Publications

The viral shunt is a fundamental ecosystem process which diverts the flux of organic carbon from grazers to heterotrophic microorganisms. Through the extracellular release of metabolites, lytic viral infections supply 2-10 we lack tools to detect the viral shunt in the natural environment and assess its ecological impact. Here, we study the use of exometabolites as biomarkers for the viral shunt by applying molecular, metabolomics, and oceanographic tools in blooms of the cosmopolitan microalga Gephyrocapsa huxleyi across the Atlantic Ocean, spanning four biogeochemical provinces between Iceland and Patagonia. We mapped the distinct metabolic footprint of its viral infections using exo- and endometabolomics approaches and detected nineteen organohalogen metabolites across the blooms, showing their global formation. Time-resolved comparison of particulate and dissolved metabolite pools during an induced mesocosm bloom indicated virocells - actively infected host cells - as the source of the halogenated metabolites. Three trichloro-iodo metabolites were present during demise of all virus-infected blooms, highlighting them as suitable metabolic biomarkers. The environmental stability of these halometabolites in the DOM pool over a few days can recapitulate viral infections at earlier stages of phytoplankton bloom succession.Competing Interest StatementThe authors have declared no competing interest.

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 hostvirus 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 hostvirus interactions and study their ecological importance and impact.

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.

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.

Ocean microbes are involved in global processes such as nutrient and carbon cycling. Recent studies indicated diverse modes of algal-bacterial interactions, including mutualism and pathogenicity, which have a substantial impact on ecology and oceanic carbon sequestration, and hence, on climate. However, the airborne dispersal and pathogenicity of bacteria in the marine ecosystem remained elusive. Here, we isolated an airborne algicidal bacterium, Roseovarius nubinhibens, emitted to the atmosphere as primary marine aerosol (referred also as sea spray aerosols) and collected above a coccolithophore bloom in the North Atlantic Ocean. The aerosolized bacteria retained infective properties and induced lysis of Gephyrocapsa huxleyi cultures.This suggests that the transport of marine bacteria through the atmosphere can effectively spread infection agents over vast oceanic regions, highlighting its significance in regulating the cell fate in algal blooms.

Dimethylsulfide (DMS) is a climatically active trace gas promoting cloud formation. The biochemical precursor of DMS, dimethylsulfoniopropionate (DMSP), is a phytoplankton metabolite and a source of reduced sulfur for many microbial species. Because of the complex interactions between their many producers and consumers, the dynamics of DMSP and DMS in the ocean are still poorly constrained. In this study we measure particulate DMSP, dissolved DMSP ((Formula presented.)), and DMS concentrations in seven mesocosms where two consecutive phytoplankton blooms (first, pico- and nano-algae; second, Emiliania huxleyi) were induced by nutrient addition, and we build a mechanistic numerical model to identify the sources and sinks that best account for the observations. The mesocosms were designed as replicates but differ from each other by their E. huxleyi virus abundance due to stochastic differences in initial conditions. The model shows that heterotrophic bacteria cannot be the only consumers of (Formula presented.). A fraction of dissolved (Formula presented.) must be consumed by phytoplankton to avoid excessive (Formula presented.) accumulation during the first bloom. The induced blooms increase DMS concentration by 220% on average, until an increase in the abundance of DMS-consuming bacteria brings DMS concentration back to its pre-bloom value, after 3 weeks of experiment. Therefore phytoplankton blooms can increase DMS emission to the atmosphere, but only during a transient regime of a few weeks. The model also shows that the DMS yield, production and emission are increased when the coccolithophore bloom is terminated by a viral infection, but decreased if the infection occurs several days before the bloom can reach its maximum.

The ability of marine bacteria to direct their movement in response to chemical gradients influences inter-species interactions, nutrient turnover, and ecosystem productivity. While many bacteria are chemotactic towards small metabolites, marine organic matter is predominantly composed of large molecules and polymers. Yet, the signalling role of these large molecules is largely unknown. Using in situ and laboratory-based chemotaxis assays, we show that marine bacteria are strongly attracted to the abundant algal polysaccharides laminarin and alginate. Unexpectedly, these polysaccharides elicited stronger chemoattraction than their oligo- and monosaccharide constituents. Furthermore, chemotaxis towards laminarin was strongly enhanced by dimethylsulfoniopropionate (DMSP), another ubiquitous algal-derived metabolite. Our results indicate that DMSP acts as a methyl donor for marine bacteria, increasing their gradient detection capacity and facilitating their access to polysaccharide patches. We demonstrate that marine bacteria are capable of strong chemotaxis towards large soluble polysaccharides and uncover a new ecological role for DMSP in enhancing this attraction. These navigation behaviours may contribute to the rapid turnover of polymers in the ocean, with important consequences for marine carbon cycling.

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.

Giant viruses infect many unicellular eukaryotes, including algae that form massive oceanic blooms. Despite the major impact of viruses on the marine ecosystem, the ability to quantify and assess active viral infection in nature remains a major challenge. We applied single-cell RNA sequencing, to profile virus and host transcriptomes of 12,000 single algal cells from a coccolithophore bloom. Viral infection was detected already at early exponential bloom phase, negatively correlating with the bloom intensity. A consistent percent of infected coccolithophores displayed the early phase of viral replication for several consecutive days, indicating a daily turnover and continuous virocell-associated metabolite production, potentially affecting the surrounding microbiome. Linking single-cell infection state to host physiology revealed that infected cells remained calcified even in the late infection stage. These findings stress the importance of studying host-virus dynamics in natural populations, at single-cell resolution, to better understand virus life cycle and its impact on microbial food webs. The dynamics of viral infection in algal blooms were deciphered using single-cell transcriptomics.

The phylum Nucleocytoviricota includes the largest and most complex viruses known. These "giant viruses"have a long evolutionary history that dates back to the early diversification of eukaryotes, and over time they have evolved elaborate strategies for manipulating the physiology of their hosts during infection. One of the most captivating of these mechanisms involves the use of genes acquired from the host - referred to here as viral homologs or "virologs"- as a means of promoting viral propagation. The best-known examples of these are involved in mimicry, in which viral machinery "imitates"immunomodulatory elements in the vertebrate defense system. But recent findings have highlighted a vast and rapidly expanding array of other virologs that include many genes not typically found in viruses, such as those involved in translation, central carbon metabolism, cytoskeletal structure, nutrient transport, vesicular trafficking, and light harvesting. Unraveling the roles of virologs during infection as well as the evolutionary pathways through which complex functional repertoires are acquired by viruses are important frontiers at the forefront of giant virus research.

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.

Phytoplankton produce the volatile dimethyl sulfide (DMS), an important infochemical mediating microbial interactions, which is also emitted to the atmosphere and affecting the global climate. Albeit the enzymatic source for DMS in eukaryotes was elucidated, namely a DMSP lyase (DL) called Alma1, we still lack basic knowledge regarding its taxonomic distribution. We defined unique sequence motifs which enable the identification of DL homologs (DLHs) in model systems and environmental populations. We used these motifs to predict DLHs in diverse algae by analyzing hundreds of genomic and transcriptomic sequences from model systems under stress conditions and from environmental samples. Our findings show that the DL enzyme is more taxonomically widespread than previously thought, as it is encoded by known algal taxa as haptophytes and dinoflagellates, but also by chlorophytes, pelagophytes and diatoms, which were conventionally considered to lack the DL enzyme. By exploring the Tara Oceans database, we showed that DLHs are widespread across the oceans and are predominantly expressed by dinoflagellates. Certain dinoflagellate DLHs were differentially expressed between the euphotic and mesopelagic zones, suggesting a functional specialization and an involvement in the metabolic plasticity of mixotrophic dinoflagellates. In specific regions as the Southern Ocean, DLH expression by haptophytes and diatoms was correlated with environmental drivers such as nutrient availability. The expanded repertoire of putative DL enzymes from diverse microbial origins and geographic niches suggests new potential players in the marine sulfur cycle and provides a foundation to study the cellular function of the DL enzyme in marine microbes.

The Tara Pacific expedition (20162018) sampled coral ecosystems around 32 islands in the Pacific Ocean and the ocean surface waters at 249 locations, resulting in the collection of nearly 58 000 samples. The expedition was designed to systematically study warm-water coral reefs and included the collection of corals, fish, plankton, and seawater samples for advanced biogeochemical, molecular, and imaging analysis. Here we provide a complete description of the sampling methodology, and we explain how to explore and access the different datasets generated by the expedition. Environmental context data were obtained from taxonomic registries, gazetteers, almanacs, climatologies, operational biogeochemical models, and satellite observations. The quality of the different environmental measures has been validated not only by various quality control steps, but also through a global analysis allowing the comparison with known environmental large-scale structures. Such publicly released datasets open the perspective to address a wide range of scientific questions.

Viruses are the most abundant biological entity in the ocean and infect a wide range of microbial life across bacteria, archaea, and eukaryotes. In this essay, we take a journey across several orders of magnitude in the scales of biological organization, time, and space of host-virus interactions in the ocean, aiming to shed light on their ecological relevance. We start from viruses infecting microbial host cells by delivering their genetic material in seconds across nanometer-size membranes, which highjack their host's metabolism in a few minutes to hours, leading to a profound transcriptomic and metabolic rewiring. The outcome of lytic infection leads to a release of virions and signaling molecules that can reach neighboring cells a few millimeters away, resulting in a population whose heterogeneous infection level impacts the surrounding community for days. These population dynamics can leave unique metabolic and biogeochemical fingerprints across scales of kilometers and over several decades. One of the biggest challenges in marine microbiology is to assess the impact of viruses across these scales, from the single cell to the ecosystem level. Here, we argue that the advent of new methodologies and conceptual frameworks represents an exciting time to pursue these efforts and propose a set of important challenges for the field. A better understanding of host-virus interactions across scales will inform models of global ocean ecosystem function in different climate change scenarios.

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.

The diversity of microbes and their transmission between ocean and atmosphere are poorly understood despite the implications for microbial global dispersion and biogeochemical processes. Here, we survey the genetic diversity of airborne and surface ocean bacterial communities sampled during springtime transects across the northwest Pacific and subtropical north Atlantic as part of the Tara Pacific Expedition. We find that microbial community composition is more variable in the atmosphere than in the surface ocean. Bacterial communities were more similar between the two surface oceans than between the ocean and the overlying atmosphere. Likewise, Pacific and Atlantic atmospheric microbial communities were more similar to each other than to those in the ocean beneath. Atmospheric community composition over the Atlantic was dominated by terrestrial and specifically, dust-associated bacteria, whereas over the Pacific there was a higher prevalence and differential abundance of marine bacteria. Our findings highlight regional differences in long-range microbial exchange and dispersal between land, ocean, and atmosphere.

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.

