Huxleyi Cells Research Articles

Structure and replication cycle of a virus infecting climate-modulating alga Emiliania huxleyi.

The globally distributed marine alga Emiliania huxleyi has cooling effect on the Earth's climate. The population density of E. huxleyi is restricted by Nucleocytoviricota viruses, including E. huxleyi virus 201 (EhV-201). Despite the impact of E. huxleyi viruses on the climate, there is limited information about their structure and replication. Here, we show that the dsDNA genome inside the EhV-201 virion is protected by an inner membrane, capsid, and outer membrane. EhV-201 virions infect E. huxleyi by using fivefold vertices to bind to and fuse the virus' inner membrane with the cell plasma membrane. Progeny virions assemble in the cytoplasm at the surface of endoplasmic reticulum-derived membrane segments. Genome packaging initiates synchronously with the capsid assembly and completes through an aperture in the forming capsid. The genome-filled capsids acquire an outer membrane by budding into intracellular vesicles. EhV-201 infection induces a loss of surface protective layers from E. huxleyi cells, which enables the continuous release of virions by exocytosis.

Cell-Penetrating Peptide Delivery of Nucleic Acid Cargo to Emiliania huxleyi, a Calcifying Marine Coccolithophore.

Coccolithophores are a group of unicellular marine phytoplankton that exhibit a prolific capacity for carbon conversion and are critical to ocean biogeochemistry. A fundamental understanding of coccolithophore biomineralization has been limited, in part, by the lack of genetic and molecular tools to investigate the organisms. In particular, it has proven to be difficult to deliver macromolecules across the coccosphere-membrane complex. To overcome this barrier, we employed cell-penetrating peptides (CPP) in the Emiliania huxleyi coccolithophores. We evaluated three established CPPs (TAT, R9, and KFF) and designed a CPP that incorporates a high proline content identified in the protein transduction domain of EhV060, an E. huxleyi virus lectin protein. To measure the delivery performance, we covalently linked CPPs to synthetic peptide nucleic acids (PNA) and attached a fluorescein marker. CPP-PNA-FITC complexes were efficiently delivered across the coccosphere-membrane complex to the cytoplasm of E. huxleyi cells. Characterization of E. huxleyi demonstrates that CPP-PNA are nontoxic and reveals specific effects of CPP-PNA on cell biology and calcification. Direct delivery and characterization of synthetic nucleic acids represent a step forward in synthetic biology to explore coccolithophore biomineralization.

A bacterial quorum sensing signal is a potent inhibitor of de novo pyrimidine biosynthesis in the globally abundant Emiliania huxleyi.

Interactions between marine phytoplankton, viruses, and bacteria drive biogeochemical cycling, shape marine trophic structures, and impact global climate. Microbially produced compounds have emerged as key players in influencing eukaryotic organismal physiology, and in turn, remodel microbial community structure. This work aimed to reveal the molecular mechanism by which the bacterial quorum sensing molecule 2-heptyl-4-quinolone (HHQ), produced by the marine gammaproteobacterium Pseudoalteromonas spp., arrests cell division and confers protection from virus-induced mortality in the bloom-forming coccolithophore Emiliania huxleyi. Previous work has established alkylquinolones as inhibitors of dihydroorotate dehydrogenase (DHODH), a fundamental enzyme catalyzing the fourth step in pyrimidine biosynthesis and a potential antiviral drug target. An N-terminally truncated version of E. huxleyi DHODH was heterologously expressed in E. coli, purified, and kinetically characterized. Here, we show HHQ is a potent inhibitor (Ki of 2.3 nM) of E. huxleyi DHODH. E. huxleyi cells exposed to brequinar, the canonical human DHODH inhibitor, experienced immediate, yet reversible cellular arrest, an effect which mirrors HHQ-induced cellular stasis previously observed. However, brequinar treatment lacked other notable effects observed in HHQ-exposed E. huxleyi including significant changes in cell size, chlorophyll fluorescence, and protection from virus-induced lysis, indicating HHQ has additional as yet undiscovered physiological targets. Together, these results suggest a novel and intricate role of bacterial quorum sensing molecules in tripartite interdomain interactions in marine ecosystems, opening new avenues for exploring the role of microbial chemical signaling in algal bloom regulation and host-pathogen dynamics.

