INTER‐ AND INTRASPECIFIC RELATIONSHIPS BETWEEN NUCLEAR DNA CONTENT AND CELL SIZE IN SELECTED MEMBERS OF THE CENTRIC DIATOM GENUS THALASSIOSIRA (BACILLARIOPHYCEAE)1

The enormous species diversity of diatoms correlates with the remarkable range of cell sizes in this group. Nuclear DNA content relates fundamentally to cell volume in other eukaryotic cells. The relationship of cell volume to G1 DNA content was determined among selected members of the genus Thalassiosira, one of the most species‐rich and well‐studied centric diatom genera. Both minimum and maximum species‐specific cell volume correlated positively with G1 DNA content. Phylogeny based on 5.8 S and ITS rDNA sequences indicated that multiple changes in G1 DNA content and cell volume occurred in Thalassiosira evolution, leading to a 1,000‐fold range in both parameters in the group. Within the Thalassiosira weissflogii (Grunow) G. A. Fryxell et Grunow species complex, G1 DNA content varied 3‐fold: differences related to geographic origin and time since isolation; doubling and tripling of G1 DNA content occurred since isolation in certain T. weissflogii isolates; and subcultures of T. weissflogii CCMP 1336 diverged in DNA content by 50% within 7 years of separation. Actin, β‐tubulin, and Spo11/TopVIA genes were selected for quantitative PCR estimation of haploid genome size in subclones of selected T. weissflogii isolates because they occur only once in the T. pseudonana Hasle et Heimdal genome. Comparison of haploid genome size estimates with G1 DNA content suggested that the most recent T. weissflogii isolate was diploid, whereas other T. weissflogii isolates appeared to be polyploid and/or aneuploid. Aberrant meiotic and mitotic cell divisions were observed, which might relate to polyploidization. The structural flexibility of diatom genomes has important implications for their evolutionary diversification and stability during laboratory maintenance.     

Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump.     

Phosphate‐limited growth of the marine diatom Thalassiosira weissflogii (Bacillariophyceae): evidence of non‐monod growth kinetics1

The marine diatom Thalassiosira weissflogii (Grunow) G. A. Fryxell & Hasle was grown in a chemostat over a series of phosphate‐limited growth rates. Ambient substrate concentrations were determined from bioassays involving picomolar spikes of ³³P‐labeled phosphate, and maximum uptake rates were determined from analogous bioassays that included the addition of micromolar concentrations of unlabeled phosphate and tracer concentrations of ³³P. The relationship between cell phosphorus quotas and growth rates was well described by the Droop equation. Maximum uptake rates of phosphate spikes were several orders of magnitude higher than steady state uptake rates. Despite the large size of the T. weissflogii cells, diffusion of phosphate through the boundary layer around the cells had little effect on growth kinetics, in part because the cellular N:P ratios exceeded the Redfield ratio at all growth rates. Fitting the Monod equation to the experimental data produced an estimate of the nutrient‐saturated growth rate that was ~50% greater than the maximum growth rate observed in batch culture. A modified hyperbolic equation with a curvature that is a maximum in magnitude at positive growth rates gave a better fit to the data and an estimate of the maximum growth rate that was consistent with observations. The failure of the Monod equation to describe the data may reflect a transition from substrate to co‐substrate limitation and/or the presence of an inducible uptake system.     

Regulation of phosphoribulokinase and glyceraldehyde 3‐phosphate dehydrogenase in a freshwater diatom,Asterionella formosa1

