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The marine microalga Emiliania huxleyi is widely distributed in the surface oceans and is prone to infection by coccolithoviruses that can terminate its blooms. However, little is known about how global change factors like solar UV radiation (UVR) and ocean warming affect the host-virus interaction. We grew the microalga at 2 temperature levels with or without the virus in the presence or absence of UVR and investigated the physiological and transcriptional responses. We showed that viral infection noticeably reduced photosynthesis and growth of the alga but was less harmful to its physiology under conditions where UVR influenced viral DNA expression. In the virus-infected cells, the combination of UVR and warming (+4°C) led to a 13-fold increase in photosynthetic carbon fixation rate, with warming alone contributing a change of about 5-7-fold. This was attributed to upregulated expression of genes related to carboxylation and light-harvesting proteins under the influence of UVR, and to warming-reduced infectivity. In the absence of UVR, viral infection downregulated the metabolic pathways of photosynthesis and fatty acid degradation. Our results suggest that solar UV exposure in a warming ocean can reduce the severity of viral attack on this ecologically important microalga, potentially prolonging its blooms., (© 2024 John Wiley & Sons Ltd.)

A dynamic reaction-diffusion model of four variables is proposed to describe the spread of lytic viruses among phytoplankton in a poorly mixed aquatic environment. The basic ecological reproductive index for phytoplankton invasion and the basic reproduction number for virus transmission are derived to characterize the phytoplankton growth and virus transmission dynamics. The theoretical and numerical results from the model show that the spread of lytic viruses effectively controls phytoplankton blooms. This validates the observations and experimental results of Emiliana huxleyi-lytic virus interactions. The studies also indicate that the lytic virus transmission cannot occur in a low-light or oligotrophic aquatic environment., (© 2024. The Author(s).)

The interactions between Emiliania huxleyi and E. huxleyi virus (EhV) regulate marine carbon and sulfur biogeochemical cycle and play a prominent role in global climate change. As a large DNA virus, EhVs have developed a novel "virocell metabolism" model to meet their higher metabolic needs. However, the regulatory mechanism of this metabolic model is still largely unclear. MicroRNAs (miRNAs) can regulate biological pathways through targeting hub genes in the metabolic processes. Here, we performed high-throughput small RNA sequencing to analyse miRNA expression in EhV99B1 infected E. huxleyi BOF92. A total of 26 miRNAs (including 2 virus-derived miRNAs) were identified, including four up-regulated and one down-regulated miRNAs. These results were further validated through quantitative real-time PCR. Functional enrichment analysis showed that five differentially-expressed miRNAs might be involved in the regulation of carbohydrate metabolism, lipid metabolism and amino acid metabolism. Moreover, the expression levels of differentially-expressed miRNAs were negatively correlated with that of several lipid metabolism-related genes, such as ACC-1, SPT, ACOX, ACAT, CERS and ACADS, indicating that these miRNAs might play an important regulatory role in virus-mediated lipid metabolism.

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., (© 2021. The Author(s).)

Aims: To search for a set of reference genes for reliable gene expression analysis in the globally important marine coccolithophore Emiliania huxleyi-virus model system., Methods and Results: Fifteen housekeeping genes (CDKA, CYP15, EFG3, POLAI, RPL30, RPL13, SAMS, COX1, GPB1-2, HSP90, TUA, TUB, UBA1, CAM3 and GAPDH) were evaluated for their stability as potential reference genes for qRT-PCR using ΔCt, geNorm, NormFinder, Bestkeeper and RefFinder software. CDKA, TUA and TUB genes were tested as loading controls for Western blot in the same sample panel. Additionally, target genes associated with cell apoptosis, that is metacaspase genes, were applied to validate the selection of reference genes. The analysis results demonstrated that putative housekeeping genes exhibited significant variations in both mRNA and protein content during virus infection. After a comprehensive analysis with all the algorithms, CDKA and GAPDH were recommended as the most stable reference genes for E huxleyi virus (EhV) infection treatments. For Western blot, significant variation was seen for TUA and TUB, whereas CDKA was stably expressed, consistent with the results of qRT-PCR., Conclusions: CDKA and GAPDH are the best choice for gene and protein expression analysis than the other candidate reference genes under EhV infection conditions., Significance and Impact of the Study: The stable internal control genes identified in this work will help to improve the accuracy and reliability of gene expression analysis and gain insight into complex E. huxleyi-EhV interaction regulatory networks., (© 2020 The Society for Applied Microbiology.)

