【作者】 付晚涛；

【导师】 张卫；

【作者基本信息】 中国科学院研究生院（大连化学物理研究所） ， 生物化工， 2006， 博士

【摘要】 养殖生物的病害和养殖水域的环境污染是制约海水养殖业健康发展的关键因素。海绵是海洋中最低等的滤食性动物,具有独特的泵水、滤食能力和免疫防御机制。本文研究中国繁茂膜海绵（Hymeniacidon perleve）滤食病原细菌、清除有机质废物和间接降低水体中无机N、P盐浓度的规律,以及海绵监测海水中病原细菌的能力,分析了海绵生物修复和生物监测近海养殖水体的可行性。为了海绵监测养殖水体中病原细菌,获得了繁茂膜海绵的18sRNA和3个14-3-3 cDNA部分序列。使用Real-Time PCR技术评价病原细菌诱导海绵14-3-3 mRNA表达量的变化。本文首次证明了海绵具有区分海水中感染型细菌和非感染型细菌的能力。海绵滤食大肠杆菌和鳗弧菌Ⅱ的实验表明:海水中大肠杆菌浓度迅速降低,鳗弧菌Ⅱ的生长受到一定程度抑制。用海绵过滤分别被荧光染料DiI染色的大肠杆菌和鳗弧菌Ⅱ,在激光共聚焦显微镜下观察发现海绵原细胞内有被染色的细菌及细菌碎片,表明海绵阻留并消化这两种细菌。对于含有多种微生物的天然海水,海绵展示了滤食总细菌和大肠杆菌、弧菌的能力。海绵清除有机质的实验证明,海绵能有效清除500 mg/L浓度以下的有机质,其饱和清除能力为25.50 mg TOC /g-新鲜海绵。首次发现海绵清除TOC和大肠杆菌的过程符合公式:K’??exp（pt） = ln（A₁– Ct）– ln（Ct– A₂）。设计并验证了10 L“微藻-海绵”集成系统清除海水中50%无机N盐和90%无机P盐。综上所述,繁茂膜海绵是修复和监测近海养殖水体的较为理想生物。更多还原

【Abstract】 Diseases of aquaculture organisms and negative environmental impacts of pollutants in aquacultural water areas are two key factors that restrain the increase in marine aquaculture production. As the oldest metazoan, sponges (Porifera) possess extreme abilities of pumping water to extract food and defense/immune mechanisms against a large number of pathogens in seawater. Using an inter-tidal marine sponge Hymeniacidon perleve as a model system, this thesis aims to (1) investigate the characteristics of sponges as bioremediators to remove pathogenic bacteria, organic matters, and inorganic nutrients (N and P); (2) clone H. perleve14-3-3 mRNA as biomarker to biomonitor pathogenic bacteria in seawater. In the laboratory we demonstrate that the feasibility of using sponges as bio-remediator and bio-monitoring specie in integrated aquaculture ecosystem.To examine a biomarker from marine sponge H. perleve in response to pathogenic bacteria in seawater, three partial 14-3-3 cDNA and partial 18s RNA of H. perleve were cloned and sequenced. The expression of 14-3-3 mRNA when exposed to non-infectious bacteria E. coli was not significantly different from that of control. However, the expression of 14-3-3 mRNA when exposed to infectious bacteria V. anguillarum II and V. alginolyticus was down-regulated respectively. The results demonstrated for the first time that sponges could differentiate the infectious bacteria from non-infectious bacteria using the 14-3-3 mRNA transcript level as a biomarker.The tests of H. perleve retaining and digesting pathogenic bacteria showed that H. perleve removed 96% of E.coli with initial density 7.0-8.3×106 cells/mL during 10.5 h, and kept the pathogens growth under control at initial density 6.3×105 cells/mL of 200 mL SNSW. E. coli and V. anguilarumⅡstained by fluorescence dye DiI were respectively fed to sponge. The laser confocal microscopy observation confirmed that the sponges filtering-retained and digested these bacteria by phagocytosis. For natural seawater (NSW) with mixture of different kinds of pathogenic bacteria, H. perleve exhibited the ability of retaining and digesting E. coli, Vibrio and Total bacteria.The tests of removing TOC in SNSW demonstrated that H. perleve could effectively removed DOMD at concentrations lower than 500 mg/L, and that the maximum removal capacity of TOC was 25.50 mg/g-fresh sponge. We firstly found that the process of sponge retaining TOC and E. coli fitted finely with the following equation: K’?exp (pt) = ln(A1– Ct)– ln(Ct– A2).We design and demonstrated that an integrated system of microalgae and sponge H. perleve can remove 50% inorganic nitrogen and 90% inorganic phosphate in seawater within 48 h.Marine sponge H. perleve is suitable organism to bioremediate and biomonitor coastal aquaculture ecosystem.更多还原