The regulation of phosphoribulokinase (PRK) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was investigated in a freshwater pennate diatom, Asterionella formosa Hassall, and compared to the well‐studied chlorophyte Chlamydomonas reinhardtii P. A. Dang. As has been reported for a marine centric diatom, in A. formosa, PRK was not regulated by reduction with dithiothreitol (DTT) apart from a weak induction in the presence of NADPH and DTT. However, NADPH‐GAPDH was strongly activated when reduced, in contrast to a previous report on a diatom. Surprisingly, it was inhibited by NADPH, unlike in C. reinhardtii, while NADH‐GAPDH was not affected. NADH‐GAPDH was also strongly activated by DTT in contrast to most other photosynthetic cells. In A. formosa, unlike C. reinhardtii, 1,3‐bisphosphoglycerate, the substrate of GAPDH, activated this enzyme, even in the absence of DTT, when using both NADH and NADPH as cofactors. Some of these kinetic behaviors are consistent with regulation by protein–protein interactions involving CP12, a small protein that links PRK and GAPDH in cyanobacteria, green algae, and higher plants. This conclusion was supported by immunodetection of CP12 in crude extracts of A. formosa, using antibodies raised against CP12 from C. reinhardtii. This is the first report of the existence of CP12 in a diatom, but CP12 may be a common feature of diatoms since a bioinformatic search suggested that it was also present in the Thalassiosira pseudonana Hasle et Heimdal genome v3.0. Despite the presence of CP12, this work provides further support for the differential regulation of Calvin cycle enzymes in diatoms compared to green algae.     

INFLUENCE OF LOW LIGHT AND A LIGHT: DARK CYCLE ON NO3– UPTAKE,INTRACELLULAR NO3–, AND NITROGEN ISOTOPE FRACTIONATION BY MARINE PHYTOPLANKTON1

The nitrogen isotope enrichment factor (ɛ) of four species of marine phytoplankton grown in batch cultures was determined during growth in continuous saturating light, continuous low light, and a 12:12‐h light:dark cycle, with nitrate as a nitrogen source. The low growth rate that resulted from low irradiance caused an increased accumulation of the intracellular nitrate pool and/or a reduction in cell volume and was correlated to a species‐specific increase in the measured ɛ value, compared with the saturating light conditions. The largest response was in the diatom Thalassiosira weissflogii (Grun.) Fryxell et Hasle, which showed a nearly 3‐fold increase between high and low light conditions (6.2–15.2‰). The smallest response was in T. pseudonana (Hustedt) Hasle et Heimdal, which showed no change in the ɛ value of approximately 5‰ in both high and low light conditions. There was significant but smaller increases in the ɛ value for the diatom T. rotula Meunier (2.7–5.6‰) and the prymnesiophyte Emiliania huxleyi (Lohm.) Hay et Mohler (4.5–9.4‰) between high and low light levels. In the light:dark experiments, all three diatoms but not the prymnesiophyte exhibited an increase in ɛ. This increase was linked to the ability of diatoms to assimilate nitrate at night. The results of the these experiments suggest that the light regime influences the relative uptake, assimilation, and efflux rates of nitrate and results in differences in the expression of the isotope effect by the enzyme nitrate reductase. Therefore, variations in nitrate isotope fractionation in nature can be more accurately interpreted when the light regime and species composition are taken into consideration.     

Diatom Vacuolar 1,6‐β‐Transglycosylases can Functionally Complement the Respective Yeast Mutants

Diatoms are unicellular photoautotrophic algae, which can be found in any aquatic habitat. The main storage carbohydrate of diatoms is chrysolaminarin, a nonlinear β‐glucan, consisting of a linear 1,3‐β‐chain with 1,6‐β‐branches, which is stored in cytoplasmic vacuoles. The metabolic pathways of chrysolaminarin synthesis in diatoms are poorly investigated, therefore we studied two potential 1,6‐β‐transglycosylases (TGS) of the diatom Phaeodactylum tricornutum which are similar to yeast Kre6 proteins and which potentially are involved in the branching of 1,3‐β‐glucan chains by adding d ‐glucose as 1,6‐side chains. We genetically fused the full‐length diatom TGS proteins to GFP and expressed these constructs in P. tricornutum, demonstrating that the enzymes are apparently located in the vacuoles, which indicates that branching of chrysolaminarin may occur in these organelles. Furthermore, we demonstrated the functionality of the diatom enzymes by expressing TGS1 and 2 proteins in yeast, which resulted in a partial complementation of growth deficiencies of a transglycosylase‐deficient ?kre6 yeast strain.     

