Emiliania Huxleyi Research Articles

Living coccolithophore communities in the central Mediterranean Sea (Summer 2016): Relations between ecology and oceanography

Coccolithophores were collected at 21 stations during summer 2016, from coastal and offshore areas of the Central Mediterranean Sea, to describe the ecology of the coccolithophore community integrating information on their abundance, environmental parameters (salinity, temperature, dissolved oxygen, and fluorescence) and oceanographic data. Emiliania huxleyi dominated the assemblage from surface to intermediate layers, while Florisphaera profunda was more abundant in the deep photic zone. Principal Component Analysis revealed that the distribution of coccolithophore taxa was influenced by environmental parameters: K-strategist taxa were related to warm surface waters, whereas lower photic zone taxa were influenced by the development of a Deep Chlorophyll Maximum and high salinity values, well below the thermocline. These results confirmed that a vertical species zonation, as a typical feature of low-middle latitude, characterizes the Central Mediterranean during summer. The distribution of F. profunda once again confirmed its use as a proxy of Deep Chlorophyll Maximum development and paleoproductivity estimates. Gephyrocapsa spp. (=total Gephyrocapsa), and in particular G. oceanica, were more abundant along the Atlantic Water pathway. Finally, the high concentration value of Helicosphaera carteri, recorded in the Ligurian Sea at an offshore station, suggested an expansion of the opportunist nature of this taxon from coastal environments to the offshore areas characterized by high turbidity and high productivity.

Discovery of Post-Translational Modifications in Emiliania huxleyi.

Emiliania huxleyi is a cosmopolitan coccolithophore that plays an essential role in global carbon and sulfur cycling, and contributes to marine cloud formation and climate regulation. Previously, the proteomic profile of Emiliania huxleyi was investigated using a three-dimensional separation strategy combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current study reuses the MS/MS spectra obtained, for the global discovery of post-translational modifications (PTMs) in this species without specific enrichment methods. Twenty-five different PTM types were examined using Trans-Proteomic Pipeline (Comet and PeptideProphet). Overall, 13,483 PTMs were identified in 7421 proteins. Methylation was the most frequent PTM with more than 2800 modified sites, and lysine was the most frequently modified amino acid with more than 4000 PTMs. The number of proteins identified increased by 22.5% to 18,780 after performing the PTM search. Compared to intact peptides, the intensities of some modified peptides were superior or equivalent. The intensities of some proteins increased dramatically after the PTM search. Gene ontology analysis revealed that protein persulfidation was related to photosynthesis in Emiliania huxleyi. Additionally, various membrane proteins were found to be phosphorylated. Thus, our global PTM discovery platform provides an overview of PTMs in the species and prompts further studies to uncover their biological functions. The combination of a three-dimensional separation method with global PTM search is a promising approach for the identification and discovery of PTMs in other species.

A Non-Destructive, Tuneable Method to Isolate Live Cells for High-Speed AFM Analysis.

Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min.

Visualizing active viral infection reveals diverse cell fates in synchronized algal bloom demise

Marine viruses are the most abundant biological entity in the ocean and are considered as major evolutionary drivers of microbial life [C. A. Suttle, Nat. Rev. Microbiol. 5, 801-812 (2007)]. Yet, we lack quantitative approaches to assess their impact on the marine ecosystem. Here, we provide quantification of active viral infection in the bloom forming single-celled phytoplankton Emiliania huxleyi infected by the large virus EhV, using high-throughput single-molecule messenger RNA in situ hybridization (smFISH) of both virus and host transcripts. In natural samples, viral infection reached only 25% of the population despite synchronized bloom demise exposing the coexistence of infected and noninfected subpopulations. We prove that photosynthetically active cells chronically release viral particles through nonlytic infection and that viral-induced cell lysis can occur without viral release, thus challenging major assumptions regarding the life cycle of giant viruses. We could also assess active infection in cell aggregates linking viral infection and carbon export to the deep ocean [C. P. Laber et al., Nat. Microbiol. 3, 537-547 (2018)] and suggest a potential host defense strategy by enrichment of infected cells in sinking aggregates. Our approach can be applied to diverse marine microbial systems, opening a mechanistic dimension to the study of biotic interactions in the ocean.

