Cell-to-cell heterogeneity drives host-virus coexistence in a bloom-forming alga.

Algal blooms drive global biogeochemical cycles of key nutrients and serve as hotspots for biological interactions in the ocean. The massive blooms of the cosmopolitan coccolithophore Emiliania huxleyi are often infected by the lytic E. huxleyi virus, which is a major mortality agent triggering bloom demise. This multi-annual "boom and bust" pattern of E. huxleyi blooms suggests that coexistence is essential for these host-virus dynamics. To investigate host-virus coexistence, we developed a new model system from an E. huxleyi culture that recovered from viral infection. The recovered population coexists with the virus, as host cells continue to divide in parallel to viral production. By applying single-molecule fluorescence in situ hybridization (smFISH) to quantify the fraction of infected cells, and assessing infection-specific lipid biomarkers, we identified a small subpopulation of cells that were infected and produced new virions, whereas most of the host population could resist infection. To further assess population heterogeneity, we generated clonal strain collections using single-cell sorting and subsequently phenotyped their susceptibility to E. huxleyi virus infection. This unraveled substantial cell-to-cell heterogeneity across a continuum of susceptibility to resistance, highlighting that infection outcome may vary depending on the individual cell. These results add a new dimension to our understanding of the complexity of host-virus interactions that are commonly assessed in bulk and described by binary definitions of resistance or susceptibility. We propose that phenotypic heterogeneity drives the host-virus coexistence and demonstrate how the coexistence with a lytic virus provides an ecological advantage for the host by killing competing strains.

Algal blooms in the ocean: hot spots for chemically mediated microbial interactions.

The cycling of major nutrients in the ocean is affected by large-scale phytoplankton blooms, which are hot spots of microbial life. Diverse microbial interactions regulate bloom dynamics. At the single-cell level, interactions between microorganisms are mediated by small molecules in the chemical crosstalk that determines the type of interaction, ranging from mutualism to pathogenicity. Algae interact with viruses, bacteria, parasites, grazers and other algae to modulate algal cell fate, and these interactions are dependent on the environmental context. Recent advances in mass spectrometry and single-cell technologies have led to the discovery of a growing number of infochemicals - metabolites that convey information - revealing the ability of algal cells to govern biotic interactions in the ocean. The diversity of infochemicals seems to account for the specificity in cellular response during microbial communication. Given the immense impact of algal blooms on biogeochemical cycles and climate regulation, a major challenge is to elucidate how microscale interactions control the fate of carbon and the recycling of major elements in the ocean. In this Review, we discuss microbial interactions and the role of infochemicals in algal blooms. We further explore factors that can impact microbial interactions and the available tools to decipher them in the natural environment.

Lipid biomarkers for algal resistance to viral infection in the ocean.

Marine viruses play a key role in regulating phytoplankton populations, greatly affecting the biogeochemical cycling of major nutrients in the ocean. Resistance to viral infection has been reported for various phytoplankton species under laboratory conditions. Nevertheless, the occurrence of resistant cells in natural populations is underexplored due to the lack of sensitive tools to detect these rare phenotypes. Consequently, our current understanding of the ecological importance of resistance and its underlying mechanisms is limited. Here, we sought to identify lipid biomarkers for the resistance of the bloom-forming alga Emiliania huxleyi to its specific virus, E. huxleyi virus (EhV). By applying an untargeted lipidomics approach, we identified a group of glycosphingolipid (GSL) biomarkers that characterize resistant E. huxleyi strains and were thus termed resistance-specific GSLs (resGSLs). Further, we detected these lipid biomarkers in E. huxleyi isolates collected from induced E. huxleyi blooms and in samples collected during an open-ocean E. huxleyi bloom, indicating that resistant cells predominantly occur during the demise phase of the bloom. Last, we show that the GSL composition of E. huxleyi cultures that recover following infection and gain resistance to the virus resembles that of resistant strains. These findings highlight the metabolic plasticity and coevolution of the GSL biosynthetic pathway and underscore its central part in this host-virus arms race.

Bacterial lifestyle switch in response to algal metabolites.

