A physiological comparison of coccolith-forming and naked cells of Coccolithus huxleyi

Summary In a naked clone of the coccolithophorid Coccolithus huxleyi, growth at light saturation was 15% slower than in a coccolith-forming clone isolated from the same parent stock. The two cell types did not differ significantly with regard to cell volume and protein content. A 10–13% smaller chlorophyll a content of the naked cells was matched by a uniform lowering of photosynthetic rates at all light intensities. It was considered that the slower growth of these cells might result from a less favourable ratio between photosynthetic output and biomass.The content of deoxyribonucleic acid was the same in coccolith-forming and naked cells, suggesting that they do not represent different phases in a sexual lifecycle.     

Incorporation of zinc into the coccoliths of the microalga <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>

The coccolithophore Emiliania huxleyi is covered with elaborated calcite plates, the so-called coccoliths, which are produced inside the cells. We investigated the incorporation of zinc into the coccoliths of E. huxleyi by applying different zinc and calcium amounts via the culture media and subsequently analyzing the zinc content in the cells and the Zn/Ca ratio of the coccoliths. To investigate the Zn/Ca ratio of coccoliths built in the manipulated media, the algae have first to be decalcified, i.e. coccolith free. We used a newly developed decalcification method to obtain ‘naked’ cells for cultivation. E. huxleyi proliferated and produced new coccoliths in all media with manipulated Zn/Ca ratios. The cells and the newly built coccoliths were investigated regarding their zinc content and their Zn/Ca ratio, respectively. High zinc amounts were taken up by the algae. The Zn/Ca ratio of the coccoliths was positively correlated to the Zn/Ca ratio of the applied media. The unique feature of the coccoliths was maintained also at high Zn/Ca ratios. We suggest the following pathway of the zinc ions into the coccoliths: first, the zinc ions are bound to the cell surface, followed by their transportation into the cytoplasm. Obviously, the zinc ions are removed afterwards into the coccolith vesicle, where the zinc is incorporated into the calcite coccoliths which are then extruded. The incorporation of toxic zinc ions into the coccoliths possibly due to a new function of the coccoliths as detoxification sites is discussed.     

ROLE OF THE LIGHT-DARK CYCLE AND MEDIUM COMPOSITION ON THE PRODUCTION OF COCCOLITHS BY EMILIANIA HUXLEYI (HAPTOPHYCEAE)1

Production of coccoliths by cells of Emiliania huxleyi (Lohmann) Hay and Mohler was measured during exposure of the cells to two diel light-dark cycles (16:8 h). During the light period about eight coccoliths per cell were formed at a constant rate of one coccolith per 2 h. Cells divided during the first half of the dark period. No coccolith production took place during the dark period. With electron microscopy we found early-stage, coccolith-production compartments in cells after mitosis while still in the dark. No calcification was observed in these compartments. Cells grown on enriched seawater (Eppley's medium) tended to produce enough coccoliths to cover the cell in a single layer. When these cells reached the stationary phase coccolith production stopped. Coccolith production was induced by removal of extracellular coccoliths. Cells grown on medium containing 2% of the nitrate and phosphate of Eppley's medium tended to produce coccoliths in the stationary phase. This resulted in the formation of multiple layers of coccoliths. The multiple covering was restored after decalcification of stationary cells. Formation of multiple layers of coccoliths may help the cells reach deeper, nutrient-rich water by increasing the sinking rate of the cells.     

COCCOLITH PRODUCTION AND DETACHMENT BY EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

Laboratory experiments were performed with the prymnesiophyte Emiliania huxleyi (Lohm.) Hay and Mohler, strain 88E, to quantify calcification per cell, coccolith detachment, and effects of coccolith production on optical scattering of individual cells. ¹⁴C incorporation into attached and detached coccoliths was measured using a bulk filtration technique. ¹⁴C-labeled cells also were sorted using a flow cytometer and analyzed for carbon incorporation into attached coccoliths. The difference between the bulk and flow cytometer analyses provided a ¹⁴C-based estimate of the rate of production of detached coccoliths. Coccolith production and detachment were separated in time in batch cultures, with most detachment happening well after calcification had stopped. Accumulation of coccoliths was maximum at the end of logarithmic growth with 50–80 coccoliths per cell (three to five complete layers of coccoliths around the cells). Net accretion rates of coccoliths were on the order of 7 coccoliths· cell^?1·d^?1 while net detachment rates were as high as 15 coccoliths· cell^?1·d^?1 for stationary phase cells. Equal numbers of coccoliths were attached and detached early in logarithmic growth, and as cells aged, the numbers of detached coccoliths exceeded the attached ones by a factor of 6. Our results demonstrate pronounced charges of forward angle light scatter and 90° light scatter of cells as they grow logarithmically and enter stationary phase. Counts of loose coccoliths in batch cultures are consistent with the detachment of coccoliths in layers rather than individual coccoliths.     