IOD-ENSO interaction with natural coccolithophore assemblages in the tropical eastern Indian Ocean

Coccolithophores, a calcifying phytoplankton group, are a major component of the oligotrophic ocean. The tropical eastern Indian Ocean (EIO) possesses a complex regional hydrological system that also impacts the global climate. Coccolithophores are a major indicator of these oceanographic and air-sea processes. Understanding coccolithophore population dynamics associated with extreme climate events is significant for predicting future ocean biogeochemical studies, and contributing to the regional and global climatic model. Therefore, we used a consecutive 7 years (2011–2018) dataset of coccolithophore assemblages and their organic carbon biomass from the tropical EIO during the spring premonsoon period to interpret the aforementioned climatic changes. Among the 33 identified species, the ecologically important species Gephyrocapsa oceanica, Emiliania huxleyi, Algirosphaera robusta, Florisphaera profunda, Umbilicosphaera sibogae, and Umbellosphaera irregularis dominated the coccolithophore assemblages. In the EIO, regional environmental factors including high-level eddy kinetic energy, equatorial jets and upwelling lead to the patchy distribution of surface coccolithophores, but no signs of interannual spatial variations were observed. Furthermore, this study revealed that variability in euphotic coccolithophore abundance and diversity indices were correlated with global climate anomalies. The variations in the interannual coccolithophore abundance and estimated coccolithophore organic carbon could be a result of global warming and other climatic variabilities. Particularly, an apparent increase in coccolithophore abundance was observed during El Niño and positive Indian Ocean Dipole (IOD) events due to their favorable thermal regimes. In contrast, the abundance of coccolithophores reduced during La Niña and negative IOD events. Overall, our pilot findings would encourage further studies on coccolithophore responses to regional EIO environments and global climatic anomalies.

Diurnally fluctuatingpCO2 enhances growth of a coastal strain ofEmiliania huxleyiunder future-projected ocean acidification conditions

AbstractThe carbonate chemistry in coastal waters is more variable compared with that of open oceans, both in magnitude and time scale of its fluctuations. However, knowledge of the responses of coastal phytoplankton to dynamic changes in pH/pCO2 has been scarcely documented. Hence, we investigated the physiological performance of a coastal isolate of the coccolithophore Emiliania huxleyi (PML B92/11) under fluctuating and stable pCO2 regimes (steady ambient pCO2, 400 μatm; steady elevated pCO2, 1200 μatm; diurnally fluctuating elevated pCO2, 600–1800 μatm). Elevated pCO2 inhibited the calcification rate in both the steady and fluctuating regimes. However, higher specific growth rates and lower ratios of calcification to photosynthesis were detected in the cells grown under diurnally fluctuating elevated pCO2 conditions. The fluctuating pCO2 regime alleviated the negative effects of elevated pCO2 on effective photochemical quantum yield and relative photosynthetic electron transport rate compared with the steady elevated pCO2 treatment. Our results suggest that growth of E. huxleyi could benefit from diel fluctuations of pH/pCO2 under future-projected ocean acidification, but its calcification was reduced by the fluctuation and the increased concentration of CO2, reflecting a necessity to consider the influences of dynamic pH fluctuations on coastal carbon cycles associated with ocean global changes.

Evaluation of actin as a reference for quantitative gene expression studies in Emiliania huxleyi (Prymnesiophyceae) under ocean acidification conditions

ABSTRACT Gene expression studies of marine phytoplankton under ocean acidification conditions are frequently based on relative measurements, with actin commonly used as a reference gene. Evidence from other organisms suggests that actin gene expression may be regulated by environmental conditions, compromising the role of actin as a reference gene. In this work the reliability of actin as a reference gene for ocean acidification experimental conditions (high CO2 vs low CO2) in two different metabolic states (acclimated metabolism vs perturbed metabolism) for the coccolithophore Emiliania huxleyi was tested. The transcriptional response of the actin (act) is compared with the expression of specific target genes associated with inorganic carbon uptake (α-carbonic anhydrase: αca1) and assimilation (RuBisCO: rbcL), which was regulated under the experimental conditions. Our results showed act expression instability in experimental conditions, evidencing that act is not a reliable reference gene for studies assessing the effect of ocean acidification on Emiliania huxleyi. Furthermore, when the act-based normalization was quantitatively tested, rbcL and αca1 expression were compromised, leading us to conclude that absolute gene expression quantification should be considered as a potentially reliable alternative for studying gene expression under ocean acidification conditions.

Strain-specific morphological response of the dominant calcifying phytoplankton species Emiliania huxleyi to salinity change.

