Marine Pennate Diatom Research Articles

Nuclear Transformation of the Marine Pennate Diatom Nitzschia sp. Strain NIES-4635 by Multi-Pulse Electroporation.

Nitzschia is one of the largest genera of diatoms found in a range of aquatic environments, from freshwater to seawater. This genus contains evolutionarily and ecologically unique species, such as those that have lost photosynthetic capacity or those that live symbiotically in dinoflagellates. Several Nitzschia species have been used as indicators of water pollution. Recently, Nitzschia species have attracted considerable attention in the field of biotechnology. In this study, a transformation method for the marine pennate diatom Nitzschia sp. strain NIES-4635, isolated from the coastal Seto Inland Sea, was established. Plasmids containing the promoter/terminator of the fucoxanthin chlorophyll a/c binding protein gene (fcp, or Lhcf) derived from Nitzschia palea were constructed and introduced into cells by multi-pulse electroporation, resulting in 500μg/mL nourseothricin-resistant transformants with transformation frequencies of up to 365 colonies per 108 cells. In addition, when transformation was performed using a new plasmid containing a promoter derived from a diatom-infecting virus upstream of the green fluorescent protein gene (gfp), 44% of the nourseothricin-resistant clones exhibited GFP fluorescence. The integration of the genes introduced into the genomes of the transformants was confirmed by Southern blotting. The Nitzschia transformation method established in this study will enable the transformation this species, thus allowing the functional analysis of genes from the genus Nitzschia, which are important species for environmental and biotechnological development.

Emerging phylogeographic perspective on the toxigenic diatom genus Pseudo-nitzschia in coastal northern European waters and gateways to eastern Arctic seas: Causes, ecological consequences and socio-economic impacts

The diatom Pseudo-nitzschia H. Peragallo is perhaps the most intensively researched genus of marine pennate diatoms, with respect to species diversity, life history strategies, toxigenicity, and biogeographical distribution. The global magnitude and consequences of harmful algal blooms (HABs) of Pseudo-nitzschia are particularly significant because of the high socioeconomic impacts and environmental and human health risks associated with the production of the neurotoxin domoic acid (DA) among populations of many (although not all) species. This has led to enhanced monitoring and mitigation strategies for toxigenic Pseudo-nitzschia blooms and their toxins in recent years. Nevertheless, human adaptive actions based on future scenarios of bloom dynamics and proposed shifts in biogeographical distribution under climate-change regimes have not been implemented on a regional scale. In the CoCliME (Co-development of climate services for adaptation to changing marine ecosystems) program these issues were addressed with respect to past, current and anticipated future status of key HAB genera such as Pseudo-nitzschia and expected benefits of enhanced monitoring. Data on the distribution and frequency of Pseudo-nitzschia blooms in relation to DA occurrence and associated amnesic shellfish toxin (AST) events were evaluated in a contemporary and historical context over the past several decades from key northern CoCliME Case Study areas. The regional studies comprised the greater North Sea and adjacent Kattegat-Skagerrak and Norwegian Sea, eastern North Atlantic marginal seas and Arctic gateways, and the Baltic Sea. The first evidence of possible biogeographical expansion of Pseudo-nitzschia taxa into frontier eastern Arctic gateways was provided from DNA barcoding signatures. Key climate change indicators, such as salinity, temperature, and water-column stratification were identified as drivers of upwelling and advection related to the distribution of regional Pseudo-nitzschia blooms. The possible influence of changing variables on bloom dynamics, magnitude, frequency and spatial and temporal distribution were interpreted in the context of regional ocean climate models. These climate change indicators may play key roles in selecting for the occurrence and diversity of Pseudo-nitzschia species within the broader microeukaryote communities. Shifts to higher temperature and lower salinity regimes predicted for the southern North Sea indicate the potential for high-magnitude Pseudo-nitzschia blooms, currently absent from this area. Ecological and socioeconomic impacts of Pseudo-nitzschia blooms are evaluated with reference to effects on fisheries and mariculture resources and coastal ecosystem function. Where feasible, effective adaptation strategies are proposed herein as emerging climate services for the northern CoCLiME region.

Biochemical Characterization of Caenorhabditis elegans Ferritins.

