Developing new approaches for the single cell manipulations and experimentation

The perception that marine microbial life is extremely diverse is well grounded based upon molecular diversity surveys and the existence of a wide variety of cultured forms. This also applies to the smallest colourless eukaryotes in planktonic systems, the heterotrophic flagellates, which are important agents in bacterial mortality through grazing. The use of extensive sampling datasets and high-throughput sequencing tools allows to identify the dominant species and better define their ecological relevance in the marine environment. In particular, Ramon Massana’s group focuses here in two model groups that represent contrasted ecological strategies in terms of growth and grazing dynamics and oceanic distribution. One of these two groups still remains uncultured, so single cell genomics is the only way to access to its genome content, which can then be used to analyse gene expression in natural communities. Opening the black box of microbial functional guilds is a necessity to really understand ecosystemic processes.

Dr Ramon Massana Institute of Marine Sciences, CSIC, Spain

Dr Ramon Massana Institute of Marine Sciences, CSIC, Spain

Ramon Massana is a Research Scientist at the ICM-CSIC with a wide experience in marine microbial ecology. His broad research line is the study of the phylogenetic and functional diversity of marine protists, especially those species that still remain uncultured. He conducts this exploration by applying simultaneously several tools, such as sequencing of environmental rDNA genes, metagenomics, single cell genomics, specific FISH counts and experiments. He is particularly interested in opening the black box of heterotrophic flagellates and exploring the role of these unpigmented protists in marine food webs as bacterial grazers and nutrient remineralisers. He is the coordinator of the EU Innovative Training Network SINGEK conceived to promote the use of single cell genomics to address critical ecological and evolutionary questions in microbial eukaryotes.

Single-cell genomics is a powerful method to get genomic data from uncultured organisms. However, the genome data generated for eukaryotes are usually less than 50% of the genome. The question is then whether this is useful for evolutionary questions. By obtaining single-cell genomic data from three choanoflagellates, Iñaki Ruiz-Trillo investigated whether this methodology can provide valuable data for evolutionary questions. In particular, and given the phylogenetic position of choanoflagellates as the sister-group to animals, the authors analysed the use of these data to address the origin of animals. The potential and the challenges will be discussed.

Dr Iñaki Ruiz-Trillo, Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Spain

Dr Iñaki Ruiz-Trillo, Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Spain

Iñaki Ruiz-Trillo is an ICREA Research Professor at the Institut de Biologia Evolutiva (CSIC-UPF) in Barcelona. His educational background includes a BS in Biology from the University of Barcelona. He earned a PhD in Biology from the University of Barcelona and completed a post-doctoral fellowship at the Dalhousie University, Canada. His current research interests include the study of the molecular mechanisms involved in the origin of multicellular animals, the analysis of genomes of different protists, the study of higher phylogenetic relationships of eukaryotes, and the understanding of the genetic repertoire of the last eukaryotic common ancestor.

Bacterial-sized unicellular eukaryotes of the class Mamiellophyceae are ubiquitous in the marine environment, where they significantly contribute to the primary production. Their ease of manipulation and compact genomes make them ideal models for benchmarking single cell approaches for phytoplanktonic micro-organisms. During this talk, Gwenaël Piganeau will explain how single cell experimentations enable to address two fundamental issues about the mode and tempo of evolution in these microalgae. First, how the set up of mutation accumulation experiment assays in liquid medium allows the segregation of deleterious and slightly deleterious mutations in individual microalgae cells. Whole genome re-sequencing of these lineages in five phytoplanktonic species enabled the estimation of the genome wide spontaneous mutation rate, the ultimate source of genetic diversity in the fate of environmental change. Second, single cell manipulation is required to estimate the phenotypic diversity within isogenic cells. This is particularly important to understand the mechanisms involved in the resistance of microalga to viruses infecting them, and how this impacts the microalga virus ratio, which influences the carbon flux in the sunlit ocean.

Dr Gwenaël Piganeau, CNRS, Integrative Biology of Marine Organisms Lab, France

Dr Gwenaël Piganeau, CNRS, Integrative Biology of Marine Organisms Lab, France

Gwenaël Piganeau has been a CNRS research fellow since 2004 and leads the Evolutionary and Environmental Genomics of Phytoplankton Lab in Banyuls sur mer, France, since 2012. She combines bioinformatic and experimental evolution approaches to unravel the evolutionary history of phytoplankton–prasinovirus interactions. Most of her research focuses on the mode and tempo of genome evolution in Ostreococus, the smallest photosynthetic eukaryote, and the large double stranded DNA viruses infecting them, for which her lab has been developing many genomic and genetic resources. Knowledge about the key processes of genome evolution, mutation and recombination, are paramount for understanding the adaptability of these species to environmental change and their amenability to domestication.

