GROWTH INHIBITION AND TOXICITY OF THE DIATOM ALDEHYDE 2‐TRANS, 4‐TRANS‐DECADIENAL ON THALASSIOSIRA WEISSFLOGII (BACILLARIOPHYCEAE)1

A common aldehyde present in marine and freshwater diatoms, 2‐trans, 4‐trans‐decadienal (A3), is involved in the wound‐activated response of diatoms to copepod grazing. Upon breakage of the diatom cell membrane, aldehydes are enzymatically produced by the rapid conversion of precursors and strongly impact copepod reproduction by impairing egg production and hatching success, inducing teratogenic embryos modifications. In this study, A3 was assayed with the marine diatom Thalassiosira weissflogii (Grunow) Fryxell et Hasle. The aldehyde concentration necessary to reduce 50% growth rate (EC₅₀) was 0.29 mg·L^?1. Decadienal was found to inhibit T. weissflogii cell growth in a dose‐ and time‐dependent manner, with irreversible effects after 24 h of exposure. Decadienal induced a degenerative process, through modifications of cell membrane characteristics, interference with cell cycle progression, and with cell metabolic activity, leading to cell death. A preferential action of A3 on dividing cells was observed. Photosynthetic efficiency significantly decreased upon exposure to the aldehyde, paralleled by an increase in diatoxanthin, suggesting a protective role of this xanthophyll, usually involved in photoprotection. Dying cells exhibited the morphological and biochemical features that bear close resemblance to apoptosis of mammalian cells, including cell shrinkage, chromatin condensation, and degradation of nuclear DNA to nucleosomal size fragments. These data are the first direct evidence to show aldehydes are toxic to diatoms. We suggest a possible nontoxic role of such compounds as chemical signals of unfavorable conditions within the phytoplankton communities, which may be relevant for the population dynamics of diatoms during blooms.     

THE MECHANISM OF ISOTOPE FRACTIONATION DURING ALGAL NITRATE ASSIMILATION AS ILLUMINATED BY THE 15N/14N OF INTRACELLULAR NITRATE1

The ¹⁵N/¹⁴N of nitrate in the external medium and intracellular pool of the cultured marine diatom Thalassiosira weissflogii (Grun.) Fryxell et Hasle was measured during nitrate assimilation under low light, a 12:12‐h light:dark cycle, low temperature, or low iron conditions. The ¹⁵N/¹⁴N of the nitrate in the medium and the particulate matter both followed the predicted Rayleigh fractionation model, and the intracellular nitrate always had a higher ¹⁵N/¹⁴N than did the medium nitrate. When the experiments were compared, the results showed a negative correlation between the isotope fractionation factor and the difference in the ¹⁵N/¹⁴N between the two pools of nitrate. These observations imply that the variations in the isotope effect result from variations in the degree to which the fractionation by nitrate reductase is expressed outside the cell, which is, in turn, controlled by the rate of nitrate efflux relative to nitrate reduction. The low iron and low temperature experiments showed relatively small isotope effects but a large intracellular‐medium difference in nitrate ¹⁵N/¹⁴N, consistent with a relative rate of efflux (compared with influx) that is small and similar to fast‐growing cells. In contrast, large isotope effects and small intracellular‐medium differences in nitrate ¹⁵N/¹⁴N were observed for low light and light:dark cycle grown cells and are explained by higher relative rates of nitrate efflux under these growth conditions.     

EFFECT OF BLUE-GREEN LIGHT ON PHOTOSYNTHETIC PIGMENTS AND CHLOROPLAST STRUCTURE IN UNICELLULAR MARINE ALGAE FROM SIX CLASSES1

The photosynthetic pigments of 17 species of unicellular marine algae grown in white and blue-green light were examined. Blue-green light (400 μW·cm^?2; 12:12 LD cycle) caused major chlorophyll increases (55–146%) in five diatoms, one dinoflagellate and one cryptomonad; minor chlorophyll increases (17–39%) in two diatoms, two dinoflagellates, one prymnesiophyte (haptophyte), one chrysophyte and one chlorophyte; and no chlorophyll increase in two diatoms and one pyrmnesiophyte (haptophyte). The relative proportions of major chlorophylls and carotenoids did not change, but in six of eight species tested small increases in the concentration of chlorophyll c occurred. Blue-green light caused a small increase in the concentration of phycoerythrin relative to chlorophyll a in the cryptomonad. A larger number of thylakoids per chloroplast were observed in six species grown in blue-green light compared to white light controls. The ultrastructure changes observed depended not only on the magnitude of the chlorophyll increase but also on the architecture of the chloroplast.     