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., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The blooming cosmopolitan coccolithophore Emiliania huxleyi and its viruses (EhVs) are a model for density-dependent virulent dynamics. EhVs commonly exhibit rapid viral reproduction and drive host death in high-density laboratory cultures and mesocosms that simulate blooms. Here we show that this system exhibits physiology-dependent temperate dynamics at environmentally relevant E. huxleyi host densities rather than virulent dynamics, with viruses switching from a long-term non-lethal temperate phase in healthy hosts to a lethal lytic stage as host cells become physiologically stressed. Using this system as a model for temperate infection dynamics, we present a template to diagnose temperate infection in other virus-host systems by integrating experimental, theoretical, and environmental approaches. Finding temperate dynamics in such an established virulent host-virus model system indicates that temperateness may be more pervasive than previously considered, and that the role of viruses in bloom formation and decline may be governed by host physiology rather than by host-virus densities.

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 μm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus), prasinophytes (chiefly Ostreococcus), fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity.

Emiliania huxleyi (Coccolithophore) plays a prominent role in the global carbon cycle and in climate processes. The annual collapse of massive E. huxleyi blooms in the marine environment has been shown to be frequently linked to viral control. These host-virus interactions shape the evolution and dynamics of oceanic microscale ecosystems, yet we still understand little of the molecular mechanism of these virus-mediated processes. Here, we present a detailed characterization of the lipidome of E. huxleyi BOF92 strain, both of uninfected cells and those infected with its specific lytic virus EhV-99B1. Non-targeted lipidomics analysis was performed in order to evaluate the dynamic alterations underlying virus-induced metabolic remodeling. The host lipidome (both lipid content and composition) significantly changed in response to the viral infection. The most statistically significant differential lipids were screened as potential biomarkers for assessing E. huxleyi population sensitivity to EhV infection. Our results reveal that the remodeling of lipid metabolism that underlies the pathogenesis of this infection primarily involved sphingolipid, glycerolipid and fatty acid metabolic pathways. Our study provides insights into how viruses shape their hosts metabolism to support their unique life cycle and a lipid-based chemical arms race during host-virus dynamic interactions in a marine environment., (Copyright © 2019. Published by Elsevier B.V.)

Two prominent characteristics of marine coccolithophores are their secretion of coccoliths and their susceptibility to infection by coccolithoviruses (EhVs), both of which display variation among cells in culture and in natural populations. We examined the impact of calcification on infection by challenging a variety of Emiliania huxleyi strains at different calcification states with EhVs of different virulence. Reduced cellular calcification was associated with increased infection and EhV production, even though calcified cells and associated coccoliths had significantly higher adsorption coefficients than non-calcified (naked) cells. Sialic acid glycosphingolipids, molecules thought to mediate EhV infection, were generally more abundant in calcified cells and enriched in purified, sorted coccoliths, suggesting a biochemical link between calcification and adsorption rates. In turn, viable EhVs impacted cellular calcification absent of lysis by inducing dramatic shifts in optical side scatter signals and a massive release of detached coccoliths in a subpopulation of cells, which could be triggered by resuspension of healthy, calcified host cells in an EhV-free, 'induced media'. Our findings show that calcification is a key component of the E. huxleyi-EhV arms race and an aspect that is critical both to the modelling of these host-virus interactions in the ocean and interpreting their impact on the global carbon cycle., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Coccolithoviruses (EhVs) are large, double-stranded DNA-containing viruses that infect the single-celled, marine coccolithophore Emiliania huxleyi. Given the cosmopolitan nature and global importance of E. huxleyi as a bloom-forming, calcifying, photoautotroph, E. huxleyi-EhV interactions play a key role in oceanic carbon biogeochemistry. Virally-encoded glycosphingolipids (vGSLs) are virulence factors that are produced by the activity of virus-encoded serine palmitoyltransferase (SPT). Here, we characterize the dynamics, diversity and catalytic production of vGSLs in an array of EhV strains in relation to their SPT sequence composition and explore the hypothesis that they are a determinant of infectivity and host demise. vGSL production and diversity was positively correlated with increased virulence, virus replication rate and lytic infection dynamics in laboratory experiments, but they do not explain the success of less-virulent EhVs in natural EhV communities. The majority of EhV-derived SPT amplicon sequences associated with infected cells in the North Atlantic derived from slower infecting, less virulent EhVs. Our lab-, field- and mathematical model-based data and simulations support ecological scenarios whereby slow-infecting, less-virulent EhVs successfully compete in North Atlantic populations of E. huxleyi, through either the preferential removal of fast-infecting, virulent EhVs during active infection or by having access to a broader host range., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist.