Unicellular algae, termed phytoplankton, greatly impact the marine environment by serving as the basis of marine food webs and by playing central roles in the biogeochemical cycling of elements. The interactions between phytoplankton and heterotrophic bacteria affect the fitness of both partners. It is becoming increasingly recognized that metabolic exchange determines the nature of such interactions, but the underlying molecular mechanisms remain underexplored. Here, we investigated the molecular and metabolic basis for the bacterial lifestyle switch, from coexistence to pathogenicity, in Sulfitobacter D7 during its interaction with Emiliania huxleyi, a cosmopolitan bloom-forming phytoplankter. To unravel the bacterial lifestyle switch, we analyzed bacterial transcriptomes in response to exudates derived from algae in exponential growth and stationary phase, which supported the Sulfitobacter D7 coexistence and pathogenicity lifestyles, respectively. In pathogenic mode, Sulfitobacter D7 upregulated flagellar motility and diverse transport systems, presumably to maximize assimilation of E. huxleyi-derived metabolites released by algal cells upon cell death. Algal dimethylsulfoniopropionate (DMSP) was a pivotal signaling molecule that mediated the transition between the lifestyles, supporting our previous findings. However, the coexisting and pathogenic lifestyles were evident only in the presence of additional algal metabolites. Specifically, we discovered that algae-produced benzoate promoted the growth of Sulfitobacter D7 and hindered the DMSP-induced lifestyle switch to pathogenicity, demonstrating that benzoate is important for maintaining the coexistence of algae and bacteria. We propose that bacteria can sense the physiological state of the algal host through changes in the metabolic composition, which will determine the bacterial lifestyle during interaction.

Viral infection switches the balance between bacterial and eukaryotic recyclers of organic matter during coccolithophore blooms.

Algal blooms are hotspots of marine primary production and play central roles in microbial ecology and global elemental cycling. Upon demise of the bloom, organic carbon is partly respired and partly transferred to either higher trophic levels, bacterial biomass production or sinking. Viral infection can lead to bloom termination, but its impact on the fate of carbon remains largely unquantified. Here, we characterize the interplay between viral infection and the composition of a bloom-associated microbiome and consequently the evolving biogeochemical landscape, by conducting a large-scale mesocosm experiment where we monitor seven induced coccolithophore blooms. The blooms show different degrees of viral infection and reveal that only high levels of viral infection are followed by significant shifts in the composition of free-living bacterial and eukaryotic assemblages. Intriguingly, upon viral infection the biomass of eukaryotic heterotrophs (thraustochytrids) rivals that of bacteria as potential recyclers of organic matter. By combining modeling and quantification of active viral infection at a single-cell resolution, we estimate that viral infection causes a 2-4 fold increase in per-cell rates of extracellular carbon release in the form of acidic polysaccharides and particulate inorganic carbon, two major contributors to carbon sinking into the deep ocean. These results reveal the impact of viral infection on the fate of carbon through microbial recyclers of organic matter in large-scale coccolithophore blooms.

Grazing on Marine Viruses and Its Biogeochemical Implications.

Viruses are the most abundant biological entities in the ocean and show great diversity in terms of size, host specificity, and infection cycle. Lytic viruses induce host cell lysis to release their progeny and thereby redirect nutrients from higher to lower trophic levels. Studies continue to show that marine viruses can be ingested by nonhost organisms. However, not much is known about the role of viral particles as a nutrient source and whether they possess a nutritional value to the grazing organisms. This review seeks to assess the elemental composition and biogeochemical relevance of marine viruses, including roseophages, which are a highly abundant group of bacteriophages in the marine environment. We place a particular emphasis on the phylum Nucleocytoviricota (NCV) (formerly known as nucleocytoplasmic large DNA viruses [NCLDVs]), which comprises some of the largest viral particles in the marine plankton that are well in the size range of prey for marine grazers. Many NCVs contain lipid membranes in their capsid that are rich carbon and energy sources, which further increases their nutritional value. Marine viruses may thus be an important nutritional component of the marine plankton, which can be reintegrated into the classical food web by nonhost organism grazing, a process that we coin the "viral sweep." Possibilities for future research to resolve this process are highlighted and discussed in light of current technological advancements.

