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

微交互 - 激光捕获微解剖，用于识别支撑海洋碳循环的浮游生物内的新型相互作用

• 批准号: NE/T009195/1
• 负责人: Glen Wheeler
• 金额: $ 37.35万
• 依托单位: Marine Biological Association of the United Kingdom
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NE%2FT009195%2F1
• 关键词: MICRO; INTERACT; Laser; capture; micro

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.

海洋生物之间的相互作用驱动了营养基团之间碳的转移，并最终确定了光合生物固定的碳的命运。有越来越多的证据表明，浮游生物内各种相互作用的特征仍然很差。例如，尽管我们对这些相互作用的程度和多样性的理解仍然有限，但浮游植物可能会被病原体（病毒和细菌）或寄生虫（例如真菌）感染。浮游植物散发的多糖在海洋中有助于大量不稳定的碳，但是回收这种碳的微生物也很差。如果没有改进的方法论来评估肠道含量或掠食性生物的食物液泡，也很难评估浮游生物中的营养相互作用。显然，需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。但是，瞬态相互作用通常很难跟踪，并且可以通过评估散装海水的技术来忽视。需要对浮游生物的直接显微镜观察来鉴定海洋生物之间的新型相互作用，例如寄生虫和营养相互作用。但是，要识别和研究这些生物，需要在技术上具有挑战性的单细胞或高度熟练的组织解剖。荧光激活的细胞分选（FACS）不允许可视化靶细胞，因此不能轻易地链接到原位观察结果，不能以靶向方式分离新物种或相互作用的细胞（例如，较少丰富的物种或在一个内的感染细胞中，因此，除非这些细胞类型可以通过其荧光特性清楚地与所有其他细胞区分开。因此，需要高吞吐量的方式进行改进的技术以识别浮游生物中许多特征性较差的相互作用。我们建议将激光捕获微分解（LCM）用于此目的。 LCM涉及将显微镜样品连接到膜上，并通过使用激光切开感兴趣细胞周围的膜，然后将其转移到收集容器中，从而分离单细胞和/或组织。这种方法的巨大优势在于，它允许观察到的细胞和组织以简单且高通量的方式直接分离。然后，收获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特征。活细胞也可以被隔离，没有污染，以进行随后的培养和生成新的细胞系。虽然LCM主要用于生物医学应用，但该技术为环境研究提供了巨大的潜力。 LCM最近已用于将特定的细胞类型与基因表达研究的棕色海藻分离，以分离单细胞藻类（例如Euglena和euglena和chlamydomonas）代谢物谱图，并分离出幼虫的肠道含量，以进行后续分子表征。 LCM在浮游生物种群中的应用将为我们表征基于海洋生态系统的关键过程的能力提供逐步变化。作为例子，我们旨在提高对浮游生物内寄生虫的理解，并确定新颖的寄生虫。我们还将通过分离单个透明的外聚合颗粒（TEP）来表征其相关的微生物组，从而研究海洋中有机碳的微生物。 LCM还将用于隔离以前未经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且高技能技术的过程。