Emiliania huxleyi is a globally important marine phytoplankton that is routinely infected by viruses. Understanding the controls on the growth and demise of E. huxleyi blooms is essential for predicting the biogeochemical fate of their organic carbon and nutrients. In this study, we show that the production of nitric oxide (NO), a gaseous, membrane-permeable free radical, is a hallmark of early-stage lytic infection in E. huxleyi by Coccolithoviruses, both in culture and in natural populations in the North Atlantic. Enhanced NO production was detected both intra- and extra-cellularly in laboratory cultures, and treatment of cells with an NO scavenger significantly reduced viral production. Pre-treatment of exponentially growing E. huxleyi cultures with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prior to challenge with hydrogen peroxide (H 2 O 2 ) led to greater cell survival, suggesting that NO may have a cellular antioxidant function. Indeed, cell lysates generated from cultures treated with SNAP and undergoing infection displayed enhanced ability to detoxify H 2 O 2 . Lastly, we show that fluorescent indicators of cellular ROS, NO, and death, in combination with classic DNA- and lipid-based biomarkers of infection, can function as real-time diagnostic tools to identify and contextualize viral infection in natural E. huxleyi blooms.

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.

Viruses that infect photoautotrophs have a fundamental relationship with light, given the need for host resources. We investigated the role of light on Coccolithovirus (EhV) infection of the globally distributed coccolithophore, Emiliania huxleyi. Light was required for EhV adsorption, and viral production was highest when host cultures were maintained in continuous light or at irradiance levels of 150-300 μmol m -2  s -1 . During the early stages of infection, photosynthetic electron transport remained high, while RuBisCO expression decreased concomitant with an induction of the pentose phosphate pathway, the primary source of de novo nucleotides. A mathematical model developed and fitted to the laboratory data supported the hypothesis that EhV replication was controlled by a trade-off between host nucleotide recycling and de novo synthesis, and that photoperiod and photon flux could toggle this switch. Laboratory results supported field observations that light was the most robust driver of EhV replication within E. huxleyi populations collected across a 2000 nautical mile transect in the North Atlantic. Collectively, these findings demonstrate that light can drive host-virus interactions through a mechanistic interplay between host metabolic processes, which serve to structure infection and phytoplankton mortality in the upper ocean., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Over the past few decades, the Arctic region has been strongly affected by global warming, leading to increased sea surface temperatures and melting of land and sea ice. Marine terminating (tide-water) glaciers are expected to show higher melting and calving rates, with an increase in the input of fine sediment particles in the coastal marine environment. We experimentally investigated whether marine viruses, which drive microbial interactions and biogeochemical cycling are removed from the water column through adsorption to glacier-delivered fine sediments. Ecologically relevant concentrations of 30, 100 and 200 mg·L -1 sediments were added to filtered lysates of 3 cultured algal viruses and to a natural marine bacterial virus community. Total virus removal increased with sediment concentration whereby the removal rate depended on the virus used (up to 88% for an Arctic algal virus), suggesting a different interaction strength with the sediment. Moreover, we observed that the adsorption of viruses to sediment is a reversible process, and that desorbed viruses are still able to infect their respective hosts. Nonetheless, the addition of sediment to infection experiments with the Arctic prasinovirus MpoV-45T substantially delayed host lysis and the production of progeny viruses. We demonstrate that glacier-derived fine sediments have the potency to alter virus availability and consequently, host population dynamics.