The future physiology of marine phytoplankton will be impacted by a range of changes in global ocean conditions, including salinity regimes that vary spatially and on a range of short- to geological timescales. Coccolithophores have global ecological and biogeochemical significance as the most important calcifying marine phytoplankton group. Previous research has shown that the morphology of their exoskeletal calcified plates (coccoliths) responds to changing salinity in the most abundant coccolithophore species, Emiliania huxleyi. However, the extent to which these responses may be strain-specific is not well established. Here we investigated the growth response of six strains of E. huxleyi under low (ca. 25) and high (ca. 45) salinity batch culture conditions and found substantial variability in the magnitude and direction of response to salinity change across strains. Growth rates declined under low and high salinity conditions in four of the six strains but increased under both low and high salinity in strain RCC1232 and were higher under low salinity and lower under high salinity in strain PLYB11. When detailed changes in coccolith and coccosphere size were quantified in two of these strains that were isolated from contrasting salinity regimes (coastal Norwegian low salinity of ca. 30 and Mediterranean high salinity of ca. 37), the Norwegian strain showed an average 26% larger mean coccolith size at high salinities compared to low salinities. In contrast, coccolith size in the Mediterranean strain showed a smaller size trend (11% increase) but severely impeded coccolith formation in the low salinity treatment. Coccosphere size similarly increased with salinity in the Norwegian strain but this trend was not observed in the Mediterranean strain. Coccolith size changes with salinity compiled for other strains also show variability, strongly suggesting that the effect of salinity change on coccolithophore morphology is likely to be strain specific. We propose that physiological adaptation to local conditions, in particular strategies for plasticity under stress, has an important role in determining ecotype responses to salinity.

Hybridisation capture allows DNA damage analysis of ancient marine eukaryotes

Marine sedimentary ancient DNA (sedaDNA) is increasingly used to study past ocean ecosystems, however, studies have been severely limited by the very low amounts of DNA preserved in the subseafloor, and the lack of bioinformatic tools to authenticate sedaDNA in metagenomic data. We applied a hybridisation capture ‘baits’ technique to target marine eukaryote sedaDNA (specifically, phyto- and zooplankton, ‘Planktonbaits1’; and harmful algal bloom taxa, ‘HABbaits1’), which resulted in up to 4- and 9-fold increases, respectively, in the relative abundance of eukaryotes compared to shotgun sequencing. We further used the bioinformatic tool ‘HOPS’ to authenticate the sedaDNA component, establishing a new proxy to assess sedaDNA authenticity, “% eukaryote sedaDNA damage”, that is positively correlated with subseafloor depth. We used this proxy to report the first-ever DNA damage profiles from a marine phytoplankton species, the ubiquitous coccolithophore Emiliania huxleyi. Our approach opens new avenues for the detailed investigation of long-term change and evolution of marine eukaryotes over geological timescales.

Glacial episodes of a freshwater Arctic Ocean covered by a thick ice shelf.

Following early hypotheses about the possible existence of Arctic ice shelves in the past1-3, the observation of specific erosional features as deep as 1,000metres below the current sea level confirmed the presence of a thick layer of ice on the Lomonosov Ridge in the central Arctic Ocean and elsewhere4-6. Recent modelling studies have addressed how an ice shelf may have built up in glacial periods, covering most of the Arctic Ocean7,8. So far, however, there is no irrefutable marine-sediment characterization of such an extensive ice shelf in the Arctic, raising doubt about the impact of glacial conditions on the Arctic Ocean. Here we provide evidence for at least two episodes during which the Arctic Ocean and the adjacent Nordic seas were not only covered by an extensive ice shelf, but also filled entirely with fresh water, causing a widespread absence of thorium-230 in marine sediments. We propose that these Arctic freshwater intervals occurred 70,000-62,000years before present and approximately 150,000-131,000years before present, corresponding to portions of marine isotope stages4 and6. Alternative interpretations of the first occurrence of the calcareousnannofossil Emiliania huxleyi in Arctic sedimentary records would suggest younger ages for the older interval. Our approach explains the unexpected minima in Arctic thorium-230 records9 that have led to divergent interpretations of sedimentation rates10,11 and hampered their use for dating purposes. About nine million cubic kilometres of fresh water is required to explain our isotopic interpretation, a calculation that we support with estimates of hydrological fluxes and altered boundary conditions. A freshwater mass of this size-stored in oceans, rather than land-suggests that a revision of sea-level reconstructions based on freshwater-sensitive stable oxygen isotopes may be required, and that large masses of fresh water could be delivered to the north Atlantic Ocean on very short timescales.