Morphofunctional study of 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA)‐induced differentiation of U937 cells under exposure to a 6 mT static magnetic field

This study deals with the morphofunctional influence of 72 h exposure to a 6 mT static magnetic field (SMF) during differentiation induced by 50 ng/ml 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA) in human leukaemia U937 cells. The cell morphology of U937 cells was investigated by optic and electron microscopy. Specific antibodies and/or molecules were used to label CD11c, CD14, phosphatidylserine, F‐actin and to investigate the distribution and activity of lysosomes, mitochondria and SER. [Ca²⁺]_i was evaluated with a spectrophotometer. The degree of differentiation in SMF‐exposed cells was lower than that of non‐exposed cells, the difference being exposure time‐dependent. SMF‐exposed cells showed cell shape and F‐actin modification, inhibition of cell attachment, appearance of membrane roughness and large blebs and impaired expression of specific macrophagic markers on the cell surface. The intracellular localization of SER and lysosomes was only partially affected by exposure. A significant localization of mitochondria with an intact membrane potential at the cell periphery in non‐exposed, TPA‐stimulated cells was observed; conversely, in the presence of SMF, mitochondria were mainly localised near the nucleus. In no case did SMF exposure affect cell viability. The sharp intracellular increase of [Ca²⁺]_i could be one of the causes of the above‐described changes. Bioelectromagnetics 30:352–364, 2009. © 2009 Wiley‐Liss, Inc.     

Transparent exopolymer particle production and aggregation by a marine planktonic diatom (Thalassiosira weissflogii) at different growth rates

Transparent exopolymer particles (TEP) play an important role in the ocean carbon cycle as they are sticky and affect particle aggregation and the biological carbon pump. We investigated the effect of growth rate on TEP production in nitrogen limited semi‐continuous cultures of the diatom Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle. Steady‐state diatom concentrations and other indicators of biomass (chl a, and total carbohydrate) were inversely related to growth rate, while individual cell volume increased with growth rate. There was no change in total TEP area with growth rate; however, individual TEP were larger at high growth rates and the number of individual TEP particles was lower. TEP concentration per cell was higher at higher growth rates. SYTOX Green staining showed that <5% of the diatom population had permeable cell membranes, with the proportion increasing at low growth rates. However, TEP production rates were greater at high growth rates, refuting our hypothesis that TEP formation is dependent on dying cells with compromised cell membranes in a diatom population. Measurements of particle size distribution in the cultures using laser scattering showed that they were most aggregated at high growth rates. These results indicate a coupling between TEP production and growth rate in diatoms under N limitation, with fast growing T. weissflogii producing more TEP and aggregates.     

Effects of concentration on the feeding of a marine copepod in algal monocultures and mixtures

High-speed microcinematography was used to examine the feedingbehavior of the marine copepod Eucalanus elongatus in a rangeof concentrations of algal monocultures and mixtures. Two celltypes were offered, the 13-µm diatom Thalassiosira weissflogii,which is primarily accumulated passively by low amplitude flappingof the second maxillae, and the 20 450-µm diatom Rhizosoleniaalata, which is actively captured by detection of and orientedresponse to individual cells. E. elongatus rapidly switchedback and forth between these two capture modes in mixtures ofboth diatoms, and flapped the second maxillae at low amplitudesregardless of the absolute or relative abundance of small andlarge cells. However, copepods in both monocultures and mixturesaltered the duration and/or rate of flapping of the feedingappendages with changes in algal concentration, with maximumactivity levels occurring at intermediate concentrations. Themarked reduction in feeding motions observed at the lowest algalconcentrations supports results from traditional grazing studiesand optimal foraging models, and may conserve energy duringprolonged perods of low food availability in continental slopewaters.     