The nematode Caenorhabditis elegans contains genes for two types of ferritin (ftn-1 and ftn-2) that express FTN-1 and FTN-2. We have expressed and purified both proteins and characterized them by X-ray crystallography, cryo-electron microscopy, transmission electron microscopy, dynamic light scattering, and kinetically by oxygen electrode and UV-vis spectroscopy. Both show ferroxidase activity, but although they have identical ferroxidase active sites, FTN-2 is shown to react approximately 10 times faster than FTN-1, with L-type ferritin character over longer time periods. We hypothesize that the large variation in rate may be due to differences in the three- and four-fold channels into the interior of the protein 24-mer. FTN-2 is shown to have a wider entrance into the three-fold channel than FTN-1. Additionally, the charge gradient through the channel of FTN-2 is more pronounced, with Asn and Gln residues in FTN-1 replaced by Asp and Glu residues in FTN-2. Both FTN-1 and FTN-2 have an Asn residue near the ferroxidase active site that is a Val in most other species, including human H ferritin. This Asn residue has been observed before in ferritin from the marine pennate diatom Pseudo-mitzchia multiseries. By replacing this Asn residue with a Val in FTN-2, we show that the reactivity decreases over long time scales. We therefore propose that Asn106 is involved in iron transport from the ferroxidase active site to the central cavity of the protein.

Haslea ostrearia Pigment Marennine Affects Key Actors of Neuroinflammation and Decreases Cell Migration in Murine Neuroglial Cell Model

Haslea ostrearia, a cosmopolitan marine pennate diatom, produces a characteristic blue pigment called marennine that causes the greening of filter-feeding organisms, such as oysters. Previous studies evidenced various biological activities of purified marennine extract, such as antibacterial, antioxidant and antiproliferative effects. These effects could be beneficial to human health. However, the specific biological activity of marennine remains to be characterized, especially regarding primary cultures of mammals. In the present study, we aimed to determine in vitro the effects of a purified extract of marennine on neuroinflammatory and cell migratory processes. These effects were assessed at non-cytotoxic concentrations of 10 and 50μg/mL on primary cultures of neuroglial cells. Marennine strongly interacts with neuroinflammatory processes in the immunocompetent cells of the central nervous system, represented by astrocytes and microglial cells. An anti-migratory activity based on a neurospheres migration assay has also been observed. These results encourage further study of Haslea blue pigment effects, particularly the identification of molecular and cellular targets affected by marennine, and strengthen previous studies suggesting that marennine has bioactivities which could be beneficial for human health applications.

Abiotic and biotic factors controlling sexual reproduction in populations of Pseudo-nitzschia pungens (Bacillariophyceae)

Pseudo-nitzschia pungens is a widely distributed marine pennate diatom. Hybrid zones, regions in which two different genotypes may interbreed, are important areas for speciation and ecology, and have been reported across the globe for this species. However, sexual reproduction between differing clades in the natural environment is yet to be observed and is difficult to predict. Here we carried out experiments using two mono-clonal cultures of P. pungens from different genotypes to measure the frequency and timing of sexual reproduction across varying biotic (growth phases and cell activity potential) and abiotic conditions (nutrients, light, turbulence). We found the mating rates and number of zygotes gradually decreased from exponential to late stationary growth phases. The maximum zygote abundance observed was 1,390 cells mL−1 and the maximum mating rate was 7.1%, both which occurred during the exponential growth phase. Conversely, only 9 cells mL−1 and a maximum mating rate of 0.1% was observed during the late stationary phase. We also found the higher the relative potential cell activity (rPCA) in parent cells, as determined by the concentration of chlorophyll a per cell and the ratio of colony formation during parent cultivations, revealed higher mating rates. Furthermore, sexual events were reduced under nutrient enrichment conditions, and mating pairs and zygotes were not formed under aphotic (dark) or shaking culture conditions (150 rpm). In order to understand the sexual reproduction of Pseudo-nitzschia in the natural environment, our results highlight that it is most likely the combination of both biotic (growth phase, Chl. a content) and abiotic factors (nutrients, light, turbulence) that will determine the successful union of intraspecific populations of P. pungens in any given region.

