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.     

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.     

A GENETIC MARKER TO SEPARATE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) MORPHOTYPES1

Emiliania huxleyi (Lohm.) Hay and Mohler is a ubiquitous unicellular marine alga surrounded by an elaborate covering of calcite platelets called coccoliths. It is an important primary producer involved in oceanic biogeochemistry and climate regulation. Currently, E. huxleyi is separated into five morphotypes based on morphometric, physiological, biochemical, and immunological differences. However, a genetic marker has yet to be found to characterize these morphotypes. With the use of sequence analysis and denaturing gradient gel electrophoresis, we discovered a genetic marker that correlates significantly with the separation of the most widely recognized A and B morphotypes. Furthermore, we reveal that the A morphotype is composed of a number of distinct genotypes. This marker lies within the 3′ untranslated region of a coccolith associated protein mRNA, which is implicated in regulating coccolith calcification. Consequently, we tentatively termed this marker the coccolith morphology motif.     

Formation and mosaicity of coccolith segment calcite of the marine algae Emiliania huxleyi

Coccolithophores belong to the most abundant calcium carbonate mineralizing organisms. Coccolithophore biomineralization is a complex and highly regulated process, resulting in a product that strongly differs in its intricate morphology from the abiogenically produced mineral equivalent. Moreover, unlike extracellularly formed biological carbonate hard tissues, coccolith calcite is neither a hybrid composite, nor is it distinguished by a hierarchical microstructure. This is remarkable as the key to optimizing crystalline biomaterials for mechanical strength and toughness lies in the composite nature of the biological hard tissue and the utilization of specific microstructures. To obtain insight into the pathway of biomineralization of Emiliania huxleyi coccoliths, we examine intracrystalline nanostructural features of the coccolith calcite in combination with cell ultrastructural observations related to the formation of the calcite in the coccolith vesicle within the cell. With TEM diffraction and annular dark‐field imaging, we prove the presence of planar imperfections in the calcite crystals such as planar mosaic block boundaries. As only minor misorientations occur, we attribute them to dislocation networks creating small‐angle boundaries. Intracrystalline occluded biopolymers are not observed. Hence, in E. huxleyi calcite mosaicity is not caused by occluded biopolymers, as it is the case in extracellularly formed hard tissues of marine invertebrates, but by planar defects and dislocations which are typical for crystals formed by classical ion‐by‐ion growth mechanisms. Using cryo‐preparation techniques for SEM and TEM, we found that the membrane of the coccolith vesicle and the outer membrane of the nuclear envelope are in tight proximity, with a well‐controlled constant gap of ~4 nm between them. We describe this conspicuous connection as a not yet described interorganelle junction, the “nuclear envelope junction”. The narrow gap of this junction likely facilitates transport of Ca²⁺ ions from the nuclear envelope to the coccolith vesicle. On the basis of our observations, we propose that formation of the coccolith utilizes the nuclear envelope–endoplasmic reticulum Ca²⁺‐store of the cell for the transport of Ca²⁺ ions from the external medium to the coccolith vesicle and that E. huxleyi calcite forms by ion‐by‐ion growth rather than by a nanoparticle accretion mechanism.     

Light-dependent coccolith formation in the two forms of Coccolithus pelagicus

Summary Two methods were employed for measuring coccolith formation and photosynthesis in coccolithophorids. The first method was based on measurements of ¹⁴C radioactivity of cells on membrane filters before and after acid treatment. The second method involved a conversion of ¹⁴C in coccoliths or whole cells to BaCO₃ prior to counting. It was observed that in determinations of photosynthetic (or total) ¹⁴C by the first method, the count rate produced by a given amount of the isotope was 30–40% lower in the non-motile and motile forms of Coccolithus pelagicus than in C. huxleyi. There was no similarly great discrepancy in determinations of coccolith ¹⁴C.Light-dependent coccolith formation was demonstrated in both forms of C. pelagicus. The non-motile form may deposit several times more carbon in its coccoliths than it assimilates photosynthetically. In the motile form, coccolith carbon amounts to less than 2% of photosynthetic carbon.     