CALCIFICATION BY COCCOLITHOPHORIDS: EFFECTS OF pH AND Sr1

Cells of Coccolithus huxleyi which fail to deposit CaCO₃ and form coccoliths often occur as unwanted components in cultures used for studies of calcification. Non-calcified cells generally cannot be made to recalcify, but they can be removed from cultures by treatment at elevated pH or by a method based on faster sinking of calcified cells. Lowering the concentrations of nitrate, phosphate, or trace metals in the medium did not restore calcifying ability of non-calcified cells. However, addition of strontium did promote recalcification of decalcified Cricosphaera carterae grown under calcium limitation. Strontium seemed to promote coccolith attachment to cells rather than to affect calcium uptake or coccolith formation itself.     

EFFECTS OF ENVIRONMENTAL VARIATION ON SINKING RATES OF MARINE PHYTOPLANKTON1

The effects of environmental variables, particularly irradiance, on the sinking rates of phytoplankton were investigated using cultures of Chaetoceros gracilis Schütt and C. flexuosum Mangin in laboratory experiments; these data were compared with results from assemblages in the open ocean and marginal ice zone of the Greenland Sea. In culture experiments both the irradiance under which the diatom was grown and culture growth rate were positively correlated with sinking rates. Sinking rates (ψ) in the Greenland Sea were smallest when determined from chlorophyll (mean ψ_chl= 0.14 m · d^?1) and biogenic silica (ψ_si= 0.14 m · d^?1) and greatest when determined from particulate carbon (ψ_c= 0.55 m · d^?1) and nitrogen (ψ_N= 0.64 m · d^?1). Field measurements indicated that variations in sinking may be associated with changes in irradiance and nitrate concentrations. Because these factors do not directly affect water density, they must be inducing physiological changes in the cell which affect buoyancy. Although a direct response to a single environmental variable was not always evident, sinking rates were positively correlated with growth rates in the marginal ice zone, further indicating a connection to physiological processes. Estimats of carbon flux at stations with vertically mixed euphotic zones indicated that approximately 30% of the daily primary production sank from the euphotic zone in the form of small particulates. Calculated carbon flux tended to increase with primary productivity.     

ON THE ROLE OF THE CYTOSKELETON IN COCCOLITH MORPHOGENESIS: THE EFFECT OF CYTOSKELETON INHIBITORS1

The coccolithophore Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler was cultured in natural seawater with the addition of either the microtubule‐inhibitor colchicine, the actin‐inhibitor cytochalasin B, or the photosynthesis inhibitor 3‐(3,4 dichlorophenyl)‐1,1‐dimethyl‐urea (DCMU). Additionally, E. huxleyi was cultured at different light intensities and temperatures. Growth rate was monitored, and coccolith morphology analyzed. While every treatment affected growth rate, the percentage of malformed coccoliths increased with colchicine, cytochalasin B, and at higher than optimal temperature. These results represent the first experimental evidence for the role of microtubules and actin microfilaments in coccolith morphogenesis.     

NEW EVIDENCE FOR MORPHOLOGICAL AND GENETIC VARIATION IN THE COSMOPOLITAN COCCOLITHOPHORE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) FROM THE COX1b‐ATP4 GENES1

Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler is a cosmopolitan coccolithophore occurring from tropical to subpolar waters and exhibiting variations in morphology of coccoliths possibly related to environmental conditions. We examined morphological characters of coccoliths and partial mitochondrial sequences of the cytochrome oxidase 1b (cox1b) through adenosine triphosphate synthase 4 (atp4) genes of 39 clonal E. huxleyi strains from the Atlantic and Pacific Oceans, Mediterranean Sea, and their adjacent seas. Based on the morphological study of culture strains by SEM, Type O, a new morphotype characterized by coccoliths with an open central area, was separated from existing morphotypes A, B, B/C, C, R, and var. corona, characterized by coccoliths with central area elements. Molecular phylogenetic studies revealed that E. huxleyi consists of at least two mitochondrial sequence groups with different temperature preferences/tolerances: a cool‐water group occurring in subarctic North Atlantic and Pacific and a warm‐water group occurring in the subtropical Atlantic and Pacific and in the Mediterranean Sea.     

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.     

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.     