CYTOPLASMIC MASSES PRESERVED IN EARLY HOLOCENE DIATOMS: A POSSIBLE TAPHONOMIC PROCESS AND ITS PALEO‐ECOLOGICAL IMPLICATIONS1

In Lake Suigetsu, central Japan, greenish/light‐brown granules identified as cytoplasmic masses had been preserved in siliceous cell walls of freshwater diatoms in annual layers of lacustrine muds since the early Holocene. The lacustrine muds consisted of alternating dark‐colored (rich in diatom valves, clay, and organic matter) and light‐colored (mainly diatom valves) laminae. The greenish/light‐brown granules were predominately preserved in frustules of the genus Aulacoseira preserved in the dark‐colored laminae. The dark‐colored laminae were inferred to have formed annually under stratified water caused by surface water warming in summer that caused the formation of an organic‐rich anoxic layer on the lake bottom that favored granule preservation. The good preservation of cytoplasmic masses in dark‐colored laminae suggested a cause for diatom assemblage periodicity, a phenomenon that was commonly noted in temperate lakes: the cells containing these masses could be potential seed stocks for subsequent spring blooms. Frustules of the most abundant granule‐containing species, Aulacoseira nipponica (Skvortzow) Tuji, in the dark‐colored laminae of the Early Holocene muds were abundant in the overlying light‐colored laminae, suggesting that these species reproduced abundantly in springtime yielding a massive diatom bloom.     

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.     

Nitrogen isotope effects in the assimilation of inorganic nitrogenous compounds by marine diatoms

Nitrogen isotope fractionation in the assimilation of inorganic nitrogenous compounds was studied using marine diatoms (Phaeodactylum tricornutum and Chaetoceros sp.). The isotopic composition (δ¹⁵N) of the diatoms ranged from 7 to ‐18‰ relative to that of the nitrogen source, i.e., ammonium, nitrite, or nitrate. When the growth was light‐limited, the isotope fractionation in nitrate assimilation was inversely correlated with the growth rate. The highest fractionation factor of 1.016 was obtained when the growth rate was as low as 0.025 day^‐1. Fractionation was negligible when the growth, rate was higher than 1 day^‐1. A steady‐state kinetic model was applied to explain the isotope fractionation in nitrate assimilation. The nitrogen isotope fractionation primarily takes place at the step of N‐O bond breaking in nitrate reduction to nitrite. The extent of the isotope fractionation associated with the nitrate uptake is very small, and barely exceeds the limit of detection.     

CELL DIVISION PERIODICITY IN 13 SPECIES OF MARINE PHYTOPLANKTON ON A LIGHT: DARK CYCLE1

The division rates of 26 clonal cultures representing 13 species of planktonic marine algae (6 diatoms, 2 flagellated chrysophytes, 2 coccolithophores, 1 cryptomonad flagellate, I dinoflagellate, 1 green alga) were determined every 2 h for 48 h during exponential growth on a 14:10 LD cycle in nutrient-replete batch culture. Cyclic oscillations in the division rate were detectable in 22 of these clones. Of 14 diatom clones examined, four displayed nearly constant division rates throughout the LD cycle and ten showed strong periodicity favoring division during the light periods. In contrast, all other algae (12 clones) exhibited division rate maxima during periods of darkness, and clearly detectable decreases in cell number for time intervals of 4–8 h during periods of illumination. Intraspecific differences in division periodicity were found among eight clones of the diatom Thalassiosira pseudonana (Hustedt) Hasle & Heimdal and six clones of the coccolithophore Emiliania huxleyi (Lohm.) Hay & Mohler.     