One-quarter of photosynthesis-derived carbon on Earth rapidly cycles through a set of short-lived seawater metabolites that are generated from the activities of marine phytoplankton, bacteria, grazers and viruses. Here we discuss the sources of microbial metabolites in the surface ocean, their roles in ecology and biogeochemistry, and approaches that can be used to analyse them from chemistry, biology, modelling and data science. Although microbial-derived metabolites account for only a minor fraction of the total reservoir of marine dissolved organic carbon, their flux and fate underpins the central role of the ocean in sustaining life on Earth.

Diatoms are frequently exposed to diverse biotic and abiotic stressors, and they rely on sophisticated sensing mechanisms to perceive and respond accordingly to fluctuations in their environment. In order to convert diverse environmental cues into activation of specific signal transduction pathways, diatoms utilize an array of second messengers such as reactive oxygen species (ROS), nitric oxide (NO), calcium (Ca2+), and cyclic nucleotides (cAMP and cGMP). Specificity in signal transduction is achieved by precise spatiotemporal control of the level of second messengers, the specific combination of these molecules, and their available cellular targets. In addition to second messengers, diatoms utilize other chemical cues that are exuded by conspecifics and other organisms in their microenvironment. These molecules, named infochemicals, convey an essential information on the producer physiological state, and serve as a chemical language that mediates marine microbial interactions. These infochemicals have specific bioactivities, and some are toxic to competitors, grazers, or pathogens. The outcomes of this chemical communication highly depend on the context, and the same signal may lead to different cell fates. In some cases, chemical cues can even initiate a programmed cell death (PCD) cascade, which was suggested to contribute to the high turnover of phytoplankton and to be involved in bloom demise. In this chapter, we discuss the current knowledge regarding diatom extracellular and intracellular signaling, mainly during biotic interactions. In addition, we discuss how cellular processes that occur at the single-cell level may alter population dynamics and even large-scale biogeochemical processes. Finally, we propose future research directions that will shed light on the ecological importance of chemical communication in diatoms.

Sea spray aerosol (SSA) formation have a major role in the climate system, but measurements at a global-scale of this micro-scale process are highly challenging. We measured high-resolution temporal patterns of SSA number concentration over the Atlantic Ocean, Caribbean Sea, and the Pacific Ocean covering over 42,000 km. We discovered a ubiquitous 24-hour rhythm to the SSA number concentration, with concentrations increasing after sunrise, remaining higher during the day, and returning to predawn values after sunset. The presence of dominating continental aerosol transport can mask the SSA cycle. We did not find significant links between the diel cycle of SSA number concentration and diel variations of surface winds, atmospheric physical properties, radiation, pollution, nor oceanic physical properties. However, the daily mean sea surface temperature positively correlated with the magnitude of the day-to-nighttime increase in SSA concentration. Parallel diel patterns in particle sizes were also detected in near-surface waters attributed to variations in the size of particles smaller than ~1 µm. These variations may point to microbial day-tonight modulation of bubble-bursting dynamics as a possible cause of the SSA cycle.

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.

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 predatorprey 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 zooplanktonalgae 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 preypredator 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.

Emiliania huxleyi is a cosmopolitan coccolithophore widespread in temperate oceans. This unicellular photoautotroph forms massive recurring blooms that play an important role in large biogeochemical cycles of carbon and sulfur, which play a role in climate change. The mechanism of bloom formation and demise, controlled by giant viruses that routinely infect these blooms, is poorly understood. We generated a pan-transcriptome of E. huxleyi, derived from three strains with different susceptibility to viral infection. Expression profiling of E. huxleyi sensitive and resistant strains showed major basal differences, including many genes that are induced upon viral infection. This suggests that basal gene expression can affect the host metabolic state and the susceptibility of E. huxleyi to viruses. Due to its ecological importance, the pan-transcriptome and its protein translation, applicable to many E. huxleyi strains, is a powerful resource for investigation of eukaryotic microbial communities.

Programmed cell death (PCD) in marine microalgae was suggested to be one of the mechanisms that facilitates bloom demise, yet its molecular components in phytoplankton are unknown. Phytoplankton are completely lacking any of the canonical components of PCD, such as caspases, but possess metacaspases. Metacaspases were shown to regulate PCD in plants and some protists, but their roles in algae and other organisms are still elusive. Here, we identified and biochemically characterized a type III metacaspase from the model diatom Phaeodactylum tricornutum, termed PtMCA-IIIc. Through expression of recombinant PtMCA-IIIc in E. coli, we revealed that PtMCA-IIIc exhibits a calcium-dependent protease activity, including auto-processing and cleavage after arginine. Similar metacaspase activity was detected in P. tricornutum cell extracts. PtMCA-IIIc overexpressing cells exhibited higher metacaspase activity, while CRISPR/Cas9-mediated knockout cells had decreased metacaspase activity compared to WT cells. Site-directed mutagenesis of cysteines that were predicted to form a disulfide bond decreased recombinant PtMCA-IIIc activity, suggesting its enhancement under oxidizing conditions. One of those cysteines was oxidized, detected in redox proteomics, specifically in response to lethal concentrations of hydrogen peroxide and a diatom derived aldehyde. Phylogenetic analysis revealed that this cysteine-pair is unique and widespread among diatom type III metacaspases. The characterization of a cell death associated protein in diatoms provides insights into the evolutionary origins of PCD and its ecological significance in algal bloom dynamics.

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 \u201cviral shunt.\u201d 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-iodinecontaining 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.

To improve our understanding of coral infection and disease, it is important to study host-pathogen interactions at relevant spatio-temporal scales. Here, we provide a dynamic microscopic view of the interaction between a coral pathogen, Vibrio coralliilyticus and its coral host Pocillopora damicornis. This was achieved using a microfluidics-based system facilitating control over flow, light and temperature conditions. Combined with time-resolved biochemical and microbial analyses of the system exudates, this approach provides novel insights into the early phases of a coral infection at unprecedented spatio-temporal resolution. We provide evidence that infection may occur through ingestion of the pathogen by the coral polyps, or following pathogen colonization of small tissue lesions on the coral surface. Pathogen ingestion invariably induced the release of pathogen-laden mucus from the gastrovascular cavity. Despite the high bacterial load used in our experiments, approximately one-third of coral fragments tested did not develop further symptoms. In the remaining two-thirds, mucus spewing was followed by the severing of calicoblastic connective tissues (coenosarc) and subsequently necrosis of most polyps. Despite extensive damage to symptomatic colonies, we frequently observed survival of individual polyps, often accompanied by polyp bail-out. Biochemical and microbial analyses of exudates over the course of symptomatic infections revealed that severing of the coenosarc was followed by an increase in matrix metaloprotease activity, and subsequent increase in both pathogen and total bacterial counts. Combined, these observations provide a detailed description of a coral infection, bringing us a step closer to elucidating the complex interactions underlying coral disease.

Turbulence is an important determinant of phytoplankton physiology, often leading to cell stress and damage. Turbulence affects phytoplankton migration both by transporting cells and by triggering switches in migratory behavior, whereby vertically migrating cells can actively invert their direction of migration upon exposure to turbulent cues. However, a mechanistic link between single-cell physiology and vertical migration of phytoplankton in turbulence is currently missing. Here, by combining physiological and behavioral experiments with a mathematical model of stress accumulation and dissipation, we show that the mechanism responsible for the switch in the direction of migration in the marine raphidophyte Heterosigma akashiwo is the integration of reactive oxygen species (ROS) signaling generated by turbulent cues. Within timescales as short as tens of seconds, the emergent downward-migrating subpopulation exhibited a twofold increase in ROS, an indicator of stress, 15% lower photosynthetic efficiency, and 35% lower growth rate over multiple generations compared to the upward-migrating subpopulation. The origin of the behavioral split as a result of a bistable oxidative stress response is corroborated by the observation that exposure of cells to exogenous stressors (H₂O₂, UV-A radiation, or high irradiance), in lieu of turbulence, caused comparable ROS accumulation and an equivalent split into the two subpopulations. By providing a mechanistic link between the single-cell mechanics of swimming and physiology on the one side and the emergent population-scale migratory response and impact on fitness on the other, the ROS-mediated early warning response we discovered contributes to our understanding of phytoplankton community composition in future ocean conditions.

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.

Aerosol size distribution has major effects on warm cloud processes. Here, we use newly acquired marine aerosol size distributions (MSDs), measured in situ over the open ocean during the Tara Pacific expedition (2016-2018), to examine how the total aerosol concentration (Ntot) and the shape of the MSDs change warm clouds' properties. For this, we used a toy model with detailed bin microphysics initialized using three different atmospheric profiles, supporting the formation of shallow to intermediate and deeper warm clouds. The changes in the MSDs affected the clouds' total mass and surface precipitation. In general, the clouds showed higher sensitivity to changes in Ntot than to changes in the MSD's shape, except for the case where the MSD contained giant and ultragiant cloud condensation nuclei (GCCN, UGCCN). For increased Ntot (for the deep and intermediate profiles), most of the MSDs drove an expected non-monotonic trend of mass and precipitation (the shallow clouds showed only the decreasing part of the curves with mass and precipitation monotonically decreasing). The addition of GCCN and UGCCN drastically changed the non-monotonic trend, such that surface rain saturated and the mass monotonically increased with Ntot. GCCN and UGCCN changed the interplay between the microphysical processes by triggering an early initiation of collision-coalescence. The early fallout of drizzle in those cases enhanced the evaporation below the cloud base. Testing the sensitivity of rain yield to GCCN and UGCCN revealed an enhancement of surface rain upon the addition of larger particles to the MSD, up to a certain particle size, when the addition of larger particles resulted in rain suppression. This finding suggests a physical lower bound can be defined for the size ranges of GCCN and UGCCN.

Phytoplankton play a central role in the regulation of global carbon and nutrient cycles, forming the basis of the marine food webs. A group of biogeochemically important phytoplankton, the coccolithophores, produce calcium carbonate scales that have been hypothesized to deter or reduce grazing by microzooplankton. Here, a meta-analysis of mesocosm-based experiments demonstrates that calcification of the cosmopolitan coccolithophore, Emiliania huxleyi, fails to deter microzooplankton grazing. The median grazing to growth ratio for E. huxleyi (0.56 ± 0.40) was not significantly different among non-calcified nano- or picoeukaryotes (0.71 ± 0.31 and 0.55 ± 0.34, respectively). Additionally, the environmental concentration of E. huxleyi did not drive preferential grazing of non-calcified groups. These results strongly suggest that the possession of coccoliths does not provide E. huxleyi effective protection from microzooplankton grazing. Such indiscriminate consumption has implications for the dissolution and fate of CaCO₃ in the ocean, and the evolution of coccoliths.

