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The marine microalga Emiliania huxleyi is widely distributed in the surface oceans and is prone to infection by coccolithoviruses that can terminate its blooms. However, little is known about how global change factors like solar UV radiation (UVR) and ocean warming affect the host-virus interaction. We grew the microalga at 2 temperature levels with or without the virus in the presence or absence of UVR and investigated the physiological and transcriptional responses. We showed that viral infection noticeably reduced photosynthesis and growth of the alga but was less harmful to its physiology under conditions where UVR influenced viral DNA expression. In the virus-infected cells, the combination of UVR and warming (+4°C) led to a 13-fold increase in photosynthetic carbon fixation rate, with warming alone contributing a change of about 5-7-fold. This was attributed to upregulated expression of genes related to carboxylation and light-harvesting proteins under the influence of UVR, and to warming-reduced infectivity. In the absence of UVR, viral infection downregulated the metabolic pathways of photosynthesis and fatty acid degradation. Our results suggest that solar UV exposure in a warming ocean can reduce the severity of viral attack on this ecologically important microalga, potentially prolonging its blooms., (© 2024 John Wiley & Sons Ltd.)

Hydrogen isotope ratios (δ 2 H) represent an important natural tracer of metabolic processes, but quantitative models of processes controlling H-fractionation in aquatic photosynthetic organisms are lacking. Here, we elucidate the underlying physiological controls of 2 H/ 1 H fractionation in algal lipids by systematically manipulating temperature, light, and CO 2 (aq) in continuous cultures of the haptophyte Gephyrocapsa oceanica . We analyze the hydrogen isotope fractionation in alkenones (α alkenone ), a class of acyl lipids specific to this species and other haptophyte algae. We find a strong decrease in the α alkenone with increasing CO 2 (aq) and confirm α alkenone correlates with temperature and light. Based on the known biosynthesis pathways, we develop a cellular model of the δ 2 H of algal acyl lipids to evaluate processes contributing to these controls on fractionation. Simulations show that longer residence times of NADPH in the chloroplast favor a greater exchange of NADPH with 2 H-richer intracellular water, increasing α alkenone . Higher chloroplast CO 2 (aq) and temperature shorten NADPH residence time by enhancing the carbon fixation and lipid synthesis rates. The inverse correlation of α alkenone to CO 2 (aq) in our cultures suggests that carbon concentrating mechanisms (CCM) do not achieve a constant saturation of CO 2 at the Rubisco site, but rather that chloroplast CO 2 varies with external CO 2 (aq). The pervasive inverse correlation of α alkenone with CO 2 (aq) in the modern and preindustrial ocean also suggests that natural populations may not attain a constant saturation of Rubisco with the CCM. Rather than reconstructing growth water, α alkenone may be a powerful tool to elucidate the carbon limitation of photosynthesis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Coccolithophores play a significant role in marine calcium carbonate production and carbon cycles, attributing to their unique feature of producing calcareous plates, coccoliths. Coccolithophores also possess a haplo-diplontic life cycle, presenting distinct morphology types and calcification states. However, differences in nutrient acquisition strategies and mixotrophic behaviors of the two life phases remain unclear. In this study, we conducted a series of phagocytosis experiments of calcified diploid and non-calcified haploid strains of coccolithophore Gephyrocapsa huxleyi under light and dark conditions. The phagocytosis capability of each strain was examined based on characteristic fluorescent signals from ingested beads using flow cytometry and fluorescence microscopy. The results show a significantly higher phagocytosis percentage on fluorescent beads in the bacterial prey surrogates of the non-calcified haploid Gephyrocapsa huxleyi strain, than the calcified diploid strain with or without light. In addition, the non-calcified diploid cells seemingly to presented a much higher phagocytosis percentage in darkness than under light. The differential phagocytosis capacities between the calcified diploid and non-calcified haploid Gephyrocapsa huxleyi strains indicate potential distinct nutritional strategies at different coccolithophore life and calcifying stages, which may further shed light on the potential strategies that coccolithophore possesses in unfavorable environments such as twilight zones and the expanding coccolithophore niches in the natural marine environment under the climate change scenario.

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.

The evolutionary advantages afforded by phytoplankton calcification remain enigmatic. In this work, fluoroelectrochemical experiments reveal that the presence of a CaCO 3 shell of a naturally calcifying coccolithophore, Coccolithus braarudii, offers protection against extracellular oxidants as measured by the time required for the switch-off in their chlorophyll signal, compared to the deshelled equivalents, suggesting the shift toward calcification offers some advantages for survival in the surface of radical-rich seawater., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

