Gephyrocapsa huxleyi (Emiliania huxleyi) as a model system for coccolithophore biology

Coccolithophores are the most abundant calcifying organisms in modern oceans and are important primary producers in many marine ecosystems. Their ability to generate a cellular covering of calcium carbonate plates (coccoliths) plays a major role in marine biogeochemistry and the global carbon cycle. Coccolithophores also play an important role in sulfur cycling through the production of the climate-active gas dimethyl sulfide. The primary model organism for coccolithophore research is Emiliania huxleyi, now named Gephyrocapsa huxleyi. G. huxleyi has a cosmopolitan distribution, occupying coastal and oceanic environments across the globe, and is the most abundant coccolithophore in modern oceans. Research in G. huxleyi has identified many aspects of coccolithophore biology, from cell biology to ecological interactions. In this perspective, we summarize the key advances made using G. huxleyi and examine the emerging tools for research in this model organism. We discuss the key steps that need to be taken by the research community to advance G. huxleyi as a model organism and the suitability of other species as models for specific aspects of coccolithophore biology.     

Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability

? To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. ? Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pH(i)). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H(+), CO(2) and HCO(3)(-). ? Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pH(i) unit. pH(i) acutely followed the pH of seawater (pH(e)) in a linear fashion between pH(e) values of 6.5 and 9 with a slope of 0.44 per pH(e) unit. pH(i) was nearly insensitive to changes in seawater CO(2) at constant pH(e) and HCO(3)(-). An increase in extracellular HCO(3)(-) resulted in a slight intracellular acidification. In the presence of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pH(e) on pH(i). ? The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pH(i) homeostasis involves a DIDS-sensitive mechanism.     

Effects of HCO-3 on Surface Calcification and CO2 Fixation in Marine Emiliania huxleyi

Emiliania huxleyi is a ubiquitous species with the largest biomass in marine planktonics. When cells of E. huxleyi were grown in ESM with additional 20 mmol/L HC03- , coccolith scales were formed on cell surface and the weight ratio of calcium carbonate fixed in coecoliths to organic substance in cells was about 2.47: 1. It was only about 0.05: 1 in cells grown in ESM without HCO3- addition, where no coccoliths were observed under scanning electron microscope, and the content of lipid reached 18.1% of dry. cell weight. It was demonstrated that the HCO3- concentration was the key factor to control the calcification on cell surface. Therefore, in addition to the pathway of photosynthesis for CO2 fixation, calcification on cell surface forming coccoliths is an alternative pathway for fixing dissolved inorganic carbon in E. huxleyi. Moreover, being rich in lipids, E. huxleyi cells produced high content of hydrocarbons including extractable organic matter, saturates and aromatics under pyrolysis at 300℃. Among those, the yield of saturates from E. huxleyi reached as high as 2.8%, 6-15 times that from other algae. All these suggest that E. huxleyi is a good experimental system for studies on the optimization of environment through carbon cycle and renewable energy in algal biomass.     

Induction of phase variation events in the life cycle of the marine coccolithophorid Emiliania huxleyi

Emiliania huxleyi is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.     

A renewable energy source — hydrocarbon gases resulting from pyrolysis of the marine nanoplanktonic alga Emiliania huxleyi

The marine coccolithophore, Emiliania huxleyi, grown in the laboratory was subjected to vacuum pyrolysis at various temperatures from 100 to 500 °C. The highest yield of pyrolytic gases (183 mL g⁻¹ dry cells) was obtained at 400 °C. The amount of total hydrocarbon gas produced at 400 °C was 129 mL, about 10 times higher than at 300 °C. CH₄ was the major component at the high gas-production stage (400–500 °C). The great increase in hydrocarbon gases at 400 °C was accompanied by a marked decrease in liquid saturates and aromatics. The results indicate that the liquid hydrocarbons (oil) produced by pyrolysis at lower temperature is a direct source for the formation of the hydrocarbon gases. Due to its large potential for the production of biomass and hydrocarbons with low energy input, E. huxleyi is suggested as one of candidates for the production of renewable fuels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of the selenite uptake mechanism in the coccolithophore Emiliania huxleyi (Haptophyta)