Two-sided effects of the organic phosphorus phytate on a globally important marine coccolithophorid phytoplankton.

Dissolved organic phosphorus (DOP) is a potential source of aquatic eutrophication and pollution because it can potentially stimulate growth in some species and inhibit growth in other species of algae, the foundation of the marine ecosystem. Inositol hexaphosphate (also named phytic acid or PA), an abundant organophosphate, is presumably ubiquitous in the marine environment, but how it affects marine primary producers is poorly understood. Here, we investigated the bioavailability of this DOP to the cosmopolitan coccolithophore Emiliania huxleyi. Our results showed that E. huxleyi cells can take up PA and dissolved inorganic phosphorus (DIP) simultaneously. Absorbed PA can efficiently support algal growth, producing cell yield between DIP and phosphorus (P)-depleted conditions. Accordingly, PA supply as the sole P source highly influences cellular metabolism and nutrient stoichiometry. Particularly, PA-grown cultures exhibited enhanced carbon fixation, increased lipid content, activated energy metabolism, and induced nitrogen assimilation. However, our data suggest that PA may also exert some levels of toxic effects on E. huxleyi. This study provides novel insights into the variable effects of a DOP on marine phytoplankton, which will inform new inquiries about how the complex DOP constituencies in the ocean will shape phytoplankton community structure and function. IMPORTANCE The dissolved organic phosphorus (DOP) utilization in phytoplankton plays vital roles in cellular P homeostasis, P-nutrient niche, and the dynamics of community structure in marine ecosystems, but its mechanisms, potentially varying with species, are far from clear. In this study, we investigated the utilization of a widespread DOP species, which is commonly produced by plants (land plants and marine macrophytes) and released into coastal areas, in a globally distributed bloom-forming coccolithophore species in various phosphorus environments. Using a combination of physiological and transcriptomic measurements and analyses, our experimental results revealed the complex mechanism and two-sided effects of DOP (major algal growth-supporting and minor toxic effects) in this species, providing a novel perspective on phytoplankton nutrient regulation.

Proton-transporting heliorhodopsins from marine giant viruses.

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here, we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-activated proton transporter. V2HeR3 absorbs blue-green light, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a PAG of the Schiff base, and mutations at these positions converted the protein into an outward proton pump. Three environmental viral HeRs from the same group as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tool, similarly to type-1 microbial rhodopsins.

MicroRNA-mediated regulation of lipid metabolism in virus-infected Emiliania huxleyi

The interactions between Emiliania huxleyi and E. huxleyi virus (EhV) regulate marine carbon and sulfur biogeochemical cycles and play a prominent role in global climate change. As a large DNA virus, EhV has developed a novel “virocell metabolism” model to meet its high metabolic needs. Although it has been widely demonstrated that EhV infection can profoundly rewire lipid metabolism, the epigenetic regulatory mechanisms of lipid metabolism are still obscure. MicroRNAs (miRNAs) can regulate biological pathways by targeting hub genes in the metabolic processes. In this study, the transcriptome, lipidome, and miRNAome were applied to investigate the epigenetic regulation of lipid metabolism in E. huxleyi cells during a detailed time course of viral infection. Combined transcriptomic, lipidomic, and physiological experiments revealed reprogrammed lipid metabolism, along with mitochondrial dysfunction and calcium influx through the cell membrane. A total of 69 host miRNAs (including 1 known miRNA) and 7 viral miRNAs were identified, 27 of which were differentially expressed. Bioinformatic prediction revealed that miRNAs involved in the regulation of lipid metabolism and a dual-luciferase reporter assay suggested that phosphatidylinositol 3-kinase (PI3K) gene might be a target of ehx-miR5. Further qPCR and western blot analysis showed a significant negative correlation between the expression of ehx-miR5 and its target gene PI3K, along with the lower activity of its downstream components (p-Akt, p-TOR, SREBP), indicating that lipid metabolism might be regulated by ehx-miR5 through the PI3K-Akt-TOR signaling pathway. Our findings reveal several novel mechanisms of viral strategies to manipulate host lipid metabolism and provide evidence that ehx-miR5 negatively modulates the expression of PI3K and disturbs lipid metabolism in the interactions between E. huxleyi and EhV.