Mono‐ and digalactosyldiacylglycerol composition of the marennine‐producing diatom,Haslea ostrearia: Comparison to a selection of pennate and centric diatoms

Diatoms are one of the largest groups of primary producers in the oceans, yet despite their environmental importance little is known about their plastidial lipid biochemistry. It has been previously reported that Skeletonema species contain primarily C₁₆/C₁₆ and C₂₀/C₁₆ forms of mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively). Likewise, it was also reported that Phaeodactylum tricornutum contains primarily C₁₆/C₁₆ and C₂₀/C₂₀ forms of MGDG and DGDG. We seek to relate their studies to other diatoms, both in the centrics and pennates, with particular focus on the marennine‐producing pennate diatom, Haslea ostrearia. To this end, the composition and positional distribution of fatty acids of MGDG and DGDG were examined using positive‐ion electrospray ionization/mass spectrometry (ESI/MS). Two centric diatoms, Skeletonema marinoi and Thalassiosira weissflogii, and the pennate diatom, P. tricornutum, contained primarily C₂₀/C₁₆ (sn‐1/sn‐2) and C₁₈/C₁₆ forms of MGDG and DGDG. The other pennate diatoms, H. ostrearia and Navicula perminuta, contained primarily C₁₈/C₁₆ or C₁₈/C₁₈ forms of MGDG and DGDG, indicating a previously unrecognized fatty acid diversity in diatom MGDG and DGDG.     

Bacteria may induce the secretion of mucin‐like proteins by the diatom Phaeodactylum tricornutum

Benthic diatoms live in photoautotrophic/heterotrophic biofilm communities embedded in a matrix of secreted extracellular polymeric substances. Closely associated bacteria influence their growth, aggregation, and secretion of exopolymers. We have studied a diatom/bacteria model community, in which a marine Roseobacter strain is able to grow with secreted diatom exopolymers as a sole source of carbon. The strain influences the aggregation of Phaeodactylum tricornutum by inducing a morphotypic transition from planktonic, fusiform cells to benthic, oval cells. Analysis of the extracellular soluble proteome of P. tricornutum in the presence and absence of bacteria revealed constitutively expressed newly identified proteins with mucin‐like domains that appear to be typical for extracellular diatom proteins. In contrast to mucins, the proline‐, serine‐, threonine‐rich (PST) domains in these proteins were also found in combination with protease‐, glucosidase‐ and leucine‐rich repeat‐domains. Bioinformatic functional predictions indicate that several of these newly identified diatom‐specific proteins may be involved in algal defense, intercellular signaling, and aggregation.     

Detection of a variable intracellular acid‐labile carbon pool in Thalassiosira weissflogii (Heterokontophyta) and Emiliania huxleyi (Haptophyta) in response to changes in the seawater carbon system

Accumulation of an intracellular pool of carbon (C_i pool) is one strategy by which marine algae overcome the low abundance of dissolved CO₂ (CO_2(aq)) in modern seawater. To identify the environmental conditions under which algae accumulate an acid‐labile C_i pool, we applied a ¹⁴C pulse‐chase method, used originally in dinoflagellates, to two new classes of algae, coccolithophorids and diatoms. This method measures the carbon accumulation inside the cells without altering the medium carbon chemistry or culture cell density. We found that the diatom Thalassiosira weissflogii [(Grunow) G. Fryxell & Hasle] and a calcifying strain of the coccolithophorid Emiliania huxleyi [(Lohmann) W. W. Hay & H. P. Mohler] develop significant acid‐labile C_i pools. Ci pools are measureable in cells cultured in media with 2–30 µmol l^?1 CO_2(aq), corresponding to a medium pH of 8.6–7.9. The absolute C_i pool was greater for the larger celled diatoms. For both algal classes, the C_i pool became a negligible contributor to photosynthesis once CO_2(aq) exceeded 30 µmol l^?1. Combining the ¹⁴C pulse‐chase method and ¹⁴C disequilibrium method enabled us to assess whether E. huxleyi and T. weissflogii exhibited thresholds for foregoing accumulation of DIC or reduced the reliance on bicarbonate uptake with increasing CO_2(aq). We showed that the C_i pool decreases with higher CO₂:HCO₃^? uptake rates.     