The scope for ecological studies of eukaryotic algal viruses has greatly improved with the development of molecular and bioinformatic approaches that do not require algal cultures. Here, we review the history and perceived future opportunities for research on eukaryotic algal viruses. We begin with a summary of the 65 eukaryotic algal viruses that are presently in culture collections, with emphasis on shared evolutionary traits (e.g., conserved core genes) of each known viral type. We then describe how core genes have been used to enable molecular detection of viruses in the environment, ranging from PCR-based amplification to community scale "-omics" approaches. Special attention is given to recent studies that have employed network-analyses of -omics data to predict virus-host relationships, from which a general bioinformatics pipeline is described for this type of approach. Finally, we conclude with acknowledgement of how the field of aquatic virology is adapting to these advances, and highlight the need to properly characterize new virus-host systems that may be isolated using preliminary molecular surveys. Researchers can approach this work using lessons learned from the Chlorella virus system, which is not only the best characterized algal-virus system, but is also responsible for much of the foundation in the field of aquatic virology.

Emiliania huxleyi produces calcium carbonate (CaCO 3 ) coccoliths and transparent exopolymer particles (TEP), sticky, acidic carbohydrates that facilitate aggregation. E. huxleyi's extensive oceanic blooms are often terminated by coccolithoviruses (EhVs) with the transport of cellular debris and associated particulate organic carbon (POC) to depth being facilitated by TEP-bound 'marine snow' aggregates. The dynamics of TEP production and particle aggregation in response to EhV infection are poorly understood. Using flow cytometry, spectrophotometry and FlowCam visualization of alcian blue (AB)-stained aggregates, we assessed TEP production and the size spectrum of aggregates for E. huxleyi possessing different degrees of calcification and cellular CaCO 3 :POC mass ratios, when challenged with two EhVs (EhV207 and EhV99B1). FlowCam imaging also qualitatively assessed the relative amount of AB-stainable TEP (i.e., blue:red ratio of each particle). We show significant increases in TEP during early phase EhV207-infection (∼ 24 h) of calcifying strains and a shift towards large aggregates following EhV99B1-infection. We also observed the formation of large aggregates with low blue:red ratios, suggesting that other exopolymer substances contribute towards aggregation. Our findings show the potential for virus infection and the associated response of their hosts to impact carbon flux dynamics and provide incentive to explore these dynamics in natural populations., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Prymnesium parvum is a toxin-producing microalga that causes harmful algal blooms globally, which often result in large-scale fish kills that have severe ecological and economic implications. Although many toxins have previously been isolated from P. parvum , ambiguity still surrounds the responsible ichthyotoxins in P. parvum blooms and the biotic and abiotic factors that promote bloom toxicity. A major fish kill attributed to P. parvum occurred in Spring 2015 on the Norfolk Broads, a low-lying set of channels and lakes (Broads) found on the East of England. Here, we discuss how water samples taken during this bloom have led to diverse scientific advances ranging from toxin analysis to discovery of a new lytic virus of P. parvum , P. parvum DNA virus (PpDNAV-BW1). Taking recent literature into account, we propose key roles for sialic acids in this type of viral infection. Finally, we discuss recent practical detection and management strategies for controlling these devastating blooms., (© 2018 The Author(s).)