The effects of ocean acidification (OA) on coccolithophore's photosynthesis, calcification rates, and growth have been extensively studied. However, how the intracellular Ca 2+ , mechanical properties and chemical composition of the coccoliths are affected by OA have not yet been investigated. This study tries to fill these gaps using Emiliania huxleyi as a model coccolithophore. When the seawater pCO 2 increased from 400 μatm to 1200 μatm, the intracellular Ca 2+ and coccolith area were reduced by 66% and 36%, respectively. Single-cell mapping by atomic force microscopy revealed that the modulus and hardness of coccolith decreased from 23.6 ± 0.2 GPa to 12.0 ± 5.5 GPa and from 0.53 ± 0.15 GPa to 0.20 ± 0.06 GPa, respectively. Additionally, the proportional organic matter and silicon in the coccolith surfaces increased with pCO 2 . The copepods Acartia pacifica fed on more E. huxleyi grown at higher pCO 2 . Our study implies that OA could change coccolithophore's competitive interactions with other phytoplankton and ultimately influence carbon export to the deep ocean., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The elemental composition of marine microorganisms (their C:N:P ratio, or stoichiometry) is central to understanding the biotic and biogeochemical processes underlying key marine ecosystem functions. Phytoplankton C:N:P is species specific and flexible to changing environmental conditions. However, bulk or fixed phytoplankton stoichiometry is usually assumed in biogeochemical and ecological models because more realistic, environmentally responsive C:N:P ratios have yet to be defined for key functional groups. Here, a comprehensive meta-analysis of experimental laboratory data reveals the variable C:N:P stoichiometry of Emiliania huxleyi, a globally significant calcifying phytoplankton species. Mean C:N:P of E. huxleyi is 124C:16N:1P under control conditions (i.e. growth not limited by one or more environmental stressors) and shows a range of responses to changes in nutrient and light availability, temperature and pCO 2 . Macronutrient limitation caused strong shifts in stoichiometry, increasing N:P and C:P under P deficiency (by 305% and 493% respectively) and doubling C:N under N deficiency. Responses to light, temperature and pCO 2 were mixed but typically shifted cellular elemental content and C:N:P stoichiometry by ca. 30% or less. Besides these independent effects, the interactive effects of multiple environmental changes on E. huxleyi stoichiometry under future ocean conditions could be additive, synergistic or antagonistic. To synthesise our meta-analysis results, we explored how the cellular elemental content and C:N:P stoichiometry of E. huxleyi may respond to two hypothetical future ocean scenarios (increased temperature, irradiance and pCO 2 combined with either N deficiency or P deficiency) if an additive effect is assumed. Both future scenarios indicate decreased calcification (which is predominantly sensitive to elevated pCO 2 ), increased C:N, and up to fourfold shifts in C:P and N:P. Our results strongly suggest that climate change will significantly alter the role of E. huxleyi (and potentially other calcifying phytoplankton species) in marine biogeochemical processes., (© 2023 The Authors. Global Change Biology published by John Wiley & Sons Ltd.)

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., (© 2022 The Authors. Journal of Phycology published by Wiley Periodicals LLC on behalf of Phycological Society of America.)

The relationship between temperature ( T ) and diversity is one of the most important issues in ecology. It provides a key direction not only for exploring the determinants of diversity's patterns, but also for understanding diversity's responses to climate change. Previous studies suggested that T -diversity relationships could be positive, negative, or unimodal. Although these studies accumulated many informative achievements, they might be unsatisfied due to (1) investigating inadequate range of T , (2) selecting incomplete diversity metrics, and (3) making insufficiently detailed analysis of correlation. In this study, species diversity is estimated by four most commonly used diversity metrics and three parameters of species abundance distribution (SAD), and two global datasets of marine phytoplankton (covering a wider range of T ) are used to evaluate the T -diversity relationships according to a piecewise model. Results show that all aspects of diversity (except evenness) have the similar relationship with T in the range of lower T , noting that diversity significantly increases as T increases. However, in the range of higher T , diversity may significantly decrease or nearly constant, which indicates that their relationships may be the unimodal or asymptotic. The asymptotic relationship found by this study is assumed that increasing diversity with T will gradually approach the Zipf's law (1:1/2:1/3…). If such assumption can be verified by future investigations, the intrinsic mechanism of the asymptotic relationship is likely to be crucial in understanding the T -diversity relationships., Competing Interests: We declare there is no conflict of interest., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Incongruent phylogenies have been widely observed between nuclear and plastid or mitochondrial genomes in terrestrial plants and animals. However, few studies have examined these patterns in microalgae or the discordance between the two organelles. Here we investigated the nuclear-mitochondrial-plastid phylogenomic incongruence in Emiliania-Gephyrocapsa, a group of cosmopolitan calcifying phytoplankton with enormous populations and recent speciations. We assembled mitochondrial and plastid genomes of 27 strains from across global oceans and temperature regimes, and analyzed the phylogenomic histories of the three compartments using concatenation and coalescence methods. Six major clades with varying morphology and distribution are well recognized in the nuclear phylogeny, but such relationships are absent in the mitochondrial and plastid phylogenies, which also differ substantially from each other. The rampant phylogenomic discordance is due to a combination of organellar capture (introgression), organellar genome recombination, and incomplete lineage sorting of ancient polymorphic organellar genomes. Hybridization can lead to replacements of whole organellar genomes without introgression of nuclear genes and the two organelles are not inherited as a single cytoplasmic unit. This study illustrates the convoluted evolution and inheritance of organellar genomes in isogamous haplodiplontic microalgae and provides a window into the phylogenomic complexity of marine unicellular eukaryotes., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

The economic viability of microalgal-derived products relies on rapid CO 2 transfer in a cost-effective manner. Many industrial gas streams contain concentrated CO 2 that, if converted to useful products, would lower greenhouse gas emissions and valorize the wasted CO 2 . Membrane carbonation (MC) uses non-porous hollow-fiber gas-transfer membranes to deliver CO 2 without bubble formation, which makes it possible to achieve a high carbon-transfer efficiency (CTE). However, inert gasses in the industrial streams (e.g., N 2 , O 2 , and H 2 O) can significantly lower the CO 2 -delivery rate. The means to overcome the buildup of inert gases in the membrane lumen is to manage the distal end of the membranes to sweep out inert gases while not wasting significant CO 2 . A MC-venting strategy was evaluated for CO 2 inputs from 5% to 100%. Abiotic tests using a restricted exit flow could achieve >95% CTE abiotic for industrial CO 2 streams. When integrated with semi-continuous cultivation of a marine coccolithophore, Emiliania huxleyi, CO 2 delivery and venting were on-demand based on a pH set points and pH-actuated feed and venting valves. MC using the venting strategy achieved 100% CTE biotic when delivering 100% and 50% CO 2 , which was better than 50% CTE biotic obtained from pH-controlled sparging of 100% CO 2 -sparging. E. huxleyi consistently fixed ~80% of the delivered CO 2 into biomass, and the remaining ~20% to calcite coccoliths. The compact size of MC modules, stable pH control, and no shear forces from bubble agitation during the CO 2 delivery made MC an ideal match for cultivation of coccolithophores, which are sensitive to shear forces and pH fluctuations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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.