CHARACTERIZATION OF DIATOM (BACILLARIOPHYCEAE) NITRATE REDUCTASE GENES AND THEIR DETECTION IN MARINE PHYTOPLANKTON COMMUNITIES1

The complete assimilatory nitrate reductase (NR) gene from the pennate diatom Phaeodactylum triconutum Bohlin was sequenced from cDNA and compared with NR sequences from fungi, green algae, vascular plants, and the recently sequenced genome of the centric diatom Thalassiosira pseudonana Hasle and Heimdal CCMP1335. In all the major eukaryotic nitrate reductase (Euk‐NR) functional domains, diatom NR gene sequences are generally 50%–60% identical to plant and alga sequences at the amino acid level. In the less conserved N‐terminal, hinge 1, and hinge 2 regions, homology to other NR sequences is weak, generally<30%. Two PCR primer sets capable of amplifying Euk‐NR from plants, algae, and diatoms were designed. One primer set was used to amplify a 750‐base pair (bp) NR fragment from the cDNA of five additional diatom strains. The PCR amplicon spans part of the well‐conserved dimer interface region, the more variable hinge 1 region, and part of the conserved cytochrome b heme binding region. The second primer set, targeted to the dimer region, was used to amplify an approximately 400‐bp fragment of the NR gene from DNA samples collected in Monterey Bay, California and in central New Jersey inner continental shelf (LEO‐15 site) waters. Only diatom‐like NR sequences were recovered from Monterey Bay samples, whereas LEO‐15 samples yielded NR sequences from a range of photosynthetic eukaryotes. The prospect of using DNA‐ and RNA‐based methods to target the NR genes of diatoms specifically is a promising approach for future physiological and ecological experiments.     

GROWTH AND PHOTOPROTECTION IN THREE DINOFLAGELLATES (INCLUDING TWO STRAINS OF ALEXANDRIUM TAMARENSE) AND ONE DIATOM EXPOSED TO FOUR WEEKS OF NATURAL AND ENHANCED ULTRAVIOLET‐B RADIATION1

Long‐term growth response to natural solar radiation with enhanced ultraviolet‐B (UVB) exposure was examined in two species of dinoflagellates [Alexandrium tamarense (M. Lebour) Balech, At, and Heterocapsa triquetra (Ehrenb.) F. Stein, Ht], including two strains of A. tamarense, one from Spain and another from UK, and one diatom species (Thalassiosira pseudonana Hasle et Heimdal). We examined whether variable photoprotection (mycosporine‐like amino acids [MAAs] and xanthophyll‐cycle pigments) affected photosynthetic performance, phytoplankton light absorption, and growth. Growth rate was significantly reduced under enhanced UVB for the UK strain of At and for Ht (both grew very little) as well as for the diatom (that maintained high growth rates), but there was no effect for the Spanish strain of At. MAA concentration was high in the dinoflagellates, but undetectable in the diatom, which instead used the xanthophyll cycle for photoprotection. The highest cell concentrations of MAAs and photoprotective pigments were observed in the UK strain of At, along with lowest growth rates and F_v/F_m, indicating high stress levels. In contrast, the Spanish strain showed progressive acclimation to the experimental conditions, with no significant difference in growth between treatments. Increase in total MAAs followed linearly the cumulative UVB of the preceding day, and both total and primary MAAs were maintained at higher constitutive levels in this strain. Acclimation to enhanced UVB in the diatom resulted in an increase in PSII activity and reduction in nonphotochemical quenching, indicating an increased resistance to photoinhibition after a few weeks. All four species showed increased phytoplankton light absorption under enhanced UVB. Large intrastrain differences suggest a need to consider more closely intraspecific variability in UV studies.     