Labyrinthula diatomea n. sp.—A Labyrinthulid Associated with Marine Diatoms

Labyrinthulomycetes are mostly fungus‐like heterotrophic protists that absorb nutrients in an osmotrophic or phagotrophic manner. Members of order Labyrinthulida produce unique membrane‐bound ectoplasmic networks for movement and feeding. Among the various types of labyrinthulids’ food substrates, diatoms play an important role due to their ubiquitous distribution and abundant biomass. We isolated and cultivated new diatom consuming Labyrinthulida strains from shallow coastal marine sediments. We described Labyrinthula diatomea n. sp. that differs from all known labyrinthulids in both molecular and morphological features. We provided strain delimitation within the genus Labyrinthula based on ITS sequences via haplotype network construction and compared it with previous phylogenetic surveys.     

ENDOBACTERIA IN THE DIATOM PINNULARIA (BACILLARIOPHYCEAE). I. “SCATTERED ct‐NUCLEOIDS” EXPLAINED: DAPI–DNA COMPLEXES STEM FROM EXOPLASTIDIAL BACTERIA BORING INTO THE CHLOROPLASTS1,2

A local strain of the pennate diatom Pinnularia cf. “nobilis” was investigated using cytochemistry and fluorescence and EM techniques. The regular perforation of the chloroplasts of P. “nobilis” and the lack of a typical diatom pyrenoid were confirmed at the ultrastructural level. Cavities and channels in the complex secondary plastid were found to harbor symbiotic bacteria, and their DNA elicited DAPI fluorescence. Wheat germ agglutinin, labeling bacteria walls, elicited a similar fluorescence pattern. Previous speculation that the apochlorotic DNA‐positive dots in the plastids of several Pinnularia species are “scattered ct‐nucleoids” is thus refuted. Bacteria were rod shaped and gram negative. They resided in the lumen of the endoplasmic reticulum (ER) during host interphase confined to the specialized ER compartment housing the secondary plastid, that is, to the space between the third and the fourth membrane profile, encircling the chloroplast. TEM images of chemically and cryofixed cells revealed that cavities resulted from the interaction of bacteria with the plastid according to the following sequence, alignment, attachment, deformation, and disintegration. This occurred without visible injury to the primary chloroplast envelope or the relict cell membrane of the reduced ancestral red alga that surrounds the chloroplast. The patterned arrangement of bacteria suggests recognition sites on the vestigial cell membrane, thought to interact with surface groups on the bacteria. The intimate association between bacteria and secondary plastid inside the common specialized ER cisterna suggests they form a functional unit. Comparison of thylakoid profiles, disrupted by bacteria in Pinnularia, with those disrupted by the pyrenoid in other pennate diatoms (e.g. Trachyneis) revealed a significant ultrastructural resemblance. No aposymbiotic Pinnularia cells were found at the sampling site.     

Pattern of Proteins after Heat Shock and UV-B Radiation of some Temperate Marine Diatoms and the Antarctic Odontella weissflogii

Synthesis of stress proteins after heat shock and different periods of UV-B radiation were investigated with marine diatom species from the North Sea Ditylum brightwellii, Lithodesmium variabile, Odontella sinensis, Thalassiosira rotula and the Antarctic diatom Odontella weissfloggii from the Weddell Sea. Algae were grown in an artifical sea-water medium under controlled laboratory conditions: light/dark regime of 12:12 h (7.2 W m^?2), normal air (0.035 vol.% CO₂) and 18° or 4 °C. All the tested diatom species can produce heat shock proteins (HSPS) of the 70 kDa family by in vivo labelling with [³⁵S]-methionine. The same results were obtained for Odontella sinensis, Ditylum brightwellii and Odontella weissflogii by estimation of the in vitro translation products with poly-A-mRNA isolated from these organisms. However, Odontella weissflogii, a species relatively insensitive to UV-B irradiance, did not synthesize UV-induced HSPS, whereas the UV-sensitive diatom Odontella sinensis, as well as Lithodesmium variabile, produced all the observed HSPS after UV-B exposure. In addition, a protein of 43 kDa was found after UV-B irradiance of the temperate Odontella sinensis. The temperate marine diatom Thalassiosira rotula synthesized 70 kDa and 5 7 kDa proteins after a heat shock and a UV-B exposure of 2 h, but a 40 kDa protein could not be detected, whereas a 60 kDa protein was found after 2 h UV-B exposure. The results are discussed in view of a possible adaptation of O. weissflogii to an enhanced UV dose.     

