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

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.

Development of the LCM facility for environmental research will have significant potential for innovation. The ability to isolate single cells for culturing or molecular identification should lead to the identification of novel phytoplankton strains or species, which could have wide-ranging implications for our understanding of marine microbial ecology. In particular, the research has the potential to identify novel associations between species and cell types (e.g. parasites/symbionts), which in turn will aid our understanding carbon fluxes in marine ecosystems.

We aim to track these direct innovations by recording all strains and cell types that are isolated by the requested equipment.

These innovations will have broad impact throughout the environmental research community by helping to revise marine ecosystem models, contributing to the development of new analytical techniques for the detection of novel cell types and aiding the analysis of marine genomics data.

A major societal benefit is the development of techniques and approaches to help improve our understanding of the marine carbon cycle. By examining and attempting to identify marine microbes responsible for poorly understood aspects of the marine carbon cycle, the requested equipment could contribute to improved understanding of global carbon cycling and management of anthropogenic carbon dioxide emissions.

The isolation of novel strains also has potential economic benefits, particularly in biotechnological applications. Novel marine microbes may be a source of high value natural products, lipids, or novel antibiotics and enzymes. Although it is not a primary aim to exploit these resources, novel strains will be deposited in the appropriate culture collections to facilitate global distribution to the scientific community. Characterisation of algal parasites may also improve our understanding of the factors limiting the large-scale cultivation of algae for biotechnology.

Examples of the outputs and the actual and potential beneficiaries are listed below:
1) Fungal parasites of diatoms. The LCM facility will identify diatoms infected with parasites. Fungal strains will be incorporated into the Marine Fungi Collection curated by Dr Michael Cunliffe (hosted at the MBA), a unique resource that will benefit a diverse range of marine scientists. Research conducted on infected diatoms will inform future studies into phytoplankton physiology and the fate of fixed carbon in the oceans. Functional assignment of specific taxa as parasites will facilitate studies into the global distribution and abundance of parasites using the wealth of marine metagenomics data that is becoming available. Ultimately, these studies will help to inform biogeochemists on processes that determine marine carbon fluxes and aid the development of improved predictive models.
2) Particle-associated microbiomes. The LCM facility will allow the identification of marine microbes associated with specific organic particles. Metagenomes of particle-associated microbiomes will allow identification of metabolic capacity, e.g. enzymes associated with degrading specific forms of organic carbon. These functional assignments will allow marine microbiologists to understand 'who does what?' in terms of carbon cycling, which will in turn inform marine biogeochemists and ecosystem modellers.
3) Isolation of uncultured organisms. A key advantage of LCM is the ability to link image data to genomic resources. The technology will help us to assign morphological identities to taxonomic groups that are abundant in molecular datasets, but remain uncultivated. Isolation of live cells for culturing may allow marine scientists to examine organisms that can't currently be studied. The deposition of novel genomes and strains from under-represented taxa in global repositories has significant potential to benefit many diverse members of the environmental research community.