Anthropogenic pollution from marine microplastic particles is a growing concern, both as a source of toxic compounds, and because they can transport pathogens and other pollutants. Airborne microplastic particles were previously observed over terrestrial and coastal locations, but not in the remote ocean. Here, we collected ambient aerosol samples in the North Atlantic Ocean, including the remote marine atmosphere, during the Tara Pacific expedition in May-June 2016, and chemically characterized them using micro-Raman spectroscopy. We detected a range of airborne microplastics, including polystyrene, polyethylene, polypropylene, and poly-silicone compounds. Polyethylene and polypropylene were also found in seawater, suggesting local production of airborne microplastic particles. Terminal velocity estimations and back trajectory analysis support this conclusion. For technical reasons, only particles larger than 5µm, at the upper end of a typical marine atmospheric size distribution, were analyzed, suggesting that our analyses underestimate the presence of airborne microplastic particles in the remote marine atmosphere.

What are algal blooms? Algal blooms are commonly defined as ephemeral events of rapid proliferation of phytoplankton, leading to the formation of dense assemblages in diverse aquatic ecosystems, including freshwater rivers and lakes, polar regions, coastal waters and open oceans. They are dominated by one or more species of phytoplankton, which are single-celled eukaryotic and prokaryotic photosynthetic microorganisms that form the basis of aquatic food webs. An algal bloom can reach a concentration of tens of thousands of cells per liter, and can vary in duration, from days to weeks, and in scale, from tens to thousands of square kilometers. Blooms often cause a change in the color of water, owing to the chlorophyll and accessory pigments that are present in photosynthetic organisms or due to other cellular components. This optical property enables detection of large-scale blooms from space by satellites, allowing quantification of blooms across the globe all year long (Figure 1A).

Marine aerosols play a significant role in the global radiative budget, in clouds' processes, and in the chemistry of the marine atmosphere. There is a critical need to better understand their production mechanisms, composition, chemical properties, and the contribution of ocean-derived biogenic matter to their mass and number concentration. Here we present an overview of a new dataset of in situ measurements of marine aerosols conducted over the 2.5-yr Tara Pacific Expedition over 110,000 km across the Atlantic and Pacific Oceans. Preliminary results are presented here to describe the new dataset that will be built using this novel set of measurements. It will characterize marine aerosols properties in detail and will open a new window to study the marine aerosol link to the water properties and environmental conditions.

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.

Diatom blooms are important features of productive marine ecosystems and are known to support higher trophic levels. However, when stressed or wounded, diatoms can produce oxylipin molecules known to inhibit the reproduction and development of copepods and decrease microzooplankton growth rates. Using oxylipin chemical treatments, lipidomic analysis and functional genomic approaches, we provide evidence that nitric oxide (NO) and oxylipin signalling pathways in diatoms respond to protist grazers, resulting in increased defence fitness and survival. Exposure of the diatom Phaeodactylum tricornutum to the dinoflagellate Oxyrrhis marina resulted in NO production by P. tricornutum and pronounced change in its dissolved oxylipin profile. Experimentally elevating levels of NO also resulted in increased oxylipin production, and lower overall grazing rates. Furthermore, O. marina preferentially grazed on P. tricornutum prey with lower levels of NO, suggesting that this molecule and its effect on oxylipin pathways play a key role in prey selection. Exposure of O. marina grazing on P. tricornutum to exogenous oxylipins also decreased grazing rates, which is consistent with a grazing deterrence role for these molecules. These results suggest that NO and oxylipin production help to structure diatom communities, in part by modulating interactions with microzooplankton predators.

Interactions between the ocean and the atmosphere occur at the air-sea interface through the transfer of momentum, heat, gases and particulate matter, and through the impact of the upper-ocean biology on the composition and radiative properties of this boundary layer. The Tara Pacific expedition, launched in May 2016 aboard the schooner Tara, was a 29-month exploration with the dual goals to study the ecology of reef ecosystems along ecological gradients in the Pacific Ocean and to assess inter-island and open ocean surface plankton and neuston community structures. In addition, key atmospheric properties were measured to study links between the two boundary layer properties. A major challenge for the open ocean sampling was the lack of ship-time available for work at "stations". The time constraint led us to develop new underway sampling approaches to optimize physical, chemical, optical, and genomic methods to capture the entire community structure of the surface layers, from viruses to metazoans in their oceanographic and atmospheric physicochemical context. An international scientific consortium was put together to analyze the samples, generate data, and develop datasets in coherence with the existing Tara Oceans database. Beyond adapting the extensive Tara Oceans sampling protocols for high-resolution underway sampling, the key novelties compared to Tara Oceans' global assessment of plankton include the measurement of (i) surface plankton and neuston biogeography and functional diversity; (ii) bioactive trace metals distribution at the ocean surface and metal-dependent ecosystem structures; (iii) marine aerosols, including biological entities; (iv) geography, nature and colonization of microplastic; and (v) high-resolution underway assessment of net community production via equilibrator inlet mass spectrometry. We are committed to share the data collected during this expedition, making it an important resource important resource to address a variety of scientific questions.

Coral reefs are the most diverse habitats in the marine realm. Their productivity, structural complexity, and biodiversity critically depend on ecosystem services provided by corals that are threatened because of climate change effects-in particular, ocean warming and acidification. The coral holobiont is composed of the coral animal host, endosymbiotic dinoflagellates, associated viruses, bacteria, and other microeukaryotes. In particular, the mandatory photosymbiosis with microalgae of the family Symbiodiniaceae and its consequences on the evolution, physiology, and stress resilience of the coral holobiont have yet to be fully elucidated. The functioning of the holobiont as a whole is largely unknown, although bacteria and viruses are presumed to play roles in metabolic interactions, immunity, and stress tolerance. In the context of climate change and anthropogenic threats on coral reef ecosystems, the Tara Pacific project aims to provide a baseline of the "-omics" complexity of the coral holobiont and its ecosystem across the Pacific Ocean and for various oceanographically distinct defined areas. Inspired by the previous Tara Oceans expeditions, the Tara Pacific expedition (2016-2018) has applied a pan-ecosystemic approach on coral reefs throughout the Pacific Ocean, drawing an east-west transect from Panama to Papua New Guinea and a south-north transect from Australia to Japan, sampling corals throughout 32 island systems with local replicates. Tara Pacific has developed and applied state-of-the-art technologies in very-high-throughput genetic sequencing and molecular analysis to reveal the entire microbial and chemical diversity as well as functional traits associated with coral holobionts, together with various measures on environmental forcing. This ambitious project aims at revealing a massive amount of novel biodiversity, shedding light on the complex links between genomes, transcriptomes, metabolomes, organisms, and ecosystem functions in coral reefs and providing a reference of the biological state of modern coral reefs in the Anthropocene.

Diatoms are photosynthetic microorganisms of great ecological and biogeochemical importance, forming vast blooms in aquatic ecosystems. However, we are still lacking fundamental understanding of how individual cells sense and respond to diverse stress conditions, and what acclimation strategies are employed during bloom dynamics. We investigated cellular responses to environmental stress at the single-cell level using the redox sensor roGFP targeted to various organelles in the diatom Phaeodactylum tricornutum. We detected cell-to-cell variability using flow cytometry cell sorting and a microfluidics system for live imaging of oxidation dynamics. Chloroplast-targeted roGFP exhibited a light-dependent, bi-stable oxidation pattern in response to H2O2 and high light, revealing distinct subpopulations of sensitive oxidized cells and resilient reduced cells. Early oxidation in the chloroplast preceded commitment to cell death, and can be used for sensing stress cues and regulating cell fate. We propose that light-dependent metabolic heterogeneity regulates diatoms' sensitivity to environmental stressors in the ocean.

Infection by large dsDNA viruses can lead to a profound alteration of host transcriptome and metabolome in order to provide essential building blocks to support the high metabolic demand for viral assembly and egress. Host response to viral infection can typically lead to diverse phenotypic outcome that include shift in host life cycle and activation of anti-viral defense response. Nevertheless, there is a major bottleneck to discern between viral hijacking strategies and host defense responses when averaging bulk population response. Here we study the interaction between Emiliania huxleyi, a bloom-forming alga, and its specific virus (EhV), an ecologically important host-virus model system in the ocean. We quantified host and virus gene expression on a single-cell resolution during the course of infection, using automatic microfluidic setup that captures individual algal cells and multiplex quantitate PCR. We revealed high heterogeneity in viral gene expression among individual cells. Simultaneous measurements of expression profiles of host and virus genes at a single-cell level allowed mapping of infected cells into newly defined infection states and allowed detection specific host response in a subpopulation of infected cell which otherwise masked by the majority of the infected population. Intriguingly, resistant cells emerged during viral infection, showed unique expression profiles of metabolic genes which can provide the basis for discerning between viral resistant and susceptible cells within heterogeneous populations in the marine environment. We propose that resolving host-virus arms race at a single-cell level will provide important mechanistic insights into viral life cycles and will uncover host defense strategies.Author summary Almost all of our current understanding of the molecular mechanisms that govern host-pathogen interactions in the ocean is derived from experiments carried out at the population level, neglecting any heterogeneity. Here we used a single cell approach to unmask the phenotypic heterogeneity produced within infected populations of the cosmopolitan bloom-forming alga Emiliania huxleyi by its specific lytic virus. We found high variability in expression of viral genes among individual cells. This heterogeneity was used to map cells into their infection state and allowed to uncover a yet unrecognized host response. We also provide evidence that variability in host metabolic states provided a sensitive tool to decipher between susceptible and resistant cells.

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.

Marine bacteria contribute substantially to nutrient cycling in the oceans and can engage in close interactions with microalgae. Many microalgae harbor characteristic satellite bacteria, many of which participate in N-acyl homoserine lactone (AHL) mediated quorum sensing. In the diffusion-controlled phycosphere, AHLs can reach high local concentrations, with some of them transforming into tetramic acids, compounds with a broad bioactivity. We tested a representative AHL, N-(3-oxododecanoyl) homoserine lactone, and its tetramic acid rearrangement product on the diatom Phaeodactylum tricornutum. While cell growth and photosynthetic efficiency of photosystem II were barely affected by the AHL, exposure to its tetramic acid rearrangement product had a negative effect on photosynthetic efficiency and led to growth inhibition and cell death in the long term, with a minimum inhibitory concentration between 20 and 50 mu M. These results strengthen the view that AHLs may play an important role in shaping the outcome of microalgae-bacteria interactions.

A Rhodobacterales bacterium, Sulfitobacter sp. strain D7, was isolated from an Emiliania huxleyi bloom in the North Atlantic and has been shown to act as a pathogen and induce cell death of E. huxleyi during lab coculturing. We report here its complete genome sequence comprising one chromosome and five low-copy-number plasmids.

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.

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides.'' Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage.