Coccolithophores are a diverse group of calcifying microalgae that have left a prominent fossil record on Earth. Various coccolithophore relics, both organic and inorganic, serve as proxies for reconstruction of past oceanic conditions. Emiliania huxleyi is the most widely distributed representative of the coccolithophores in modern oceans and is known to engage in dynamic interactions with bacteria. Algal-bacterial interactions influence various aspects of algal physiology and alter algal alkenone unsaturation (U K' ), a frequently used organic coccolithophore-derived paleo-temperature proxy. Whether algal-bacterial interactions influence inorganic coccolithophore-derived paleo-proxies is yet unknown. A commonly used inorganic proxy for past productivity and sea surface temperature is the Sr/Ca ratio of the coccolith calcite. Interestingly, during interactions between bacteria and a population of calcifying algae, bacteria were shown to physically attach only to non-calcified algal cells, suggesting an influence on algal calcification. In this study, we explore the effects of algal-bacterial interactions on calcification and coccolith Sr/Ca ratios. We find that while bacteria attach only to non-calcified algal cells, coccolith cell coverage and overall calcite production in algal populations with and without bacteria is similar. Furthermore, we find that Sr/Ca values are impacted only by water temperature and algal growth rate, regardless of bacterial influences on algal physiology. Our observations reinforce the robustness of coccolith Sr/Ca ratios as a paleo-proxy independent of microbial interactions and highlight a fundamental difference between organic and inorganic paleo-proxies.37 ), a frequently used organic coccolithophore-derived paleo-temperature proxy. Whether algal-bacterial interactions influence inorganic coccolithophore-derived paleo-proxies is yet unknown. A commonly used inorganic proxy for past productivity and sea surface temperature is the Sr/Ca ratio of the coccolith calcite. Interestingly, during interactions between bacteria and a population of calcifying algae, bacteria were shown to physically attach only to non-calcified algal cells, suggesting an influence on algal calcification. In this study, we explore the effects of algal-bacterial interactions on calcification and coccolith Sr/Ca ratios. We find that while bacteria attach only to non-calcified algal cells, coccolith cell coverage and overall calcite production in algal populations with and without bacteria is similar. Furthermore, we find that Sr/Ca values are impacted only by water temperature and algal growth rate, regardless of bacterial influences on algal physiology. Our observations reinforce the robustness of coccolith Sr/Ca ratios as a paleo-proxy independent of microbial interactions and highlight a fundamental difference between organic and inorganic paleo-proxies., (© 2022 The Authors. Geobiology published by John Wiley & Sons Ltd.)

To investigate effects of UV radiation (UVR, 280-400 nm) on coccolithophorids under nutrient-limited conditions, we grew Gephyrocapsa oceanica to determine its resilience to consecutive daily short-term exposures to +UVR (irradiances >295 nm) under a range of nitrate availabilities (100, 24, 12, 6 and 3 μM). +UVR alone significantly hampered the growth of G. oceanica, with the synergistic negative effects of +UVR and N-limitation being about 58% and 22% greater than under UVR or N-limitation alone, respectively. Most 3 μM nitrate cultures died, but those exposed to UVR succumbed sooner. This was due to a failure of photoprotection and repair mechanisms under low N-availability with exposures to UVR. Additionally, the UVR-induced inhibition of the effective quantum yield of photosystem II (PSII) was significantly higher and was further aggravated by N limitation. The algal cells increased photoprotective pigments and UV-absorbing compounds as a priority rather than using calcification for defense against UVR, indicating a trade-off in energy and resource allocation. Our results indicate the negative effects of UVR on coccolithophorid growth and photosynthesis, and highlight the important role of N availability in defense against UVR as well as high PAR. We predict that enhanced N-limitation in future surface oceans due to warming-induced stratification will exacerbate the sensitivity of G. oceanica to UVR, while coccolithophores can be potentially more susceptible to other environmental stresses due to increased levels of nutrient limitation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Elemental contents change with shifts in macromolecular composition of marine phytoplankton. Recent studies focus on the responses of elemental contents of coccolithophores, a major calcifying phytoplankton group, to changing carbonate chemistry, caused by the dissolution of anthropogenically derived CO 2 into the surface ocean. However, the effects of changing carbonate chemistry on biomacromolecules, such as protein and carbohydrate of coccolithophores, are less documented. Here, we disentangled the effects of elevated dissolved inorganic carbon (DIC) concentration (900 to 4,930μmolkg -1 ) and reduced pH value (8.04 to 7.70) on physiological rates, elemental contents, and macromolecules of the coccolithophore Emiliania huxleyi . Compared to present DIC concentration and pH value, combinations of high DIC concentration and low pH value (ocean acidification) significantly increased pigments content, particulate organic carbon (POC), and carbohydrate content and had less impact on growth rate, maximal relative electron transport rate ( rETR max ), particulate organic nitrogen (PON), and protein content. In high pH treatments, elevated DIC concentration significantly increased growth rate, pigments content, rETR max , POC, particulate inorganic carbon (PIC), protein, and carbohydrate contents. In low pH treatments, the extents of the increase in growth rate, pigments and carbohydrate content were reduced. Compared to high pH value, under low DIC concentration, low pH value significantly increased POC and PON contents and showed less impact on protein and carbohydrate contents; however, under high DIC concentration, low pH value significantly reduced POC, PON, protein, and carbohydrate contents. These results showed that reduced pH counteracted the positive effects of elevated DIC concentration on growth rate, rETR max , POC, PON, carbohydrate, and protein contents. Elevated DIC concentration and reduced pH acted synergistically to increase the contribution of carbohydrate-carbon to POC, and antagonistically to affect the contribution of protein-nitrogen to PON, which further shifted the carbon/nitrogen ratio of E. huxleyi ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2021 Xie, Xu, Li, Li, Yi, Li, Han, Zhang and Zhang.)