What Was Old Is New Again: The Pennate Diatom Haslea ostrearia (Gaillon) Simonsen in the Multi-Omic Age

The marine pennate diatom Haslea ostrearia has long been known for its characteristic blue pigment marennine, which is responsible for the greening of invertebrate gills, a natural phenomenon of great importance for the oyster industry. For two centuries, this taxon was considered unique; however, the recent description of a new blue Haslea species revealed unsuspected biodiversity. Marennine-like pigments are natural blue dyes that display various biological activities—e.g., antibacterial, antioxidant and antiproliferative—with a great potential for applications in the food, feed, cosmetic and health industries. Regarding fundamental prospects, researchers use model organisms as standards to study cellular and physiological processes in other organisms, and there is a growing and crucial need for more, new and unconventional model organisms to better correspond to the diversity of the tree of life. The present work, thus, advocates for establishing H. ostrearia as a new model organism by presenting its pros and cons—i.e., the interesting aspects of this peculiar diatom (representative of benthic-epiphytic phytoplankton, with original behavior and chemodiversity, controlled sexual reproduction, fundamental and applied-oriented importance, reference genome, and transcriptome will soon be available); it will also present the difficulties encountered before this becomes a reality as it is for other diatom models (the genetics of the species in its infancy, the transformation feasibility to be explored, the routine methods needed to cryopreserve strains of interest).

The Sexual Reproduction and Life Cycle of the Pennate Diatom Entomoneis cf. paludosa (W. Smith) Reimer (Bacillariophyta)

The sexual reproduction and life history of the marine pennate diatom Entomoneis cf. paludosa have been described for the first time. The sexual process has been found to involve two gametangia, each producing two gametes. This species is characterized by morphological and behavioral isogamy. Its mating system includes the heterothallic mode of reproduction, with no cases of homothallic reproduction recorded. The type of sexual process is categorized as IC in Geitler’s system.

Changes in excitation relaxation of diatoms in response to fluctuating light, probed by fluorescence spectroscopies.

A marine pennate diatom Phaeodactylum tricornutum (Pt) and a marine centric diatom Chaetoceros gracilis (Cg) possess unique light-harvesting complexes, fucoxanthin chlorophyll a/c-binding proteins (FCPs). FCPs have dual functions: light harvesting in the blue to green regions and quenching of excess energy. So far, excitation dynamics including FCPs have been studied by altering continuous light conditions. In the present study, we examined responses of the diatom cells to fluctuating light (FL) conditions. Excitation dynamics in the cells incubated under the FL conditions were analyzed by time-resolved fluorescence measurements followed by global analysis. As responses common to the Pt and Cg cells, quenching behaviors were observed in photosystem (PS) II with time constants of hundreds of picoseconds. The PSII → PSI energy transfer was modified only in the Pt cells, whereas quenching in FCPs was suggested only in the Cg cells, indicating different strategy for the dissipation of excess energy under the FL conditions.

Associated Bacteria Affect Sexual Reproduction by Altering Gene Expression and Metabolic Processes in a Biofilm Inhabiting Diatom.

Diatoms are unicellular algae with a fundamental role in global biogeochemical cycles as major primary producers at the base of aquatic food webs. In recent years, chemical communication between diatoms and associated bacteria has emerged as a key factor in diatom ecology, spurred by conceptual and technological advancements to study the mechanisms underlying these interactions. Here, we use a combination of physiological, transcriptomic, and metabolomic approaches to study the influence of naturally co-existing bacteria, Maribacter sp. and Roseovarius sp., on the sexual reproduction of the biofilm inhabiting marine pennate diatom Seminavis robusta. While Maribacter sp. severely reduces the reproductive success of S. robusta cultures, Roseovarius sp. slightly enhances it. Contrary to our expectation, we demonstrate that the effect of the bacterial exudates is not caused by altered cell-cycle regulation prior to the switch to meiosis. Instead, Maribacter sp. exudates cause a reduced production of diproline, the sexual attraction pheromone of S. robusta. Transcriptomic analyses show that this is likely an indirect consequence of altered intracellular metabolic fluxes in the diatom, especially those related to amino acid biosynthesis, oxidative stress response, and biosynthesis of defense molecules. This study provides the first insights into the influence of bacteria on diatom sexual reproduction and adds a new dimension to the complexity of a still understudied phenomenon in natural diatom populations.