On culture artefacts in coccolith morphology

Coccolith malformations occur more frequently in cultured specimens than in specimens from natural samples, a phenomenon commonly termed ‘culture artefacts’. The causes of culture artefacts are unknown. Here, we tested the effect of culture flask shape, mixing, and cell density on the morphology of Emiliania huxleyi coccoliths. While there was no effect of different culture flask types typically used in coccolithophore culturing, continuous mixing reduced the percentage of malformations by ca. 11 % in exponential-phase cells (cell density ca. 80 × 10³ cells per ml) and ca. 17 % in stationary-phase cells (cell density ca. 2 × 10⁶ cells per ml). Stationary-phase cells displayed 19 % more malformations than mid-exponential-phase cells when not mixed at all and 20 % more malformations when continuously mixed. It is concluded that the lack of mixing and unnaturally high cell densities, typical for coccolithophore stock cultures, are partly responsible for culture artefacts.     

ADAPTATION OF CERATIUM FURCA AND GONYAULAX POLYEDRA (DINOPHYCEACE) TO DIFFERENT TEMPERATURES AND IRRADIANCES: GROWTH RATES AND CELL VOLUMES1

Growth rates and cell volumes of Ceratium furca Ehrenberg and Gonyaulax polyedra Stein were determined during the log phase of growth in cultures which had been extensively adapted to one of three temperatures and five irradiances. At each temperature, curves for the growth rate vs. irradiance for both species had light-limited and light-saturated regions. Three properties of these curves characterized the response of each species to temperature: the light-saturated growth rate, the irradiance at which growth became light-saturated and the compensation irradiance for growth. For both species, the first two properties generally decreased with declining growth temperature, while the compensation irradiance declined for Ceratium but had a V-shaped response pattern for Gonyaulax. The light-saturated growth rates were generally higher for Ceratium than for Gonyaulax, while the irradiance at which growth became saturated and the compensation irradiance were lower for Ceratium. The changes in cell volume associated with the irradiance and temperature of growth were very different for Ceratium and Gonyaulax. The cell size of Gonyaulax increased as irradiance and temperature decreased, while cell volumes of Ceratium did not change with temperature but were smallest at the highest and lowest growth irradiances. In general, the growth rate patterns were similar for Ceratium and Gonyaulax, while those for cell size were different. The maximum growth rate, the irradiance at which growth became saturated, the compensation irradiance, and the cell volume all showed that Ceratium grew at the same rate or faster than Gonyaulax over the entire range of irradiances and temperatures examined.     

Inter-strain differences in nitrogen use by the coccolithophore Emiliania huxleyi, and consequences for predation by a planktonic ciliate

Four strains of the coccolithophore Emiliania huxleyi (CCMP strains 370, 373, 374, 379) were tested for their ability to grow on various nitrogen sources. All strains grew on ammonium, nitrate, and urea, although growth of CCMP379 on urea was low. Responses to other dissolved organic nitrogen (DON) sources varied. CCMP379 did not grow on any DON source other than urea. All other strains grew on one of the two tested amino acids: CCMP370 and CCMP373 on glutamine, and CCMP374 on alanine. All three of these strains also grew on hypoxanthine; in addition, two grew well on acetamide and one on ethanolamine. E. huxleyi strains also differed in their susceptibility to predation by the ciliate Strobilidium sp. CCMP374 was ingested at substantially higher rates than CCMP373 regardless of E. huxleyi growth condition. Ciliate feeding rates also depended on E. huxleyi growth condition. For CCMP374, feeding rates were 2× higher on growing E. huxleyi cells than on non-growing cells (average 27.5 versus 15.6 cells ciliate⁻¹ h⁻¹, respectively). For CCMP373, a relationship between E. huxleyi growth rate and ciliate feeding rate was not evident, but E. huxleyi grown on some N sources (ammonium, nitrate, urea) were ingested at consistently higher rates than E. huxleyi grown on other sources (ethanolamine, glutamine). Interstrain differences in the ability to utilize DON and resist predation may contribute to maintenance of high genetic diversity within this cosmopolitan, bloom-forming species.     