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., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Photophysiological responses of phytoplankton to changing multiple environmental drivers are essential in understanding and predicting ecological consequences of ocean climate changes. In this study, we investigated the combined effects of two CO 2 levels (410 and 925 μatm) and five light intensities (80 to 480 μmol photons m -2  s -1 ) on cellular pigments contents, photosynthesis and calcification of the coccolithophore Emiliania huxleyi grown under nutrient replete and limited conditions, respectively. Our results showed that high light intensity, high CO 2 level and nitrate limitation acted synergistically to reduce cellular chlorophyll a and carotenoid contents. Nitrate limitation predominantly enhanced calcification rate; phosphate limitation predominantly reduced photosynthetic carbon fixation rate, with larger extent of the reduction under higher levels of CO 2 and light. Reduced availability of both nitrate and phosphate under the elevated CO 2 concentration decreased saturating light levels for the cells to achieve the maximal relative electron transport rate (rETR max ). Light-saturating levels for rETR max were lower than that for photosynthetic and calcification rates under the nutrient limitation. Regardless of the culture conditions, rETR under growth light levels correlated linearly and positively with measured photosynthetic and calcification rates. Our findings imply that E. huxleyi cells acclimated to macro-nutrient limitation and elevated CO 2 concentration decreased their light requirement to achieve the maximal electron transport, photosynthetic and calcification rates, indicating a photophysiological strategy to cope with CO 2 rise/pH drop in shoaled upper mixing layer above the thermocline where the microalgal cells are exposed to increased levels of light and decreased levels of nutrients., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The recently characterized single-domain voltage-gated ion channels from eukaryotic protists (EukCats) provide an array of novel channel proteins upon which to test the pharmacology of both clinically and environmentally relevant marine toxins. Here, we examined the effects of the hydrophilic µ-CTx PIIIA and the lipophilic brevetoxins PbTx-2 and PbTx-3 on heterologously expressed EukCat ion channels from a marine diatom and coccolithophore. Surprisingly, none of the toxins inhibited the peak currents evoked by the two EukCats tested. The lack of homology in the outer pore elements of the channel may disrupt the binding of µ-CTx PIIIA, while major structural differences between mammalian sodium channels and the C-terminal domains of the EukCats may diminish interactions with the brevetoxins. However, all three toxins produced significant negative shifts in the voltage dependence of activation and steady state inactivation, suggesting alternative and state-dependent binding conformations that potentially lead to changes in the excitability of the phytoplankton themselves.

Coccolithophores are unique primary producers in the ocean with the ability to calcify. They are known to produce calcareous scales, which form the significant part of calcite oozes or chalk deposits on the seafloor. Coccolithophores are very noteworthy and they are explored to a great extent as nannofossils to reconstruct the past climate. Calcite plates in coccolithophores make them a vital tool in global climate change studies specifically with ocean acidification. These microscopic plants are the major contributor of the carbonate rain that controls the inorganic carbon pump in the ocean, which in turn influences both carbon and carbonate cycles. The emergence of advanced techniques enables us to study the biological aspects of this pelagic calcifier with improved precision. But still, they are understudied world over compared to any other phytoplankton groups. The northern Indian Ocean, being landlocked in three sides and vulnerable to climate change and ocean acidification, severely lacks focused studies on coccolithophores, though the US JGOFS in the 1990s have outlined the ecological significance of coccolithophores in the Arabian Sea. This paper reviews and outlines our understanding of coccolithophores as well as the nix in the northern Indian Ocean.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Emiliania huxleyi is a globally important coccolithophore and one of the most successful eukaryotic organisms in the modern oceans. Despite a large body of work on this organism, including the sequencing of its genome, the tools required for forward and reverse functional genetic studies are still undeveloped. Here we present an optimized method for the clonal isolation of E. huxleyi by plating on solid medium. We demonstrate the utility of this method for a variety of strains including haploid, calcifying-diploid, and noncalcifying diploid strains. We show that, in contrast to previous studies, no changes in cell ploidy status occur when the cells are plated. Our method will greatly aid attempts to elucidate the genetic basis of the remarkable physiology of E. huxleyi by forward and reverse genetic approaches., (© 2019 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America.)

