Single-cell RNA-seq of the rare virosphere reveals the native hosts of giant viruses in the marine environment.

Giant viruses (phylum Nucleocytoviricota) are globally distributed in aquatic ecosystems. They play fundamental roles as evolutionary drivers of eukaryotic plankton and regulators of global biogeochemical cycles. However, we lack knowledge about their native hosts, hindering our understanding of their life cycle and ecological importance. In the present study, we applied a single-cell RNA sequencing (scRNA-seq) approach to samples collected during an induced algal bloom, which enabled pairing active giant viruses with their native protist hosts. We detected hundreds of single cells from multiple host lineages infected by diverse giant viruses. These host cells included members of the algal groups Chrysophycae and Prymnesiophycae, as well as heterotrophic flagellates in the class Katablepharidaceae. Katablepharids were infected with a rare Imitervirales-07 giant virus lineage expressing a large repertoire of cell-fate regulation genes. Analysis of the temporal dynamics of these host-virus interactions revealed an important role for the Imitervirales-07 in controlling the population size of the host Katablepharid population. Our results demonstrate that scRNA-seq can be used to identify previously undescribed host-virus interactions and study their ecological importance and impact.

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.

Homing in on the rare virosphere reveals the native host of giant viruses.

Giant viruses (phylum Nucleocytoviricota) are globally distributed in aquatic ecosystems1,2. They play major roles as evolutionary drivers of eukaryotic plankton3 and regulators of global biogeochemical cycles4. Recent metagenomic studies have significantly expanded the known diversity of marine giant viruses1,5-7, but we still lack fundamental knowledge about their native hosts, thereby hindering our understanding of their lifecycle and ecological importance. Here, we aim to discover the native hosts of giant viruses using a novel, sensitive single-cell metatranscriptomic approach. By applying this approach to natural plankton communities, we unraveled an active viral infection of several giant viruses, from multiple lineages, and identified their native hosts. We identify a rare lineage of giant virus (Imitervirales-07) infecting a minute population of protists (class Katablepharidaceae) and revealed the prevalence of highly expressed viral-encoded cell-fate regulation genes in infected cells. Further examination of this host-virus dynamics in a temporal resolution suggested this giant virus controls its host population demise. Our results demonstrate how single-cell metatranscriptomics is a sensitive approach for pairing viruses with their authentic hosts and studying their ecological significance in a culture-independent manner in the marine 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.

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.

Complete Genome Sequence of Emiliania huxleyi Virus Strain M1, Isolated from an Induced E. huxleyi Bloom in Bergen, Norway.

Emiliania huxleyi virus strain M1 (EhVM1), a large double-stranded DNA virus from the family Phycodnaviridae, was isolated from an Emiliania huxleyi bloom during a mesocosm experiment in Raunefjorden, Bergen, Norway. Here, we report its complete genome, composed of one full contig.

Dimethyl sulfide mediates microbial predator-prey interactions between zooplankton and algae in the ocean.

Phytoplankton are key components of the oceanic carbon and sulfur cycles1. During bloom events, some species can emit large amounts of the organosulfur volatile dimethyl sulfide (DMS) into the ocean and consequently the atmosphere, where it can modulate aerosol formation and affect climate2,3. In aquatic environments, DMS plays an important role as a chemical signal mediating diverse trophic interactions. Yet, its role in microbial predator-prey interactions remains elusive with contradicting evidence for its role in either algal chemical defence or in the chemo-attraction of grazers to prey cells4,5. Here we investigated the signalling role of DMS during zooplankton-algae interactions by genetic and biochemical manipulation of the algal DMS-generating enzyme dimethylsulfoniopropionate lyase (DL) in the bloom-forming alga Emiliania huxleyi6. We inhibited DL activity in E. huxleyi cells in vivo using the selective DL-inhibitor 2-bromo-3-(dimethylsulfonio)-propionate7 and overexpressed the DL-encoding gene in the model diatom Thalassiosira pseudonana. We showed that algal DL activity did not serve as an anti-grazing chemical defence but paradoxically enhanced predation by the grazer Oxyrrhis marina and other microzooplankton and mesozooplankton, including ciliates and copepods. Consumption of algal prey with induced DL activity also promoted O. marina growth. Overall, our results demonstrate that DMS-mediated grazing may be ecologically important and prevalent during prey-predator dynamics in aquatic ecosystems. The role of algal DMS revealed here, acting as an eat-me signal for grazers, raises fundamental questions regarding the retention of its biosynthetic enzyme through the evolution of dominant bloom-forming phytoplankton in the ocean.

