Modelling phytoplankton-virus interactions: phytoplankton blooms and lytic virus transmission.

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.

Analysis of microRNA expression profile in Emiliania huxleyi in response to virus infection.

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.

An Emiliania huxleyi pan-transcriptome reveals basal strain specificity in gene expression patterns.

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.

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

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

Validation of superior reference genes for qRT-PCR and Western blot analyses in marine Emiliania huxleyi-virus model system.

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.

Temperate infection in a virus-host system previously known for virulent dynamics.

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.

Diel transcriptional response of a California Current plankton microbiome to light, low iron, and enduring viral infection.

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.

Emerging lipidome patterns associated with marine Emiliania huxleyi-virus model system.

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.

Biochemical diversity of glycosphingolipid biosynthesis as a driver of Coccolithovirus competitive ecology.

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.

Unmasking cellular response of a bloom-forming alga to viral infection by resolving expression profiles at a single-cell level.

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.

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

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

Sediments from Arctic Tide-Water Glaciers Remove Coastal Marine Viruses and Delay Host Infection.

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.

Nitric oxide production and antioxidant function during viral infection of the coccolithophore Emiliania huxleyi.

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 (H2O2) 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 H2O2. 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.

The mutual interplay between calcification and coccolithovirus infection.

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.

Light regulation of coccolithophore host-virus interactions.

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.

Viruses of Eukaryotic Algae: Diversity, Methods for Detection, and Future Directions.

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.

Dynamics of transparent exopolymer particle production and aggregation during viral infection of the coccolithophore, Emiliania huxleyi.

Emiliania huxleyi produces calcium carbonate (CaCO3 ) 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 CaCO3 :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.

Coccolithovirus facilitation of carbon export in the North Atlantic.

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 (~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.

Insights into toxic Prymnesium parvum blooms: the role of sugars and algal viruses.

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.