Pleurochrysis genus algae are widely distributed in ocean waters. Pleurochrysis sp. algae are popularly known for its coccolithophores. Calcium carbonate (CaCO 3 ) shells are major components of the coccolithophore, and they are key absorbers of carbondioxide. In this study, we have reported the effects of potassium nitrate (KNO 3 ) concentration on calcium accumulation and total lipid, carbohydrate and protein contents of Pleurochrysis dentata. Results obtained from complexometric titration and scanning electron microscopy analysis showed higher rates of CaCO 3 accumulation on Pleurochrysis dentata cell surface . We have also observed that overall cell size of Pleurochrysis dentata reached maximum when it was cultured at 0.75 mmol L -1 of KNO 3 . During 10 days of Pleurochrysis dentata culture total lipids and carbohydrate contents decreased, with slightly increased protein content. Results obtained from Fourier-Transform Infrared Spectroscopy (FTIR) also reported an increase in protein and decrease in lipids and carbohydrate contents, respectively. Similarly, Pleurochrysis dentata cultured at 1 mmol L -1 concentration of KNO 3 exhibited the lowest carbohydrate (21.08%) and highest protein (32.87%) contents. Interestingly, Pleurochrysis dentata cultured without KNO 3 exhibited 33.61% of total lipid content which reduced to a total lipid content of 13.67% when cultured at 1 mmol L -1 concentration of KNO 3 . Thus, culture medium containing higher than 1 mmol L -1 of KNO 3 could inhibit the cell size of Pleurochrysis dentata and CaCO 3 accumulation in shells but it could promote its cell growth. For the first time we have reported a relatively complete coccolith structure devoid of its protoplast. In this study, we have also described about the special planar structure of Pleurochrysis dentata CaCO 3 shells present on its inner tube of the R unit and parallel to the outer tube of the V unit which we named it as "doornail structure". We believe that this doornail structure provides structural stability and support to the developing coccoliths in Pleurochrysis dentata . Also, we have discussed about the "double-disc" structure of coccoliths which are closely arranged and interlocked with each other. The double-disc structure ensures fixation of each coccolith and objecting its free horizontal movement and helps in attaining a complementary coccolith structure., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Coccolithophores are marine phytoplankton that are among the most prolific calcifiers widespread in Earth's oceans, playing a crucial role in the carbon cycle and in the transport of organic matter to the deep sea. These organisms produce highly complex mineralized scales that are composed of hierarchical assemblies of nano-crystals of calcium carbonate in the form of calcite. Coccolith formation in vivo occurs within compartmentalized mineralisation vesicles derived from the Golgi body, which contain coccolith-associated polysaccharides ('CAPs') providing polymorph selection and mediating crystal growth kinetics, and oval organic mineralisation templates, also known as base plates, which promote heterogenous nucleation and further mechanical interlocking of calcite single crystals. Although the function of coccolith base plates in controlling crystal nucleation have been widely studied, their 3D spatial organization and the chemical functional groups present on the crystal nucleation sites, which are two crucial features impacting biomineralization, remain unsolved. Utilising cryo-electron tomography we show that base plates derived from an exemplary coccolithophore Pleurochrysis carterae (Pcar) in their native hydrated state have a complex 3-layered structure. We further demonstrate, for the first time, the edge and rim of the base plate - where the crystals nucleate - are rich in primary amine functionalities that provide binding targets for negatively charged complexes composed of synthetic macromolecules and Ca 2+ ions. Our results indicate that electrostatic interactions between the negatively charged biogenic CAPs and the positively charged rim of the base plate are sufficient to mediate the transport of Ca 2+ cations to the mineralization sites., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare there are no competing interests.

Brevetoxin (PbTx) is a neurotoxic secondary metabolite of the dinoflagellate Karenia brevis. We used a novel, fluorescent BODIPY-labeled conjugate of brevetoxin congener PbTx-2 (B-PbTx) to track absorption of the metabolite into a variety of marine microbes. The labeled toxin was taken up and brightly fluoresced in lipid-rich regions of several marine microbes including diatoms and coccolithophores. The microzooplankton (20-200 μm) tintinnid ciliate Favella sp. and the rotifer Brachionus rotundiformis also took up B-PbTx. Uptake and intracellular fluorescence of B-PbTx was weak or undetectable in phytoplankton species representative of dinoflagellates, cryptophytes, and cyanobacteria over the same (4 h) time course. The cellular fate of two additional BODIPY-conjugated K. brevis associated secondary metabolites, brevenal (B-Bn) and brevisin (B-Bs), were examined in all the species tested. All taxa exhibited minimal or undetectable fluorescence when exposed to the former conjugate, while most brightly fluoresced when treated with the latter. This is the first study to observe the uptake of fluorescently-tagged brevetoxin conjugates in non-toxic phytoplankton and zooplankton taxa, demonstrating their potential in investigating whether marine microbes can serve as a significant biological sink for algal toxins. The highly variable uptake of B-PbTx observed among taxa suggests some may play a more significant role than others in vectoring lipophilic toxins in the marine environment., (© 2018 Phycological Society of America.)

Calcium storage organelles are common to all eukaryotic organisms and play a pivotal role in calcium signaling and cellular calcium homeostasis. In most organelles, the intraorganellar calcium concentrations rarely exceed micromolar levels. Acidic organelles called acidocalcisomes, which concentrate calcium into dense phases together with polyphosphates, are an exception. These organelles have been identified in diverse organisms, but, to date, only in cells that do not form calcium biominerals. Recently, a compartment storing molar levels of calcium together with phosphorous was discovered in an intracellularly calcifying alga, the coccolithophore Emiliania huxleyi , raising a possible connection between calcium storage organelles and calcite biomineralization. Here we used cryoimaging and cryospectroscopy techniques to investigate the anatomy and chemical composition of calcium storage organelles in their native state and at nanometer-scale resolution. We show that the dense calcium phase inside the calcium storage compartment of the calcifying coccolithophore Pleurochrysis carterae and the calcium phase stored in acidocalcisomes of the noncalcifying alga Chlamydomonas reinhardtii have common features. Our observations suggest that this strategy for concentrating calcium is a widespread trait and has been adapted for coccolith formation. The link we describe between acidocalcisomal calcium storage and calcium storage in coccolithophores implies that our physiological and molecular genetic understanding of acidocalcisomes could have relevance to the calcium pathway underlying coccolithophore calcification, offering a fresh entry point for mechanistic investigations on the adaptability of this process to changing oceanic conditions., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

