尾白虾卵黄蛋白原受体基因(VgR)cDNA全长，开放阅读框5661bp，编码1886个氨基酸。亲缘关系分析，脊尾白虾VgR与虾类的亲缘关系最近，其次为昆虫，而与鱼类等脊椎动物关系较远。脊尾白虾VgR含4种结构域，LDL-receptor classAdomain，Low-density lipoprotein-receptor YWTD domain，EGF domain和Transmembraneregion，是一种跨膜转运蛋白。通过荧光定量PCR对该基因在各组织及卵巢发育期肝胰腺和卵巢内的表达分析显示，脊尾白虾VgR在肝胰腺、卵巢、鳃和肌肉中均有表达，但主要在卵巢内表达，其次为肝胰腺。在卵巢发育过程中，卵巢内的VgR的表达量呈现先升高后降低又升高趋势，在III期的表达量显著高于I、II期(P<0.05)，IV期的表达量最低(P<0.05)，而在V期时表达量达到最大(P<0.05)；随着卵巢的发育，肝胰腺内VgR的表达量在IV时显著高于其它时期的表达量(P<0.05)，I和V期的表达量相近(P>0.05)，II和III的表达量最低。在卵巢整个发育过程中，卵巢内VgR的表达量始终显著高于肝胰腺内的表达量(P<0.05)。结果表明，脊尾白虾VgR对Vg的转运可能存在一个平衡，即当卵巢成熟时由肝胰腺分泌到血液中的部分Vg被肝胰腺VgR又重新转移至肝胰腺内，而为下次卵巢发育准备，在排卵后的恢复期肝胰腺中VgR表达量的降低和卵巢中VgR表达量的升高也说明，VgR对Vg的转运在不同阶段是有方向性的。六、脊尾白虾蜕皮激素受体基因的克隆及其在卵巢和胚胎发育中表达分析首次克隆得到了脊尾白虾蜕皮激素受体(ECR)基因cDNA，全长2638bp，开放阅读框1713bp，编码570个氨基酸。系统进化分析，与已知的甲壳动物ECR亲缘关系最近，而与昆虫亲缘关系较远。荧光定量分析显示，脊尾白虾ECR除在血细胞中不表达外，在其它各组织器官中均有分布，主要在受精卵和肝胰腺中表达，而在肌肉、卵巢、大额腺和鳃等组织中的表达却相对较低。卵巢发育不同阶段，肝胰腺中ECR和HSP90的表达量变化与Vg的表达量呈正相关性，推测在肝胰腺中ECR与HSP90主要参与了Vg的合成；卵巢中ECR与性腺成熟相关，ECR的表达量在III期达到最高(P<0.05)，而III期是生殖蜕皮前蜕皮激素含量高峰期。脊尾白虾胚胎发育过程中，ECR的表达量随着胚胎发育呈逐渐上升趋势，在Zoea I达到最大(P<0.05)；HSP90的表达量随着胚胎发育逐渐升高，在前溞状幼体期(Protozoea)达到最大，而后稍有下降，但仍维持在较高水平；Vg在受精卵和十六细胞期均无表达，囊胚期和无节幼体有微量表达，前溞状幼体表达量显著升高，后溞状幼体表达量达到最大，但与溞状幼体I期表达量无显著性差异(P>0.05)。更多还原

【Abstract】 1. The artifical culturing of Exopalaemon carinicauda parents(1) Morphological and histological observation on ovary development of ExopalaemoncarinicaudaThe anatomy and histology of reproductive system of female Exopalaemon carinicaudawere observed. The results showed that the reproductive system of female Exopalaemoncarinicauda was “⊥” or “Rugby” shaped and contained an oviduct. The ovary was composedof the left and right parts connected at front and end. The ovary development could be dividedinto five stages: inactive stage (I stage), minor growth stage (stage II), major growth stage(stage III), mature stage (stage IV), and spawn stage (stage V). The gonadosomatic index(GSI) increased with the ovary development, and the value of GSI was the higest at matureatage, and lowest at the spawn stage. One or two nucleolus were observed clearly and thenumber of follicle cells increased from stage I to stage III, however, the nucleus were notobserved at mature stage, the follicle cells and oocytes were fused in late ovarian stage. Theconcentration of vitellin was the highest at mature stage. Cortical rods were not observedfrom stage I to stage V.(2) The artifical culturing of Exopalaemon carinicauda parentsA technique of artifical culturing of Exopalaemon carinicauda parents was introduced inthe part, which can cutted off the spread of pathogen. Exopalaemon carinicauda can maturesynchronously under the control of manual.2. The eggs incubation of Exopalaemon carinicauda(1) Effects of temperature and salinity on the embryonic development of ExopalaemoncarinicaudaThe purpose of the present study was to determine the effects of temperature on the eggincubation period of E. carinicauda larvae reared in the laboratory. The number of days from spawning to hatching and mean rearing temperature were determined from25females. Theegg incubation period decreased exponentially from41to10days with increasing meantemperatures of15.3to28.1C, but at30C, embryonic development could not be completed.The relationship between mean temperature (T) and egg incubation period was analyzed usingthe equation T KV C, based on heat summation theory. The lower critical temperature forembryonic development represented by parameter C was12.18C, and the sum of effectivetemperature for embryonic development was3828.27C h. To conclude, water temperaturesbetween12.18C and28C were most suitable for embryonic development. There was nosignificant effect of salinity on the hatching of E. carinicauda.(2) The eggs incubation and selection of larvae of Exopalaemon carinicaudaThis study proposes a method for optimization of eggs incubation and larval selcetion ofExopalaemon carinicauda. It included the regulation of embryonic development, the bestlarval selection. And each female shrimp was reared in a container alone, so the relationshipwas very clear among different famliys.3. The rearing of Exopalaemon carinicauda larvae(1) Effects of water temperature on the survival and development period of larvae ofExopalaemon carinicaudaThe purpose of the present study was to determine the effects of temperature on thesurvival and development period of Exopalaemon carinicauda larvae reared in the laboratory.The larvae from each female were reared in1.5L