Ecological significance of extracellular vesicles in modulating host-virus interactions during algal blooms.

Extracellular vesicles are produced by organisms from all kingdoms and serve a myriad of functions, many of which involve cell-cell signaling, especially during stress conditions and host-pathogen interactions. In the marine environment, communication between microorganisms can shape trophic level interactions and population succession, yet we know very little about the involvement of vesicles in these processes. In a previous study, we showed that vesicles produced during viral infection by the ecologically important model alga Emiliania huxleyi, could act as a pro-viral signal, by expediting infection and enhancing the half-life of the virus in the extracellular milieu. Here, we expand our laboratory findings and show the effect of vesicles on natural populations of E. huxleyi in a mesocosm setting. We profile the small-RNA (sRNA) cargo of vesicles that were produced by E. huxleyi during bloom succession, and show that vesicles applied to natural assemblages expedite viral infection and prolong the half-life of this major mortality agent of E. huxleyi. We subsequently reveal that exposure of the natural assemblage to E. huxleyi-derived vesicles modulates not only host-virus dynamics, but also other components of the microbial food webs, thus emphasizing the importance of extracellular vesicles to microbial interactions in the marine environment.

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.

Visualizing active viral infection reveals diverse cell fates in synchronized algal bloom demise.

Marine viruses are the most abundant biological entity in the ocean and are considered as major evolutionary drivers of microbial life [C. A. Suttle, Nat. Rev. Microbiol. 5, 801-812 (2007)]. Yet, we lack quantitative approaches to assess their impact on the marine ecosystem. Here, we provide quantification of active viral infection in the bloom forming single-celled phytoplankton Emiliania huxleyi infected by the large virus EhV, using high-throughput single-molecule messenger RNA in situ hybridization (smFISH) of both virus and host transcripts. In natural samples, viral infection reached only 25% of the population despite synchronized bloom demise exposing the coexistence of infected and noninfected subpopulations. We prove that photosynthetically active cells chronically release viral particles through nonlytic infection and that viral-induced cell lysis can occur without viral release, thus challenging major assumptions regarding the life cycle of giant viruses. We could also assess active infection in cell aggregates linking viral infection and carbon export to the deep ocean [C. P. Laber et al., Nat. Microbiol. 3, 537-547 (2018)] and suggest a potential host defense strategy by enrichment of infected cells in sinking aggregates. Our approach can be applied to diverse marine microbial systems, opening a mechanistic dimension to the study of biotic interactions in the ocean.

A single-cell view on alga-virus interactions reveals sequential transcriptional programs and infection states.

The discovery of giant viruses infecting eukaryotes from diverse ecosystems has revolutionized our understanding of the evolution of viruses and their impact on protist biology, yet knowledge on their replication strategies and transcriptome regulation remains limited. Here, we profile single-cell transcriptomes of the globally distributed microalga Emiliania huxleyi and its specific giant virus during infection. We detected profound heterogeneity in viral transcript levels among individual cells. Clustering single cells based on viral expression profiles enabled reconstruction of the viral transcriptional trajectory. Reordering cells along this path unfolded highly resolved viral genetic programs composed of genes with distinct promoter elements that orchestrate sequential expression. Exploring host transcriptome dynamics across the viral infection states revealed rapid and selective shutdown of protein-encoding nuclear transcripts, while the plastid and mitochondrial transcriptomes persisted into later stages. Single-cell RNA-seq opens a new avenue to unravel the life cycle of giant viruses and their unique hijacking strategies.