Photosynthesis and calcification of the coccolithophore Emiliania huxleyi are more sensitive to changed levels of light and CO2 under nutrient limitation

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 CO2 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 CO2 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 CO2 and light. Reduced availability of both nitrate and phosphate under the elevated CO2 concentration decreased saturating light levels for the cells to achieve the maximal relative electron transport rate (rETRmax). Light-saturating levels for rETRmax 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 CO2 concentration decreased their light requirement to achieve the maximal electron transport, photosynthetic and calcification rates, indicating a photophysiological strategy to cope with CO2 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.

Group 2i Isochrysidales produce characteristic alkenones reflecting sea ice distribution

Alkenones are biomarkers produced solely by algae in the order Isochrysidales that have been used to reconstruct sea surface temperature (SST) since the 1980s. However, alkenone-based SST reconstructions in the northern high latitude oceans show significant bias towards warmer temperatures in core-tops, diverge from other SST proxies in down core records, and are often accompanied by anomalously high relative abundance of the C37 tetra-unsaturated methyl alkenone (%C37:4). Elevated %C37:4 is widely interpreted as an indicator of low sea surface salinity from polar water masses, but its biological source has thus far remained elusive. Here we identify a lineage of Isochrysidales that is responsible for elevated C37:4 methyl alkenone in the northern high latitude oceans through next-generation sequencing and lab-culture experiments. This Isochrysidales lineage co-occurs widely with sea ice in marine environments and is distinct from other known marine alkenone-producers, namely Emiliania huxleyi and Gephyrocapsa oceanica. More importantly, the %C37:4 in seawater filtered particulate organic matter and surface sediments is significantly correlated with annual mean sea ice concentrations. In sediment cores from the Svalbard region, the %C37:4 concentration aligns with the Greenland temperature record and other qualitative regional sea ice records spanning the past 14 kyrs, reflecting sea ice concentrations quantitatively. Our findings imply that %C37:4 is a powerful proxy for reconstructing sea ice conditions in the high latitude oceans on thousand- and, potentially, on million-year timescales.

PHYTOPLANKTON IN THE PORT OF NOVOROSSIYSK AND OPEN PART OF THE BAY IN 2018

The article presents research results of taxonomic composition and quantitative development of the phytoplankton in area of Novorossiysk port and in the open part of the bay for the period of May-September 2018. In total 52 species of the phytoplankton were found. Coefficient of flora similarity in 2017 and 2018 was 62%. Average values of abundance and biomass in the port were 190 thousands cell/L and 250 mg/m3 , in the open areas of the Novorossiysk bay – 266 thousands cell/L и 303 mg/m3 . Dominated in the study area was Bacillariophyta – 70–58 % of the total abundance and 78–75% of the phytoplankton biomass. The small mesosaprobic species diatoms (Thalassionema nitzschioides, Leptocylindrus minimus, Chaetoceros compressus), Euglenophyta (Euglena sp.), Cryptophyta (Plagioselmis punctata) developed significantly in the port. The Chrysophyta (Emiliania huxleyi) and diatoms Proboscia alata и Pseudosolenia calcar-avis prevailed in the open area of the bay

Reduced growth with increased quotas of particulate organic and inorganic carbon in the coccolithophore <i>Emiliania huxleyi</i> under future ocean climate change conditions

Abstract. Effects of ocean acidification and warming on marine primary producers can be modulated by other environmental factors, such as levels of nutrients and light. Here, we investigated the interactive effects of five oceanic environmental drivers (CO2, temperature, light, dissolved inorganic nitrogen and phosphate) on the growth rate, particulate organic carbon (POC) and particulate inorganic carbon (PIC) quotas of the cosmopolitan coccolithophore Emiliania huxleyi. The population growth rate increased with increasing temperature (16 to 20 ∘C) and light intensities (60 to 240 µmolphotonsm-2s-1) but decreased with elevated pCO2 concentrations (370 to 960 µatm) and reduced availability of nitrate (24.3 to 7.8 µmol L−1) and phosphate (1.5 to 0.5 µmol L−1). POC quotas were predominantly enhanced by the combined effects of increased pCO2 and decreased availability of phosphate. PIC quotas increased with decreased availability of nitrate and phosphate. Our results show that concurrent changes in nutrient concentrations and pCO2 levels predominantly affected the growth, photosynthetic carbon fixation and calcification of E. huxleyi and imply that plastic responses to progressive ocean acidification, warming, and decreasing availability of nitrate and phosphate reduce the population growth rate while increasing cellular quotas of particulate organic and inorganic carbon of E. huxleyi, ultimately affecting coccolithophore-related ecological and biogeochemical processes.