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., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Coccolithophores are unicellular marine phytoplankton, which produce intricate, tightly regulated, exoskeleton calcite structures. The formation of biogenic calcite occurs either intracellularly, forming 'wheel-like' calcite plates, or extracellularly, forming 'tiled-like' plates known as coccoliths. Secreted coccoliths then self-assemble into multiple layers to form the coccosphere, creating a protective wall around the organism. The cell wall hosts a variety of unique species-specific inorganic morphologies that cannot be replicated synthetically. Although biomineralisation has been extensively studied, it is still not fully understood. It is becoming more apparent that biologically controlled mineralisation is still an elusive goal. A key question to address is how nature goes from basic building blocks to the ultrafine, highly organised structures found in coccolithophores. A better understanding of coccolithophore biomineralisation will offer new insight into biomimetic and bioinspired synthesis of advanced, functionalised materials for bone tissue regeneration. The purpose of this review is to spark new interest in biomineralisation and gain new insight into coccolithophores from a material science perspective, drawing on existing knowledge from taxonomists, geologists, palaeontologists and phycologists.

Coccolithophores are important oceanic primary producers not only in terms of photosynthesis but also because they produce calcite plates called coccoliths. Ongoing ocean acidification associated with changing seawater carbonate chemistry may impair calcification and other metabolic functions in coccolithophores. While short-term ocean acidification effects on calcification and other properties have been examined in a variety of coccolithophore species, long-term adaptive responses have scarcely been documented, other than for the single species Emiliania huxleyi. Here, we investigated the effects of ocean acidification on another ecologically important coccolithophore species, Gephyrocapsa oceanica, following 1,000 generations of growth under elevated CO 2 conditions (1,000 μatm). High CO 2 -selected populations exhibited reduced growth rates and enhanced particulate organic carbon (POC) and nitrogen (PON) production, relative to populations selected under ambient CO 2 (400 μatm). Particulate inorganic carbon (PIC) and PIC/POC ratios decreased progressively throughout the selection period in high CO 2 -selected cell lines. All of these trait changes persisted when high CO 2 -grown populations were moved back to ambient CO 2 conditions for about 10 generations. The results suggest that the calcification of some coccolithophores may be more heavily impaired by ocean acidification than previously predicted based on short-term studies, with potentially large implications for the ocean's carbon cycle under accelerating anthropogenic influences., (© 2018 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.)

The Arctic Ocean and its surrounding shelf seas are warming much faster than the global average, which potentially opens up new distribution areas for temperate-origin marine phytoplankton. Using over three decades of continuous satellite observations, we show that increased inflow and temperature of Atlantic waters in the Barents Sea resulted in a striking poleward shift in the distribution of blooms of Emiliania huxleyi, a marine calcifying phytoplankton species. This species' blooms are typically associated with temperate waters and have expanded north to 76°N, five degrees further north of its first bloom occurrence in 1989. E. huxleyi's blooms keep pace with the changing climate of the Barents Sea, namely ocean warming and shifts in the position of the Polar Front, resulting in an exceptionally rapid range shift compared to what is generally detected in the marine realm. We propose that as the Eurasian Basin of the Arctic Ocean further atlantifies and ocean temperatures continue to rise, E. huxleyi and other temperate-origin phytoplankton could well become resident bloom formers in the Arctic Ocean., (© 2018 John Wiley & Sons Ltd.)

Background: The globally abundant coccolithophore, Emiliania huxleyi , plays an important ecological role in oceanic carbon biogeochemistry by forming a cellular covering of plate-like CaCO 3 crystals (coccoliths) and fixing CO 2 . It is unknown how the cells arrange different-sized coccoliths to maintain full coverage, as the cell surface area of the cell changes during daily cycle., Methods: We used Euler's polyhedron formula and CaGe simulation software, validated with the geometries of coccoliths, to analyze and simulate the coccolith topology of the coccosphere and to explore the arrangement mechanisms., Results: There were only small variations in the geometries of coccoliths, even when the cells were cultured under variable light conditions. Because of geometric limits, small coccoliths tended to interlock with fewer and larger coccoliths, and vice versa. Consequently, to sustain a full coverage on the surface of cell, each coccolith was arranged to interlock with four to six others, which in turn led to each coccosphere contains at least six coccoliths., Conclusion: The number of coccoliths per coccosphere must keep pace with changes on the cell surface area as a result of photosynthesis, respiration and cell division. This study is an example of natural selection following Euler's polyhedral formula, in response to the challenge of maintaining a CaCO 3 covering on coccolithophore cells as cell size changes., Competing Interests: The authors declare that they have no competing interests.

Viruses are thought to be fundamental in driving microbial diversity in the oceanic planktonic realm. That role and associated emerging infection patterns remain particularly elusive for eukaryotic phytoplankton and their viruses. Here we used a vast number of strains from the model system Emiliania huxleyi /Emiliania huxleyi Virus to quantify parameters such as growth rate (µ), resistance (R), and viral production (Vp) capacities. Algal and viral abundances were monitored by flow cytometry during 72-h incubation experiments. The results pointed out higher viral production capacity in generalist EhV strains, and the virus-host infection network showed a strong co-evolution pattern between E. huxleyi and EhV populations. The existence of a trade-off between resistance and growth capacities was not confirmed.