In plaque-mass spectrometry imaging of a bloom-forming alga during viral infection reveals a metabolic shift towards odd-chain fatty acid lipids.

Tapping into the metabolic crosstalk between a host and its virus can reveal unique strategies employed during infection. Viral infection is a dynamic process that generates an evolving metabolic landscape. Gaining a continuous view into the infection process is highly challenging and is limited by current metabolomics approaches, which typically measure the average of the entire population at various stages of infection. Here, we took an innovative approach to study the metabolic basis of host-virus interactions between the bloom-forming alga Emiliania huxleyi and its specific virus. We combined a classical method in virology, the plaque assay, with advanced mass spectrometry imaging (MSI), an approach we termed 'in plaque-MSI'. Taking advantage of the spatial characteristics of the plaque, we mapped the metabolic landscape induced during infection in a high spatiotemporal resolution, unfolding the infection process in a continuous manner. Further unsupervised spatially aware clustering, combined with known lipid biomarkers, revealed a systematic metabolic shift during infection towards lipids containing the odd-chain fatty acid pentadecanoic acid (C15:0). Applying 'in plaque-MSI' may facilitate the discovery of bioactive compounds that mediate the chemical arms race of host-virus interactions in diverse model systems.

Bacterial virulence against an oceanic bloom-forming phytoplankter is mediated by algal DMSP.

Emiliania huxleyi is a bloom-forming microalga that affects the global sulfur cycle by producing large amounts of dimethylsulfoniopropionate (DMSP) and its volatile metabolic product dimethyl sulfide. Top-down regulation of E. huxleyi blooms has been attributed to viruses and grazers; however, the possible involvement of algicidal bacteria in bloom demise has remained elusive. We demonstrate that a Roseobacter strain, Sulfitobacter D7, that we isolated from a North Atlantic E. huxleyi bloom, exhibited algicidal effects against E. huxleyi upon coculturing. Both the alga and the bacterium were found to co-occur during a natural E. huxleyi bloom, therefore establishing this host-pathogen system as an attractive, ecologically relevant model for studying algal-bacterial interactions in the oceans. During interaction, Sulfitobacter D7 consumed and metabolized algal DMSP to produce high amounts of methanethiol, an alternative product of DMSP catabolism. We revealed a unique strain-specific response, in which E. huxleyi strains that exuded higher amounts of DMSP were more susceptible to Sulfitobacter D7 infection. Intriguingly, exogenous application of DMSP enhanced bacterial virulence and induced susceptibility in an algal strain typically resistant to the bacterial pathogen. This enhanced virulence was highly specific to DMSP compared to addition of propionate and glycerol which had no effect on bacterial virulence. We propose a novel function for DMSP, in addition to its central role in mutualistic interactions among marine organisms, as a mediator of bacterial virulence that may regulate E. huxleyi blooms.

Extracellular vesicles - new players in cell-cell communication in aquatic environments.

Communication between microorganisms in aquatic environments can influence ecosystem function and determine the structure and composition of microbial populations. This microbial cross talk can be mediated by excretion of specialized metabolites or extracellular vesicles (EVs). Recently it has become apparent that cells across all domains of life produce EVs that may convey specific targeted signals that can modulate cell fate, morphology and susceptibility to viruses. The vast majority of knowledge about EVs is derived from studies of mammalian tissues, parasitic host-pathogen interactions and model bacterial systems. Very little is known about the role of EVs in aquatic environments, although they have potential to influence community structure and trophic-level interactions. We propose functions and ecological implications of communication via EVs in aquatic microbial ecosystems.

Expression profiling of host and virus during a coccolithophore bloom provides insights into the role of viral infection in promoting carbon export.