Ocean-related global change alters lipid biomarker production in common marine phytoplankton

Abstract. Lipids, in their function as trophic markers in food webs and organic matter source indicators in the water column and sediments, provide a tool for reconstructing the complexity of global change effects on aquatic ecosystems. It remains unclear how ongoing changes in multiple environmental drivers affect the production of key lipid biomarkers in marine phytoplankton. Here, we tested the responses of sterols, alkenones and fatty acids (FAs) in the diatom Phaeodactylum tricornutum, the cryptophyte Rhodomonas sp. and the haptophyte Emiliania huxleyi under a full-factorial combination of three temperatures (12, 18 and 24 ∘C), three N : P supply ratios (molar ratios 10 : 1, 24 : 1 and 63 : 1) and two pCO2 levels (560 and 2400 µatm) in semicontinuous culturing experiments. Overall, N and P deficiency had a stronger effect on per-cell contents of sterols, alkenones and FAs than warming and enhanced pCO2. Specifically, P deficiency caused an overall increase in biomarker production in most cases, while N deficiency, warming and high pCO2 caused nonsystematic changes. Under future ocean scenarios, we predict an overall decrease in carbon-normalized contents of sterols and polyunsaturated fatty acids (PUFAs) in E. huxleyi and P. tricornutum and a decrease in sterols but an increase in PUFAs in Rhodomonas sp. Variable contents of lipid biomarkers indicate a diverse carbon allocation between marine phytoplankton species in response to changing environments. Thus, it is necessary to consider the changes in key lipids and their consequences for food-web dynamics and biogeochemical cycles, when predicting the influence of global change on marine ecosystems.

The Possession of Coccoliths Fails to Deter Microzooplankton Grazers

Phytoplankton play a central role in the regulation of global carbon and nutrient cycles, forming the basis of the marine food webs. A group of biogeochemically important phytoplankton, the coccolithophores, produce calcium carbonate scales that have been hypothesized to deter or reduce grazing by microzooplankton. Here, a meta-analysis of mesocosm-based experiments demonstrates that calcification of the cosmopolitan coccolithophore, Emiliania huxleyi, fails to deter microzooplankton grazing. The median grazing to growth ratio for E. huxleyi (0.56 ± 0.40) was not significantly different among non-calcified nano- or picoeukaryotes (0.71 ± 0.31 and 0.55 ± 0.34, respectively). Additionally, the environmental concentration of E. huxleyi did not drive preferential grazing of non-calcified groups. These results strongly suggest that the possession of coccoliths does not provide E. huxleyi effective protection from microzooplankton grazing. Such indiscriminate consumption has implications for the dissolution and fate of CaCO3 in the ocean, and the evolution of coccoliths.

Pliocene-Pleistocene Calcareous Nannoplankton Biostratigraphy, Section Banyuurip, Rembang Zone, East Java Basin, Indonesia

The present study was carried out at the Banyuurip area in the East Java Basin, Indonesia, a site contains nannofossil assemblages of Pliocene to Pleistocene, with lithology is suitable for nannoplankton research. Methods used in this research include making a Stratigraphical section Measurement, collecting 41 samples and preparing the samples with the smear slides method to be observed using a polarizing microscope with 1000x magnification, and preparing several rock samples using SEM analysis. Analysis of nannofossil resulted in identifying 19 genus and 51 species. Biostratigraphic zone of this study can be arranged into 9 biostratigraphic zones. The zone order from older to younger are: a) Sphenolithus neoabies Zone /NN12/Late Miocene to Early Pliocene, b) Ceratolithus rugosus Zone /NN13/Early Pliocene, c) Discoaster asymmetricus–Reticulofenestra pseudoumbulicus Range Zone/NN14-NN15/Early Pliocene to Middle Pliocene, d) Discoaster surculus Zone/NN16/Middle Pliocene, e) Discoaster pentaradiatus Zone/NN17/Late Pliocene, f) Discoaster brouweri Zone/NN18/Late Pliocene, g) Gephyrocapsa caribbeanica Zone/NN19 Zone/Early Pleistocene, h) Gephyrocapsa oceanica Zone/NN20 Zone/Middle Pleistocene, and i) Emiliania huxleyi Zone/NN21 Zone/Late Pleistocene.