Micro‐photoluminescence of single living diatom cells

Diatoms are single‐celled microalgae that possess a nanostructured, porous biosilica shell called a frustule. This study characterized the micro‐photoluminescence (μ‐PL) emission of single living cells of the photosynthetic marine diatom Thalassiosira pseudonana in response to UV laser irradiation at 325 nm using a confocal Raman microscope. The photoluminescence (PL) spectrum had two primary peaks, one centered at 500–510 nm, which was attributed to the frustule biosilica, and a second peak at 680 nm, which was attributed to auto‐fluorescence of photosynthetic pigments. The portion of the μ‐PL emission spectrum associated with biosilica frustule in the single living diatom cell was similar to that from single biosilica frustules isolated from these diatom cells. The PL emission by the biosilica frustule in the living cell emerged only after cells were cultivated to silicon depletion. The discovery of the discovery of PL emission by the frustule biosilica within a single living diatom itself, not just its isolated frustule, opens up future possibilities for living biosensor applications, where the interaction of diatom cells with other molecules can be probed by μ‐PL spectroscopy. Copyright © 2016 John Wiley & Sons, Ltd.     

Ocean acidification stimulates particulate organic carbon accumulation in two Antarctic diatom species under moderate and high natural solar radiation

Impacts of rising atmospheric CO₂ concentrations and increased daily irradiances from enhanced surface water stratification on phytoplankton physiology in the coastal Southern Ocean remain still unclear. Therefore, in the two Antarctic diatoms Fragilariopsis curta and Odontella weissflogii, the effects of moderate and high natural solar radiation combined with either ambient or future pCO₂ on cellular particulate organic carbon (POC) contents and photophysiology were investigated. Results showed that increasing CO₂ concentrations had greater impacts on diatom physiology than exposure to increasing solar radiation. Irrespective of the applied solar radiation regime, cellular POC quotas increased with future pCO₂ in both diatoms. Lowered maximum quantum yields of photochemistry in PSII (F_v/F_m) indicated a higher photosensitivity under these conditions, being counteracted by increased cellular concentrations of functional photosynthetic reaction centers. Overall, our results suggest that both bloom‐forming Antarctic coastal diatoms might increase carbon contents under future pCO₂ conditions despite reduced physiological fitness. This indicates a higher potential for primary productivity by the two diatom species with important implications for the CO₂ sequestration potential of diatom communities in the future coastal Southern Ocean.     

Determination and quantification of alpha,beta,gamma,delta-unsaturated aldehydes as pentafluorobenzyl-oxime derivates in diatom cultures and natural phytoplankton populations: application in marine field studies

Reactive alpha,beta,gamma,delta-unsaturated aldehydes and oxo-acids produced by marine diatoms upon cell damage interfere negatively with the reproduction success of their grazers. A simple, sensitive and specific method based on gas-chromatography coupled to mass spectrometry (EI or CI/EC) was developed for the quantification of these deleterious substances in laboratory diatom cultures and in natural phytoplankton populations. For aldehyde quantification, diatom containing samples are damaged in the presence of O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA.HCl) which leads to an in situ derivatisation without inhibition of the biosynthesis of the aldehydes. The oxime derivates of oxo-acids were in addition reacted with N-tert-butyldimethylsilyl-N-methyl-trifluoracetamide (MTBSTFA).