Physiological and metabolic effects of glyphosate as the sole P source on a cosmopolitan phytoplankter and biogeochemical implications

Nutrient conditions influence the physiology and stoichiometry of marine phytoplankton. While extensive studies have documented the effects of abundances and types of nutrients such as nitrogen (N) and phosphorus (P), the effect of phosphonates as a P source is less understood and underexplored. Here, with the cosmopolitan coccolithophorid Emiliania huxleyi as a model phytoplankter, we investigated the effect of the phosphonate type of herbicide glyphosate as the sole P source in comparison with the P-depleted and P-replete (with 36 μM dissolved inorganic phosphate [DIP]) cultures. We measured changes in cellular C (carbon):P and N:P ratios and physiological performance and documented the corresponding transcriptomic and miRNAomic responses in E. huxleyi to glyphosate treatment. We found that glyphosate supported population growth but not to the full scale relative to DIP, and this was under the concerted regulation of DNA replication and cell cycle arrest genes as well as the growth-regulating miRNA. Furthermore, our data suggest that E. huxleyi took up glyphosate directly, bypassing extracellular hydrolysis, and this involved ABC transporters. Meanwhile, glyphosate-grown cultures displayed marked increases in cellular particulate organic C (POC) and PON contents, cell size, and transcription of genes for CO2 fixation and citrate cycle, nitrate transport, and protein biosynthesis. However, compared to DIP, the maximum absorption rate of glyphosate was only 33%, and glyphosate-grown E. huxleyi cells exhibited a mild P-stress symptom and elevated cellular C:P and N:P ratios. Interestingly, glyphosate-grown cells showed an increased sinking rate, suggesting that glyphosate as the sole P source might enhance the efficiency of C export by E. huxleyi, which would compensate for the expected decline in primary productivity (and hence carbon efflux) in the future more nutrient-depleted ocean. This biogeochemical implication needs to be further studied and verified, however.

Raman Microspectroscopy Goes Viral: Infection Dynamics in the Cosmopolitan Microalga, Emiliania huxleyi.

Emiliania huxleyi is a cosmopolitan member of the marine phytoplankton. This species’ capacities for carbon sequestration and sulfur mobilization make it a key player in oceanic biogeochemical cycles that influence climate on a planetary scale. Seasonal E. huxleyi blooms are abruptly terminated by viral epidemics caused by a clade of large DNA viruses collectively known as coccolithoviruses (EhVs). EhVs thereby mediate a significant part of material and energy fluxes associated with E. huxleyi population dynamics. In this study, we use spontaneous Raman microspectroscopy to perform label-free and non-invasive measurements of the macromolecular composition of individual virions and E. huxleyi host cells. Our novel autofluorescence suppression protocol enabled spectroscopic visualization of evolving macromolecular redistributions in individual E. huxleyi cells at different stages of EhV infection. Material transfer from E. huxleyi hosts to single EhV-163 virions was confirmed by combining stable isotope probing (SIP) experiments with Raman microspectroscopy. Inheritance of the host cells’ 13C-enriched isotopic signature was quantified based on red shifts of Raman peaks characteristic of phenylalanine’s phenyl ring. Two-dimensional Raman mapping of EhV-infected E. huxleyi cells revealed that the compact region producing an intense Raman DNA signal (i.e., the nucleus) in healthy E. huxleyi cells becomes diffuse during the first hours of infection. Raman DNA emissions integrated throughout individual cells decreased during the infection cycle. Our observations are consistent with EhV-163 degrading the host’s nuclear DNA, scavenging released nucleotides for its own genome replication, and shedding newly-produced virions prior to host lysis via budding.