EFFECTS OF LIGHT AND GLYCEROL ON THE ORGANIZATION OF THE PHOTOSYNTHETIC APPARATUS IN THE FACULTATIVE HETEROTROPH PYRENOMONAS SALINA (CRYPTOPHYCEAE)1

The marine cryptophyte Pyrenomonas salina Santore is capable of autotrophic and heterotrophic nutrition. We studied the physiological and ultrastructural changes that accompany the shift between these nutritional modes. The addition of glycerol to batch cultures of P. salina, grown at an irradiance limiting for photoautotrophic growth, increased its growth rate and induced specific biochemical and structural changes in its photosynthetic system. Results from extracted pigment analyses, thin-section electron microscopy, and freeze-fracture electron microscopy indicated that glycerol addition reduced the cell phycoerythrin content, phycoerythrin to chlorophyll a ratio, degree of thylakoid packing, number of thylakoids · cell^?1, and PSII particle size. These properties were reduced to a similar extent in cells grown photoautotrophically under an irradiance saturating for growth. These results are consistent with the hypothesis that enhancement of heterotrophic potential occurs at the expense of light-harvesting ability in glycerol-grown P. salina.     

Phytoplankton sinking rate determination: Technical and theoretical aspects, an improved methodology

An analysis of technical and theoretical problems associated with the measurement of phytoplankton sinking rates by several commonly used methods indicates that these methods are both inaccurate and imprecise. Sinking rates estimated by the discrete sample layer (DSL) method tend systematically to over-estimate the mean sinking rate of the population due to the effect of inefficient detection of cells on the bottom of the settling chamber. Sinking rates estimated using the homogeneous sample (HS) method and so-called t_0–5 technique systematically under-estimate the mean sinking rate if any cells in the population sink faster than twice the estimated average sinking rate.

A rigorous mathematical framework is developed which, in conjunction with certain modifications in settling chamber design, allows calculation of the mean sinking rate (assuming that no cells are positively buoyant) for both the DSL and HS methods. These theoretical and technical modifications permit mean sinking rates to be estimated more rapidly and with greater accuracy and precision than has been possible by previous methods.     

Turbulent mixing mediates the photosynthetic activities and biochemical composition of <Emphasis Type="Italic">Anabaena</Emphasis>: implications for bioengineering

Cyanobacteria exhibit a variety of adaptive strategies that allow them to thrive in ever-changing aquatic environments. Here, successive steady states of continuous cultures were used to investigate the effects of quantified turbulence on the biochemical compounds and physiological processes of Anabaena flos-aquae in a photobioreactor under different dilution rates. A rapid increase in cell density was clearly observed following an increase in the turbulent dissipation rate at all growth rates of A. flos-aquae. The photosynthetic response to irradiance curves showed that the turbulence-treated strains exhibited lower photosynthetic oxygen evolution and saturating irradiance as well as higher respiration in rapidly growing young cells, indicating that they might not be very adaptable to high turbulent dissipation rates. Additionally, there was an increase in the protein levels of A. flos-aquae with increasing turbulence at all growth rates, whereas carbohydrate formation and lipid accumulation demonstrated the opposite trends. At a high growth rate, the level of carbohydrates decreased whereas that of lipids increased, which was interpreted as reflecting an adaptation to the turbulent environment. These findings suggest that turbulence sensitivity is shear regimen- and growth rate-dependent in A. flos-aquae. The high respiration capacity, low saturating irradiance, and conversion of carbohydrates to lipids represent effective measures for revealing the adaptive strategies of rapidly growing young cells under hydrodynamic regimes. The results regarding optimum lipid accumulation with specific growth traits have important implications for the design of cultivation methods of cyanobacteria resource utilization with respect to regulating turbulence.     

Coccolithogenesis In Scyphosphaera apsteinii (Prymnesiophyceae)

Coccolithophores are the most significant producers of marine biogenic calcite, although the intracellular calcification process is poorly understood. In the case of Scyphosphaera apsteinii Lohmann 1902, flat ovoid muroliths and bulky, vase‐shaped lopadoliths with a range of intermediate morphologies may be produced by a single cell. This polymorphic species is within the Zygodiscales, a group that remains understudied with respect to ultrastructure and coccolith ontogeny. We therefore undertook an analysis of cell ultrastructure, morphology, and coccolithogenesis. The cell ultrastructure showed many typical haptophyte features, with calcification following a similar pattern to that described for other heterococcolith bearing species including Emiliania huxleyi. Of particular significance was the reticular body role in governing fine‐scale morphology, specifically the central pore formation of the coccolith. Our observations also highlighted the essential role of the inter‐ and intracrystalline organic matrix in growth and arrangement of the coccolith calcite. S. apsteinii secreted mature coccoliths that attached to the plasma membrane via fibrillar material. Time‐lapse light microscopy demonstrated secretion of lopadoliths occurred base first before being actively repositioned at the cell surface. Significantly, growth irradiance influenced the coccosphere composition with fewer lopadoliths being formed relative to muroliths at higher light intensities. Overall, our observations support dynamic metabolic (i.e., in response to growth irradiance), sensory and cytoskeletal control over the morphology and secretion of polymorphic heterococcoliths. With a basic understanding of calcification established, S. apsteinii could be a valuable model to further study coccolithophore calcification and cell physiological responses to ocean acidification.     