The marine coccolithophore Emiliania huxleyi (Haptophyta) requires selenium as an essential element for growth, and the active species absorbed is selenite, not selenate. This study characterized the selenite uptake mechanism using ??Se as a tracer. Kinetic analysis of selenite uptake showed the involvement of both active and passive transport processes. The active transport was suppressed by 0.5 mM vanadate, a membrane-permeable inhibitor of H?-ATPase, at pH 8.3. When the pH was lowered from 8.3 to 5.3, the selenite uptake activity greatly increased, even in the presence of vanadate, suggesting that the H? concentration gradient may be a motive force for selenite transport. [??Se]Selenite uptake at selenite-limiting concentrations was hardly affected by selenate, sulfate and sulfite, even at 100 μM. In contrast, 3 μM orthophosphate increased the K(m) 5-fold. These data showed that HSeO??, a dominant selenite species at acidic pH, is the active species for transport through the plasma membrane and transport is driven by ΔpH energized by H?-ATPase. Kinetic analysis showed that the selenite uptake activity was competitively inhibited by orthophosphate. Furthermore, the active selenite transport mechanism was shown to be induced de novo under Se-deficient conditions and induction was suppressed by the addition of either sufficient selenite or cycloheximide, an inhibitor of de novo protein synthesis. These results indicate that E. huxleyi cells developed an active selenite uptake mechanism to overcome the disadvantages of Se limitation in ecosystems, maintaining selenium metabolism and selenoproteins for high viability.     

Dynamics and genotypic composition of Emiliania huxleyi and their co-occurring viruses during a coccolithophore bloom in the North Sea

We studied the temporal succession of vertical profiles of Emiliania huxleyi and their specific viruses (EhVs) during the progression of a natural phytoplankton bloom in the North Sea in June 1999. Genotypic richness was assessed by exploiting the variations in a gene encoding a protein with calcium-binding motifs (GPA) for E.?huxleyi and in the viral major capsid protein gene for EhVs. Using denaturing gradient gel electrophoresis and sequencing analysis, we showed at least three different E.?huxleyi and EhV genotypic profiles during the period of study, revealing a complex, and changing assemblage at the molecular level. Our results also indicate that the dynamics of EhV genotypes reflect fluctuations in abundance of potential E.?huxleyi host cells. The presence and concentration of specific EhVs in the area prior to the bloom, or EhVs transported into the area by different water masses, are significant factors affecting the structure and intraspecific succession of E.?huxleyi during the phytoplankton bloom.     

Distribution of living coccolithophores in the California Current system,southern California borderland

The distribution of living coccolithophores in the California Current system of southern California at 10 m water depth was investigated on two dates in March and June, 1982. Six closely spaced stations were sampled in March, of which three were resampled in June. Thirty-six euphotic species were identified of which four,Emiliania huxleyi, Umbilicosphaera sibogae, Gephyrocapsa oceanica, andRhabdosphaera longistylis, respectively, were the most abundant. Both the “cold” and “warm” morphotypes ofE. huxleyi were present, in varying proportions. Large ranges in community structure, diversity (0.35–2.64 natural bels), and standing crop (1.0 × 10⁴–6.2 × 10⁵cells/l) were recorded. This range of end-member values is approximately that found in the open ocean from 0° to about 65° latitude.The distributions of four coccolithophore assemblages recognized in March samples from the Borderland area appear to reflect the following distinct water masses: (1) California Current; (2) Southern California Counter Current; (3) Transitional Zone; (4) Near Shore. The coccolithophore assemblages from the June stations were more uniform, indicating that the Borderland was experiencing more stable conditions than in March.     