The cosmopolitan coccolithophore Emiliania huxleyi is a unicellular eukaryotic alga that forms vast blooms in the oceans impacting large biogeochemical cycles. These blooms are often terminated due to infection by the large dsDNA virus, E. huxleyi virus (EhV). It was recently established that EhV-induced modulation of E. huxleyi metabolism is a key factor for optimal viral infection cycle. Despite the huge ecological importance of this host-virus interaction, the ability to assess its spatial and temporal dynamics and its possible impact on nutrient fluxes is limited by current approaches that focus on quantification of viral abundance and biodiversity. Here, we applied a host and virus gene expression analysis as a sensitive tool to quantify the dynamics of this interaction during a natural E. huxleyi bloom in the North Atlantic. We used viral gene expression profiling as an index for the level of active infection and showed that the latter correlated with water column depth. Intriguingly, this suggests a possible sinking mechanism for removing infected cells as aggregates from the E. huxleyi population in the surface layer into deeper waters. Viral infection was also highly correlated with induction of host metabolic genes involved in host life cycle, sphingolipid, and antioxidant metabolism, providing evidence for modulation of host metabolism under natural conditions. The ability to track and quantify defined phases of infection by monitoring co-expression of viral and host genes, coupled with advance omics approaches, will enable a deeper understanding of the impact that viruses have on the environment.

Communication via extracellular vesicles enhances viral infection of a cosmopolitan alga.

Communication between microorganisms in the marine environment has immense ecological impact by mediating trophic-level interactions and thus determining community structure 1 . Extracellular vesicles (EVs) are produced by bacteria 2,3 , archaea 4 , protists 5 and metazoans, and can mediate pathogenicity 6 or act as vectors for intercellular communication. However, little is known about the involvement of EVs in microbial interactions in the marine environment 7 . Here we investigated the signalling role of EVs produced during interactions between the cosmopolitan alga Emiliania huxleyi and its specific virus (EhV, Phycodnaviridae) 8 , which leads to the demise of these large-scale oceanic blooms 9,10 . We found that EVs are highly produced during viral infection or when bystander cells are exposed to infochemicals derived from infected cells. These vesicles have a unique lipid composition that differs from that of viruses and their infected host cells, and their cargo is composed of specific small RNAs that are predicted to target sphingolipid metabolism and cell-cycle pathways. EVs can be internalized by E. huxleyi cells, which consequently leads to a faster viral infection dynamic. EVs can also prolong EhV half-life in the extracellular milieu. We propose that EVs are exploited by viruses to sustain efficient infectivity and propagation across E. huxleyi blooms. As these algal blooms have an immense impact on the cycling of carbon and other nutrients 11,12 , this mode of cell-cell communication may influence the fate of the blooms and, consequently, the composition and flow of nutrients in marine microbial food webs.

Phosphorus starvation induces membrane remodeling and recycling in Emiliania huxleyi.

Nutrient availability is an important factor controlling phytoplankton productivity. Phytoplankton contribute c. 50% of the global photosynthesis and possess efficient acclimation mechanisms to cope with nutrient stress. We investigate the cellular response of the bloom-forming coccolithophore Emiliania huxleyi to phosphorus (P) scarcity, which is often a limiting factor in marine ecosystems. We combined mass spectrometry, fluorescence microscopy, transmission electron microscopy (TEM) and gene expression analyses in order to assess diverse cellular features in cells exposed to P limitation and recovery. Early starvation-induced substitution of phospholipids in the cells' membranes with galacto- and betaine lipids. Lipid remodeling was rapid and reversible upon P resupply. The PI3K inhibitor wortmannin reduced phospholipid substitution, suggesting a possible involvement of PI3K- signaling in this process. In addition, P limitation enhanced the formation and acidification of membrane vesicles in the cytoplasm. Intracellular vesicles may facilitate the recycling of cytoplasmic content, which is engulfed in the vesicles and delivered to the main vacuole. Long-term starvation was characterized by a profound increase in cell size and morphological alterations in cellular ultrastructure. This study provides cellular and molecular basis for future ecophysiological assessment of natural E. huxleyi populations in oligotrophic regions.