DOES CARBOHYDRATE CONTENT AFFECT THE SINKING RATES OF MARINE DIATOMS?1

We tested the hypothesis that positive relationships between sinking rate and irradiance were due to increases in cell density caused by accumulations of carbohydrate. In semicontinuous batch cultures of Thalassiosira weissflogii (Gru.) Fryxell el Hasle and Ditylum brightwellii (t. West) Grunow in Van Huerk, carbohydrate content was varied by growing cells under diel cycles of high or low light. Sinking rate was measured at the end of the light period and the end of the dark period, on live and heat-killed cells. No positive correlations were found between sinking rate (which varied from – 0.060 to 0.13 m·d^?1) and carbohydrate content (which varied from 10 to 950 pg · cell^?1), indicating that accumulations of carbohydrate did not significantly affect sinking rate. There were no large diel variations in the sinking rate of T. weissflogii, but sinking rates of D. brightwellii grown under high light ranged from being negative (i.e. cells were floating) at the end of the light period to positive at the end of the dark period. This is the first report of positive buoyancy in vegetative D. brightwellii, a phenomenon that may only occur in D. brightwellii grown under diel cycles.     

Coccolithophore (–CaCO3) flux in the Sea of Okhotsk: seasonality, settling and alteration processes

Coccolithophore fluxes were determined in the Sea of Okhotsk using samples from a 1 year experiment (12 August 1990 to 12 August 1991) with sediment traps at 258 and 1061 m depth. A special study was made on Coccolithus pelagicus, using fragmentation and the degree of etching, as indicators of transport mechanisms. A Corrosion Index for C. pelagicus is developed. The coccolithophore flux pattern at 258 m depth was characterised by a strong seasonality, with flux peaks during autumn 1990 (late November to early December) and spring 1991 (March). The assemblage consisted almost entirely of the two species C. pelagicus and Emiliania huxleyi. During autumn, coccolithophore transportation to 258 m depth mainly occurred within cylindrical fecal pellets and marine snow aggregates of silicoflagellates, and through agglutination on tintinnids. Grazing caused severe fragmentation of coccoliths and disintegration of coccospheres. Marine snow aggregates contained many intact coccospheres of C. pelagicus. During spring, coccolithophores were probably removed from the euphotic zone by the ballast effect of sinking diatoms. The coccolithophore flux peak in spring occurred immediately after the ice had retreated from the trap station, and the trapped assemblage included coccoliths of subtropical species. These features indicate drifting from an ice-free location to the south or east.The coccolith and coccosphere flux at 1061 m was respectively 7 and 12 times lower than at 258 m depth, and maximum fluxes were recorded 2 months later. Increasing carbonate dissolution from 258 to 1061 m depth is expressed in the coccolithophore–CaCO₃ flux reduction of 82%, and in the increasing percentage of etched coccoliths of Coccolithus pelagicus from 32 to >90%.     

Effects of elevated CO2 on growth,calcification, and spectral dependence of photoinhibition in the coccolithophore Emiliania huxleyi (Prymnesiophyceae)1

We studied the effects of elevated CO₂ concentrations on cell growth, calcification, and spectral variation in the sensitivity of photosynthesis to inhibition by solar radiation in the globally important coccolithophore Emiliania huxleyi. Growth rates and chlorophyll a content per cell showed no significant differences between elevated (800 ppmv) and ambient (400 ppmv) CO₂ conditions. However, the production of organic carbon and the cell quotas for both carbon and nitrogen, increased under elevated CO₂ conditions, whilst particulate inorganic carbon production rates decreased under the same conditions. Biometric analyses of cells showed that coccoliths only presented significant differences due to treatments in the central area width. Most importantly, the size of the coccosphere decreased under elevated CO₂ conditions. The susceptibility of photosynthesis to inhibition by ultraviolet radiation (UVR) was estimated using biological weighting functions (BWFs) and a model that predicts photosynthesis under photosynthetically active radiation and UVR exposures. BWF results demonstrated that the sensitivity of photosynthesis to UVR was not significantly different between E. huxleyi cells grown under elevated and present CO₂ concentrations. We propose that the acclimation to elevated CO₂ conditions involves a physiological mechanism of regulation and allocation of energy and metabolites in the cell, which is also responsible for altering the sensitivity to UVR. In coccolithophores, this mechanism might be affected by the decrease in the calcification rates.