【作者】 衣启麟；

【导师】 宋林生；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2014， 博士

【摘要】 虾蟹养殖是世界水产养殖业的重要组成部分，但病毒和细菌性疫病的频繁爆发严重影响了该产业的健康发展，发展有效的疫病防控策略迫在眉睫。本研究选取凡纳滨对虾（Litopenaes vannamei）和中华绒螯蟹（Eriocheir sinensis）为实验对象，研究了CpG寡聚核苷酸（CpG ODN）、表面展示VP28的解脂耶罗维亚酵母、嗜酸乳杆菌、长双歧杆菌、地衣次级代谢产物红粉苔酸对虾蟹的免疫保护作用及其作用机制，利用碳纳米管和壳聚糖优化了免疫活性物质的导入途径，探讨了不同免疫增强剂在养殖虾蟹疫病防控中的应用前景。将大规模发酵制备的CpG ODN和表面展示VP28的解脂耶罗维亚酵母（VP28-yl）分别拌饲投喂凡纳滨对虾15天，发现在WSSV侵染后，CpG ODN和VP28-yl投喂组对虾的死亡率和体内病毒拷贝数均显著低于对照组。CpGODN投喂组对虾的细胞凋亡水平呈现先升高后降低的趋势，而VP28-yl投喂组对虾与对照组相比则一直维持较低水平，提示VP28-yl能抑制由WSSV侵染而引起的细胞凋亡。同时CpG ODN投喂组对虾血淋巴细胞的呼吸爆发水平和Dicer、STAT、prophenoloxidase的mRNA表达量显著增加，VP28-yl投喂组对虾LGBP表达量显著增加，以上结果表明CpG ODN和表面展示VP28的耶罗维亚酵母能通过不同的作用机制增强凡纳滨对虾抵御WSSV侵染的能力。田间试验发现投喂CpG ODN和表面展示VP28的解脂耶罗维亚酵母30天后，凡纳滨对虾的相对存活率显著提高，且CpG ODN投喂组对虾的生长速率显著增加，进一步证明CpG ODN和VP28-yl对养殖对虾病毒性疫病的防控具有显著作用，具有在养殖生产中推广使用的前景。发现经嗜酸乳杆菌和长双歧杆菌拌饲投喂21天的中华绒螯蟹在嗜水气单胞菌注射处理后的死亡率较对照投喂组显著降低，其血细胞吞噬能力及酚氧化酶、过氧化氢酶和溶菌酶活力水平显著增强，C型凝集素、prophenoloxidase、peroxinectin和LGBP基因的mRNA表达量显著上升。这表明嗜酸乳杆菌和长双歧杆菌能激活中华绒螯蟹的固有免疫系统，可作为候选的免疫增强剂，应用于养殖虾蟹类细菌性疫病的免疫防控。体外抑菌实验发现光肺衣（Lobaria kurokawae）次级代谢产物红粉苔酸（lecanoric acid）对嗜水气单胞菌具有显著的抑制作用。发现10μg mL-1红粉苔酸注射处理中华绒螯蟹后，其新生血淋巴细胞数量显著增加，血淋巴细胞吞噬活性和酚氧化酶、溶菌酶的活性显著上升，而一氧化氮的含量显著降低，表明红粉苔酸具有较强的抗氧化作用，能增强中华绒螯蟹免疫防御能力，有效抵御嗜水气单胞菌的侵染。利用碳纳米管和壳聚糖等纳米载药载体，对免疫活性物质的导入途径进行了优化。发现碳纳米管-病毒灵在对虾原代血淋巴细胞中发挥了良好的抗病毒作用，与WSSV共孵育后，检测到100μg mL-1碳纳米管-病毒灵处理组血细胞中病毒拷贝数达到(1.20±0.21)×106copies ng-1，显著低于对照组的(1.95±0.16)×106copies ng-1，能在凡纳滨对虾和中华绒螯蟹细胞中实现自由穿膜，导入效率接近于100%；壳聚糖-CpG ODN和壳聚糖-dsRNAie1通过内吞作用被摄入到对虾细胞内并在胞内呈点状分布，且壳聚糖载体具有良好的缓释作用。表明碳纳米管和壳聚糖可辅助将免疫活性物质高效导入虾蟹细胞，不产生明显细胞毒性，因而可作为虾蟹类免疫增强剂的候选导入载体。综上所述，CpG ODN、表面展示VP28的解脂耶罗维亚酵母、嗜酸乳杆菌、长双歧杆菌、红粉苔酸等免疫活性物质能激活虾蟹类固有免疫系统，增强机体抵御WSSV和嗜水气单胞菌侵染的能力，可作为候选免疫增强剂应用于养殖甲壳动物疫病的免疫防控；碳纳米管和壳聚糖等纳米载药载体的应用能实现免疫活性物质在虾蟹细胞中的高效导入。研究结果对于发展虾蟹疫病免疫防控新技术具有重要理论和实践意义。更多还原

