An uneven distribution of strontium in the coccolithophore Scyphosphaera apsteinii revealed by nanoscale X-ray fluorescence tomography.

Coccolithophores are biogeochemically and ecologically important phytoplankton that produce a composite calcium carbonate-based exoskeleton - the coccosphere - comprised of individual platelets, known as coccoliths. Coccoliths are stunning examples of biomineralization; their formation featuring exceptional control over both biomineral chemistry and shape. Understanding how coccoliths are formed requires information about minor element distribution and chemical environment. Here, the first high-resolution 3D synchrotron X-ray fluorescence (XRF) mapping of a coccolith is presented, showing that the lopadoliths of Scyphosphaera apsteinii display stripes of different Sr concentration. The presence of Sr stripes is unaffected by elevated Sr in the culture medium, macro-nutrient concentration, and light intensity, indicating that the observed stripiness is an expression of the fundamental coccolith formation process in this species. Current Sr fractionation models, by contrast, predict an even Sr distribution and will have to be modified to account for this stripiness. Additionally, nano-XANES analyses show that Sr resides in a Ca site in the calcite lattice in both high and low Sr stripes, confirming a central assumption of current Sr fractionation models.

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 Effect of cytoskeleton inhibitors on coccolith morphology in Coccolithus braarudii and Scyphosphaera apsteinii.

The calcite platelets of coccolithophores (Haptophyta), the coccoliths, are among the most elaborate biomineral structures. How these unicellular algae accomplish the complex morphogenesis of coccoliths is still largely unknown. It has long been proposed that the cytoskeleton plays a central role in shaping the growing coccoliths. Previous studies have indicated that disruption of the microtubule network led to defects in coccolith morphogenesis in Emiliania huxleyi and Coccolithus braarudii. Disruption of the actin network also led to defects in coccolith morphology in E. huxleyi, but its impact on coccolith morphology in C. braarudii was unclear, as coccolith secretion was largely inhibited under the conditions used. A more detailed examination of the role of actin and microtubule networks is therefore required to address the wider role of the cytoskeleton in coccolith morphogenesis. In this study, we have examined coccolith morphology in C. braarudii and Scyphosphaera apsteinii following treatment with the microtubule inhibitors vinblastine and colchicine (S. apsteinii only) and the actin inhibitor cytochalasin B. We found that all cytoskeleton inhibitors induced coccolith malformations, strongly suggesting that both microtubules and actin filaments are instrumental in morphogenesis. By demonstrating the requirement for the microtubule and actin networks in coccolith morphogenesis in diverse species, our results suggest that both of these cytoskeletal elements are likely to play conserved roles in defining coccolith morphology.

Characterization of the molecular mechanisms of silicon uptake in coccolithophores.

Coccolithophores are an important group of calcifying marine phytoplankton. Although coccolithophores are not silicified, some species exhibit a requirement for Si in the calcification process. These species also possess a novel protein (SITL) that resembles the SIT family of Si transporters found in diatoms. However, the nature of Si transport in coccolithophores is not yet known, making it difficult to determine the wider role of Si in coccolithophore biology. Here, we show that coccolithophore SITLs act as Na+ -coupled Si transporters when expressed in heterologous systems and exhibit similar characteristics to diatom SITs. We find that CbSITL from Coccolithus braarudii is transcriptionally regulated by Si availability and is expressed in environmental coccolithophore populations. However, the Si requirement of C. braarudii and other coccolithophores is very low, with transport rates of exogenous Si below the level of detection in sensitive assays of Si transport. As coccoliths contain only low levels of Si, we propose that Si acts to support the calcification process, rather than forming a structural component of the coccolith itself. Si is therefore acting as a micronutrient in coccolithophores and natural populations are only likely to experience Si limitation in circumstances where dissolved silicon (DSi) is depleted to extreme levels.

Novel combination coccospheres from Helicosphaera spp indicate complex relationships between species.

Coccolithophores play an important role in global biogeochemical cycling, but many aspects of their ecology remain poorly understood, including their heteromorphic haplo-diplontic life cycle. The presence of combination coccospheres in environmental samples, which represent a transition between the lightly calcified haploid (HOL) and heavily calcified diploid (HET) life phases, provides crucial evidence linking the two life cycle phases of a particular species. Here, we describe combination coccospheres from the Southern Ocean that show a novel association between Helicosphaera hyalina (HET) and Helicosphaera HOL catilliferus type. The ability of Helicosphaera HET and HOL morphospecies to form multiple different combinations indicates a substantial complexity in the relationships between life cycle phases in this group. The findings suggest recent divergence within the Helicosphaera lineage may have resulted in significant inter- and intra-specific variability, with cryptic speciation in one or both life cycle phases contributing to their ability to form multiple HET/HOL associations.