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

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

A Non-Destructive, Tuneable Method to Isolate Live Cells for High-Speed AFM Analysis.

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min.

The Calcium Carbonate Shell of Emiliania huxleyi Provides Limited Protection Against Viral Infection

Coccolithophores are an important group of marine phytoplankton which cover themselves with the coccosphere – a shell composed of numerous calcium carbonate (CaCO3) platelets. Despite more than a century of coccolithophore research, it remains speculative why coccolithophores calcify. Resolving this question is essential to assess the competitive fitness of coccolithophores in the future ocean where changes in calcification are expected. Here, we used the Emiliania huxleyi – Emiliania huxleyi Virus 86 host-virus model system to test the hypothesis that the coccosphere serves as a physical barrier reducing viral infection. Therefore, we removed the coccosphere from living E. huxleyi cells and compared the infection progress relative to calcified cells in a series of 6 experiments under different growth conditions. We found that the coccosphere does not constitute an effective physical barrier against viral penetration, since non-growing calcified cells were susceptible to viral infection and lysis (growth stopped by light limitation). However, we also found that protection against the virus may depend on the daily growth cycle. E. huxleyi reached higher peak abundances when decalcified cells were allowed to rebuild their coccosphere before entering cell division phase and being exposed to the virus, thereby suggesting that rates of viral infection could be reduced by the coccosphere during the critical phase in the cell cycle. However, the benefit of this potential protection is arguably of limited ecological significance since the concentrations of both, calcified and decalcified E. huxleyi approached similar values until the end of the bloom. We conclude that the coccosphere provides at best a limited protection against infection with the EhV86.

Li Partitioning Into Coccoliths of Emiliania huxleyi: Evaluating the General Role of “Vital Effects” in Explaining Element Partitioning in Biogenic Carbonates

AbstractEmiliania huxleyi cells were grown in artificial seawater of different Li and Ca concentrations and coccolith Li/Ca ratios determined. Coccolith Li/Ca ratios were positively correlated to seawater Li/Ca ratios only if the seawater Li concentration was changed, not if the seawater Ca concentration was changed. This Li partitioning pattern of E. huxleyi was previously also observed in the benthic foraminifer Amphistegina lessonii and inorganically precipitated calcite. We argue that Li partitioning in both E. huxleyi and A. lessonii is dominated by a coupled transmembrane transport of Li and Ca from seawater to the site of calcification. We present a refined version of a recently proposed transmembrane transport model for Li and Ca. The model assumes that Li and Ca enter the cell via Ca channels, the Li flux being dependent on the Ca flux. While the original model features a linear function to describe the experimental data, our refined version uses a power function, changing the stoichiometry of Li and Ca. The version presented here accurately predicts the observed dependence of DLi on seawater Li/Ca ratios. Our data demonstrate that minor element partitioning in calcifying organisms is partly mediated by biological processes even if the partitioning behavior of the calcifying organism is indistinguishable from that of inorganically precipitated calcium carbonate.

De novo transcriptome profile of coccolithophorid alga Emiliania huxleyi CCMP371 at different calcium concentrations with proteome analysis.