Effects of excess light energy on excitation-energy dynamics in a pennate diatom Phaeodactylum tricornutum.

Controlling excitation energy flow is a fundamental ability of photosynthetic organisms to keep a better performance of photosynthesis. Among the organisms, diatoms have unique light-harvesting complexes, fucoxanthin chlorophyll (Chl) a/c-binding proteins. We have recently investigated light-adaptation mechanisms of a marine centric diatom, Chaetoceros gracilis, by spectroscopic techniques. However, it remains unclear how pennate diatoms regulate excitation energy under different growth light conditions. Here, we studied light-adaptation mechanisms in a marine pennate diatom Phaeodactylum tricornutum grown at 30µmol photons m-2s-1 and further incubated for 24h either in the dark, or at 30 or 300µmol photons m-2s-1 light intensity, by time-resolved fluorescence (TRF) spectroscopy. The high-light incubated cells showed no detectable oxygen-evolving activity of photosystem II, indicating the occurrence of a severe photodamage. The photodamaged cells showed alterations of steady-state absorption and fluorescence spectra and TRF spectra compared with the dark and low-light adapted cells. In particular, excitation-energy quenching is significantly accelerated in the photodamaged cells as shown by mean lifetime analysis of the Chl fluorescence. These spectral changes by the high-light treatment may result from arrangements of pigment-protein complexes to maintain the photosynthetic performance under excess light illumination. These growth-light dependent spectral properties in P. tricornutum are largely different from those in C. gracilis, thus providing insights into the different light-adaptation mechanisms between the pennate and centric diatoms.

Sexual reproduction of Entomoneis cf. paludosa (Bacillariophyta)

Sexual reproduction and the life cycle of the marine pennate diatom Entomoneis cf. paludosa are described. The reproduction in this species is characterized by morphological and behavioral isogamy. Two gametangia are involved in the sexual process, each of which produces two gametes.

Molecular aspects of the biophysical CO2-concentrating mechanism and its regulation in marine diatoms.

Diatoms operate a CO2-concentrating mechanism (CCM) that drives upwards of 20% of annual global primary production. Recent progress in CCM research in the marine pennate diatom Phaeodactylum tricornutum revealed that this diatom directly takes up HCO3- from seawater through low-CO2-inducible plasma membrane HCO3- transporters, which belong to the solute carrier (SLC) 4 family. Apart from this, studies of carbonic anhydrases (CAs) in diatoms have revealed considerable diversity in classes and localization among species. This strongly suggests that the CA systems, which control permeability and flux of dissolved inorganic carbon (DIC) by catalysing reversible CO2 hydration, have evolved from diverse origins. Of particular interest is the occurrence of low-CO2-inducible external CAs in the centric marine diatom Thalassiosira pseudonana, offering a strategy of CA-catalysed initial CO2 entry via passive diffusion, contrasting with active DIC transport in P. tricornutum. Molecular mechanisms to transport DIC across chloroplast envelopes are likely also through specific HCO3- transporters, although details have yet to be elucidated. Furthermore, recent discovery of a luminal θ-CA in the diatom thylakoid implied a common strategy in the mechanism to supply CO2 to RubisCO in the pyrenoid, which is conserved among green algae and some heterokontophytes. These results strongly suggest an occurrence of convergent coevolution between the pyrenoid and thylakoid membrane in aquatic photosynthesis.

Photosynthetic activity of Arctic Vaucheria (Xanthophyceae) measured in microcosmos