Electron paramagnetic resonance (EPR) is one of the few methods that allows for the unambiguous detection of nitric oxide (NO). However, the dithiocarbamate-iron spin traps employed with this method inhibit the activity of nitric oxide synthase and catalyze NO production from nitrite. These disadvantages limit EPR's application to biological NO detection. We present a liposome-encapsulated spin-trap (LEST) method for the capture and in situ detection of NO by EPR. The method shows a linear response for [NO]≥4µM and can detect [NO]≥40nM in a 500µL sample (≥20 pmol). The kinetics of NO production can be followed in real time over minutes to hours. LEST does not inhibit the activity of inducible nitric oxide synthase or nitrate reductase and shows minimal abiotic NO production in the presence of nitrite and NADH. Nitrate reductase-like activity is detected in cell lysates of the coccolithophore Emiliania huxleyi and is elevated in virus-infected culture. This method shows particular promise for NO detection in cell lysates and crude preparations of NO-producing tissues., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Temperature has a profound effect on the species composition and physiology of marine phytoplankton, a polyphyletic group of microbes responsible for half of global primary production. Here, we ask whether and how thermal reaction norms in a key calcifying species, the coccolithophore Emiliania huxleyi , change as a result of 2.5 years of experimental evolution to a temperature ≈2°C below its upper thermal limit. Replicate experimental populations derived from a single genotype isolated from Norwegian coastal waters were grown at two temperatures for 2.5 years before assessing thermal responses at 6 temperatures ranging from 15 to 26°C, with pCO 2 (400/1100/2200 μatm) as a fully factorial additional factor. The two selection temperatures (15°/26.3°C) led to a marked divergence of thermal reaction norms. Optimal growth temperatures were 0.7°C higher in experimental populations selected at 26.3°C than those selected at 15.0°C. An additional negative effect of high pCO 2 on maximal growth rate (8% decrease relative to lowest level) was observed. Finally, the maximum persistence temperature ( T max ) differed by 1-3°C between experimental treatments, as a result of an interaction between pCO 2 and the temperature selection. Taken together, we demonstrate that several attributes of thermal reaction norms in phytoplankton may change faster than the predicted progression of ocean warming.

Ocean acidification (OA), induced by rapid anthropogenic CO2 rise and its dissolution in seawater, is known to have consequences for marine organisms. However, knowledge on the evolutionary responses of phytoplankton to OA has been poorly studied. Here we examined the coccolithophore Gephyrocapsa oceanica, while growing it for 2000 generations under ambient and elevated CO2 levels. While OA stimulated growth in the earlier selection period (from generations ~700 to ~1550), it reduced it in the later selection period up to 2000 generations. Similarly, stimulated production of particulate organic carbon and nitrogen reduced with increasing selection period and decreased under OA up to 2000 generations. The specific adaptation of growth to OA disappeared in generations 1700 to 2000 when compared with that at 1000 generations. Both phenotypic plasticity and fitness decreased within selection time, suggesting that the species' resilience to OA decreased after 2000 generations under high CO2 selection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In many marine ecosystems, diatoms dominate in nutrient-rich coastal waters while coccolithiophores are found offshore in areas where nutrients may be limiting. In lab-controlled batch cultures, mixed-species competition between the diatom Phaeodactylum tricornutum and the coccolithophore Emiliana huxleyi and the response of each species were examined under nitrate (N) and phosphate (P) starvation. Based on the logistic growth model and the Lotka-Volterra competition model, E. huxleyi showed higher competitive abilities than P. tricornutum under N and P starvation. For both species, cell growth was more inhibited by P starvation, while photosynthetic functions (chl a fluorescence parameters) and cellular constituents (pigments) were impaired by N starvation. The decline of photosynthetic functions occurred later in E. huxleyi (day 12) than in P. tricornutum (day 9); this time difference was associated with greater damage of the photosynthetic apparatus in P. tricornutum compared with E. huxleyi. Xanthophyll cycle pigment accumulation and the transformation from diadinoxanthin to diatoxanthin was more active in E. huxleyi than P. tricornutum, under similar N and P starvation. We concluded that E. huxleyi and P. tricornutum have different mechanisms to allocate resources and energy under nutrient starvation. It appears that E. huxleyi has a more economic strategy to adapt to nutrient depleted environments than P. tricornutum. These findings provided additional evidence explaining how N versus P limitation differentially support diatom and coccolithophore blooms in natural environments., (© 2015 Phycological Society of America.)

The response of N (nitrate) starved cells of the diatom Phaeodactylum tricornutum and the coccolithophore Emiliania huxleyi to a pulse of new N were measured to investigate rapid cellular and photosynthetic recovery kinetics. The changes of multiple parameters were followed over 24 h. In P. tricornutum, the recovery of Fv /Fm (the maximum quantum yield of PS II) and σPSII (the functional absorption cross-section for PSII) started within the first hour, much earlier than other parameters. Cellular pigments did not recover during the 24 h but the chlorophyll (chl) a/carotenoid ratios increased to levels measured in the controls. Cell division was independent of the recovery of chl a. In E. huxleyi, the recovery of Fv /Fm and σPSII started after an hour, synchronous with the increase in cellular organic N and chl a with pigments fully recovered within 14 h. P. tricornutum prioritized the recovery of its photosynthetic functions and cell divisions while E. huxleyi did not follow this pattern. We hypothesize that the different recovery strategies between the two species allow P. tricornutum to be more competitive when N pulses are introduced into N-limited water while E. huxleyi is adapted to N scarce waters where such pulses are infrequent. These findings are consistent with successional patterns observed in coastal environments. This is one of only a few studies exploring recovery kinetics of cellular functions and photosynthesis after nitrogen stress in phytoplankton. Our results can be used to enhance ecological models linking phytoplankton traits to species diversity and community structure., (© 2015 Phycological Society of America.)