Prevalence of chytrid parasitism among diatom populations in the lower Columbia River (2009–2013)

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EFFECTS OF LIGHT AND SUBSTRATE ON THE BENTHIC DIATOMS IN AN OLIGOTROPHIC LAKE: A COMPARISON BETWEEN NATURAL AND ARTIFICIAL SUBSTRATES1

Benthic diatoms form a particularly important community in oligotrophic lakes, but factors influencing their distribution are not well known. This study reports the depth distribution of living motile and total diatoms (living plus dead diatoms) on both natural (from sand to fine organic mud) and artificial substrates in an oligotrophic lake. On artificial substrates, motile diatom densities peaked in abundance (24–30 cells · mm^?2) between 0.6 and 1.9 m depth; on natural sediment surfaces, motile diatoms were generally more numerous and peaked in abundance (925 cells · mm^?2) at 1.3 m depth. Total diatom densities on artificial substrates were highest (1260 valves · mm^?2) at 0.6 m depth, with very low values below 3 m depth; on natural sediment surfaces, total diatom abundances were generally much higher (21600 valves · mm^?2) at 3 m depth and declined gradually with depth. Significant relationships were found between light and diatom densities on the artificial substrate. Ordination analysis indicated that substrate type significantly correlated with the variation of diatom composition on artificial and natural substrates. Our results suggest that in oligotrophic lakes, light influences benthic diatom abundance, whereas substrate type has more influence on benthic diatom composition.     

COPPER‐UPTAKE KINETICS OF COASTAL AND OCEANIC DIATOMS1

We investigated copper (Cu) acquisition mechanisms and uptake kinetics of the marine diatoms Thalassiosira oceanica Hasle, an oceanic strain, and Thalassiosira pseudonana Hasle et Heimdal, a coastal strain, grown under replete and limiting iron (Fe) and Cu availabilities. The Cu‐uptake kinetics of these two diatoms followed classical Michaelis–Menten kinetics. Biphasic uptake kinetics as a function of Cu concentration were observed, suggesting the presence of both high‐ and low‐affinity Cu‐transport systems. The half‐saturation constants (K_m) and the maximum Cu‐uptake rates (V_max) of the high‐affinity Cu‐transport systems (～7–350 nM and 1.5–17 zmol · μm^?2 · h^?1, respectively) were significantly lower than those of the low‐affinity systems (>800 nM and 30–250 zmol · μm^?2 · h^?1, respectively). The two Cu‐transport systems were controlled differently by low Fe and/or Cu. The high‐affinity Cu‐transport system of both diatoms was down‐regulated under Fe limitation. Under optimal‐Fe and low‐Cu growth conditions, the K_m of the high‐affinity transport system of T. oceanica was lower (7.3 nM) than that of T. pseudonana (373 nM), indicating that T. oceanica had a better ability to acquire Cu at subsaturating concentrations. When Fe was sufficient, the low‐affinity Cu‐transport system of T. oceanica saturated at 2,000 nM Cu, while that of T. pseudonana did not saturate, indicating different Cu‐transport regulation by these two diatoms. Using CuEDTA as a model organic complex, our results also suggest that diatoms might be able to access Cu bound within organic Cu complexes.     

MICROSATELLITE MARKER DEVELOPMENT AND GENETIC VARIATION IN THE TOXIC MARINE DIATOM PSEUDO‐NITZSCHIA MULTISERIES (BACILLARIOPHYCEAE)1

The genetic structure of phytoplankton populations is largely unknown. In this study we developed nine polymorphic microsatellite markers for the domoic acid–producing marine diatom Pseudo‐nitzschia multiseries (Hasle) Hasle. We then used them in the genotyping of 25 physiologically diverse field isolates and six of their descendants: 22 field isolates originated from eastern Canadian waters, two from European waters, and one from Russian waters. The numbers of alleles per locus ranged from three to seven and the observed heterozygosities from 0.39 to 0.70. A substantial degree of genetic variation was observed within the field isolates, with 23 different genotypes detected. The Russian isolate was the most genetically distinct, although there was also evidence of genetic differentiation at a more local scale. Mating experiments demonstrated that alleles were inherited in a Mendelian manner. Pseudo‐nitzschia multiseries primer pairs were tested on DNA from four congeners: P. calliantha Lundholm, Moestrup et Hasle; P. fraudulenta (P. T. Cleve) Hasle; P. pungens (Grunow ex P. T. Cleve) Hasle; and P. seriata (P. T. Cleve) H. Peragallo. Cross‐reactivity was only observed in P. pungens. Our results are a first step in understanding the genetic variation present at the Pseudo‐nitzschia“species” level and in determining the true biogeographic extent of Pseudo‐nitzschia species.     