Reduced H+ channel activity disrupts pH homeostasis and calcification in coccolithophores at low ocean pH.

Coccolithophores are major producers of ocean biogenic calcite, but this process is predicted to be negatively affected by future ocean acidification scenarios. Since coccolithophores calcify intracellularly, the mechanisms through which changes in seawater carbonate chemistry affect calcification remain unclear. Here we show that voltage-gated H+ channels in the plasma membrane of Coccolithus braarudii serve to regulate pH and maintain calcification under normal conditions but have greatly reduced activity in cells acclimated to low pH. This disrupts intracellular pH homeostasis and impairs the ability of C. braarudii to remove H+ generated by the calcification process, leading to specific coccolith malformations. These coccolith malformations can be reproduced by pharmacological inhibition of H+ channels. Heavily calcified coccolithophore species such as C. braarudii, which make the major contribution to carbonate export to the deep ocean, have a large intracellular H+ load and are likely to be most vulnerable to future decreases in ocean pH.

Role of silicon in the development of complex crystal shapes in coccolithophores.

The development of calcification by the coccolithophores had a profound impact on ocean carbon cycling, but the evolutionary steps leading to the formation of these complex biomineralized structures are not clear. Heterococcoliths consisting of intricately shaped calcite crystals are formed intracellularly by the diploid life cycle phase. Holococcoliths consisting of simple rhombic crystals can be produced by the haploid life cycle stage but are thought to be formed extracellularly, representing an independent evolutionary origin of calcification. We use advanced microscopy techniques to determine the nature of coccolith formation and complex crystal formation in coccolithophore life cycle stages. We find that holococcoliths are formed in intracellular compartments in a similar manner to heterococcoliths. However, we show that silicon is not required for holococcolith formation and that the requirement for silicon in certain coccolithophore species relates specifically to the process of crystal morphogenesis in heterococcoliths. We therefore propose an evolutionary scheme in which the lower complexity holococcoliths represent an ancestral form of calcification in coccolithophores. The subsequent recruitment of a silicon-dependent mechanism for crystal morphogenesis in the diploid life cycle stage led to the emergence of the intricately shaped heterococcoliths, enabling the formation of the elaborate coccospheres that underpin the ecological success of coccolithophores.

Coccolithophore calcification: Changing paradigms in changing oceans.

Coccolithophores represent a major component of the marine phytoplankton and contribute to the bulk of biogenic calcite formation on Earth. These unicellular protists produce minute calcite scales (coccoliths) within the cell, which are secreted to the cell surface. Individual coccoliths and their arrangements on the cell surface display a wide range of morphological variations. This review explores some of the recent evidence that points to similarities and differences in the mechanisms of calcification, focussing on the transport mechanisms that bring substrates to, and remove products from the site of calcification, together with new findings on factors that regulate coccolith morphology. We argue that better knowledge of these mechanisms and their variations is needed to inform more generally how different species of coccolithophore are likely to respond to changes in ocean chemistry. STATEMENT OF SIGNIFICANCE: Coccolithophores, minute single celled phytoplankton are the major producers of biogenic carbonate on Earth. They also represent an important component of the ocean's biota and contribute significantly to global carbon fluxes. Coccolithophores produce intricate calcite scales (coccoliths) internally that they secrete onto their external surface. This review presents some recent key findings on the mechanisms underlying the production of coccoliths. It also considers the factors that regulate the rate of production as well as the variety of shapes of individual coccoliths and their arrangements at the cell surface. Understanding these processes is needed to allow better predictions of how coccolithophores may respond to changing ocean chemistry associated with climate change.

The requirement for calcification differs between ecologically important coccolithophore species.

