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

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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.