Viral infection of algal blooms leaves a unique metabolic footprint on the dissolved organic matter in the ocean.

Algal blooms are hotspots of primary production in the ocean, forming the basis of the marine food web and fueling the dissolved organic matter (DOM) pool. Viruses are key players in controlling algal demise, thereby diverting biomass from higher trophic levels to the DOM pool, a process termed the "viral shunt." To decode the metabolic footprint of the viral shunt in the environment, we induced a bloom of Emiliania huxleyi and followed its succession using untargeted exometabolomics. We show that bloom succession induces dynamic changes in the exometabolic landscape. We found a set of chlorine-iodine-containing metabolites that were induced by viral infection and released during bloom demise. These metabolites were further detected in virus-infected oceanic E. huxleyi blooms. Therefore, we propose that halogenation with both chlorine and iodine is a distinct hallmark of the virus-induced DOM of E. huxleyi, providing insights into the metabolic consequences of the viral shunt.

Metabolomics-derived marker metabolites to characterize Phaeocystis pouchetii physiology in natural plankton communities.

Phaeocystis pouchetii (Hariot) Lagerheim, 1893 regularly dominates phytoplankton blooms in higher latitudes spanning from the English Channel to the Arctic. Through zooplankton grazing and microbial activity, it is considered to be a key resource for the entire marine food web, but the actual relevance of biomass transfer to higher trophic levels is still under discussion. Cell physiology and algal nutritional state are suggested to be major factors controlling the observed variability in zooplankton grazing. However, no data have so far yielded insights into the metabolic state of Phaeocystis populations that would allow testing this hypothesis. Therefore, endometabolic markers of different growth phases were determined in laboratory batch cultures using comparative metabolomics and quantified in different phytoplankton blooms in the field. Metabolites, produced during exponential, early and late stationary growth of P. pouchetii, were profiled using gas chromatography-mass spectrometry. Then, metabolites were characterized that correlate with the growth phases using multivariate statistical analysis. Free amino acids characterized the exponential growth, whereas the early stationary phase was correlated with sugar alcohols, mono- and disaccharides. In the late stationary phase, free fatty acids, sterols and terpenes increased. These marker metabolites were then traced in Phaeocystis blooms during a cruise in the Barents Sea and North Norwegian fjords. About 50 endometabolites of P. pouchetii were detected in natural phytoplankton communities. Mannitol, scyllo-inositol, 24-methylcholesta-5,22-dien-3ß-ol, and several free fatty acids were characteristic for Phaeocystis-dominated blooms but showed variability between them. Distinct metabolic profiles were detected in the nutrient-depleted community in the inner Porsangerfjord (< 0.5 µM NO3-, < 0.1 µM PO 4 3- ), with high relative amounts of free mono- and disaccharides indicative for a limited culture. This study thereby shows how the variable physiology of phytoplankton can alter the metabolic landscape of entire plankton communities.

Metabolomics in chemical ecology.

Chemical ecology elucidates the nature and role of natural products as mediators of organismal interactions. The emerging techniques that can be summarized under the concept of metabolomics provide new opportunities to study such environmentally relevant signaling molecules. Especially comparative tools in metabolomics enable the identification of compounds that are regulated during interaction situations and that might play a role as e.g. pheromones, allelochemicals or in induced and activated defenses. This approach helps overcoming limitations of traditional bioassay-guided structure elucidation approaches. But the power of metabolomics is not limited to the comparison of metabolic profiles of interacting partners. Especially the link to other -omics techniques helps to unravel not only the compounds in question but the entire biosynthetic and genetic re-wiring, required for an ecological response. This review comprehensively highlights successful applications of metabolomics in chemical ecology and discusses existing limitations of these novel techniques. It focuses on recent developments in comparative metabolomics and discusses the use of metabolomics in the systems biology of organismal interactions. It also outlines the potential of large metabolomics initiatives for model organisms in the field of chemical ecology.