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.

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.

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.

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.

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.

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.