beakers at six temperatures (18,20,22,24,26and28C) and fed Artemia sp. nauplii. The number of days from hatching to theattainment of each larval stage decreased with increasing temperature, the period over which90%of larvae from the T18—T28groups developed into juvenile shrimps was21,18,15,14,11and11days, respectively. The survival rates of larvae decreased with development time. Inparticular, the survival rate of the T28group decreased sharply over time. The survival rates,however, from incubation to juvenile shrimp showed no significant differences (P>0.05)between the six groups. The individual dry weight of post-larva from the T18group was thehighest of all of the six groups (P<0.05), and the individual dry weight of post-larva from theT28group was the lowest, but there was no significant difference (P>0.05) between the five groups from the T20group to the T28group. To conclude, water temperatures from22°C to26°C were most suitable for rearing E. carinicauda larvae.(2) Effects of salinity on the metamrphosis rate, survival rate and specific growth rate oflarvae and post-larvae of Exopalaemon carinicaudaThe female and male E. carinicauda were selcted from the laboratory, the ovary was atstage II of femal E. carinicauda, and then reared at different salinity water until hatching, soas to anayze the effect of salinity on metamrphosis rate, survival rate and specific growth rateof larvae and post-larvae. The results showed that there were no significant effects of salinityon metamrphosis rate and survival rate of larvae (P>0.05), but the individual dry weightsignificantly affected by salinity (P<0.05). The body length and body wet weight increasedwith salintity from5to20ppt, and the salinity of20was the optimum salinity to20-days ageE. carinicauda. When the salinity is beyond20, the growth was decreased.The expression level of Na+-K+-ATPase mRNA was investigated in gill of60-days age E.carinicauda. The result showed that the Na+-K+-ATPase mRNA level was lowest at20ofsalinity. According to correlation equations and calculation results, the optimum salinity was17.53. It indicated that the salinity17.53ppt might be equal to the hemolymph osmoticpressure of E. carinicauda.The above results suggested that the optimum salinity was from15to20ppt to larva andjuvenile growth of E. carinicauda.(3) Acute toxicity of ammonia nitrogen to different ages Exopalaemon carinicaudaAcute toxicity test of ammonia nitrogen to E. carinicauda was made by using themethod of usual biological toxicity test under the condition of seawater T=24, S=31, pH8.1.The result showed that the24,48,73,96h median lethal of total ammonia nitrogen were155.81,116.71,92.55,80.40mg/L to juvenile of E. carinicauda, respectively, and178.80,156.37,140.28,120.86mg/L to adult of E. carinicauda, respectively. The “safety level” forrearing E. carinicauda juveniles was estimated to be8.04mg/L for total ammonia nitrogen(0.26mg/L for nonionic ammonia nitrogen), and for E. carinicauda adults to be12.09mg/Lfor total ammonia nitrogen (0.50mg/L for nonionic ammonia nitrogen).4. Molecular characterization and expression analysis of vitellogenin (Vg) in Exopalaemon carinicaudaThe technique of homplgy cloning and anchored PCR were used to clone the Vg genefrom Exopalaemon carinicauda. The full length cDNA of Exopalaemon carinicauda Vg was7841bp, which contained an ORF (open reading frame) of7632bp encoding a polypeptide of2543amino acids with an estimated molecular mass of287763.7Da. The sequence of thecoding region shared33-75%identity with other known crustacean Vgs. Bioinformaticsanalysis revealed seven putative N-glycosylation sites in E. carinicauda Vg, the glycosylationof seven putative sites of E. carinicauda Vg (N151, N159, N168, N614, N660, N936and N2306) wasconfirmed. However, the Dendrobranchiata exhibited a remarkable deficiency in putativeN-glycosylation sites. E. carinicauda belonged to Pleocyemata, which were egg holdingspecies, while the Dendrobranchiata were releasing species. It seemed that there was a strongselective force against the occurrence of N-glycosylation sites in the Dendrobranchiatasuborder.The expression of the E. carinicauda Vg was observed in the most of the examinedtissues, but the expression level varied significantly among the tissues. There was a high levelin hepatopancreas, lower in ovary, while lowest in muscle and gill. In hepatopancreas, therelative level of Vg mRNA increased with the ovarian development, the