Viral infection of the marine alga Emiliania huxleyi triggers lipidome remodeling and induces the production of highly saturated triacylglycerol.

Viruses that infect marine photosynthetic microorganisms are major ecological and evolutionary drivers of microbial food webs, estimated to turn over more than a quarter of the total photosynthetically fixed carbon. Viral infection of the bloom-forming microalga Emiliania huxleyi induces the rapid remodeling of host primary metabolism, targeted towards fatty acid metabolism. We applied a liquid chromatography-mass spectrometry (LC-MS)-based lipidomics approach combined with imaging flow cytometry and gene expression profiling to explore the impact of viral-induced metabolic reprogramming on lipid composition. Lytic viral infection led to remodeling of the cellular lipidome, by predominantly inducing the biosynthesis of highly saturated triacylglycerols (TAGs), coupled with a significant accumulation of neutral lipids within lipid droplets. Furthermore, TAGs were found to be a major component (77%) of the lipidome of isolated virions. Interestingly, viral-induced TAGs were significantly more saturated than TAGs produced under nitrogen starvation. This study highlights TAGs as major products of the viral-induced metabolic reprogramming during the host-virus interaction and indicates a selective mode of membrane recruitment during viral assembly, possibly by budding of the virus from specialized subcellular compartments. These findings provide novel insights into the role of viruses infecting microalgae in regulating metabolism and energy transfer in the marine environment and suggest their possible biotechnological application in biofuel production.

Infection of phytoplankton by aerosolized marine viruses.

Marine viruses constitute a major ecological and evolutionary driving force in the marine ecosystems. However, their dispersal mechanisms remain underexplored. Here we follow the dynamics of Emiliania huxleyi viruses (EhV) that infect the ubiquitous, bloom-forming phytoplankton E. huxleyi and show that EhV are emitted to the atmosphere as primary marine aerosols. Using a laboratory-based setup, we showed that the dynamic of EhV aerial emission is strongly coupled to the host-virus dynamic in the culture media. In addition, we recovered EhV DNA from atmospheric samples collected over an E. huxleyi bloom in the North Atlantic, providing evidence for aerosolization of marine viruses in their natural environment. Decay rate analysis in the laboratory revealed that aerosolized viruses can remain infective under meteorological conditions prevailing during E. huxleyi blooms in the ocean, allowing potential dispersal and infectivity over hundreds of kilometers. Based on the combined laboratory and in situ findings, we propose that atmospheric transport of EhV is an effective transmission mechanism for spreading viral infection over large areas in the ocean. This transmission mechanism may also have an important ecological impact on the large-scale host-virus "arms race" during bloom succession and consequently the turnover of carbon in the ocean.

Early perturbation in mitochondria redox homeostasis in response to environmental stress predicts cell fate in diatoms.

Diatoms are ubiquitous marine photosynthetic eukaryotes that are responsible for about 20% of global photosynthesis. Nevertheless, little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a redox-sensitive green fluorescent protein sensor targeted to various subcellular organelles in the marine diatom Phaeodactylum tricornutum, to map the spatial and temporal oxidation patterns in response to environmental stresses. Specific organelle oxidation patterns were found in response to various stress conditions such as oxidative stress, nutrient limitation and exposure to diatom-derived infochemicals. We found a strong correlation between the mitochondrial glutathione (GSH) redox potential (EGSH) and subsequent induction of cell death in response to the diatom-derived unsaturated aldehyde 2E,4E/Z-decadienal (DD), and a volatile halocarbon (BrCN) that mediate trophic-level interactions in marine diatoms. Induction of cell death in response to DD was mediated by oxidation of mitochondrial EGSH and was reversible by application of GSH only within a narrow time frame. We found that cell fate can be accurately predicted by a distinct life-death threshold of mitochondrial EGSH (-335 mV). We propose that compartmentalized redox-based signaling can integrate the input of diverse environmental cues and will determine cell fate decisions as part of algal acclimation to stress conditions.