The haptophyte alga Emiliania huxleyi is the most abundant coccolithophore in the modern ocean and produces elaborate calcite crystals, called coccolith, in a separate intracellular compartment known as the coccolith vesicle. Despite the importance of biomineralization in coccolithophores, the molecular mechanism underlying it remains unclear. Understanding this precise machinery at the molecular level will provide the knowledge needed to enable further manipulation of biomineralization. In our previous study, altering the calcium concentration modified the calcifying ability of E. huxleyi CCMP371. Therefore in this study, we tested E. huxleyi cells acclimated to three different calcium concentrations (0, 0.1, and 10 mM). To understand the whole transcript profile at different calcium concentrations, RNA-sequencing was performed and used for de novo assembly and annotation. The differentially expressed genes (DEGs) among the three different calcium concentrations were analyzed. The functional classification by gene ontology (GO) revealed that ‘intrinsic component of membrane’ was the most enriched of the GO terms at the ambient calcium concentration (10 mM) compared with the limited calcium concentrations (0 and 0.1 mM). Moreover, the DEGs in those comparisons were enriched mainly in ‘secondary metabolites biosynthesis, transport and catabolism’ and ‘signal transduction mechanisms’ in the KOG clusters and ‘processing in endoplasmic reticulum’, and ‘ABC transporters’ in the KEGG pathways. Furthermore, metabolic pathways involved in protein synthesis were enriched among the differentially expressed proteins. The results of this study provide a molecular profile for understanding the expression of transcripts and proteins in E. huxleyi at different calcium concentrations, which will help to identify the detailed mechanism of its calcification.

Physiological responses of Oxyrrhis marina to a diet of virally infected Emiliania huxleyi.

The coccolithophore Emiliania huxleyi forms some of the largest phytoplankton blooms in the ocean. The rapid demise of these blooms has been linked to viral infections. E. huxleyi abundance, distribution, and nutritional status make them an important food source for the heterotrophic protists which are classified as microzooplankton in marine food webs. In this study we investigated the fate of E. huxleyi (CCMP 374) infected with virus strain EhV-86 in a simple predator-prey interaction. The ingestion rates of Oxyrrhis marina were significantly lower (between 26.9 and 50.4%) when fed virus-infected E. huxleyi cells compared to non-infected cells. Despite the lower ingestion rates, O. marina showed significantly higher growth rates (between 30 and 91.3%) when fed infected E. huxleyi cells, suggesting higher nutritional value and/or greater assimilation of infected E. huxleyi cells. No significant differences were found in O. marina cell volumes or fatty acids profiles. These results show that virally infected E. huxleyi support higher growth rates of single celled heterotrophs and in addition to the “viral shunt” hypothesis, viral infections may also divert more carbon to mesozooplankton grazers.

Morphological switch to a resistant subpopulation in response to viral infection in the bloom-forming coccolithophore Emiliania huxleyi.

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.

Virus-induced apoptosis and phosphorylation form of metacaspase in the marine coccolithophorid Emiliania huxleyi.

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 ≈ 23kDa; 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.

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

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

Trace-Element Incorporation into Intracellular Pools Uncovers Calcium-Pathways in a Coccolithophore.

Many organisms form minerals from precursor phases that crystallize under strict biological control. The dynamic intracellular processes of formation, transport, and deposition of these precursor phases are challenging to identify. An unusual situation is recently revealed for the calcifying alga Emiliania huxleyi, as the cells contain a compartment filled with a concentrated Ca and P phase but the final calcite crystals, which are nucleated in a different compartment, are P‐free. Thus, the connection of the Ca–P‐rich pool to the mineralization process remains unclear. Here, pulse‐chase experiments are used with Sr to label the Ca–P‐rich phase in E. huxleyi cells, and cryo X‐ray absorption spectroscopy and analytical transmission electron microscopy to follow the Sr within cells. It is found that Sr is first found in the Ca–P‐rich phase and then becomes incorporated into the calcite. This demonstrates that the calcium used by the cells to build calcite originates from the Ca–P‐rich pool.