The xanthophycean alga Vaucheria inhabits very variable, and hence extreme, ecosys-tem of polar river estuaries. Therefore, it represents a unique system for the study of adaptation/acclimatization mechanisms. Since the ecophysiological measurements cannot be performed in the field directly, a piece of Vaucheria community collected in the Adventelva estuary, Longyearbyen, Svalbard, was taken to a microcosmos in order to study it. The set of instrumentation for measurement of ecophysiological parameters was constructed for such a field study. The community structure and photosynthetic activity expressed as the dissolved oxygen concentration and effective quantum yield (and hence relative electron transfer rate) were measured. The study defined relations between encountered environmental conditions and Vaucheria photosynthetic activity in late Arctic summer. The community consisted almost entirely of Vaucheria thalli; small marine pennate diatoms were rare. The microcosmos was proved functional since the structure of the community remained unaffected. Both methods of photosynthetic activity measurement were able to record its diel changes during 10 days lasting incubation. Although only weak correlation was found between the oxygen concentration and variable chlorophyll, probably due to the time necessary for oxygen diffusion, data from fluorometers were highly correlated. The Vaucheria expressed diel cycles in the photosynthetic activity, and the photosynthetically active radiation was the main driving factor. The incubation in microcosmos proved to be suitable for ecophysiological studies which will include the addition of tidal cycles and more sophisticated protocols for photosynthetic activity measurement.

Морфологическая изменчивость двух видов морских диатомовых водорослей Nitzschia ventricosa и Ardissonea crystallina (Bacillariophyta)

Актуальность работы обусловлена недостаточной изученностью проблемы понимания объёма видов диатомовых водорослей, особенно в условиях in vitro. Цель исследования – изучить морфологическую изменчивость в культурах двух видов морских пеннатных диатомовых водорослей Nitzschia ventricosa Kitton и Ardissonea crystallina (C. Agardh) Grunow при помощи методов световой и сканирующей электронной микроскопии, а также оценить в эксперименте воздействие солёности на морфологические показатели панциря A. crystallina, использующиеся при диагностике вида; сделать сопутствующие таксономические замечания. Материалом послужили изоляты (клоны и альгологически чистые культуры), полученные в условиях in vitro: изоляты N. ventricosa – из природных популяций прибрежных районов Канарских островов, о-ва Мартиника и штата Техас (Мексиканский залив), изоляты A. crystallinа – из бентосных проб в бухте Казачья (Севастополь, Чёрное море). Морфологическая изменчивость в культурах N. ventricosa и A. crystallinа оказалась сопоставимой с данными, полученными на материале из природных популяций. Обнаруженная вариабельность изученных изолятов позволила уточнить морфологические особенности N. ventricosa. Варьирование в одном клоне N. ventricosa по числу штрихов и фибул оказалось выше, чем обнаруженное в альгологически чистых культурах этого вида. Широкая фенотипическая вариация створок в культурах N. ventricosa и A. crystallinа сопоставима с данными, полученными in situ. Выявлена тенденция возрастания плотности расположения штрихов и ареол и уменьшения ширины клеток A. crystallinа с увеличением солёности в эксперименте. При солёности 8 ‰ вегетативное деление замедлялось, культура погибала. С повышением уровня солёности увеличивались диапазоны варьирования числа штрихов A. crystallinа.

A Diatom Ferritin Optimized for Iron Oxidation but Not Iron Storage

Ferritin from the marine pennate diatom Pseudo-nitzschia multiseries (PmFTN) plays a key role in sustaining growth in iron-limited ocean environments. The di-iron catalytic ferroxidase center of PmFTN (sites A and B) has a nearby third iron site (site C) in an arrangement typically observed in prokaryotic ferritins. Here we demonstrate that Glu-44, a site C ligand, and Glu-130, a residue that bridges iron bound at sites B and C, limit the rate of post-oxidation reorganization of iron coordination and the rate at which Fe(3+) exits the ferroxidase center for storage within the mineral core. The latter, in particular, severely limits the overall rate of iron mineralization. Thus, the diatom ferritin is optimized for initial Fe(2+) oxidation but not for mineralization, pointing to a role for this protein in buffering iron availability and facilitating iron-sparing rather than only long-term iron storage.