Sea spray aerosols (SSA), have a profound effect on the climate; however, the contribution of oceanic microbial activity to SSA is not fully established. We assessed aerosolization of the calcite units (coccoliths) that compose the exoskeleton of the cosmopolitan bloom-forming coccolithophore, Emiliania huxleyi. Airborne coccolith emission occurs in steady-state conditions and increases by an order of magnitude during E. huxleyi infection by E. huxleyi virus (EhV). Airborne to seawater coccolith ratio is 1:10⁸, providing estimation of airborne concentrations from seawater concentrations. The coccoliths' unique aerodynamic structure yields a characteristic settling velocity of ∼0.01 cm s^-1, ∼25 times slower than average sea salt particles, resulting in coccolith fraction enrichment in the air. The calculated enrichment was established experimentally, indicating that coccoliths may be key contributors to coarse mode SSA surface area, comparable with sea salt aerosols. This study suggests a coupling between key oceanic microbial interactions and fundamental atmospheric processes like SSA formation.

The complex structures and morphologies of coral skeletons make it difficult to study the construction of individual skeleton components. This is especially true regarding micro-structures deposited during different stages of skeletogenesis. As such structures serve as the basis for subsequent skeleton development, they are often obscured by additional skeletal deposition and growth. Recently we described a new model system, coral-on-a-chip, for studying coral biology, and skeletogenesis. This system utilizes micropropagates of the Indo-Pacific coral Pocillopora damicornis maintained within a microfluidic environment, allowing us to follow early stages of skeletogenesis over time and under controlled environmental conditions. Our findings reveal that, following settlement onto glass slides, the micropropagates initially form a thin, almost two dimensional skeleton, which subsequently develops into a robust three-dimensional form with features resembling those of the mother colony. Studying the early stages of skeleton accretion in our micropropagates using high-resolution scanning electron microscopy and Energy Dispersive X-ray Spectroscopy (EDS) revealed a magnesium-rich layer deposited directly on the glass surface. This layer, which has a typical Mg/Ca ratio of 1.43 +/- 0.78, forms a dense lattice with a typical pore size of 100 nm. Microscopic observations indicate that this lattice serves as the basis for subsequent growth of fibrous aragonite. Examination of the underside of a skeleton from a small P. damicornis colony growing on a glass surface revealed a similar high-magnesium lattice at the interface between the glass and aragonitic skeleton in association with fibrous aragonite deposition. These observations suggest a role for this magnesium-rich lattice in the deposition of the fibrous aragonite forming the bulk of the coral skeleton.

Diatoms are one of the key phytoplankton groups in the ocean, forming vast oceanic blooms and playing a significant part in global primary production. To shed light on the role of redox metabolism in diatom's acclimation to light-dark transition and its interplay with cell fate regulation, we generated transgenic lines of the diatom Thalassiosira pseudonana that express the redox-sensitive green fluorescent protein targeted to various subcellular organelles. We detected organelle-specific redox patterns in response to oxidative stress, indicating compartmentalized antioxidant capacities. Monitoring the GSH redox potential (E-GSH) in the chloroplast over diurnal cycles revealed distinct rhythmic patterns. Intriguingly, in the dark, cells exhibited reduced basal chloroplast E-GSH but higher sensitivity to oxidative stress than cells in the light. This dark-dependent sensitivity to oxidative stress was a result of a depleted pool of reduced glutathione which accumulated during the light period. Interestingly, reduction in the chloroplast E-GSH was observed in the light phase prior to the transition to darkness, suggesting an anticipatory phase. Rapid chloroplast E-GSH re-oxidation was observed upon re-illumination, signifying an induction of an oxidative signaling during transition to light that may regulate downstream metabolic processes. Since light-dark transitions can dictate metabolic capabilities and susceptibility to a range of environmental stress conditions, deepening our understanding of the molecular components mediating the light-dependent redox signals may provide novel insights into cell fate regulation and its impact on oceanic bloom successions.

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 trophiclevel interactions. We propose functions and ecological implications of communication via EVs in aquatic microbial ecosystems.

Marine phytoplankton account for approximately half of global primary productivity(1), making their fate an important driver of the marine carbon cycle. Viruses are thought to recycle more than one-quarter of oceanic photosynthetically fixed organic carbon(2), which can stimulate nutrient regeneration, primary production and upper ocean respiration(2) via lytic infection and the 'virus shunt'. Ultimately, this limits the trophic transfer of carbon and energy to both higher food webs and the deep ocean(2). Using imagery taken by the Moderate Resolution Imaging Spectroradiometer (MODIS) onboard the Aqua satellite, along with a suite of diagnostic lipid- and gene-based molecular biomarkers, in situ optical sensors and sediment traps, we show that Coccolithovirus infections of mesoscale (similar to 100 km) Emiliania huxleyi blooms in the North Atlantic are coupled with particle aggregation, high zooplankton grazing and greater downward vertical fluxes of both particulate organic and particulate inorganic carbon from the upper mixed layer. Our analyses captured blooms in different phases of infection (early, late and post) and revealed the highest export flux in 'early-infected blooms' with sinking particles being disproportionately enriched with infected cells and subsequently remineralized at depth in the mesopelagic. Our findings reveal viral infection as a previously unrecognized ecosystem process enhancing biological pump efficiency.

Background: Global warming has triggered an increase in the prevalence and severity of coral disease, yet little is known about coral/pathogen interactions in the early stages of infection. The point of entry of the pathogen and the route that they take once inside the polyp is currently unknown, as is the coral's capacity to respond to infection. To address these questions, we developed a novel method that combines stable isotope labelling and microfluidics with transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS), to monitor the infection process between Pocillopora damicornis and Vibrio coralliilyticus under elevated temperature.Results: Three coral fragments were inoculated with N-15-labeled V. coralliilyticus and then fixed at 2.5, 6 and 22 h post-inoculation (hpi) according to the virulence of the infection. Correlative TEM/NanoSIMS imaging was subsequently used to visualize the penetration and dispersal of V. coralliilyticus and their degradation or secretion products. Most of the V. coralliilyticus cells we observed were located in the oral epidermis of the fragment that experienced the most virulent infection (2.5 hpi). In some cases, these bacteria were enclosed within electron dense host-derived intracellular vesicles. N-15-enriched pathogen-derived breakdown products were visible in all tissue layers of the coral polyp (oral epidermis, oral gastrodermis, aboral gastrodermis), at all time points, although the relative N-15-enrichment depended on the time at which the corals were fixed. Tissues in the mesentery filaments had the highest density of N-15-enriched hotspots, suggesting these tissues act as a "collection and digestion" site for pathogenic bacteria. Closer examination of the sub-cellular structures associated with these N-15-hotspots revealed these to be host phagosomal and secretory cells/vesicles.Conclusions: This study provides a novel method for tracking bacterial infection dynamics at the levels of the tissue and single cell and takes the first steps towards understanding the complexities of infection at the microscale, which is a crucial step towards understanding how corals will fare under global warming.

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.

Recognizing the life cycle of an organism is key to understanding its biology and ecological impact. Emiliania huxleyi is a cosmopolitan marine microalga, which displays a poorly understood biphasic sexual life cycle comprised of a calcified diploid phase and a morphologically distinct biflagellate haploid phase. Diploid cells (2N) form large-scale blooms in the oceans, which are routinely terminated by specific lytic viruses (EhV). In contrast, haploid cells (1N) are resistant to EhV. Further evidence indicates that 1N cells may be produced during viral infection. A shift in morphology, driven by meiosis, could therefore constitute a mechanism for E. huxleyi cells to escape from EhV during blooms. This process has been metaphorically coined the 'Cheshire Cat' (CC) strategy. We tested this model in two E. huxleyi strains using a detailed assessment of morphological and ploidy-level variations as well as expression of gene markers for meiosis and the flagellate phenotype. We showed that following the CC model, production of resistant cells was triggered during infection. This led to the rise of a new subpopulation of cells in the two strains that morphologically resembled haploid cells and were resistant to EhV. However, ploidy-level analyses indicated that the new resistant cells were diploid or aneuploid. Thus, the CC strategy in E. huxleyi appears to be a life-phase switch mechanism involving morphological remodeling that is decoupled from meiosis. Our results highlight the adaptive significance of morphological plasticity mediating complex host-virus interactions in marine phytoplankton.

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.

Biotic interactions underlie lifes diversity and are the lynchpin to understanding its complexity and resilience within an ecological niche. Algal biologists have embraced this paradigm, and studies building on the explosive growth in omics and cell biology methods have facilitated the in-depth analysis of nonmodel organisms and communities from a variety of ecosystems. In turn, these advances have enabled a major revision of our understanding of the origin and evolution of photosynthesis in eukaryotes, bacterialalgal interactions, control of massive algal blooms in the ocean, and the maintenance and degradation of coral reefs. Here, we review some of the most exciting developments in the field of algal biotic interactions and identify challenges for scientists in the coming years. We foresee the development of an algal knowledgebase that integrates ecosystem-wide omics data and the development of molecular tools/resources to perform functional analyses of individuals in isolation and in populations. These assets will allow us to move beyond mechanistic studies of a single species towards understanding the interactions amongst algae and other organisms in both the laboratory and the field.

The redox-sensitive proteome (RSP) consists of protein thiols that undergo redox reactions, playing an important role in coordinating cellular processes. Here, we applied a large-scale phylogenomic reconstruction approach in the model diatom Phaeodactylum tricornutum to map the evolutionary origins of the eukaryotic RSP. The majority of P. tricornutum redox-sensitive cysteines (76%) is specific to eukaryotes, yet these are encoded in genes that are mostly of a prokaryotic origin (57%). Furthermore, we find a threefold enrichment in redox-sensitive cysteines in genes that were gained by endosymbiotic gene transfer during the primary plastid acquisition. The secondary endosymbiosis event coincides with frequent introduction of reactive cysteines into existing proteins. While the plastid acquisition imposed an increase in the production of reactive oxygen species, our results suggest that it was accompanied by significant expansion of the RSP, providing redox regulatory networks the ability to cope with fluctuating environmental conditions.

Spatial characteristics of phytoplankton blooms often reflect the horizontal transport properties of the oceanic turbulent flow in which they are embedded. Classically, bloom response to horizontal stirring is regarded in terms of generation of patchiness following large-scale bloom initiation. Here, using satellite observations from the North Pacific Subtropical Gyre and a simple ecosystem model, we show that the opposite scenario of turbulence dispersing and diluting fine-scale (similar to 1-100 km) nutrient-enriched water patches has the critical effect of regulating the dynamics of nutrients-phytoplankton-zooplankton ecosystems and enhancing accumulation of photosynthetic biomass in low-nutrient oceanic environments. A key factor in determining ecological and biogeochemical consequences of turbulent stirring is the horizontal dilution rate, which depends on the effective eddy diffusivity and surface area of the enriched patches. Implementation of the notion of horizontal dilution rate explains quantitatively plankton response to turbulence and improves our ability to represent ecological and biogeochemical processes in oligotrophic oceans.