Predatory protists have long been recognised for their role in aquatic food webs, often exerting control on microbial abundance and community structure. Nevertheless, little is known about the relative importance and activity of different predatory groups, let alone the specific predator-prey interactions that drive population dynamics of individual taxa in the ocean. Resolving such interactions is complicated by the extreme diversity of the predators involved, both in terms of their distant evolutionary histories spanning the eukaryotic tree of life, as well as their functional diversity that includes the combination of photosynthetic with predatory nutrition in a mixotrophic lifestyle. Here, single-cell and population level cell sorting based on the presence of acidic food vacuoles were used to identify actively feeding microbes in the NE Pacific Ocean. Prey assimilation by predators feeding on the most abundant photosynthetic microbe Prochlorococcus was further verified by RNA-stable isotope probing. Both of these approaches suggested the quantitative importance of choanoflagellates as microbial predators in both productive coastal and nutrient-poor oceanic waters. Finally, the suitability of transcriptional patterns to indicate a predatory, phagotrophic nutrition will be discussed. Collectively, these approaches help to dissect the diverse trophic roles of microbial eukaryotes that shape the marine carbon cycle.

Dr Susanne Wilken, University of Amsterdam, The Netherlands

Dr Susanne Wilken, University of Amsterdam, The Netherlands

Susanne Wilken is an Assistant Professor of Aquatic Microbial Ecology at the University of Amsterdam’s Institute for Biodiversity and Ecosystem Dynamics. She carried out her PhD research at the Netherlands Institute of Ecology followed by postdoctoral work at the Monterey Bay Aquarium Research Institute, USA, and the Institute of Marine Sciences (ICM-CSIC) in Barcelona. Her research spans from the physiological basis to the ecosystem level consequences of resource acquisition strategies in microbial eukaryotes and their integration into the microbial interaction networks that drive biogeochemical cycles in aquatic ecosystems. A major focus is on mixotrophic organisms that combine photosynthesis with heterotrophic nutrition by predation on other microbes. This lifestyle is abundant in nature and challenges traditional concepts of microbial food web dynamics and carbon cycling in the ocean.

Most eukaryotic microbial diversity is uncultivated, under-studied, and lacks nuclear genome data. Mitochondrial sampling is more comprehensive, yet still groups that occupy important phylogenetic positions remain unexplored. Using photopigment exclusion and tubulin-specific fluorescence staining, the group sorted single-cell ‘heterotrophic’ flagellates directly from eastern North Pacific waters for genome sequencing. They recovered 206 single amplified genomes (SAGs) predominantly from under-represented protistan branches on the tree of life. 69 SAGs contained mitochondrial contigs including 21 unique complete, or near-complete, mitochondrial genomes from cryptic phylogenetic branches including telonemids, katablepharids and marine stramenopiles (MASTs). Collectively, these data point to a dynamic history of mitochondrial genome evolution including intron gain/loss, extensive patterns of genetic code variation, and complex patterns of convergent gene loss and endosymbiotic gene transfer. Surprisingly, mitochondrial coding content variation in heterotrophic stramenopiles resembles patterns previously only observed in plants.

Dr Jeremy Wideman, Dalhousie University, Canada

Dr Jeremy Wideman, Dalhousie University, Canada

Jeremy Wideman is an evolutionary cell biologist whose major research interest is understanding how eukaryotic cell complexity and diversity evolved. To do this he focuses on investigating how eukaryotic cell phenotypes have changed over evolutionary time using single-cell genomics, cell biological, and biochemical approaches. He is currently a postdoctoral fellow in the Department of Biochemistry and Molecular Biology at Dalhousie University, Halifax, Canada.

The Darby lab currently has a good understanding of how bacteria form partnerships with arthropods, but very little is known about eukaryotic microbes and their roles in arthropod biology. There are obviously exceptions as some parasites have been extensively studied, such as Plasmodium and Trypanosomes. However, these examples only represent the tip of the iceberg in terms of the possible diversity and interactions between microeukaryotes and arthropods. The main reason that we still don’t understand much about this microeukaryote diversity is that they are non-culture viable and are extremely rare in arthropod populations. Therefore, there is a need to develop high-throughput tools targeting microeukaryotes to describe their diversity in arthropods and to sequence their genomes so we can understand their functions. In this talk Alistair Darby will give examples of how harassing cell generics can be used to describe diversity and function, but also highlight some of the challenges still to be solved in studying these systems.

Dr Alistair Darby, University of Liverpool, UK

Dr Alistair Darby, University of Liverpool, UK

Alistair is a genome scientist with research interests in the symbionts and pathogens of arthropod pests and disease vectors. From his PhD to present he has worked on non-culture viable microbes and used molecular/genomic approaches to describe and understand the biology of interactions between the host arthropod and the microbial partner. He is Co-Director at the Centre for Genomics Research, University of Liverpool, where he heads the single cell sequencing lab.