Phylogeny and Fatty Acid Profiles of New Pinnularia (Bacillariophyta) Species from Soils of Vietnam.

We studied the morphology, ultrastructure, and phylogeny of eight soil diatom strains assigned to the Pinnularia genus. Six of these strains, identified by us as new species, are described for the first time. We provide a comprehensive comparison with related species and include ecological data. Molecular phylogeny reconstruction using 18S rDNA and rbcL affiliates the new strains with different subclades within Pinnularia, including 'borealis', 'grunowii' and 'stomatophora'. We also studied the fatty acid profiles in connection with the emerging biotechnological value of diatoms as a source of lipids. Stearic (36.0-64.4%), palmitic (20.1-30.4%), and palmitoleic (up to 20.8%) acids were the dominant fatty acids in the algae cultured on Waris-H + Si medium. High yields of saturated and monounsaturated fatty acids position the novel Pinnularia strains as a promising feedstock for biofuel production.

In Situ Manipulation and Micromechanical Characterization of Diatom Frustule Constituents Using Focused Ion Beam Scanning Electron Microscopy.

Biocomposite structures are difficult to characterize by bulk approaches due to their morphological complexity and compositional heterogeneity. Therefore, a versatile method is required to assess, for example, the mechanical properties of geometrically simple parts of biocomposites at the relevant length scales. Here, it is demonstrated how a combination of Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and micromanipulators can be used to isolate, transfer, and determine the mechanical properties of frustule constituents of diatom Thalassiosira pseudonana (T.p.). Specifically, two parts of the diatom frustule, girdle bands and valves, are separated by FIB milling and manipulated using a sharp tungsten tip without compromising their physical or chemical integrity. In situ mechanical studies on isolated girdle bands combined with Finite Element Method (FEM) simulations, enables the quantitative assessment of the Young's modulus of this biosilica; E = 40.0 GPa. In addition, the mechanical strength of isolated valves could be measured by transferring and mounting them on top of premilled holes in the sample support. This approach may be extended to any hierarchical biocomposite material, regardless of its chemical composition, to isolate, transfer, and investigate the mechanical properties of selected constituents or specific regions.

Structural evidence for extracellular silica formation by diatoms.

The silica cell wall of diatoms, a widespread group of unicellular microalgae, is an exquisite example for the ability of organisms to finely sculpt minerals under strict biological control. The prevailing paradigm for diatom silicification is that this is invariably an intracellular process, occurring inside specialized silica deposition vesicles that are responsible for silica precipitation and morphogenesis. Here, we study the formation of long silicified extensions that characterize many diatom species. We use cryo-electron tomography to image silica formation in situ, in 3D, and at a nanometer-scale resolution. Remarkably, our data suggest that, contradictory to the ruling paradigm, these intricate structures form outside the cytoplasm. In addition, the formation of these silica extensions is halted at low silicon concentrations that still support the formation of other cell wall elements, further alluding to a different silicification mechanism. The identification of this unconventional strategy expands the suite of mechanisms that diatoms use for silicification.

Extreme Enlargement of the Inverted Repeat Region in the Plastid Genomes of Diatoms from the Genus Climaconeis.

We sequenced the plastid genomes of three diatoms from the genus Climaconeis, including two strains formerly designated as Climaconeis scalaris. At 208,097 and 216,580 bp, the plastid genomes of the latter strains are the largest ever sequenced among diatoms and their increased size is explained by the massive expansion of the inverted repeat region. Important rearrangements of gene order were identified among the two populations of Climaconeis cf. scalaris. The other sequenced Climaconeis chloroplast genome is 1.5 times smaller compared with those of the Climaconeis cf. scalaris strains and it features an usual quadripartite structure. The extensive structural changes reported here for the genus Climaconeis are compared with those previously observed for other algae and plants displaying large plastid genomes.

Revealing the architecture of the photosynthetic apparatus in the diatom Thalassiosira pseudonana.

