MICRO-INTERACT - Laser capture micro-dissection for identification of novel interactions within the plankton that underpin marine carbon cycling

海洋生物之间的相互作用驱动了营养群之间的碳转移，并最终决定了光合作用生物固定碳的命运。越来越多的证据表明，浮游生物内部存在各种各样的相互作用，但这些相互作用的特征仍然很差。例如，浮游植物可能被病原体（病毒和细菌）或寄生虫（例如真菌）感染，尽管我们对这些相互作用的程度和多样性的了解仍然有限。浮游植物分泌的多糖为海洋中大量的不稳定碳做出了贡献，但回收这种碳的微生物也没有得到很好的描述。如果没有改进的方法来评估肠道内容物或掠食性生物的食物液泡，浮游生物的营养相互作用也很难评估。显然有必要更详细地研究这些不同的相互作用，以提高我们对海洋生态系统功能的理解。然而，瞬态相互作用通常难以跟踪，并且可能被评估散装海水的技术所忽略。浮游生物的直接显微观察需要确定海洋生物之间的新相互作用，如寄生虫和营养相互作用。然而，要识别和研究这些生物，需要在技术上具有挑战性和艰苦的单细胞采摘或高度熟练的组织解剖。荧光激活细胞分选（FACS）不允许目标细胞的可视化，因此不能轻易地与原位观察联系起来，也不能用于以靶向方式分离新物种或相互作用的细胞（例如，数量较少的物种或群体中的感染细胞），除非这些细胞类型可以通过其荧光特性与所有其他细胞明确区分开来。因此，需要改进技术以高通量的方式识别浮游生物内部许多特征不明显的相互作用。为此，我们建议使用激光捕获显微解剖（LCM）。LCM包括将显微镜样品附着在膜上，使用激光切割感兴趣细胞周围的膜，然后将其转移到收集容器中，从而分离单个细胞和/或组织。这种方法的巨大优势在于，它允许以简单和高通量的方式直接分离观察到的细胞和组织。然后，收集的细胞或组织可以通过基因组学、蛋白质组学或代谢物分析方法进一步表征。活细胞也可以分离出来，不受污染，用于后续培养和新细胞系的产生。虽然LCM主要用于生物医学应用，但该技术在环境研究方面具有巨大潜力。LCM最近被用于从褐藻（Ectocarpus）中分离特定细胞类型进行基因表达研究，用于分离单细胞藻类（例如Euglena和Chlamydomonas）进行代谢物分析，并用于分离鱼类幼虫的肠道内容物以进行随后的分子表征。将LCM应用于浮游生物种群将为我们描述支撑海洋生态系统的关键过程的能力提供一个阶段性的变化。作为例子，我们的目标是提高对浮游生物寄生的理解，并识别新的寄生虫。我们还将研究海洋中降解有机碳的微生物，通过分离单个透明外聚合物颗粒（TEP）来表征其相关微生物群。LCM还将用于分离以前未培养的浮游植物物种。LCM为多个用户提供了极大的灵活性，并将大大加快以前需要费力和高技能技术的流程。

Interactions between marine organisms drive the transfer of carbon between trophic groups and ultimately determine the fate of carbon fixed by photosynthetic organisms. There is mounting evidence for a diverse array of interactions within the plankton that remain poorly characterised. For example, phytoplankton may become infected by pathogens (viruses and bacteria) or parasites (e.g. fungi), although our understanding of the extent and diversity of these interactions remains limited. Polysaccharides exuded by phytoplankton contribute to a large pool of labile carbon in the oceans, but the micro-organisms that recycle this carbon are also poorly characterised. Trophic interactions in the plankton are also difficult to assess without improved methodologies to assess gut contents or food vacuoles from predatory organisms.There is a clear need to study these diverse interactions in greater detail to improve our understanding of marine ecosystem function. However, transient interactions are often difficult to track and may be overlooked by techniques that assess bulk seawater. Direct microscopic observations of planktonic organisms is required to identify novel interactions between marine organisms, such as parasites and trophic interactions. However, to identify and study these organisms requires technically challenging and laborious picking of single cells or highly skilled tissue dissection. Fluorescence-activated cell sorting (FACS) do not allow visualisation of target cells and therefore cannot be easily linked to in situ observations and cannot be used to isolate novel species or interacting cells in a targeted manner (e.g. less abundant species or infected cells within a population) unless these cell types can be clearly discriminated from all of the other cells by their fluorescent properties.Improved technologies are therefore required to identify the many poorly characterised interactions within the plankton in a high throughput manner. We propose to use laser capture microdissection (LCM) for this purpose. LCM involves attaching microscopy samples to a membrane and isolating single cells and/or tissue by using a laser to cut the membrane around the cells of interest and then transfer them to a collecting vessel. The huge advantage of this approach is that it allows observed cells and tissue to be directly isolated in a simple and high-throughput manner. Harvested cells or tissue can then be further characterised by genomics, proteomics or metabolite profiling approaches. Live cells may be also isolated, free from contamination, for subsequent culturing and generation of novel cell lines.While LCM has been employed primarily in biomedical applications, the technique offers huge potential for environmental research. LCM has recently been used to isolate specific cell types from a brown seaweed (Ectocarpus) for gene expression studies, to isolate unicellular algae (e.g. Euglena and Chlamydomonas) for metabolite profiling, and to isolate the gut contents of fish larvae for subsequent molecular characterisation.The application of LCM to the plankton populations will provide a step-change in our ability to characterise key processes that underpin marine ecosystems. As examples, we aim to improve understanding of parasitism within the plankton and to identify novel parasites. We will also investigate the micro-organisms that degrade organic carbon in the oceans, by isolating individual transparent exopolymeric particles (TEP) for characterisation of their associated microbiomes. LCM will also be used to isolate previously uncultured phytoplankton species.LCM offers great flexibility for multiple users and will greatly speed up processes that have previously required laborious and highly skilled techniques.