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.     

VARIATIONS IN THE SUBSTITUTED 3‐LINKED MANNANS CLOSELY ASSOCIATED WITH THE SILICIFIED WALLS OF DIATOMS1

The polysaccharides from cleaned frustules of the diatoms Pinnularia viridis (Nitzsch) Ehrenberg, Craspedostauros australis Cox, Thalassiosira pseudonana Hasle et Heimdal, and Nitzschia navis‐varingica Lundholm et Moestrup were extracted with hot alkali that dissolved the silica and were characterized by constituent sugar and linkage analyses. The polysaccharides from P. viridis were investigated further by permethylation, partitioning according to solubility, desulfation, and CD₃I‐methylation. Yields of carbohydrate in the hot alkali extracts ranged from 0.9% to 1.8% w/w based on the dry weight of the silica. Mannose was the dominant sugar in the polysaccharides from all four species (54–69 mol% of constituent sugars), although 14 other monosaccharides, including neutral sugars (glucose, galactose, xylose, arabinose, rhamnose, fucose), acidic sugars (glucuronic acid, galacturonic acid, 2‐O‐methylglucuronic acid), and O‐methylated neutral sugars (2‐O‐methylrhamnose, 3‐O‐methylrhamnose, 2,3‐di‐O‐methylrhamnose, 3‐O‐methylxylose, 4‐O‐methylxylose) were also detected in varying proportions among the four samples. The polysaccharides were predominantly composed of a 3‐linked mannopyranose backbone with a prevalence of linkage and/or substitution at O‐2 of the 3‐linked mannopyranosyl residues, and they were polyanionic, bearing uronic acid residues and/or sulfate esters. There were, however, species‐specific differences in the degree and position of substitution on the mannan backbone, the type and substitution patterns of the anionic substituents, and the type and linkage patterns of sugars other than mannose. Although definitive functions for these polysaccharides in diatom biology remain uncertain, a possible role in biosilicification is discussed.     

SPECIFICITY AND FUNCTION OF GLYCERALDEHYDE‐3‐PHOSPHATE DEHYDROGENASE IN A FRESHWATER DIATOM,ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE)1

The plastidic glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) catalyzes the only reductive step in the Calvin cycle and exists as different forms of which GapC1 enzyme is present in chromalveolates, such as diatoms. Biochemical studies on diatoms are still fragmentary, and, thus, in this report, GAPDH from the freshwater diatom Asterionella formosa Hassall has been purified and kinetically characterized. It is a homotetrameric enzyme with a molecular mass of ~150 ± 15 kDa. The enzyme showed Michaelis–Menten kinetics with respect to both cofactors, NADPH and NADH, with a 16‐fold greater catalytic constant for NADPH. The K_m for NADPH was 140 μM, the lowest affinity reported, while the catalytic constant, 815 s^?1, is the highest reported. The K_m for NADH was 93 μM, and the catalytic constant was 50 s^?1, both are similar to reported values for other types of GAPDH. The GapC1 enzyme, like the Chlamydomonas reinhardtii A₄ GAPDH, exhibits a cooperative behavior toward the substrate, 1,3‐bisphosphoglyceric acid (BPGA), with both cofactors. Mass spectrometry analysis showed that when GapC1 enzyme was purified without reducing agents, it copurified with a small protein with a mass of 8.2 kDa. This protein was recognized by antibodies against CP12. When associated with this protein, GAPDH displayed a lag that disappeared upon incubation with reducing agent in the presence of either BPGA or NADPH as a consequence of dissociation of the GAPDH/CP12 complex. Thus, as in other species of algae and higher plants, regulation of GapC1 enzyme in A. formosa may occur through association‐dissociation processes linked to dark‐light transitions.