【Abstract】 Crustacean farming is an important part in world aquaculture, however, thefrequent outbreaks of viral and bacterial diseases have caused severe economiclosses. It is urgently necessary to launch researches regarding to the exploration ofnew approaches for the immune control of diseases, which is meaningful for thedevelopment of aquaculture industry. In the present study, the CpG oligonucleotides(CpG ODN), surface-displayed VP28Yarrowia lipolytica (VP28-yl), Lactobacillusacidophilus，Bifidobacterium longum and lichen secondary metabolites lecanoricacid were applied and their protective effects for Litopenaes vannamei and Eriocheirsinensis in the defense against white spot syndrome virus (WSSV) or Aeromonashydrophila infection were investigated, and the importing route for immunoreactivesubstances into crustaceans were optimized by using Nano drug delivery vectors likecarbon nanotubes and chitosan, as well as the further applications of CpG ODN andVP28-yl in the shrimp aquaculture industry were evaluated.CpG ODN prepared by large-scale fermentation and VP28-yl were mixed intodiets for the feeding of L. vannamei with a15day oral administration. After WSSVchallenge, the mortalities and the numbers of viral copies in CpG ODN and VP28-ylfeeding shrimps were significantly lower than those in control group. The apoptosislevel in heamocytes of CpG ODN feeding group increased at the beginning and thendecreased, whereas remained relatively low in VP28-yl feeding shrimps, suggestingthat CpG ODN and VP28-yl exerted immune protective roles against WSSVinfection via different mechanisms in shrimp.The mRNA expression of Dicer, STAT,prophenoloxidase in shrimp heamocytes increased in CpG ODN feeding group,while that of LGBP increased in VP28-yl feeding group. It indicated that theantiviral abilities against WSSV enhanced by CpG ODN and VP28-yl were viadifferent menchanisms. Meanwhile, after the feeding tiral for30days in ponds, thesignificantly higher growth parameters were observed in CpG ODN feeding shrimps. And the relative survival rates in CpG ODN and VP28-yl feeding shrimps were alsosignificantly higher than that in control groups after WSSV infection, which furtherconfirmed the application of these two immunoactvie substances in shrimpaquaculture industry had marked effects for the control of viral diseases.A21-day oral administration of L. acidophilus and B.longum was performed incrab E.sinensis. After A. hydrophila infection, in comparition with the control group,the cumulative mortalities in lactic acid feeding crabs were significantly decreased.In addition, cellular and humoral immunity parameters such as phagocytosis, oxygenspecies (ROS) production and the activity of catalase, lysozyme were significantlyenhanced, and the mRNA expression levels of prophenoloxidase，peroxinectin andC-lectin were also remarkably up-regualted, suggested that L. acidophilus andB.longum could active the immune defense system of E.sinensis and exertedprotective effects against A. hydrophila infection. Therefore, they could be used aspromising immunostimulant candidates in the development of therapeutic agents forthe immune control of the bacterial diseases in crustacean aquaculture.In vitro antibacterial experiment showed that lecanoric acid, which was isolatedfrom the acetone extracts of Lobaria kurokawae, could significantly inhibit thegrowth of A. hydrophila. After lecanoric acid was injected into crab E.sinensis, itsprotective effects against A. hydrophila infection were investigated. It showed that10μg mL-1lecanoric acid could not only promote regeneration of circulatingheamocytes and the phagocytosis, enhance the phenoloxidase and lysozyme activity,but also decrease NO production, which suggested lecanoric acid had antioxidantproperties. All of above were contributed to the boosted immunity of E.sinensiscasused by lecanoric acid against the infection of pathogen A. hydrophila.Nano drug delivery vectors like carbon nanotubes and chitosan were used tooptimize the importing route for immunoreactive substances into crustaceans.CNT-ribavirin exerted antiviral effects in primary culture heamocytes，after WSSVinfection，the viral number of WSSV copies in10μg mL-1treated group was (1.20±0.21)×106copies ng-1, which was significantly lower than that in the control group of (1.95±0.16)×106copies ng-1. And CNT-ribavirin could enter intoheamocytes via free diffusion, while the importing efficiency was reach to100%. Inaddition, chitosan-CpG ODN and chitosan-dsRNAie1were internalized directly byshrimp and crab haemocytes, and sustained release effect caused by chitosan wasalso observed. Because carbon nanotubes and chitosan drug carriers wereimmunoreactive substances can be efficiently introduced into cells of crabs, and theyhad no obvious cytotoxicity, they could serve as candidate methods to optimize theimporting route.In conclusion, CpG oligonucleotides (CpG ODN), surface-displayed VP28Y.lipolytica, L.acidophilus and B.longum and lichen secondary metabolites lecanoricacid could activate the innate immunity of crustaceans and played important roles inprotecting the animals against WSSV and A.hydrophlia. They might be possiblepractical supplements applicated in crustacean aquaculture industry. Meanwhile,carbon nanotubes and chitosan nanoparticle drug delivery system could optimize theimporting routes of immune active substance into crustaceans, which provided newstrategies for future industrial application. The findings were helpful for developingnovel immunostimulants in the disease control of aquaculture crustaceans.更多还原