highest level wasobserved at mature stage (GSI7.15%), but decreased during later stage maturation. In ovary,the relative level of Vg expression increased with the ovarian development, the highest levelwea observed at spawn stage (GSI0.67%). It indicated that the hepatopancreas was the majorsite of Vg synthesis, and the ovary might play a minor role in Vg synthesis of E. carinicauda.It might be an important reason that E. carinicauda can spawn many times in its life.5. Molecular cloning and expression analysis of vitellogenin receptor (VgR)in Exopalaemon carinicaudaA full-length cDNA encoding vitellogenin receptor (VgR) was cloned from E.carinicauda using RACE method. The full-length cDNA consist of5892bp nucleotidesincluding5661bp open reading frame, which encodes1886amino acid residues. Aphylogenetic tree was constructed by the programs of CLUSTAL and MEGA. Thecrustaceans, insect, and vertebrate were clustered together and formed a group, respectively. In the tree, E. carinicauda showed the closest relationship with Macrobrachium rosenbergii,the result was similar with the result of the BLAST. So the relationships displayed in thephylogenic tree corresponded to their classification position. Analysis of the deduced proteinsequence of VgR with SMART algorithm revealed several conserved domains similar to thelow density lipoprotein receptor family. These domains included LDL-receptor class Adomain, Low-density lipoprotein-receptor YWTD domain, EGF domain and Transmembraneregion.The expression of the E. carinicauda VgR was observed in the most of the examinedtissues by quantitative Real-time PCR, but the expression level varied significantly among thetissues(P<0.05). There was a high level in ovary, lower in hepatopancreas, while lowest inmuscle and gill. With the ovarian development, expression of the VgR in the ovary was low atthe early vitellogenic stage, and significantly higher at major growth stage (stage III),however, it slightly decreased at mature stage (stage IV), and was the highest at spawn stage(stage V)(P<0.05). The highest level of VgR mRNA expression was found in hepatopancreasat mature stage, a significant decrease of VgR mRNA expression occurred at stage I, II, IIIand V. It indicated that the main site of E. carinicauda VgR mRNA expression was the ovary,and mediated the uptake of vitellogenin (Vg) into developing oocytes. However, the excessiveVg from blood was carried into the hepatopancreas at mature stage, as the nutrients ready forovarian development next time.6. ECR cDNA characterization and its expression during ovarian andembryonic development of Exopalaemon carinicaudaEcdysteroid are steroid hormones that play an important role in development, growth,molting of larva, and reproduction in crustacean. The effect of ecdysteroids is mediated by itsbinding to ecdysteroid recptor (ECR). To investigate the role of ECR during development ofovary and embryo of E. carinicauda, we isolated and characterized cDNA of E. carinicaudaECR, and studied mRNA expression pattern. The full-length cDNA seqience was2638bp andthe open reading frame encoded570amino acids. Sequence alignment showed that the E.carinicauda ECR shared high identity with other known crustacean. There was a high level inhepatopancreas, lower in eggs, while lowest in muscle and gill, ovary and mandibular gland, not detected in blood cells.In different development stages of E. carinicauda ovary, the ECR mRNA expression wasagreed with the HSP90mRNA expression in hepatopancreas, the ECR mRNA expression wasnot agreed with the HSP90mRNA expression in ovary, and was much higher at stage III, andkeeping relative highexpression level at later development of ovary. It indicated that ECR inovary played an important role in molting before spawning, while in synthetizing vitellogeninin hepatopancreas.In differdent development stage of E. carinicauda embryo, the the relative level of ECRmRNA increased with the embryonic development, the highest level was observed at Zoea I.The HSP90mRNA increased with the embryonic development, the highest level wasobserved at Protozoea stage, and then maintained high expression levels at later developmentstage. The Vg mRNA was not observed in oosperm and sixteen cells stage, the highest levelwas observed at Metazoea stage. It indicated that ECR and HSP90were involved inembryonic development, while Vg was involved in innate immune response of embryo andlarva of E. carinicauda.更多还原