Atmospheric dimethylsulfide (DMS) is massively produced in the oceans by bacteria, algae, and corals. To enable identification of DMS sources, we developed a potent mechanism-based inhibitor of the algal Alma dimethylsulfoniopropionate lyase family that does not inhibit known bacterial lyases. Its application to coral holobiont indicates that DMS originates from Alma lyase(s). This biochemical profiling may complement meta-genomics and transcriptomics to provide better understanding of the marine sulfur Cycle.

Diatoms are single-celled, photosynthetic, bloom-forming algae that are responsible for at least 20% of global primary production. Nevertheless, more than 30% of the oceans are considered "ocean deserts" due to iron limitation. We used the diatom Phaeodactylum tricornutum as a model system to explore diatom's response to iron limitation and its interplay with susceptibility to oxidative stress. By analyzing physiological parameters and proteome profiling, we defined two distinct phases: short-term (5 d, phase II) iron limitation. While at phase I no significant changes in physiological parameters were observed, molecular markers for iron starvation, such as Iron Starvation Induced Protein and flavodoxin, were highly up-regulated. At phase II, down-regulation of numerous iron-containing proteins was detected in parallel to reduction in growth rate, chlorophyll content, photosynthetic activity, respiration rate, and antioxidant capacity. Intriguingly, while application of oxidative stress to phase I and II iron-limited cells similarly oxidized the reduced glutathione (GSH) pool, phase II iron limitation exhibited transient resistance to oxidative stress, despite the down regulation of many antioxidant proteins. By comparing proteomic profiles of P. tricornutum under iron limitation and metatranscriptomic data of an iron enrichment experiment conducted in the Pacific Ocean, we propose that iron-limited cells in the natural environment resemble the phase II metabolic state. These results provide insights into the trade-off between optimal growth rate and susceptibility to oxidative stress in the response of diatoms to iron quota in the marine environment.

Marine viruses are considered to be major ecological, evolutionary, and biogeochemical drivers of the marine environment, responsible for nutrient recycling and determining species composition. Viruses can re-shape their host's metabolic network during infection, generating the virocell-a unique metabolic state that supports their specific requirement. Here we discuss the concept of 'virocell metabolism' and its formation by rewiring of host-encoded metabolic networks, or by introducing virus-encoded auxiliary metabolic genes which provide the virocell with novel metabolic capabilities. The ecological role of marine viruses is commonly assessed by their relative abundance and phylogenetic diversity, lacking the ability to assess the dynamics of active viral infection. The new ability to define a unique metabolic state of the virocell will expand the current virion-centric approaches in order to quantify the impact of marine viruses on microbial food webs.

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.

The cosmopolitan coccolithophore Emiliania huxleyi is a unicellular eukaryotic alga responsible for vast blooms in the ocean. These blooms have immense impact on large biogeochemical cycles and are terminated by a specific large double-stranded DNA E. huxleyi virus (EhV, Phycodnaviridae). EhV infection is accompanied by induction of hallmarks of programmed cell death and production of reactive oxygen species (ROS). Here we characterized alterations in ROS metabolism and explored its role during infection. Transcriptomic analysis of ROS-related genes predicted an increase in glutathione (GSH) and H₂O₂ production during infection. In accordance, using biochemical assays and specific fluorescent probes we demonstrated the overproduction of GSH during lytic infection. We also showed that H₂O₂ production, rather than superoxide, is the predominant ROS during the onset of the lytic phase of infection. Using flow cytometry, confocal microscopy and multispectral imaging flow cytometry, we showed that the profound co-production of H₂O₂ and GSH occurred in the same subpopulation of cells but at different subcellular localization. Positively stained cells for GSH and H₂O₂ were highly infected compared with negatively stained cells. Inhibition of ROS production by application of a peroxidase inhibitor or an H₂O₂ scavenger inhibited host cell death and reduced viral production. We conclude that viral infection induced remodeling of the host antioxidant network that is essential for a successful viral replication cycle. This study provides insight into viral replication strategy and suggests the use of specific cellular markers to identify and quantify the extent of active viral infection during E. huxleyi blooms in the ocean.

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 selectivemodeofmembrane 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.

Coral reefs, and the unique ecosystems they support, are facing severe threats by human activities and climate change. Our understanding of these threats is hampered by the lack of robust approaches for studying the micro-scale interactions between corals and their environment. Here we present an experimental platform, coral-on-a-chip, combining micropropagation and microfluidics to allow direct microscopic study of live coral polyps. The small and transparent coral micropropagates are ideally suited for live-imaging microscopy, while the microfluidic platform facilitates long-term visualization under controlled environmental conditions. We demonstrate the usefulness of this approach by imaging coral micropropagates at previously unattainable spatio-temporal resolutions, providing new insights into several micro-scale processes including coral calcification, coral-pathogen interaction and the loss of algal symbionts (coral bleaching). Coral-on-a-chip thus provides a powerful method for studying coral physiology in vivo at the micro-scale, opening new vistas in coral biology.

Marine viruses are the most abundant biological entities in the oceans shaping community structure and nutrient cycling. The interaction between the bloom-forming alga Emiliania huxleyi and its specific large dsDNA virus (EhV) is a major factor determining the fate of carbon in the ocean, thus serving as a key host-pathogen model system. The EhV genome encodes for a set of genes involved in the de novo sphingolipid biosynthesis, not reported in any viral genome to date. We combined detailed lipidomic and biochemical analyses to characterize the functional role of this virus-encoded pathway during lytic viral infection. We identified a major metabolic shift, mediated by differential substrate specificity of virus-encoded serine palmitoyltransferase, a key enzyme of sphingolipid biosynthesis. Consequently, unique viral glycosphingolipids, composed of unusual hydroxylated C17 sphingoid bases (t17:0) were highly enriched in the infected cells, and their synthesis was found to be essential for viral assembly. These findings uncover the biochemical bases of the virus-induced metabolic rewiring of the host sphingolipid biosynthesis during the chemical "arms race" in the ocean.

Marine viruses that infect phytoplankton strongly influence the ecology and evolution of their hosts. Emiliania huxleyi is characterized by a biphasic life cycle composed of a diploid (2N) and haploid (1N) phase; diploid cells are susceptible to infection by specific coccolithoviruses, yet haploid cells are resistant. Glycosphingolipids (GSLs) play a role during infection, but their molecular distribution in haploid cells is unknown. We present mass spectrometric analyses of lipids from cultures of uninfected diploid, infected diploid, and uninfected haploid E. huxleyi. Known viral GSLs were present in the infected diploid cultures as expected, but surprisingly, trace amounts of viral GSLs were also detected in the uninfected haploid cells. Sialic-acid GSLs have been linked to viral susceptibility in diploid cells, but were found to be absent in the haploid cultures, suggesting a mechanism of haploid resistance to infection. Additional untargeted high-resolution mass spectrometry data processed via multivariate analysis unveiled a number of novel biomarkers of infected, non-infected, and haploid cells. These data expand our understanding on the dynamics of lipid metabolism during E. huxleyi host/virus interactions and highlight potential novel biomarkers for infection, susceptibility, and ploidy.

Algal blooms produce large amounts of dimethyl sulfide (DMS), a volatile with a diverse signaling role in marine food webs that is emitted to the atmosphere, where it can affect cloud formation. The algal enzymes responsible for forming DMS from dimethylsulfoniopropionate (DMSP) remain unidentified despite their critical role in the global sulfur cycle. We identified and characterized Alma1, a DMSP lyase from the bloom-forming algae Emiliania huxleyi. Alma1 is a tetrameric, redox-sensitive enzyme of the aspartate racemase superfamily. Recombinant Alma1 exhibits biochemical features identical to the DMSP lyase in E. huxleyi, and DMS released by various E. huxleyi isolates correlates with their Alma1 levels. Sequence homology searches suggest that Alma1 represents a gene family present in major, globally distributed phytoplankton taxa and in other marine organisms.

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.

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 (E GSH) 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 E GSH 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 E GSH (-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.

Viruses infecting marine phytoplankton are key biogeochemical engines of the oceans, regulating the dynamics of algal populations and the fate of their extensive blooms. In addition they are important ecological and evolutionary drivers of microbial diversification. Yet, little is known about mechanisms influencing viral dispersal in aquatic systems, enabling the rapid infection and demise of vast phytoplankton blooms. In a recent study we showed that migrating zooplankton as copepods that graze on marine phytoplankton can act as transmission vectors for algal viruses. We demonstrated that these grazers can concentrate virions through topical adsorption and by ingesting infected cells and then releasing back to the medium, via detachment or defecation, high viral titers that readily infect host populations. We proposed that this zooplankton-driven process can potentially boost viral dispersal over wide oceanic scales and enhance bloom termination. Here, we highlight key results and further discuss the ecological and evolutionary consequences of our findings.

Aquatic photosynthetic eukaryotes represent highly diverse groups (green, red, and chromalveolate algae) derived from multiple endosymbiosis events, covering a wide spectrum of the tree of life. They are responsible for about 50% of the global photosynthesis and serve as the foundation for oceanic and fresh water food webs. Although the ecophysiology and molecular ecology of some algal species are extensively studied, some basic aspects of algal cell biology are still underexplored. The recent wealth of genomic resources from algae has opened new frontiers to decipher the role of cell signaling pathways and their function in an ecological and biotechnological context. Here, we took a bioinformatic approach to explore the distribution and conservation of TOR and autophagy-related (ATG) proteins (Atg in yeast) in diverse algal groups. Our genomic analysis demonstrates conservation of TOR and ATG proteins in green algae. In contrast, in all 5 available red algal genomes, we could not detect the sequences that encode for any of the 17 core ATG proteins examined, albeit TOR and its interacting proteins are conserved. This intriguing data suggests that the autophagy pathway is not conserved in red algae as it is in the entire eukaryote domain. In contrast, chromalveolates, despite being derived from the red-plastid lineage, retain and express ATG genes, which raises a fundamental question regarding the acquisition of ATG genes during algal evolution. Among chromalveolates, Emiliania huxleyi (Haptophyta), a bloom-forming coccolithophore, possesses the most complete set of ATG genes, and may serve as a model organism to study autophagy in marine protists with great ecological significance.