Ocean acidification effects on haploid and diploid Emiliania huxleyi strains: Why changes in cell size matter

The effects of ocean acidification (OA) on Emiliania huxleyi non-calcifying haploid strain CCMP 374 (s374) and calcifying diploid strain CCMP 2668 (s2668) were investigated in this study. Both E. huxleyi strains were cultured semi-continuously for ~20 generations under three pCO2 levels, one representing equilibrium with contemporary atmospheric pCO2 and two representing different OA scenarios projected for the end the century. During the course of the experiments, measurements were made for cell size in s374 and coccosphere size in strain s2668, cellular intrinsic growth rates, and the per cell (s374) and per coccosphere (s2668) concentrations of particulate organic carbon (POC) and nitrogen (PON), particulate inorganic carbon (PIC), chlorophyll a (chl a) and particulate dimethylsulfoniopropionate (DMSPp). In agreement with other studies, elevated pCO2 led to a small increase in POC and PON in the diploid strain, but not in the haploid strain. Although the PIC was static across pCO2 treatments in the diploid calcifier, PIC:POC was reduced under OA due to the disproportionate increase in POC. The concentration of E. huxleyi DMSPp cell−1 increased (s2668) or responded non-linearly (s374) to OA. Exposure to OA caused cell and coccosphere volumes to increase by 18% and 37% from the ambient to high pCO2 treatment in the haploid and diploid strain, respectively, despite equivalent cross-treatment growth rates. Therefore, when normalized to cell and coccosphere volume, small to negligible increases in cellular constituents under OA translated into decreased cellular and coccosphere densities (e.g. mass vol−1) of those same constituents. This produces seemingly contradictory cellular responses to OA depending on data normalization, and highlights the need to cautiously interpret OA experimental data when cell and coccosphere size is not reported. A consistent OA-induced increase in cell and coccosphere size has implications for E. huxleyi cellular physiology, ecology and trophic interactions, as cell size is a master characteristic that governs cellular rate processes, predator encounter rates, and predator ingestion efficiencies. Further, because POC and PON did not scale with volume increases, there is a dilution in the carbon and nitrogen concentration in E. huxleyi cells under OA. This result could have cascading effects on planktonic food webs as predator ingestion rates and growth efficiencies adjust.

Influence of physiological states of Emiliania huxleyi cells on their lipids and associated molecular isotopic compositions during microbial degradation

The goal of this study was to examine the potential effect of physiological states of phytoplankton cells on their lipids (phytol, sterols, alkenones, and fatty acids) and associated stable carbon isotopic compositions during microbial degradation. Cells of Emiliania huxleyi were cultivated using batch cultures with the same medium but over different growth stages: exponential growth phase (culture-1) and stationary phase (culture-2), respectively. There were remarkable differences in cellular lipid contents and relative compositions between these two cultures: ~8 times higher lipid content with higher percentages of poly- and mono-unsaturated fatty acids, and alkenones but lower percentages of saturated fatty acids, phytol, and sterols in culture-2 than in culture-1. Dead phytoplankton cells (killed by freezing) from the two cultures were incubated in the dark with addition of natural seawater (containing natural microbial communities) under aerobic conditions at 17°C for about one month. Most phytoplankton fatty acids (except for 16:0) from the two cultures were almost completely degraded while most neutral lipids (phytol, sterols and alkenones) (except for 27Δ5 from culture-1) were partially degraded with relatively higher percentages of these compounds remaining in culture-2 than in culture-1. Along with degradation of lipids, their stable carbon isotopic compositions varied differently among compound classes: positive shifts (+1.7‰ to +3.9‰) in δ13C values for fatty acids; little change for sterols; and for alkenones, either little change in most cases or a negative shift (−3‰) in one case (37:2 alkenone in culture-1). Moreover, relatively larger isotopic shifts (~+1.5‰ each) for fatty acids 16:0 and 18:1(ω9) were observed in culture-1 compared to those in culture-2. The results from these experiments imply that the variability in isotopic compositions of phytoplankton lipid compounds during microbial degradation is critically affected by physiological states of phytoplankton cells due to heterogeneity in isotopic compositions of cellular lipids and differential reactivities of various lipid compounds bound in different intracellular components, which are synthesized through different cell growth phases.