Lytic viral infection and programmed cell death (PCD) are thought to represent two distinct death mechanisms in phytoplankton, unicellular photoautotrophs that drift with ocean currents. PCD (apoptosis) is mainly brought about by the activation of caspases, a protease family with unique substrate selectivity. Here, we demonstrated that virus infection induced apoptosis of marine coccolithophorid Emiliania huxleyi BOF92 involving activation of metacaspase. E. huxleyi cells exhibited cell death process akin to that of apoptosis when exposed to virus infection. We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes and DNA fragmentation. Immunoblotting revealed that antibody against human active-caspase-3 shared epitopes with a protein of ≈ 23 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, analysis on two-dimensional gel electrophoresis revealed that two spots of active caspase-3 co-migrated with the different isoelectric points. Phosphatase treatment of cytosolic extracts containing active caspases-3 showed a mobility shift, suggesting that phosphorylated form of this enzyme might be present in the extracts. Computational prediction of phosphorylation sites based on the amino acid sequence of E. huxleyi metacaspase showed multiple phosphorylated sites for serine, threonine and tyrosine residues. This is the first report showing that phosphorylation modification of metacaspase in E. huxleyi might be required for certain biochemical and morphological changes during virus induced apoptosis.

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.

Viruses influence the ecology and diversity of phytoplankton in the ocean. Most studies of phytoplankton host-virus interactions have focused on bloom-forming species like Emiliania huxleyi or Phaeocystis spp. The role of viruses infecting phytoplankton that do not form conspicuous blooms have received less attention. Here we explore the dynamics of phytoplankton and algal viruses over several sequential seasons, with a focus on the ubiquitous and diverse phytoplankton division Haptophyta, and their double-stranded DNA viruses, potentially with the capacity to infect the haptophytes. Viral and phytoplankton abundance and diversity showed recurrent seasonal changes, mainly explained by hydrographic conditions. By 454 tag-sequencing we revealed 93 unique haptophyte operational taxonomic units (OTUs), with seasonal changes in abundance. Sixty-one unique viral OTUs, representing Megaviridae and Phycodnaviridae , showed only distant relationship with currently isolated algal viruses. Haptophyte and virus community composition and diversity varied substantially throughout the year, but in an uncoordinated manner. A minority of the viral OTUs were highly abundant at specific time-points, indicating a boom-bust relationship with their host. Most of the viral OTUs were very persistent, which may represent viruses that coexist with their hosts, or able to exploit several host species.

Viruses are thought to be fundamental in driving microbial diversity in the oceanic planktonic realm. That role and associated emerging infection patterns remain particularly elusive for eukaryotic phytoplankton and their viruses. Here we used a vast number of strains from the model system Emiliania huxleyi /Emiliania huxleyi Virus to quantify parameters such as growth rate (µ), resistance (R), and viral production (Vp) capacities. Algal and viral abundances were monitored by flow cytometry during 72-h incubation experiments. The results pointed out higher viral production capacity in generalist EhV strains, and the virus-host infection network showed a strong co-evolution pattern between E. huxleyi and EhV populations. The existence of a trade-off between resistance and growth capacities was not confirmed.

Coccolithoviruses (Phycodnaviridae) infect and lyse the most ubiquitous and successful coccolithophorid in modern oceans, Emiliania huxleyi. So far, the genomes of 13 of these giant lytic viruses (i.e., Emiliania huxleyi viruses-EhVs) have been sequenced, assembled, and annotated. Here, we performed an in-depth comparison of their genomes to try and contextualize the ecological and evolutionary traits of these viruses. The genomes of these EhVs have from 444 to 548 coding sequences (CDSs). Presence/absence analysis of CDSs identified putative genes with particular ecological significance, namely sialidase, phosphate permease, and sphingolipid biosynthesis. The viruses clustered into distinct clades, based on their DNA polymerase gene as well as full genome comparisons. We discuss the use of such clustering and suggest that a gene-by-gene investigation approach may be more useful when the goal is to reveal differences related to functionally important genes. A multi domain "Best BLAST hit" analysis revealed that 84% of the EhV genes have closer similarities to the domain Eukarya. However, 16% of the EhV CDSs were very similar to bacterial genes, contributing to the idea that a significant portion of the gene flow in the planktonic world inter-crosses the domains of life.