Increased coccolith production by Emiliania huxleyi cultures enriched with dissolved inorganic carbon

Cells of Emiliania huxleyi grown on Eppley's medium enriched with dissolved inorganic carbon (DIC) developed multiple layers of coccoliths. The maximum diameter of cells grown in the presence of 13.2 mM DIC was 12.3 m, whereas that of cells grown in the presence of 1.5 mM DIC was 8.0 m. Although enrichment of Eppley's medium with DIC increased both coccolith production and cell growth, coccolith production was enhanced to a greater extent than cell growth. The enrichment of Eppley's medium with DIC was used to enhance production of coccolith particles by E. huxleyi. Repeated-batch culture, in which DIC, Ca²⁺, nitrate and phosphate concentrations in the medium were maintained by replacing the culture medium, was carried out in a closed photobioreactor. During repeated-batch culture, a maximum coccolith yield of 560 mg/l for 2 days and a maximum biomass yield of 810 mg/l for 2 days were achieved. Enrichment and maintenance of DIC is therefore an efficient method for the production of large quantities of coccoliths.     

Calcareous nannoplankton in surface sediments of the East China Sea

Twenty-eight species of coccoliths were identified in surface sediments of the East China Sea. The species composition of the coccolith assemblages is similar to that of the North Pacific Water Mass, but differs from the latter by a greater dominance ofEmiliania huxleyi andGephyrocapsa oceanica (together making up over 90% specimens). The coccolith species composition shows considerable differences between the continental shelf and the Okinawa Trough areas, reflecting different water masses. On the other hand, the coccolith content in sediments is apparently controlled largely by the deposition rate of terrigenous material.     

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.     

Action Spectrum of Coccolith Formation

The action spectrum of coccolith formation in Coccolithus huxleyi was determined by measuring the uptake of carbon-14 in coccoliths in four-hour experiments as a function of light intensity at each of seven wavelengths. An action spectrum | of photosynthetic carbon assimilation was obtained at the same time. The coccolith action spectrum had peaks at wavelengths of about 440 nm and 670 nm. probably corresponding to the regions of maximum cellular absorption and carbon assimilation. However, blue light appeared to be relatively more efficient in coccolith formation than in carbon assimilation. The results suggest that light-dependent coccolith formation may be catalyzed by two photochemical reactions, one mediated by chloroplast pigments and the other by some pigment absorbing specifically in the blue part of the spectrum.     

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.     

COCCOLITH FUNCTION AND MORPHOGENESIS: INSIGHTS FROM APPENDAGE‐BEARING COCCOLITHOPHORES OF THE FAMILY SYRACOSPHAERACEAE (HAPTOPHYTA)1

The morphology of three remarkable genera of coccolithophores, Ophiaster, Michaelsarsia, and Calciopappus, is reviewed based on new images using field emission scanning electron microscopy. Each of these genera characteristically forms coccospheres with long appendages formed of highly modified coccoliths, which radiate from either the circum‐flagellar pole of the coccosphere (Calciopappus and Michaelsarsia) or the antapical pole (Ophiaster). For each genus, it is shown that the appendage coccoliths can also occur in an alternative orientation appressed to the main coccosphere. It is hypothesized that the appendage coccoliths are initially deployed in the appressed orientation and that extension of the appendages is a dynamic response to environmental stress. The observations suggest that coccoliths are more sophisticatedly adapted to specific functions than has been assumed and that the cytoskeleton plays more active roles in coccolith morphogenesis and deployment than has previously been inferred.     

Adjustment to Light and Dark Rates of Coccolith Formation

Coccolith formation in Coccolithus huxleyi was investigated in a semiquantitative manner by microscopic enumeration of coccolith-carrying cells appearing, after varying periods in normal growth medium, following an initial removal of all coccoliths by treatment with a decalcifying medium. Coccoliths were formed both in the light and in the dark, but the rate of coccolith formation was very much greater in the light. Time-course experiments on the uptake of carbon-14 in coccoliths demonstrated that after a period in darkness, the cells required half an hour of illumination in order to adjust to the light rate of coccolith formation. Coccoliths were produced at a maximum rate only as long as the illumination lasted, although about one hour in the dark was needed before the transition back to the original dark rate was complete.     

Low Temperature Stimulates Cell Enlargement and Intracellular Calcification of Coccolithophorids

Temperature effect on growth, cell size, calcium uptake activity, coccolith production was studied in coccolith-producing haptophytes, Emiliania huxleyi (Lohmann) Hay & Mohler (strain EH2) and Gephyrocapsa oceanica Kamptner (strain GO1) (Coccolithophorales, Prymnesiophyceae). E. huxleyi grew at a wider temperature range (10°–25°C), while G. oceanica growth was limited to warmer temperatures (20°–25°C). Cell size was inversely correlated with temperature. At low temperature, the enlargement of chloroplasts and cells and the stimulation of coccolith production were morphologically confirmed under fluorescent and polarization microscopes, respectively. ⁴⁵ Ca uptake by E. huxleyi at 10°C was greatly increased after a 5-day lag and exceeded that at 20°C. These results clearly showed that low temperature suppressed coccolithophorid growth but induced cell enlargement and as stimulated the intracellular calcification that produces coccoliths.     