Marine photosynthetic microorganisms are the basis of marine food webs and are responsible for nearly 50% of the global primary production. Emiliania huxleyi forms massive oceanic blooms that are routinely terminated by large double-stranded DNA coccolithoviruses. The cellular mechanisms that govern the replication cycle of these giant viruses are largely unknown. We used diverse techniques, including fluorescence microscopy, transmission electron microscopy, cryoelectron tomography, immunolabeling and biochemical methodologies to investigate the role of autophagy in host-virus interactions. Hallmarks of autophagy are induced during the lytic phase of E.huxleyi viral infection, concomitant with up-regulation of autophagy-related genes (ATG genes). Pretreatment of the infected cells with an autophagy inhibitor causes a major reduction in the production of extracellular viral particles, without reducing viral DNA replication within the cell. The host-encoded Atg8 protein was detected within purified virions, demonstrating the pivotal role of the autophagy-like process in viral assembly and egress. We show that autophagy, which is classically considered as a defense mechanism, is essential for viral propagation and for facilitating a high burst size. This cellular mechanism may have a major impact on the fate of the viral-infected blooms, and therefore on the cycling of nutrients within the marine ecosystem. 10.1111/(ISSN)1469-8137

Summary Marine viruses are recognized as a major driving force regulating phytoplankton community composition and nutrient cycling in the oceans [1, 2]. Yet, little is known about mechanisms that influence viral dispersal in aquatic systems, other than physical processes, and that lead to the rapid demise of large-scale algal blooms in the oceans [3, 4]. Here, we show that copepods, abundant migrating crustaceans that graze on phytoplankton [5, 6], as well as other zooplankton can accumulate and mediate the transmission of viruses infecting Emiliania huxleyi, a bloom-forming coccolithophore that plays an important role in the carbon cycle [7, 8]. We detected by PCR that >80% of copepods collected during a North Atlantic E. huxleyi bloom carried E. huxleyi virus (EhV) DNA. We demonstrated by isolating a new infectious EhV strain from a copepod microbiome that these viruses are infectious. We further showed that EhVs can accumulate in high titers within zooplankton guts during feeding or can be adsorbed to their surface. Subsequently, EhV can be dispersed by detachment or via viral-dense fecal pellets over a period of 1 day postfeeding on EhV-infected algal cells, readily infecting new host populations. Intriguingly, the passage through zooplankton guts prolonged EhV's half-life of infectivity by 35%, relative to free virions in seawater, potentially enhancing viral transmission. We propose that zooplankton, swimming through topographically adjacent phytoplankton micropatches and migrating daily over large areas across physically separated water masses [9-11], can serve as viral vectors, boosting host-virus contact rates and potentially accelerating the demise of large-scale phytoplankton blooms.

Phytoplankton blooms are ephemeral events of exceptionally high primary productivity that regulate the flux of carbon across marine food webs [1-3]. Quantification of bloom turnover [4] is limited by a fundamental difficulty to decouple between physical and biological processes as observed by ocean color satellite data. This limitation hinders the quantification of bloom demise and its regulation by biological processes [5, 6], which has important consequences on the efficiency of the biological pump of carbon to the deep ocean [7-9]. Here, we address this challenge and quantify algal blooms turnover using a combination of satellite and in situ data, which allows identification of a relatively stable oceanic patch that is subject to little mixing with its surroundings. Using a newly developed multisatellite Lagrangian diagnostic, we decouple the contributions of physical and biological processes, allowing quantification of a complete life cycle of a mesoscale (w10-100 km) bloom of coccolithophores in the North Atlantic, from exponential growth to its rapid demise. We estimate the amount of organic carbon produced during the bloom to be in the order of 24,000 tons, of which two-thirds were turned over within 1 week. Complimentary in situ measurements of the same patch area revealed high levels of specific viruses infecting coccolithophore cells, therefore pointing at the importance of viral infection as a possible mortality agent. Application of the newly developed satellite-based approaches opens the way for large-scale quantification of the impact of diverse environmental stresses on the fate of phytoplankton blooms and derived carbon in the ocean.

Dimethyl sulfide (DMS) is produced in oceans in vast amounts (>10 ⁷ tons/year) and mediates a wide range of processes from regulating marine life forms to cloud formation. Nonetheless, none of the enzymes that produce DMS from dimethylsulfoniopropionate (DMSP) has been adequately characterized. We describe the expression and purification of DddD from the marine bacterium Marinomonas sp. MWYL1 and its biochemical characterization. We identified DMSP and acetyl-coenzyme A to be DddD's native substrates and Asp602 as the active site residue mediating the CoA-transferase prior to lyase activity. These findings shed light on the biochemical utilization of DMSP in the marine environment.

The exchange of nutrients and dissolved gasses between corals and their environment is a critical determinant of the growth of coral colonies and the productivity of coral reefs. To date, this exchange has been assumed to be limited by molecular diffusion through an unstirred boundary layer extending 1-2 mm from the coral surface, with corals relying solely on external flow to overcome this limitation. Here, we present direct microscopic evidence that, instead, corals can actively enhance mass transport through strong vortical flows driven by motile epidermal cilia covering their entire surface. Ciliary beating produces quasi-steady arrays of counterrotating vortices that vigorously stir a layer of water extending up to 2 mm from the coral surface. We show that, under low ambient flow velocities, these vortices, rather than molecular diffusion, control the exchange of nutrients and oxygen between the coral and its environment, enhancing mass transfer rates by up to 400%. This ability of corals to stir their boundary layer changes the way that we perceive the microenvironment of coral surfaces, revealing an active mechanism complementing the passive enhancement of transport by ambient flow. These findings extend our understanding of mass transport processes in reef corals and may shed new light on the evolutionary success of corals and coral reefs.

Marine viruses are major ecological and evolutionary drivers of microbial food webs regulating the fate of carbon in the ocean. We combined transcriptomic and metabolomic analyses to explore the cellular pathways mediating the interaction between the bloom-forming coccolithophore Emiliania huxleyi and its specific coccolithoviruses (E. huxleyi virus [EhV]). We show that EhV induces profound transcriptome remodeling targeted toward fatty acid synthesis to support viral assembly. A metabolic shift toward production of viral-derived sphingolipids was detected during infection and coincided with downregulation of host de novo sphingolipid genes and induction of the viral-encoded homologous pathway. The depletion of host-specific sterols during lytic infection and their detection in purified virions revealed their novel role in viral life cycle. We identify an essential function of the mevalonate-isoprenoid branch of sterol biosynthesis during infection and propose its downregulation as an antiviral mechanism. We demonstrate how viral replication depends on the hijacking of host lipid metabolism during the chemical "arms race" in the ocean.

Summary: Viruses play a key role in controlling the population dynamics of algae, including Emiliania huxleyi, a globally distributed haptophyte with calcite coccoliths that comprise ca. 50% of the sinking carbonate flux from the surface ocean. Emiliania huxleyi viruses (EhVs) routinely infect and terminate E.huxleyi blooms. EhVs are surrounded by a lipid envelope, which we found to be comprised largely of glycosphingolipids (GSLs) with lesser amounts of polar glycerolipids. Infection appears to involve membrane fusion between the virus and host, and we hypothesized that specific polar lipids may facilitate virus attachment. We identified three novel intact polar lipids in E.huxleyi strain CCMP 374 and EhV86, including a GSL with a monosaccharide sialic acid headgroup (sGSL); for all 11 E.huxleyi strains we tested, there was a direct relationship between sGSL content and sensitivity to infection by EhV1, EhV86 and EhV163. In mesocosms, the E.huxleyi population with greatest initial sGSL content had the highest rate of virus-induced mortality. We propose potential physiological roles for sGSL that would be beneficial for growth but leave cells susceptible to infection, thus furthering the discussion of Red Queen-based co-evolution and the cost(s) of sensitivity and resistance in the dynamic E.huxleyi-EhV system.

Background: The advent of Next Generation Sequencing technologies and corresponding bioinformatics tools allows the definition of transcriptomes in non-model organisms. Non-model organisms are of great ecological and biotechnological significance, and consequently the understanding of their unique metabolic pathways is essential. Several methods that integrate de novo assembly with genome-based assembly have been proposed. Yet, there are many open challenges in defining genes, particularly where genomes are not available or incomplete. Despite the large numbers of transcriptome assemblies that have been performed, quality control of the transcript building process, particularly on the protein level, is rarely performed if ever. To test and improve the quality of the automated transcriptome reconstruction, we used manually defined and curated genes, several of them experimentally validated.Results: Several approaches to transcript construction were utilized, based on the available data: a draft genome, high quality RNAseq reads, and ESTs. In order to maximize the contribution of the various data, we integrated methods including de novo and genome based assembly, as well as EST clustering. After each step a set of manually curated genes was used for quality assessment of the transcripts. The interplay between the automated pipeline and the quality control indicated which additional processes were required to improve the transcriptome reconstruction. We discovered that E. huxleyi has a very high percentage of non-canonical splice junctions, and relatively high rates of intron retention, which caused unique issues with the currently available tools. While individual tools missed genes and artificially joined overlapping transcripts, combining the results of several tools improved the completeness and quality considerably. The final collection, created from the integration of several quality control and improvement rounds, was compared to the manually defined set both on the DNA and protein levels, and resulted in an improvement of 20% versus any of the read-based approaches alone.Conclusions: To the best of our knowledge, this is the first time that an automated transcript definition is subjected to quality control using manually defined and curated genes and thereafter the process is improved. We recommend using a set of manually curated genes to troubleshoot transcriptome reconstruction.

Diatoms are ubiquitous marine photosynthetic eukaryotes responsible for approximately 20% of global photosynthesis. Little is known about the redox-based mechanisms that mediate diatom sensing and acclimation to environmental stress. Here we used a quantitative mass spectrometry-based approach to elucidate the redox-sensitive signaling network (redoxome) mediating the response of diatoms to oxidative stress. We quantified the degree of oxidation of 3,845 cysteines in the Phaeodactylum tricornutum proteome and identified approximately 300 redox-sensitive proteins. Intriguingly, we found redox-sensitive thiols in numerous enzymes composing the nitrogen assimilation pathway and the recently discovered diatom urea cycle. In agreement with this finding, the flux from nitrate into glutamine and glutamate, measured by the incorporation of 15N, was strongly inhibited under oxidative stress conditions. Furthermore, by targeting the redox-sensitive GFP sensor to various subcellular localizations, we mapped organellespecific oxidation patterns in response to variations in nitrogen quota and quality. We propose that redox regulation of nitrogen metabolism allows rapid metabolic plasticity to ensure cellular homeostasis, and thus is essential for the ecological success of diatoms in the marine ecosystem.

Using shipboard and satellite measurements we explore the environmental factors affecting the number concentration of aerosols with diameter 100

Marine viruses are major evolutionary and biogeochemical drivers in marine microbial foodwebs. However, an in-depth understanding of the cellular mechanisms and the signal transduction pathways mediating host-virus interactions during natural bloomdynamics has remained elusive. We used field-based mesocosms to examine the "arms race" between natural populations of the coccolithophore Emiliania huxleyi and its double-stranded DNA-containing coccolithoviruses (EhVs). Specifically,we examined the dynamics of EhV infection and its regulation of cell fate over the course of bloom development and demise using a diverse suite of molecular tools and in situ fluorescent staining to target different levels of subcellular resolution.We demonstrate the concomitant induction of reactive oxygen species, caspase-specific activity, metacaspase expression, and programmed cell death in response to the accumulation of virus-derived glycosphingolipids upon infection of natural E. huxleyi populations. These subcellular responses to viral infection simultaneously resulted in the enhanced production of transparent exopolymer particles,which can facilitate aggregation and stimulate carbon flux. Our results not only corroborate the critical role for glycosphingolipids and programmed cell death in regulating E. huxleyi-EhV interactions, but also elucidate promising molecular biomarkers and lipid-based proxies for phytoplankton host-virus interactions in natural systems.