Emiliania huxleyi is the main calcite producer on Earth and is routinely infected by a virus (EhV); a double stranded DNA (dsDNA) virus belonging to the family Phycodnaviridae . E. huxleyi exhibits a haplodiploid life cycle; the calcified diploid stage is non-motile and forms extensive blooms. The haploid phase is a non-calcified biflagellated cell bearing organic scales. Haploid cells are thought to resist infection, through a process deemed the "Cheshire Cat" escape strategy; however, a recent study detected the presence of viral lipids in the same haploid strain. Here we report on the application of an E. huxleyi CCMP1516 EhV-86 combined tiling array (TA) that further confirms an EhV infection in the RCC1217 haploid strain, which grew without any signs of cell lysis. Reverse transcription polymerase chain reaction (RT-PCR) and PCR verified the presence of viral RNA in the haploid cells, yet indicated an absence of viral DNA, respectively. These infected cells are an alternative stage of the virus life cycle deemed the haplococcolithovirocell. In this instance, the host is both resistant to and infected by EhV, i.e., the viral transcriptome is present in haploid cells whilst there is no evidence of viral lysis. This superimposed state is reminiscent of Schrödinger's cat; of being simultaneously both dead and alive.

Prymnesium parvum is a toxin-producing haptophyte that causes harmful algal blooms globally, leading to large-scale fish kills that have severe ecological and economic implications. For the model haptophyte, Emiliania huxleyi , it has been shown that large dsDNA viruses play an important role in regulating blooms and therefore biogeochemical cycling, but much less work has been done looking at viruses that infect P. parvum , or the role that these viruses may play in regulating harmful algal blooms. In this study, we report the isolation and characterization of a lytic nucleo-cytoplasmic large DNA virus (NCLDV) collected from the site of a harmful P. parvum bloom. In subsequent experiments, this virus was shown to infect cultures of Prymnesium sp. and showed phylogenetic similarity to the extended Megaviridae family of algal viruses.

Effects of elevated p CO₂ on Emiliania huxleyi genetic diversity and the viruses that infect E. huxleyi (EhVs) have been investigated in large volume enclosures in a Norwegian fjord. Triplicate enclosures were bubbled with air enriched with CO₂ to 760 ppmv whilst the other three enclosures were bubbled with air at ambient p CO₂; phytoplankton growth was initiated by the addition of nitrate and phosphate. E. huxleyi was the dominant coccolithophore in all enclosures, but no difference in genetic diversity, based on DGGE analysis using primers specific to the calcium binding protein gene ( gpa ) were detected in any of the treatments. Chlorophyll concentrations and primary production were lower in the three elevated p CO₂ treatments than in the ambient treatments. However, although coccolithophores numbers were reduced in two of the high- p CO₂ treatments; in the third, there was no suppression of coccolithophores numbers, which were very similar to the three ambient treatments. In contrast, there was considerable variation in genetic diversity in the EhVs, as determined by analysis of the major capsid protein ( mcp ) gene. EhV diversity was much lower in the high- p CO₂ treatment enclosure that did not show inhibition of E. huxleyi growth. Since virus infection is generally implicated as a major factor in terminating phytoplankton blooms, it is suggested that no study of the effect of ocean acidification in phytoplankton can be complete if it does not include an assessment of viruses.

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., (Copyright © 2016. Published by Elsevier Ltd.)

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 selective mode of membrane recruitment during viral assembly, possibly by budding of the virus from specialized subcellular compartments. These findings provide novel insights into the role of viruses infecting microalgae in regulating metabolism and energy transfer in the marine environment and suggest their possible biotechnological application in biofuel production., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

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.