Viruses infecting marine phytoplankton are key biogeochemical 'engines' of the oceans, regulating the dynamics of algal populations and the fate of their extensive blooms. In addition they are important ecological and evolutionary drivers of microbial diversification. Yet, little is known about mechanisms influencing viral dispersal in aquatic systems, enabling the rapid infection and demise of vast phytoplankton blooms. In a recent study we showed that migrating zooplankton as copepods that graze on marine phytoplankton can act as transmission vectors for algal viruses. We demonstrated that these grazers can concentrate virions through topical adsorption and by ingesting infected cells and then releasing back to the medium, via detachment or defecation, high viral titers that readily infect host populations. We proposed that this zooplankton-driven process can potentially boost viral dispersal over wide oceanic scales and enhance bloom termination. Here, we highlight key results and further discuss the ecological and evolutionary consequences of our findings.

In phytoplankton a high species diversity of microalgae co-exists at a given time. But diversity is not only reflected by the species composition. Within these species different life phases as well as different metabolic states can cause additional diversity. One important example is the coccolithophore Emiliania huxleyi. Diploid cells play an important role in marine ecosystems since they can form massively abundant algal blooms but in addition the less abundant haploid life phase of E. huxleyi occurs in lower quantities. Both life phases may fulfill different functions in the plankton. We hypothesize that in addition to the functional diversity caused by this life phase transition the growth stage of cells can also influence the metabolic composition and thus the ecological impact of E. huxleyi. Here we introduce a metabolomic survey in dependence of life phases as well as different growth phases to reveal such changes. The comparative metabolomic approach is based on the extraction of intracellular metabolites from intact microalgae, derivatization and analysis by gas chromatography coupled to mass spectrometry (GC-MS). Automated data processing and statistical analysis using canonical analysis of principal coordinates (CAP) revealed unique metabolic profiles for each life phase. Concerning the correlations of metabolites to growth phases, complex patterns were observed. As for example the saccharide mannitol showed its highest concentration in the exponential phase, whereas fatty acids were correlated to stationary and sterols to declining phase. These results are indicative for specific ecological roles of these stages of E. huxleyi and are discussed in the context of previous physiological and ecological studies., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

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., (© 2013 Phycological Society of America.)

Increased atmospheric pCO2 is expected to render future oceans warmer and more acidic than they are at present. Calcifying organisms such as coccolithophores that fix and export carbon into the deep sea provide feedbacks to increasing atmospheric pCO2. Acclimation experiments suggest negative effects of warming and acidification on coccolithophore calcification, but the ability of these organisms to adapt to future environmental conditions is not well understood. Here, we tested the combined effect of pCO2 and temperature on the coccolithophore Emiliania huxleyi over more than 700 generations. Cells increased inorganic carbon content and calcification rate under warm and acidified conditions compared with ambient conditions, whereas organic carbon content and primary production did not show any change. In contrast to findings from short-term experiments, our results suggest that long-term acclimation or adaptation could change, or even reverse, negative calcification responses in E. huxleyi and its feedback to the global carbon cycle. Genome-wide profiles of gene expression using RNA-seq revealed that genes thought to be essential for calcification are not those that are most strongly differentially expressed under long-term exposure to future ocean conditions. Rather, differentially expressed genes observed here represent new targets to study responses to ocean acidification and warming.

The ongoing ocean acidification associated with a changing carbonate system may impose profound effects on marine planktonic calcifiers. Here, we show that a coccolithophore, Gephyrocapsa oceanica, evolved in response to an elevated CO2 concentration of 1000 μatm (pH reduced to 7.8) in a long-term (∼670 generations) selection experiment. The high CO2 -selected cells showed increases in photosynthetic carbon fixation, growth rate, cellular particulate organic carbon (POC) or nitrogen (PON) production, and a decrease in C:N elemental ratio, indicating a greater upregulation of PON than of POC production under the ocean acidification condition. Cells from the low CO2 selection process shifted to high CO2 exposure showed an enhanced cellular POC and PON production rates. Our data suggest that the coccolithophorid could adapt to ocean acidification with enhanced assimilations of carbon and nitrogen but decreased C:N ratios., (© 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.)

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., (© 2012 Phycological Society of America.)

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., (© 2011 Phycological Society of America.)

Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an 'in house' enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase.

A new combination coccosphere of the heterococcolithophore Helicosphaera wallichii (Lohman 1902) Okada and McIntyre 1977 and holococcolithophore Syracolithus ponticuliferus (Kamptner 1941) Kleijne and Jordan 1990 is documented. This combination coccosphere was observed within a rich and diverse assemblage collected during late summer at a 20 m water depth from the NW Mediterranean Sea., (© 2009 Phycological Society of America.)

Dissolved organic nitrogen (DON) can account for a significant portion of total nitrogen in some aquatic environments, and many species of phytoplankton are able to scavenge nitrogen from this pool especially when inorganic nitrogen is limiting. Emiliania huxleyi (Lohmann) H. W. Hay et H. Mohler is able to use various forms of DON for growth, including several amino acids, purines, and pyrimidines. A cell-surface protein up-regulated in the absence of inorganic nitrogen, NRP1, is hypothesized to play a role in the metabolism of one or more of these organic nitrogen forms. Here, the genomic and cDNA sequence of NRP1 is reported. Structural predictions based on the amino acid sequence suggest a pyridoxal-5'-phosphate-dependent enzyme that may have a role in acquiring nitrogen from amino acids. Further evidence for the function of NRP1 is measured in spent media from nitrogen-limited cultures, which contain NRP1 and have glutaminase and formamidase activity. Field studies using an antibody to NRP1 show that it is expressed in E. huxleyi during bloom conditions in a Norwegian fjord., (© 2009 Phycological Society of America.)

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., (© 2009 Phycological Society of America.)