Despite the critical importance of viruses in shaping marine microbial ecosystems and lubricating upper ocean biogeochemical cycles, relatively little is known about the molecular mechanisms mediating phytoplankton host-virus interactions. Recent work in algal host-virus systems has begun to shed novel insight into the elegant strategies of viral infection and subcellular regulation of cell fate, which not only reveal tantalizing aspects of viral replication and host resistance strategies but also provide new diagnostic tools toward elucidating the impact of virus-mediated processes in the ocean. Widespread lateral gene transfer between viruses and their hosts plays a prominent role in host-virus diversification and in the regulation of host-virus infection mechanisms by allowing viruses to manipulate and 'rewire' host metabolic pathways to facilitate infection.

Background: Diatoms represent the predominant group of eukaryotic phytoplankton in the oceans and are responsible for around 20% of global photosynthesis. Two whole genome sequences are now available. Notwithstanding, our knowledge of diatom biology remains limited because only around half of their genes can be ascribed a function based onhomology-based methods. High throughput tools are needed, therefore, to associate functions with diatom-specific genes.Results: We have performed a systematic analysis of 130,000 ESTs derived from Phaeodactylum tricornutum cells grown in 16 different conditions. These include different sources of nitrogen, different concentrations of carbon dioxide, silicate and iron, and abiotic stresses such as low temperature and low salinity. Based on unbiased statistical methods, we have catalogued transcripts with similar expression profiles and identified transcripts differentially expressed in response to specific treatments. Functional annotation of these transcripts provides insights into expression patterns of genes involved in various metabolic and regulatory pathways and into the roles of novel genes with unknown functions. Specific growth conditions could be associated with enhanced gene diversity, known gene product functions, and over-representation of novel transcripts. Comparative analysis of data from the other sequenced diatom, Thalassiosira pseudonana, helped identify several unique diatom genes that are specifically regulated under particular conditions, thus facilitating studies of gene function, genome annotation and the molecular basis of species diversity.Conclusions: The digital gene expression database represents a new resource for identifying candidate diatom-specific genes involved in processes of major ecological relevance.

Diatoms are the most successful group of eukaryotic phytoplankton in the modern ocean and have risen to dominance relatively quickly over the last 100 million years. Recently completed whole genome sequences from two species of diatom, Thalassiosira pseudonana and Phaeodactylum tricornutum, have revealed a wealth of information about the evolutionary origins and metabolic adaptations that have led to their ecological success. A major finding is that they have incorporated genes both from their endosymbiotic ancestors and by horizontal gene transfer from marine bacteria. This unique melting pot of genes encodes novel capacities for metabolic management, for example, allowing the integration of a urea cycle into a photosynthetic cell. In this review we show how genome-enabled approaches are being leveraged to explore major phenomena of oceanographic and biogeochemical relevance, such as nutrient assimilation and life histories in diatoms. We also discuss how diatoms may be affected by climate change-induced alterations in ocean processes.

Background: Transposable elements (TEs) are mobile DNA sequences present in the genomes of most organisms. They have been extensively studied in animals, fungi, and plants, and have been shown to have important functions in genome dynamics and species evolution. Recent genomic data can now enlarge the identification and study of TEs to other branches of the eukaryotic tree of life. Diatoms, which belong to the heterokont group, are unicellular eukaryotic algae responsible for around 40% of marine primary productivity. The genomes of a centric diatom, Thalassiosira pseudonana, and a pennate diatom, Phaeodactylum tricornutum, that likely diverged around 90 Mya, have recently become available.Results: In the present work, we establish that LTR retrotransposons (LTR-RTs) are the most abundant TEs inhabiting these genomes, with a much higher presence in the P. tricornutum genome. We show that the LTR-RTs found in diatoms form two new phylogenetic lineages that appear to be diatom specific and are also found in environmental samples taken from different oceans. Comparative expression analysis in P. tricornutum cells cultured under 16 different conditions demonstrate high levels of transcriptional activity of LTR retrotransposons in response to nitrate limitation and upon exposure to diatom-derived reactive aldehydes, which are known to induce stress responses and cell death. Regulatory aspects of P. tricornutum retrotransposon transcription also include the occurrence of nitrate limitation sensitive cis-regulatory components within LTR elements and cytosine methylation dynamics. Differential insertion patterns in different P. tricornutum accessions isolated from around the world infer the role of LTR-RTs in generating intraspecific genetic variability.Conclusion: Based on these findings we propose that LTR-RTs may have been important for promoting genome rearrangements in diatoms.

Marine viruses that infect phytoplankton are recognized as a major ecological and evolutionary driving force, shaping community structure and nutrient cycling in the marine environment. Little is known about the signal transduction pathways mediating viral infection. We show that viral glycosphingolipids regulate infection of Emiliania huxleyi, a cosmopolitan coccolithophore that plays a major role in the global carbon cycle. These sphingolipids derive from an unprecedented cluster of biosynthetic genes in Coccolithovirus genomes, are synthesized de novo during lytic infection, and are enriched in virion membranes. Purified glycosphingolipids induced biochemical hallmarks of programmed cell death in an uninfected host. These lipids were detected in coccolithophore populations in the North Atlantic, which highlights their potential as biomarkers for viral infection in the oceans.

The results of two published genome sequences from marine diatoms provide basic insights into how these remarkable organisms evolved to become one of the most successful groups of eukaryotic algae in the contemporary ocean.

Diatoms are photosynthetic secondary endosymbionts found throughout marine and freshwater environments, and are believed to be responsible for around one-fifth of the primary productivity on Earth. The genome sequence of the marine centric diatom Thalassiosira pseudonana was recently reported, revealing a wealth of information about diatom biology. Here we report the complete genome sequence of the pennate diatom Phaeodactylum tricornutum and compare it with that of T. pseudonana to clarify evolutionary origins, functional significance and ubiquity of these features throughout diatoms. In spite of the fact that the pennate and centric lineages have only been diverging for 90 million years, their genome structures are dramatically different and a substantial fraction of genes (

Marine photosynthetic microorganisms (phytoplankton) are the basis of marine foodwebs and are responsible for nearly 50% of the global annual carbon-based primary production.1 Phytoplankton can grow rapidly and form massive blooms that can be regulated by environmental factors such as nutrients and light availability and biotic interaction with grazers and viruses.2,3 Their crucial role in drawing down atmospheric CO2 and their potential use for future biofuel production4 raises the critical need for better understanding of fundamental features of their biology.5 Although traditionally phytoplankton were considered passive drifters with the currents (from Greek- \u201cPlanktos\u201d), our recent reports demonstrate how cells employ a complex mechanism to sense changes in environmental cues and activate chemical-based defense strategies.

Two monogalactosyl diacylglycerols, 1 and 2, were isolated from the marine diatom Phaeodactylum tricornutum, using the patented ApopScreen cell-based screen for apoptosis-inducing, potential anticancer compounds. The molecular structures of the galactolipids were determined using a combination of NMR, mass spectrometry, and chemical degradation. The bioactivities were confirmed using a specific apoptosis induction assay based on genetically engineered mammalian cell lines with differential, defined capacities for apoptosis. The galactolipids induce apoptosis in micromolar concentrations. This is the first report of apoptosis induction by galactolipids.

Diatoms are unicellular phytoplankton accounting for ∼40% of global marine primary productivity [1], yet the molecular mechanisms underlying their ecological success are largely unexplored. We use a functional-genomics approach in the marine diatom Phaeodactylum tricornutum to characterize a novel protein belonging to the widely conserved YqeH subfamily [2] of GTP-binding proteins thought to play a role in ribosome biogenesis [3], sporulation [4], and nitric oxide (NO) generation [5]. Transgenic diatoms overexpressing this gene, designated PtNOA, displayed higher NO production, reduced growth, impaired photosynthetic efficiency, and a reduced ability to adhere to surfaces. A fused YFP-PtNOA protein was plastid localized, distinguishing it from a mitochondria-localized plant ortholog. PtNOA was upregulated in response to the diatom-derived unsaturated aldehyde 2E,4E/Z-decadienal (DD), a molecule previously shown to regulate intercellular signaling, stress surveillance [6], and defense against grazers [7]. Overexpressing cell lines were hypersensitive to sublethal levels of this aldehyde, manifested by altered expression of superoxide dismutase and metacaspases, key components of stress and death pathways [8, 9]. NOA-like sequences were found in diverse oceanic regions, suggesting that a novel NO-based system operates in diatoms and may be widespread in phytoplankton, providing a biological context for NO in the upper ocean [10].

Estimates of phytoplankton sticking efficiency (α) were made in the laboratory within a 1500 1 annular flume mesocosm over the initiation, maintenance and senescence phases of a bloom of the diatom Thalassiosira pseudonana. The spatially weighted mean turbulence kinetic energy dissipation rate in the annular flume was comparable to values found at the ocean's surface on a calm day. The α of T. pseudonana varied as a result of physiological state, and ranged from 0 ± 0.08 during the bloom initiation to 0.26 and 0.73 ± 0.16, respectively, during bloom maintenance and senescence phases. During the periods of high α, physiological changes included (1) diminished phytoplankton photosynthetic quantum efficiency, (2) an increase in super-oxide dismutase protein expression, reflecting oxidative stress, and (3) the induction of a biochemical cascade initiating autocatalytic programmed cell death. Additionally, during the period of high physiological stress on the diatoms, there was an increase in the presence of transparent exopolymer particles and bacteria. Applying a variable α to a 1-dimensional export flux model shows that carbon export can be increased by at least 2-fold compared to simulations assuming a typically modeled value of α = 1 (100% sticking efficiency). The model using a physiologically dependent α had a low initial sticking efficiency that allowed a significant increase in the critical concentration of algal cells. Such an increase in the number of cells during bloom initiation followed by an increase in α during the maintenance and senescence phases resulted in enhanced export fluxes during the latter, 'stickier', stages of the bloom.