Viruses are a major cause of coccolithophore bloom demise in both temperate and sub-temperate oceanic regions. Most infection studies on coccolithoviruses have been conducted with a single virus strain, and the effect of intragenus competition by closely related coccolithoviruses has been ignored. Here we conducted combined infection experiments, infecting Emiliania huxleyi CCMP 2090 with two coccolithoviruses: EhV-86 and EhV-207 both simultaneously and independently. EhV-207 displayed a shorter lytic cycle and increased production potential than EhV-86 and was remarkably superior under competitive conditions. Although the viruses displayed identical adsorption kinetics in the first 2 h post infection, EhV-207 gained a numerical advantage as early as 8 h post infection. Quantitative polymerase chain reaction (PCR) revealed that when infecting in combination, EhV-207 was not affected by the presence of EhV-86, whereas EhV-86 was quickly out-competed, and a significant reduction in free and cell-associated EhV-86 was seen as early as 2 days after the initial infection. The observation of such clear phenotypic differences between genetically distinct, yet similar, coccolithovirus strains, by flow cytometry and quantitative real-time PCR allowed tentative links to the burgeoning genomic, transcriptomic and metabolic data to be made and the factors driving their selection, in particular to the de novo coccolithovirus-encoded sphingolipid biosynthesis pathway. This work illustrates that, even within a family, not all viruses are created equally, and the potential exists for relatively small genetic changes to infer disproportionately large competitive advantages for one coccolithovirus over another, ultimately leading to a few viruses dominating the many., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Water samples from Lake Ontario, Canada were tested for lytic activity against the freshwater haptophyte algae Chrysochromulina parva. A filterable lytic agent was isolated and identified as a virus via transmission electron microscopy and molecular methods. The virus, CpV-BQ1, is icosahedral, ca. 145nm in diameter, assembled within the cytoplasm, and has a genome size of ca. 485kb. Sequences obtained through PCR-amplification of DNA polymerase (polB) genes clustered among sequences from the family Phycodnaviridae, whereas major capsid protein (MCP) sequences clustered among sequences from either the Phycodnaviridae or Mimiviridae. Based on quantitative molecular assays, C. parva׳s abundance in Lake Ontario was relatively stable, yet CpV-BQ1׳s abundance was variable suggesting complex virus-host dynamics. This study demonstrates that CpV-BQ1 is a member of the proposed order Megavirales with characteristics of both phycodnaviruses and mimiviruses indicating that, in addition to its complex ecological dynamics, it also has a complex evolutionary history., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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.

Both ocean acidification and viral infection bring about changes in marine phytoplankton physiological activities and community composition. However, little information is available on how the relationship between phytoplankton and viruses may be affected by ocean acidification and what impacts this might have on photosynthesis-driven marine biological CO2 pump. Here, we show that when the harmful bloom alga Phaeocystis globosa is infected with viruses under future ocean conditions, its photosynthetic performance further decreased and cells became more susceptible to stressful light levels, showing enhanced photoinhibition and reduced carbon fixation, up-regulation of mitochondrial respiration and decreased virus burst size. Our results indicate that ocean acidification exacerbates the impacts of viral attack on P. globosa, which implies that, while ocean acidification directly influences marine primary producers, it may also affect them indirectly by altering their relationship with viruses. Therefore, viruses as a biotic stressor need to be invoked when considering the overall impacts of climate change on marine productivity and carbon sequestration., (© 2014 John Wiley & Sons Ltd.)

We have isolated three novel lytic dsDNA-viruses from Raunefjorden (Norway) that are putative members of the Mimiviridae family, namely Haptolina ericina virus RF02 (HeV RF02), Prymnesium kappa virus RF01 (PkV RF01), and Prymnesium kappa virus RF02 (PkV RF02). Each of the novel haptophyte viruses challenges the common conceptions of algal viruses with respect to host range, phylogenetic affiliation and size. PkV RF01 has a capsid of ~310 nm and is the largest algal virus particle ever reported while PkV RF01 and HeV RF02 were able to infect different species, even belonging to different genera. Moreover, PkV RF01 and HeV RF02 infected the same hosts, but phylogenetic analysis placed them in different groups. Our results reveal large variation among viruses infecting closely related microalgae, and challenge the common conception that algal viruses have narrow host range, and phylogeny reflecting their host affiliation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Annual Emiliania huxleyi blooms (along with other coccolithophorid species) play important roles in the global carbon and sulfur cycles. E. huxleyi blooms are routinely terminated by large, host-specific dsDNA viruses, (Emiliania huxleyi Viruses; EhVs), making these host-virus interactions a driving force behind their potential impact on global biogeochemical cycles. Given projected increases in sea surface temperature due to climate change, it is imperative to understand the effects of temperature on E. huxleyi's susceptibility to viral infection and its production of climatically active dimethylated sulfur species (DSS). Here we demonstrate that a 3°C increase in temperature induces EhV-resistant phenotypes in three E. huxleyi strains and that successful virus infection impacts DSS pool sizes. We also examined cellular polar lipids, given their documented roles in regulating host-virus interactions in this system, and propose that alterations to membrane-bound surface receptors are responsible for the observed temperature-induced resistance. Our findings have potential implications for global biogeochemical cycles in a warming climate and for deciphering the particular mechanism(s) by which some E. huxleyi strains exhibit viral resistance.