DISTRIBUTION OF TWO TYPES OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) IN THE NORTHEAST ATLANTIC REGION AS DETERMINED BY IMMUNOFLUORESCENCE AND COCCOLITH MORPHOLOGY1

Culture strains of Emiliania huxleyi (Lohmann 1902) Hay et al. 1967 were placed into two groups designated E. huxleyi type A and type B on the basis of coccolith morphology and immunological properties of the coccolith polysaccharide. We studied the distribution of these types in the North Atlantic region using an indirect immunofluorescence assay with antisera directed against the coccolith polysaccharide of E. huxleyi type A and type B and epifluorescence microscopy. In field samples taken in the Northeast Atlantic Ocean, E. huxleyi type A was found exclusively. In contrast, type B was dominant in the North Sea. Scanning electron microscopy of the samples revealed the same unequal distribution of the two types as found with the immunofluorescent-labelling assay.     

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.     

Seasonal variation in Emiliania huxleyi coccolith morphology and calcification in the Aegean Sea (Eastern Mediterranean)

A seasonal morphological variability is observed in Emiliania huxleyi var. huxleyi specimens, collected from discrete water samples in the Aegean Sea. Biometric analyses reveal a consistent pattern of increase in the size of coccoliths and coccospheres, including the thickness of the inner tube elements (INT), in winter/spring time low sea surface temperature and moderate productivity samples when compared with summer time high temperature-low productivity samples. The small range of salinity change in the Aegean Sea and the absence of seasonal pattern in nutrient content do not support any association with the observed increase in E. huxleyi coccolith size. A relatively increased [HCO₃^-] content is observed during spring-time interval related with the increase in the coccolith size, however it remains unclear which parameter of the carbonate system causes the observed effects.     

The last 100,000 years in the western Mediterranean: sea surface water and frontal dynamics as revealed by coccolithophores

A quantitative analysis was carried out on coccolith assemblages from two Pleistocene cores (K1 and K10) from the western Mediterranean. The distribution of selected coccolithophore species provides new paleoclimatic and paleoceano-graphic data. A continuous sequence from the top of Isotope Stage 5 to the Holocene was recorded. The reversal in dominance between Gephyrocapsa muellerae and Emiliania huxleyi was dated in both cores at ca. 73 ka. At about 47 ka, E. huxleyi shows a regular increase, whereas G. muellerae progressively decreases in abundance. During interglacial periods, high concentrations of coccoliths are observed, whereas in glacial times, coccoliths are more diluted and the percentage of reworked forms increases as a consequence of the higher terrigenous input. After taking careful account of the dilution factor, we conclude that the production of coccolithophores was higher during warm periods. Maxima in coccolith concentrations coincide with highstand episodes, probably as a result of the intensification of the Atlantic flux into the Mediterranean across the Gibraltar Strait. This intensification could have produced an increase in nutrient content in the surface Mediterranean waters. During cold periods, the western Mediterranean front underwent a reduction in activity, probably due to an increase in the saline and/or thermal gradients between the superficial waters, and intermediate waters in the Liguro Provençal basin.     

Disruption of planar cell polarity activity leads to developmental biliary defects

Planar cell polarity (PCP) establishes polarity within an epithelial sheet. Defects in PCP are associated with developmental defects involving directional cell growth, including defects in kidney tubule elongation that lead to formation of kidney cysts. Given the strong association between kidney cyst formation and developmental biliary defects in patients and in animal models, we investigated the importance of PCP in biliary development. Here we report that in zebrafish, morpholino antisense oligonucleotide-mediated knockdown of PCP genes including prickle-1a (pk1a) led to developmental biliary abnormalities, as well as localization defects of the liver and other digestive organs. The defects in biliary development appear to be mediated via downstream PCP targets such as Rho kinase, Jun kinase (JNK), and both actin and microtubule components of the cytoskeleton. Knockdown of pk1a led to decreased expression of vhnf1, a homeodomain gene previously shown to be involved in biliary development and in kidney cyst formation; forced expression of vhnf1 mRNA led to rescue of the pk1a morphant phenotype. Our results demonstrate that PCP plays an important role in vertebrate biliary development, interacting with other factors known to be involved in biliary morphogenesis.