Diatoms are unicellular brown algae that likely arose from the endocytobiosis of a red alga into a single-celled heterotroph and that constitute an algal class of major importance in phytoplankton communities around the globe. The first whole-genome sequence from a diatom species, Thalassiosira pseudonana Hasle et Heimdal, was recently reported, and features that are central to diatom physiology and ecology, such as silicon and nitrogen metabolism, iron uptake, and carbon concentration mechanisms, were described. Following this initial study, the basic cellular systems controlling cell signaling, gene expression, cytoskeletal structures, and response to stress have been cataloged in an attempt to obtain a global view of the molecular foundations that sustain such an ecologically successful group of organisms. Comparative analysis with several microbial, plant, and metazoan complete genome sequences allowed the identification of putative membrane receptors, signaling proteins, and other components of central interest to diatom ecophysiology and evolution. Thalassiosira pseudonana likely perceives light through a novel phytochrome and several cryptochrome photoreceptors; it may lack the conserved RHO small-GTPase subfamily of cell-polarity regulators, despite undergoing polarized cell-wall synthesis; and it possesses an unusually large number of heat-shock transcription factors, which may indicate the central importance of transcriptional responses to environmental stress. The availability of the complete gene repertoire will permit a detailed biochemical and genetic analysis of how diatoms prosper in aquatic environments and will contribute to the understanding of eukaryotic evolution.

Microcystins constitute a serious threat to the quality of drinking water worldwide. These protein phosphatase inhibitors are formed by various cyanobacterial species, including Microcystis sp. Microcystins are produced by a complex microcystin synthetase, composed of peptide synthetases and polyketide synthases, encoded by the mcyA-J gene cluster. Recent phylogenetic analysis suggested that the microcystin synthetase predated the metazoan lineage, thus dismissing the possibility that microcystins emerged as a means of defence against grazing, and their original biological role is not clear. We show that lysis of Microcystis cells, either mechanically or because of various stress conditions, induced massive accumulation of McyB and enhanced the production of microcystins in the remaining Microcystis cells. A rise in McyB content was also observed following exposure to microcystin or the protease inhibitors micropeptin and microginin, also produced by Microcystis. The extent of the stimulation by cell extract was strongly affected by the age of the treated Microcystis culture. Older cultures, or those recently diluted from stock cultures, hardly responded to the components in the cell extract. We propose that lysis of a fraction of the Microcystis population is sensed by the rest of the cells because of the release of non-ribosomal peptides. The remaining cells respond by raising their ability to produce microcystins thereby enhancing their fitness in their ecological niche, because of their toxicity.

Regulated programmed cell death (PCD) processes have been documented in several phytoplankton species and are hypothesized to play a role in population dynamics. However, the mechanisms leading to the coordinated collapse of phytoplankton blooms are poorly understood. We showed that the collapse of the annual bloom of Peridinium gatunense, an abundant dinoflagellate in Lake Kinneret, Israel, is initiated by CO₂ limitation followed by oxidative stress that triggers a PCD-like cascade. We provide evidences that a protease excreted by senescing P. gatunense cells sensitizes younger cells to oxidative stress and may consequently trigger synchronized cell death of the population. Ageing of the P. gatunense cultures was characterized by a remarkable rise in DNA fragmentation and enhanced sensitivity to H ₂O₂. Exposure of logarithmic phase (young) cultures to conditioning media from stationary phase (old) cells sensitized them to H ₂O₂ and led to premature massive cell death. We detected the induction of specific extracellular protease activity, leupeptin-sensitive, in ageing cultures and in lake waters during the succession of the P. gatunense bloom. Partial purification of the conditioned media revealed that this protease activity is responsible for the higher susceptibility of young cells to oxidative stress. Inhibition of the protease activity lowered the sensitivity to oxidative stress, whereas application of papain to logarithmic phase P. gatunense cultures mimicked the effect of the spent media and enhanced cell death. We propose a novel mechanistic framework by which a population of unicellular phytoplankton orchestrates a coordinated response to stress, thereby determine the fate of its individuals.

Whole-genome sequence analysis has revealed that diatoms contain genes and pathways that are novel in photosynthetic eukaryotes. More generally, the unique evolutionary footprint of the chromalveolates, which includes a genome fusion between a heterotrophic protist and a red alga in addition to a major prokaryotic influence, has fostered their inheritance of a unique complement of metabolic capabilities. Many aspects of nitrogen metabolism and cell signaling appear to be linked in diatoms. This new perspective provides a basis for understanding the ecological dominance of diatoms in contemporary oceans.

Diatoms are an important group of eukaryotic phytoplankton, responsible for about 20% of global primary productivity. Study of the functional role of chemical signaling within phytoplankton assemblages is still in its infancy although recent reports in diatoms suggest the existence of chemical-based defense strategies. Here, we demonstrate how the accurate perception of diatom-derived reactive aldehydes can determine cell fate in diatoms. In particular, the aldehyde (2E,4E/Z)-decadienal (DD) can trigger intracellular calcium transients and the generation of nitric oxide (NO) by a calcium-dependent NO synthase-like activity, which results in cell death. However, pretreatment of cells with sublethal doses of aldehyde can induce resistance to subsequent lethal doses, which is reflected in an altered calcium signature and kinetics of NO production. We also present evidence for a DD-derived NO-based intercellular signaling system for the perception of stressed bystander cells. Based on these findings, we propose the existence of a sophisticated stress surveillance system in diatoms, which has important implications for understanding the cellular mechanisms responsible for acclimation versus death during phytoplankton bloom successions. Copyright:

Diatoms are unicellular algae with plastids acquired by secondary endosymbiosis. They are responsible for ∼20% of global carbon fixation. We report the 34 million - base pair draft nuclear genome of the marine diatom Thalassiosira pseudonana and its 129 thousand - base pair plastid and 44 thousand - base pair mitochondrial genomes. Sequence and optical restriction mapping revealed 24 diploid nuclear chromosomes. We identified novel genes for silicic acid transport and formation of silica-based cell walls, high-affinity iron uptake, biosynthetic enzymes for several types of polyunsaturated fatty acids, use of a range of nitrogenous compounds, and a complete urea cycle, all attributes that allow diatoms to prosper in aquatic environments.

We describe a novel allelopathic interaction whereby the cyanobacterium Microcystis sp. inhibits photosynthesis in the freshwater dinoflagellate Peridinium gatunense by abolishing its internal carbonic anhydrase activity. Our analysis indicated a positive correlation between the winter presence of Microcystis and the timing of the annual spring bloom of Peridinium in Lake Kinneret (Sea of Galilee, Israel). Microcystis severely inhibited the growth of Peridinium in mixed laboratory cultures. This was attributed to the excretion of allelopathic substances rather than to successful competition for nutrients. Microcystis-free spent medium (MFSM) inhibited steady-state photosynthesis of Peridinium, as indicated by the reduced rate of oxygen evolution and by a suppressed fluorescence signal. Boosting the inorganic carbon concentration to 25 mmol L^-1, i.e., 10-fold higher than essential to saturate photosynthesis in the absence of MFSM, partially restored the photosynthetic capacity. MFSM-treated Peridinium cells accumulated a larger internal inorganic carbon pool concomitantly with reduced accumulation of photosynthetic products. The MFSM slowed the hydration rate of CO₂ (assessed from the rate of ¹⁸O exchange between ¹³C¹⁸O₂ and water), indicating that the activity of an internal carbonic anhydrase was severely depressed. Although the nature of the active component in the MFSM that inhibits CA activity has yet to be identified, the ecological consequences of its excretion to the water body are emerging as a potent allelopathic substance that may control the development of phytoplankton competitors. We propose that its allelopathic activity could control the fate and succession of the P. gatunense bloom in Lake Kinneret.

The reasons for annual variability in the composition of phytoplankton assemblages are poorly understood but may include competition for resources and allelopathic interactions [1-4]. We show that domination by the patch-forming dinoflagellate, Peridinium gatunense, or, alternatively, a bloom of a toxic cyanobacterium, Microcystis sp., in the Sea of Galilee [5] may be accounted for by mutual density-dependent allelopathic interactions. Over the last 11 years, the abundance of these species in the lake displayed strong negative correlation. Laboratory experiments showed reciprocal, density-dependent, but nutrient-independent, inhibition of growth. Application of spent P. gatunense medium induced sedimentation and, subsequently, massive lysis of Microcystis cells within 24 hr, and sedimentation and lysis were concomitant with a large rise in the level of McyB, which is involved in toxin biosynthesis by Microcystis [6]. P. gatunense responded to the presence of Microcystis by a species-specific pathway that involved a biphasic oxidative burst and activation of certain protein kinases. Blocking this recognition by MAP-kinase inhibitors abolished the biphasic oxidative burst and affected the fate (death or cell division) of the P. gatunense cells. We propose that patchy growth habits may confer enhanced defense capabilities, providing ecological advantages that compensate for the aggravated limitation of resources in the patch. Cross-talk via allelochemicals may explain the phytoplankton assemblage in the Sea of Galilee.

The phytoplankton assemblage in Lake Kinneret is dominated in spring by a bloom of the dinoflagellate Peridinium gatunense, which terminates sharply in summer [1]. The pH in Peridinium patches rises during the bloom to values higher than pH9 [2] and results in CO₂ limitation. Here we show that depletion of dissolved CO₂ (CO₂(dis)) stimulated formation of reactive oxygen species (ROS) and induced cell death in both natural and cultured Peridinium populations. In contrast, addition of CO₂ prevented ROS formation. Catalase inhibited cell death in culture, implicating hydrogen peroxide (H₂O₂) as the active ROS. Cell death was also blocked by a cysteine protease inhibitor, E-64, a treatment which stimulated cyst formation. Intracellular ROS accumulation induced protoplast shrinkage and DNA fragmentation prior to cell death. We propose that CO₂ limitation resulted in the generation of ROS to a level that induced programmed cell death, which resembles apoptosis in animal and plant cells. Our results also indicate that cysteine protease(s) are involved in processes that determine whether a cell is destined to die or to form a cyst.

The first appearance of Aphanizomenon ovalisporum in Lake Kinneret in August 1994 was apparently boosted by relatively high concentrations of total dissolved phosphorus (12 μg P l^-1 as compared to an average of 8 μg P l^-1). The increasing Aphanizomenon biomass in a lake in which phytoplankton are generally phosphate limited in summer and autumn was accompanied by high enzymatic activity of alkaline phosphatase, reaching values of 2830 nmol MU l^-1 h^-1, suggesting a great demand for phosphorus. In addition, the nitrogen requirement of the developing population of Aphanizomenon was partly provided by nitrogen fixation, as indicated by a high percentage of heterocysts. Laboratory experiments demonstrated that filtrate from an old Peridinium gatunense culture enhanced Aphanizomenon growth. Thus, it is postulated that the degradation of the massive Peridinium bloom in spring and early summer supported the development of A. ovalisporum. The high pH and alkalinity during the bloom of Aphanizomenon indicate that A.ovalisporum is probably a HCO₃^- user. After 1994, akinetes of A. ovalisporum were left in sediments and the water column, and could be a source for the next year's bloom. This possibility was demonstrated by inoculation of lake water and sediments into nitrogen-depleted BG-11 medium, resulting in the dominance of A.ovalisporum.