Scleractinian coral response to microbial biofilms, Great Barrier Reef

Glass microscope slides placed in square polyvinyl chloride frames (112 per grid), exposing the top surface to the seawater, were deployed at Davies Reef, on the Great Barrier Reef at two depths - 4 m (shallow) and 10 m (deep) - within each of 2 sites. To allow biofilm development, slides were maintained at site 1 for 2, 4, and 8 weeks, and at site 2 for 8 weeks prior to the November 2000 mass coral spawning. Slides were used in larval metamorphosis assays, with a minimum of 20 replicate biofilm slides randomly selected for each treatment. To determine biofilm community composition, an additional 30 slides were randomly selected for DNA extraction, 30 slides for fluorescence in situ hybridization (FISH), and 10 slides for scanning electron microscopy (SEM).Coral larvae used in the metamorphosis assays were raised from gametes collected from live colonies of the reef-building coral Acropora microphthalma, from a depth of 6 to 8 m on a fringing reef of Pelorus Island, Great Barrier Reef. Slides were sorted according to whether or not they induced larval metamorphosis, and replicate slides were processed for SEM, FISH, and denaturing gradient gel electrophoresis (DGGE). Sequences of oligonucleotide probes used for FISH. DGGE: the 16S rRNA genes were amplified by PCR with bacterial and eukaryotic primers.Estimates were made of the relative densities of specific probe-targeted bacteria and archaea (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes, Cytophaga-Flavobacterium, Bacteroidetes, Planctomycetales, Archaea). The importance of three factors (depth, time, and CCA presence) in inducing larval metamorphosis was investigated in an unbalanced repeated-measures analysis of variance (type IV SS; SPSS version 11).Quantitative estimates of biofilm community composition were made using group-specific FISH probes. All densities are expressed as a percentage of total bacterial numbers obtained using dual hybridization reactions with the bacterium-specific probe EUB338. A principal-component analysis (PCA) was used to summarize biofilm community composition using FISH data in which depth of biofilm formation and exposure time of slides were the variables. Cluster analysis was used to identify replicates that generated similar DGGE profiles. To examine the community structure of developing coral reef biofilms and their ability to induce the metamorphosis of coral larvae.To examine the role microorganisms not associated with calcareous coralline algae in coral metamorphosis.

将顶面暴露于海水中、置于方形聚氯乙烯（polyvinyl chloride）框架内的玻璃载玻片（每栅格112片），部署于大堡礁戴维斯礁（Davies Reef）的2个采样点，每个采样点设置4米（浅水区）与10米（深水区）两个水深梯度。为促进生物膜形成，玻片于站点1分别放置2周、4周及8周，于站点2放置8周，实验开展于2000年11月大规模珊瑚产卵事件之前。玻片用于幼虫变态实验，每个处理组随机选取至少20张重复生物膜玻片开展测试。为测定生物膜群落组成，额外随机选取30张玻片用于DNA提取、30张用于荧光原位杂交（fluorescence in situ hybridization, FISH）、10张用于扫描电子显微镜（scanning electron microscopy, SEM）。本实验所用的珊瑚幼虫，取自大堡礁佩洛鲁斯岛（Pelorus Island）岸礁6至8米水深处的造礁珊瑚微小眼鹿角珊瑚（Acropora microphthalma）活体群落的配子培育而来。根据玻片是否诱导幼虫变态进行分组，对重复玻片分别进行SEM、FISH以及变性梯度凝胶电泳（denaturing gradient gel electrophoresis, DGGE）处理。本研究列出了用于FISH的寡核苷酸探针序列。在DGGE实验中，利用细菌与真核生物引物通过聚合酶链式反应（polymerase chain reaction, PCR）扩增16S rRNA基因。对特定探针靶向的细菌与古菌（包括α-变形菌门、β-变形菌门、γ-变形菌门、放线菌门、厚壁菌门、噬纤维黄杆菌群（拟杆菌门）、浮霉菌门、古菌）的相对密度进行了估算。采用非平衡重复测量方差分析（IV型平方和；SPSS 11.0版本），探究水深、培养时长以及钙化藻壳（crustose coralline algae, CCA）存在与否三个因素对幼虫变态的诱导效应。利用组特异性FISH探针对生物膜群落组成进行定量估算，所有密度均以使用细菌特异性探针EUB338的双重杂交反应所得总细菌数的百分比表示。采用主成分分析（principal-component analysis, PCA），以生物膜形成水深与玻片暴露时长为变量，基于FISH数据总结生物膜群落组成特征。采用聚类分析对DGGE图谱相似的重复样本进行归类。本研究旨在两方面：其一，探究发育中的珊瑚礁生物膜的群落结构及其诱导珊瑚幼虫变态的能力；其二，探究与钙化珊瑚藻无关的微生物在珊瑚幼虫变态过程中的调控作用。