Ultrastructure and Membrane Traffic During Cell Division in the Marine Pennate Diatom Phaeodactylum tricornutum

The marine pennate diatom Phaeodactylum tricornutum has become a model for diatom biology, due to its ease of culture and accessibility to reverse genetics approaches. While several features underlying the molecular mechanisms of cell division have been described, morphological analyses are less advanced than they are in other diatoms. We therefore examined cell ultrastructure changes prior to and during cytokinesis. Following chloroplast division, cleavage furrows are formed at both longitudinal ends of the cell and are accompanied by significant vesicle transport. Although neither spindle nor microtubules were observed, the nucleus appeared to be split by the furrow after duplication of the Golgi apparatus. Finally, centripetal cytokinesis was completed by fusion of the furrows. Additionally, F-actin formed a ring structure and its diameter became smaller, accompanying the ingrowing furrows. To further analyse vesicular transport during cytokinesis, we generated transgenic cells expressing yellow fluorescent protein (YFP) fusions with putative diatom orthologs of small GTPase Sec4 and t-SNARE protein SyntaxinA. Time-lapse observations revealed that SyntaxinA-YFP localization expands from both cell tips toward the center, whereas Sec4-YFP was found in the Golgi and subsequently relocalizes to the future division plane. This work provides fundamental new information about cell replication processes in P. tricornutum.

Synthesizing CdSe nanoparticles by using a low concentration of cadmium ions and the apoferritin protein cage of marine pennate diatoms

Cadmium selenide (CdSe) nanoparticles (NPs) were first synthesized in the cavity of recombinant cage-shaped protein from marine pennate diatoms. Our constructed recombinant apoferritin (FerA-dCys) from the diatoms revealed that the removal of cysteine residues on the surface of the recombinant apoferritin enhanced the CdSe NPs synthesis in the apoferritin cavity. The CdSe NPs were synthesized in the FerA-dCys with a very low concentration of cadmium ions (0.02mM). The core formation ratio of CdSe NPs showed more than 20%. The average diameter of synthesized CdSe NPs was 7nm with a small size distribution. The synthesized NPs were analyzed by energy-dispersive X-ray spectroscopy (EDX) and high-resolution transmission electron microscopy (HR-TEM), and it was confirmed that the synthesized NPs in the FerA-dCys were consistent with Cd and Se ions.

New records of the marine pennate diatoms in Korea

New records of the marine pennate diatoms in Korea

Morphological and molecular phylogenetic analysis of the high triglyceride‐producing marine diatom, Fistulifera solaris sp. nov. (Bacillariophyceae)

SummaryFistulifera sp. strain JPCC DA0580, a marine pennate diatom, contains extremely high levels of intracellular triglyceride and has been suggested as a promising source of feedstock for biodiesel fuels. JPCC DA0580 was isolated from a mangrove swamp located in Sumiyo Bay below the mouths of the Sumiyo and Yakugachi Rivers in Amami‐Ohshima, Kagoshima, Japan. In this study, the morphology and the 18S rDNA sequence of JPCC DA0580 were compared with those of other Fistulifera strains. JPCC DA0580 possesses morphological characters of the genus Fistulifera, namely lightly silicified frustules, a distinct median costa (raphe sternum), and a wart‐like central pore (fistula). Morphometric analysis revealed that JPCC DA0580 differs from other Fistulifera species by the presence of a valve with coarser striation and coarser areolation. On the basis of 18S rDNA phylogeny, JPCC DA0580 formed a well‐supported clade with other members of the Fistulifera species complex, although the number of nucleotide substitutions was highest in JPCC DA0580. Our results led us to propose the taxonomic name Fistulifera solaris sp. nov. for JPCC DA0580.

Marennine, promising blue pigments from a widespread Haslea diatom species complex.

In diatoms, the main photosynthetic pigments are chlorophylls a and c, fucoxanthin, diadinoxanthin and diatoxanthin. The marine pennate diatom Haslea ostrearia has long been known for producing, in addition to these generic pigments, a water-soluble blue pigment, marennine. This pigment, responsible for the greening of oysters in western France, presents different biological activities: allelopathic, antioxidant, antibacterial, antiviral, and growth-inhibiting. A method to extract and purify marennine has been developed, but its chemical structure could hitherto not be resolved. For decades, H. ostrearia was the only organism known to produce marennine, and can be found worldwide. Our knowledge about H. ostrearia-like diatom biodiversity has recently been extended with the discovery of several new species of blue diatoms, the recently described H. karadagensis, H. silbo sp. inedit. and H. provincialis sp. inedit. These blue diatoms produce different marennine-like pigments, which belong to the same chemical family and present similar biological activities. Aside from being a potential source of natural blue pigments, H. ostrearia-like diatoms thus present a commercial potential for aquaculture, cosmetics, food and health industries.