Stickiness of extracellular polymeric substances on different surfaces via magnetic tweezers

Organic particle dynamics in the surface ocean plays a critical part in the marine carbon cycle. Aggregation of marine organic particles drives their downward transport to support various marine organisms on their transit to the sediments. Extracellular polymeric substances (EPS) from various microbes are a major contributor to the oceanic organic particle pool. The stickiness of EPS is expected to play a determining role in the aggregation process of particles; however, stickiness parameters are usually indirectly estimated through data fitting without direct assessment. Here a magnetic tweezer method was developed to quantitatively assess the stickiness of three model EPS produced by: Amphora sp., (diatom), Emiliania huxleyi (coccolithophore), and Sagittula stellata (bacteria), under different in vitro environmental conditions (salinity or EDTA complexed cations) and surface matrices (EPS-EPS and bare glass). Our results showed the stickiness of three microbial EPS decreasing for S. stellata > E. huxleyi > Amphora sp., in line with their decreasing protein-to-carbohydrate (P/C) ratios (related to their relative hydrophobicity). The data not only emphasize the importance of hydrophobicity on EPS stickiness, but also demonstrates that salinity and the nature of the substrate surface can influence the stickiness. Furthermore, we investigated stickiness between various types of EPS, and the observed selective stickiness of EPS between species may shed light on the interactions among heterogeneous marine microorganisms. Overall, this newly developed system provides a platform to assess the EPS stickiness to advance our understanding of the aggregation and sedimentation process of organic particles that are critical for the fate of organic carbon as well as for biofilm formation and microbial colonization of surfaces in the ocean.

Coccolithophore calcification studied by single-cell impedance cytometry: Towards single-cell PIC:POC measurements

Since the industrial revolution 30% of the anthropogenic CO2 is absorbed by oceans, resulting in ocean acidification, which is a threat to calcifying algae. As a result, there has been profound interest in the study of calcifying algae, because of their important role in the global carbon cycle. The coccolithophore Emiliania huxleyi is considered to be globally the most dominant calcifying algal species, which creates a unique exoskeleton from inorganic calcium carbonate platelets. The PIC (particulate inorganic carbon): POC (particulate organic carbon) ratio describes the relative amount of inorganic carbon in the algae and is a critical parameter in the ocean carbon cycle.In this research we explore the use of microfluidic single-cell impedance spectroscopy in the field of calcifying algae. Microfluidic impedance spectroscopy enables us to characterize single-cell electrical properties in a non-invasive and label-free way. We use the ratio of the impedance at high frequency vs. low frequency, known as opacity, to discriminate between calcified coccolithophores and coccolithophores with a calcite exoskeleton dissolved by acidification (decalcified).We have demonstrated that using opacity we can discriminate between calcified and decalcified coccolithophores with an accuracy of 94.1%. We have observed a correlation between the measured opacity and the cell height in the channel, which is supported by FEM simulations. The difference in cell density between calcified and decalcified cells can explain the difference in cell height and therefore the measured opacity.

Transformation of coccolithophorid Emiliania huxleyi harboring a marine virus (Coccolithoviruses) serine palmitoyltransferase (SPT) gene by electroporation

Emiliania huxleyi is the most prominent modern coccolithophore, a group of marine unicellular eukaryotes that play a critical role in ocean biogeochemistry. Coccolithoviruses are large double stranded DNA viruses, which is responsible for the demise of large oceanic blooms formed by E. huxleyi. E. huxleyi virus (EhVs) acquired a series of enzyme-coding genes predicted to be involved in the sphingolipid biosynthesis by horizontal gene transfer between virus-host. Currently, there is limited experimental validation identifying the functions of these genes in EhV. Genetic transformation of eukaryotic cells is a powerful tool to get an insight into gene functions of the studied organisms. Serine palmitoyltransferase (SPT) catalyzes the first committed step in de novo sphingolipid biosynthetic pathway. Here, a novel vector system for the transformation of E. huxleyi was designed. It contained fragments of promoter and terminator sequences of E. huxleyi endogenic fucoxanthin chlorophyll a/c-binding protein gene “fcp” and harbored EhV-99B1 spt gene. The resultant recombinant transformation vectors pEhux-I-spt and pEhux-II were co-transferred into E. huxleyi BOF92 by electroporation. Transformants were obtained upon glufosinate-ammonium selection, and confirmed by Southern hybridization, genome PCR, qRT-PCR and Western blot screening of spt gene, which indicated that spt gene was integrated into the nuclear genome and was expressed at the mRNA and protein levels. The expression of the viral spt gene led to differences in lipid compositions analyzed using thin-layer chromatography (TLC). The results present the genetic transformation system for E. huxleyi, providing additional genetic resource with potential for exploring basic biological questions such as the virus-host interactions.