BIOCOMPLEXITY (IDEA): In Situ Measurement of Marine Microbes to Investigate Mechanisms of Community Structure Regulation

该项目由环境中的生物复杂性计划，环境活动仪器开发子计划（BE-IDEA）支持。目的是开发用于自动化就地监测海洋微生物群落的仪器和软件。 微生物几乎占海洋中所有的初级生产力和大部分生物量，微生物群落的结构影响到较高的营养水平，包括人类消费的物种。 要从根本上了解调节群落结构的物理和生物因素之间的复杂相互作用，需要进行比现有设备更详细和更持久的观察。 该项目将开发和部署现场仪器，以监测微观浮游生物的单个细胞。 计划中的方法将结合联合收割机原位流式细胞术和流动成像技术，以测量从最小的浮游植物（小于1微米）到大链硅藻（100微米）的细胞（和其他颗粒）的丰度和特性。将使用自动图像分析系统来确定较大尺寸类别的每个细胞的分类从属关系。 较小的细胞（通常缺乏独特的形态学特征）将通过单个细胞光散射和荧光的脉冲形状分析进行评价。 为了更详细地调查特定群体或物种，将开发一个能够应用群体特异性rRNA探针（通过肽核酸原位杂交）的模块。 将通过原位流式细胞仪对探测样本进行真实的实时分析。同一模块还将用于应用荧光探针以检测其他特性，如DNA含量和细胞活力。 还将开发近实时分析和整合流式细胞仪和图像数据的软件。 该仪器将以WHOI开发的原位流式细胞仪（加上目前正在开发的杂交技术）为基础，并与市售的流动成像系统相结合。完成后，该仪器包将在Marthas Vineyard Coastal Observatory（MVCO）附近进行现场测试。结合物理海洋学、气象学和大量荧光数据，单个粒子数据将有助于理解影响沿海生态系统的过程，包括调节浮游植物水华、物种演替和营养相互作用的机制。计划中的工作将为天然微生物种群的前所未有的长期高分辨率时间序列奠定基础，其成员的大小变化了许多数量级。这类观测对于评估和开发长期生态变化模型至关重要。

This project is supported by the program Biocomplexity in the Environment, subprogram Instrumentation Development for Environmental Activities (BE-IDEA). The objective is to develop instrumentation and software for automated in situ monitoring of marine microbial communities. Microbes account for almost all the primary productivity and a majority of the biomass in the ocean, and the structure of the microbial community affects higher trophic levels, including species consumed by humans. A fundamental understanding of the complex interaction between physical and biological factors that regulate community structure requires more detailed and sustained observations than are possible with current equipment. This project will develop and deploy in situ instrumentation to monitor individual cells of microscopic plankton. The planned approach will combine in situ flow cytometry and imaging-in-flow techniques to measure the abundance and properties of cells (and other particles) ranging from the smallest phytoplankton (less than 1 um) to large chain diatoms (100 um). An automated image analysis system will be used to determine taxonomic affiliation of each cell for the larger size classes. Smaller cells (which often lack distinctive morphological features) will be evaluated by pulse-shape analysis of individual cell light scattering and fluorescence. For more detailed investigation of particular groups or species, a module capable of applying group-specific rRNA probes (via Peptide Nucleic Acid in situ hybridization) will be developed. The probed samples will be analyzed in real time by the in situ flow cytometer. The same module will also be used to apply fluorescent probes for other properties such as DNA content and cell viability. Software to analyze and integrate the flow cytometric and image data in near-real time will also be developed. The instrument will be based on an in situ flow cytometer developed at WHOI (plus hybridization technology currently under development) and integrated with a commercially available imaging-in-flow system. When completed, the instrument package will be field tested near the Marthas Vineyard Coastal Observatory (MVCO).In combination with physical oceanographic, meteorological, and bulk fluorescence data, individual particle data will facilitate an understanding of processes affecting coastal ecosystems, including the mechanisms regulating phytoplankton blooms, species succession, and trophic interactions. The planned work will form the foundation for an unprecedented long term high resolution time series of natural microbial populations, whose members vary in size by many orders of magnitude. These kinds of observations are critical for evaluating and developing models of long-term ecological change.