Marine viruses are recognized as a major driving force regulating phytoplankton community composition and nutrient cycling in the oceans. 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. Here, we show that copepods, abundant migrating crustaceans that graze on phytoplankton, 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. 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, can serve as viral vectors, boosting host-virus contact rates and potentially accelerating the demise of large-scale phytoplankton blooms., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Giant viruses are known to be significant mortality agents of phytoplankton, often being implicated in the terminations of large Emiliania huxleyi blooms. We have previously shown the high temporal variability of E. huxleyi-infecting coccolithoviruses (EhVs) within a Norwegian fjord mesocosm. In the current study we investigated EhV dynamics within a naturally-occurring E. huxleyi bloom in the Western English Channel. Using denaturing gradient gel electrophoresis and marker gene sequencing, we uncovered a spatially highly dynamic Coccolithovirus population that was associated with a genetically stable E. huxleyi population as revealed by the major capsid protein gene (mcp) and coccolith morphology motif (CMM), respectively. Coccolithoviruses within the bloom were found to be variable with depth and unique virus populations were detected at different stations sampled indicating a complex network of EhV-host infections. This ultimately will have significant implications to the internal structure and longevity of ecologically important E. huxleyi blooms., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

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 (∼10-100 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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Coccolithoviruses infect the marine coccolithophorid microalga Emiliania huxleyi. Here, we describe the genomes of four new coccolithoviruses isolated from UK coastal locations. Of particular interest, EhV-18 and EhV-145 encode serine palmitoyltransferase function via two distinct genes, whereas all other coccolithoviruses have SPT as a gene fusion of LCB1/LCB2 domains., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein-protein interactions, likely play a fundamental role in host-virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E. huxleyi cells and those infected with EhV86, the type strain for Coccolithovirus. Lipid raft-enriched fractions were isolated and purified as buoyant, detergent-resistant membranes (DRMs) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography-triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV86-encoded C-type lectin-containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft-associated proteins., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ∼20% transfer of organic matter derived from the infected (13)C- and (15)N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate (13)C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.

Emiliania huxleyi, a key player in the global carbon cycle is one of the best studied coccolithophores with respect to biogeochemical cycles, climatology, and host-virus interactions. Strains of E. huxleyi show phenotypic plasticity regarding growth behaviour, light-response, calcification, acidification, and virus susceptibility. This phenomenon is likely a consequence of genomic differences, or transcriptomic responses, to environmental conditions or threats such as viral infections. We used an E. huxleyi genome microarray based on the sequenced strain CCMP1516 (reference strain) to perform comparative genomic hybridizations (CGH) of 16 E. huxleyi strains of different geographic origin. We investigated the genomic diversity and plasticity and focused on the identification of genes related to virus susceptibility and coccolith production (calcification). Among the tested 31940 gene models a core genome of 14628 genes was identified by hybridization among 16 E. huxleyi strains. 224 probes were characterized as specific for the reference strain CCMP1516. Compared to the sequenced E. huxleyi strain CCMP1516 variation in gene content of up to 30 percent among strains was observed. Comparison of core and non-core transcripts sets in terms of annotated functions reveals a broad, almost equal functional coverage over all KOG-categories of both transcript sets within the whole annotated genome. Within the variable (non-core) genome we identified genes associated with virus susceptibility and calcification. Genes associated with virus susceptibility include a Bax inhibitor-1 protein, three LRR receptor-like protein kinases, and mitogen-activated protein kinase. Our list of transcripts associated with coccolith production will stimulate further research, e.g. by genetic manipulation. In particular, the V-type proton ATPase 16 kDa proteolipid subunit is proposed to be a plausible target gene for further calcification studies.