Export production of coccolithophores in an upwelling region: Results from San Pedro Basin, Southern California Borderlands

A seven month-long time series sediment trap project was carried out in San Pedro Basin (Southern California Borderlands) in order to evaluate the response of calcareous nannoplankton to seasonal hydrographic changes. This region is periodically influenced by upwelling, particularly during the spring and early summer. The highest fluxes of both whole coccospheres and individual coccoliths occurred during winter (January-February), a period when the fluxes of diatoms and planktic foraminifera were low. The highest coccolithophore fluxes were recorded in the mid-February with 860 × 10⁶ coccoliths m⁻² day⁻¹, 8 × 10⁶ whole coccospheres m⁻² day⁻¹, and 80 mg of coccolith carbonate m⁻² day⁻¹. Coccolith carbonate fluxes in January and February account for most of the total carbonate fluxes measured during this period. The season of maximum coccolithophore production in this region (winter) is correlated with weak stratification of the upper water column, low total primary production, low nutrient contents, and low temperatures.Emiliania huxleyi and Florisphaera profunda are the two most abundant species in this region. While E. huxleyi displays no distinct seasonal changes in flux, F. profunda shows a clear preference for cold, low nutrient water conditions and low light levels. Helicosphaera spp. flux is positively correlated to the total coccosphere fluxes and is indicative of high coccolithophore productivity.     

Molecular dynamics of Emiliania huxleyi and cooccurring viruses during two separate mesocosm studies

In this study we used denaturing gradient gel electrophoresis, sequencing analysis, and analytical flow cytometry to monitor the dynamics and genetic richness of Emiliania huxleyi isolates and cooccurring viruses during two mesocosm experiments in a Norwegian fjord in 2000 and 2003. We exploited variations in a gene encoding a protein with calcium-binding motifs (GPA) and in the major capsid protein (MCP) gene to assess allelic and genotypic richness within E. huxleyi and E. huxleyi-specific viruses (EhVs), respectively. To our knowledge, this is the first report that shows the effectiveness of the GPA gene for analysis of natural communities of E. huxleyi. Our results revealed the existence of a genetically rich, yet stable E. huxleyi and EhV community in the fjordic environment. Incredibly, the same virus and host genotypes dominated in separate studies conducted 3 years apart. Both E. huxleyi-dominated blooms contained the same six E. huxleyi alleles. In addition, despite the presence of at least six and four EhV genotypes at the start of the blooms in 2000 and 2003, respectively, the same two virus genotypes dominated the naturally occurring infections during the exponential and termination phases of the blooms in both years.     

Virus succession observed during an Emiliania huxleyi bloom

Denaturing gradient gel electrophoresis was used as a molecular tool to determine the diversity and to monitor population dynamics of viruses that infect the globally important coccolithophorid Emiliania huxleyi. We exploited variations in the major capsid protein gene from E. huxleyi-specific viruses to monitor their genetic diversity during an E. huxleyi bloom in a mesocosm experiment off western Norway. We reveal that, despite the presence of several virus genotypes at the start of an E. huxleyi bloom, only a few virus genotypes eventually go on to kill the bloom.     

Genetic delineation between and within the widespread coccolithophore morpho‐species Emiliania huxleyi and Gephyrocapsa oceanica (Haptophyta)

Emiliania huxleyi and Gephyrocapsa oceanica are abundant coccolithophore morpho‐species that play key roles in ocean carbon cycling due to their importance as both primary producers and cal‐cifiers. Global change processes such as ocean acidification impact these key calcifying species. The physiology of E. huxleyi, a developing model species, has been widely studied, but its genetic delineation from G. oceanica remains unclear due to a lack of resolution in classical genetic markers. Using nuclear (18S rDNA and 28S rDNA), mitochondrial (cox1, cox2, cox3, rpl16, and dam), and plastidial (16S rDNA, rbcL, tufA, and petA) DNA markers from 99 E. huxleyi and 44 G. oceanica strains, we conducted a multigene/multistrain survey to compare the suitability of different markers for resolving phylogenetic patterns within and between these two morpho‐species. The nuclear genes tested did not provide sufficient resolution to discriminate between the two morpho‐species that diverged only 291Kya. Typical patterns of incomplete lineage sorting were generated in phylogenetic analyses using plastidial genes. In contrast, full morpho‐species delineation was achieved with mitochondrial markers and common intra‐morpho‐species phylogenetic patterns were observed despite differing rates of DNA substitution. Mitochondrial genes are thus promising barcodes for distinguishing these coccolithophore morpho‐species, in particular in the context of environmental monitoring.     

Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens

The utilization of archived, formalin-fixed paraffin-embedded (FFPE) tumor samples for massive parallel sequencing has been challenging due to DNA damage and contamination with normal stroma. Here, we perform whole genome sequencing of DNA isolated from two triple-negative breast cancer tumors archived for >11 years as 5 μm FFPE sections and matched germline DNA. The tumor samples show differing amounts of FFPE damaged DNA sequencing reads revealed as relatively high alignment mismatch rates enriched for C · G > T · A substitutions compared to germline samples. This increase in mismatch rate is observable with as few as one million reads, allowing for an upfront evaluation of the sample integrity before whole genome sequencing. By applying innovative quality filters incorporating global nucleotide mismatch rates and local mismatch rates, we present a method to identify high-confidence somatic mutations even in the presence of FFPE induced DNA damage. This results in a breast cancer mutational profile consistent with previous studies and revealing potentially important functional mutations. Our study demonstrates the feasibility of performing genome-wide deep sequencing analysis of FFPE archived tumors of limited sample size such as residual cancer after treatment or metastatic biopsies.     

Intragenomic Spread of Plastid-Targeting Presequences in the Coccolithophore Emiliania huxleyi

Nucleus-encoded plastid-targeted proteins of photosynthetic organisms are generally equipped with an N-terminal presequence required for crossing the plastid membranes. The acquisition of these presequences played a fundamental role in the establishment of plastids. Here, we report a unique case of two non-homologous proteins possessing completely identical presequences consisting of a bipartite plastid-targeting signal in the coccolithophore Emiliania huxleyi. We further show that this presequence is highly conserved in five additional proteins that did not originally function in plastids, representing de novo plastid acquisitions. These are among the most recent cases of presequence spreading from gene to gene and shed light on important evolutionary processes that have been usually erased by the ancient history of plastid evolution. We propose a mechanism of acquisition involving genomic duplications and gene replacement through non-homologous recombination that may have played a more general role for equipping proteins with targeting information.     

The sensitivity of Emiliania huxleyi (Prymnesiophyceae) to ultraviolet-b radiation

Emiliania huxleyi (Lohm.) Hay et Miller is an important component of the phytoplankton in open ocean waters. The sensitivity of this cosmopolitan alga to natural levels of UVB radiation has never been tested. Since DNA is believed to be a major target of natural UVB radiation (UVBR: 280–315 nm) in living cells, experiments with E. huxleyi were performed using growth rate reduction and DNA damage as indicators of UVBR stress. Specific growth rate, cell volume, pigment content, and CPD (cyclobutane pyrimidine dimer) formation (a measure for DNA damage) were followed during and after prolonged exposure of a series of cultures to a range of UVBR levels. E. huxleyi was found to be very sensitive to UVBR: at a daily weighted UVBR dose of only 400 J·m⁻² ·d⁻¹ (BED_DNA300nm), growth was halted. At this UVBR level, both cell volume and contents of the major photosynthetic and photoprotective pigments had increased. The UVBR vulnerability of E. huxleyi cannot be explained by a high potential for cyclobutane thymine dimer formation (the most abundant CPD type) due to a high T content of nuclear DNA: the CG content of this E. huxleyi strain is high (68%) compared with other species. The high UVBR sensitivity may be related to the stage of the cell cycle during UVBR exposure, in combination with low repair capacity. It is concluded that E. huxleyi may experience UVBR stress through the formation of cyclobutane pyrimidine dimers, with subsequent low repair capacity and thereby arrest of the cell cycle.