Coccolithophores are globally distributed unicellular marine algae that are characterized by their covering of calcite coccoliths. Calcification by coccolithophores contributes significantly to global biogeochemical cycles. However, the physiological requirement for calcification remains poorly understood as non-calcifying strains of some commonly used model species, such as Emiliania huxleyi, grow normally in laboratory culture. To determine whether the requirement for calcification differs between coccolithophore species, we utilized multiple independent methodologies to disrupt calcification in two important species of coccolithophore: E. huxleyi and Coccolithus braarudii. We investigated their physiological response and used time-lapse imaging to visualize the processes of calcification and cell division in individual cells. Disruption of calcification resulted in major growth defects in C. braarudii, but not in E. huxleyi. We found no evidence that calcification supports photosynthesis in C. braarudii, but showed that an inability to maintain an intact coccosphere results in cell cycle arrest. We found that C. braarudii is very different from E. huxleyi as it exhibits an obligate requirement for calcification. The identification of a growth defect in C. braarudii resulting from disruption of the coccosphere may be important in considering their response to future changes in ocean carbonate chemistry.

Temperature effects on sinking velocity of different Emiliania huxleyi strains.

The sinking properties of three strains of Emiliania huxleyi in response to temperature changes were examined. We used a recently proposed approach to calculate sinking velocities from coccosphere architecture, which has the advantage to be applicable not only to culture samples, but also to field samples including fossil material. Our data show that temperature in the sub-optimal range impacts sinking velocity of E. huxleyi. This response is widespread among strains isolated in different locations and moreover comparatively predictable, as indicated by the similar slopes of the linear regressions. Sinking velocity was positively correlated to temperature as well as individual cell PIC/POC over the sub-optimum to optimum temperature range in all strains. In the context of climate change our data point to an important influence of global warming on sinking velocities. It has recently been shown that seawater acidification has no effect on sinking velocity of a Mediterranean E. huxleyi strain, while nutrient limitation seems to have a small negative effect on sinking velocity. Given that warming, acidification, and lowered nutrient availability will occur simultaneously under climate change scenarios, the question is what the net effect of different influential factors will be. For example, will the effects of warming and nutrient limitation cancel? This question cannot be answered conclusively but analyses of field samples in addition to laboratory culture studies will improve predictions because in field samples multi-factor influences and even evolutionary changes are not excluded. As mentioned above, the approach of determining sinking rate followed here is applicable to field samples. Future studies could use it to analyse not only seasonal and geographic patterns but also changes in sinking velocity over geological time scales.

Effect of different seawater Mg2 + concentrations on calcification in two benthic foraminifers.

Magnesium, incorporated in foraminiferal calcite (Mg/CaCC), is used intensively to reconstruct past seawater temperatures but, in addition to temperature, the Mg/CaCC of foraminiferal tests also depends on the ratio of Mg and Ca in seawater (Mg/CaSW). The physiological mechanisms responsible for these proxy relationships are still unknown. This culture study investigates the impact of different seawater [Mg2 +] on calcification in two benthic foraminiferal species precipitating contrasting Mg/CaCC: Ammonia aomoriensis, producing low-Mg calcite and Amphistegina lessonii, producing intermediate-Mg calcite. Foraminiferal growth and test thickness were determined and, Mg/Ca was analyzed using Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS). Results show that at present-day seawater Mg/CaSW of ~ 5, both species have highest growth rates, reflecting their adaptation to modern seawater element concentrations. Test thickness is not significantly affected by different Mg/CaSW. The relationship between Mg/CaSW and Mg/CaCC shows a distinct positive y-axis intercept, possibly reflecting at least two processes involved in foraminiferal biomineralization. The associated Mg partition (DMg) changes non-linearly with increasing Mg/CaSW, hence suggesting that the DMg is best described by an exponential function approaching an asymptote.

Substrate supply for calcite precipitation in Emiliania huxleyi: assessment of different model approaches.

Over the last four decades, different hypotheses of Ca(2+) and dissolved inorganic carbon transport to the intracellular site of calcite precipitation have been put forth for Emiliania huxleyi (Lohmann) Hay & Mohler. The objective of this study was to assess these hypotheses by means of mathematical models. It is shown that a vesicle-based Ca(2+) transport would require very high intravesicular Ca(2+) concentrations, high vesicle fusion frequencies as well as a fast membrane recycling inside the cell. Furthermore, a kinetic model for the calcification compartment is presented that describes the internal chemical environment in terms of carbonate chemistry including calcite precipitation. Substrates for calcite precipitation are transported with different stoichiometries across the compartment membrane. As a result, the carbonate chemistry inside the compartment changes and hence influences the calcification rate. Moreover, the effect of carbonic anhydrase (CA) activity within the compartment is analyzed. One very promising model version is based on a Ca(2+) /H(+) antiport, CO2 diffusion, and a CA inside the calcification compartment. Another promising model version is based on an import of Ca(2+) and HCO3 (-) and an export of H(+) .