Diatoms are a large group of marine algae that are responsible for about one-quarter of global carbon fixation. Light-harvesting complexes of diatoms are formed by the fucoxanthin chlorophyll a/c proteins and their overall organization around core complexes of photosystems (PSs) I and II is unique in the plant kingdom. Using cryo-electron tomography, we have elucidated the structural organization of PSII and PSI supercomplexes and their spatial segregation in the thylakoid membrane of the model diatom species Thalassiosira pseudonana. 3D sub-volume averaging revealed that the PSII supercomplex of T. pseudonana incorporates a trimeric form of light-harvesting antenna, which differs from the tetrameric antenna observed previously in another diatom, Chaetoceros gracilis. Surprisingly, the organization of the PSI supercomplex is conserved in both diatom species. These results strongly suggest that different diatom classes have various architectures of PSII as an adaptation strategy, whilst a convergent evolution occurred concerning PSI and the overall plastid structure.

Biogeography of the cosmopolitan terrestrial diatom Hantzschia amphioxys sensu lato based on molecular and morphological data.

Until now, the reported diversity of representatives from the genus Hantzschia inhabiting soils from different parts of Eurasia was limited to the few species H. amphioxys, H. elongata and H. vivax and some of their infraspecific taxa. We have studied the morphology, ultrastructure and phylogeny of 25 soil diatom strains, which according to published description would be assigned to "H. amphioxys sensu lato" using 18S rDNA, 28S rDNA and rbcL. We show that strains are made up of seven different species of Hantzschia, including five new for science. Five strains were identified as H. abundans. This species has a slight curvature of the raphe near its external proximal ends. Four of the examined strains were represented by different populations of H. amphioxys and their morphological characteristics fully correspond with its isolectotype and epitype. The main specific features of this species include 21-25 striae in 10 µm, 6-11 fibulae in 10 µm, 40-50 areolae in 10 µm and internal proximal raphe endings bent in opposite directions. H. attractiva sp. nov., H. belgica sp. nov., H. parva sp. nov., H. pseudomongolica sp. nov. and H. stepposa sp. nov. were described based on differences in the shape of the valves, significant differences in dimensions, a lower number of striae and areolae in 10 µm and the degree and direction of deflection of the internal central raphe endings. Based on the study of the morphological variability and phylogeny of soil Hantzschia-species from different geographical locations we conclude that while some species such as H. amphioxys are truly cosmopolitan in their distributions, some sympatric populations of pseudocryptic taxa exist in the Holarctic.

It's elemental! Siliceous diatom frustules producing sarcoid-like granulomas in the subcutis.

Silica granulomas have been described on the skin and are rare; however, this is the first report of a sarcoid-like granulomatous reaction because of siliceous diatom frustules in the subcutis, making this an unprecedented case. A 41-year-old female presented with a subcutaneous nodule on the right forearm clinically suggestive of cyst, foreign body, or lipoma. Excisional biopsy revealed subcutis with a sarcoid-like granulomatous reaction in a background of fibrosis, containing abundant semitransparent, exquisitely geometric particles, mildly refringent under polarized light, highlighted by phase contrast microscopy; special stains were negative for microorganisms. Definitive characterization of the peculiar fragments was accomplished by confocal laser microscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy, revealing them as diatom frustules made of silicon dioxide (SiO2 ) or silica. Diatoms are unicellular algae, their skeletons (frustules) made of silica have collected on the bottom of rivers, lakes, and oceans for thousands or millions of years and form what we know as diatomite or diatomaceous earth, which is widely used in different industries and easily available in the market. The mechanism whereby diatom frustules gained access to the patient's subcutis is enigmatic.

First Evidence of the Toxin Domoic Acid in Antarctic Diatom Species.

The Southern Ocean is one of the most productive ecosystems in the world. It is an area heavily dependent on marine primary production and serving as a feeding ground for numerous seabirds and marine mammals. Therefore, the phytoplankton composition and presence of toxic species are of crucial importance. Fifteen monoclonal strains of Pseudo-nitzschia subcurvata, a diatom species endemic to the Southern Ocean, were established, which were characterized by morphological and molecular data and then analysed for toxin content. The neurotoxins domoic acid and iso-domoic acid C were present in three of the strains, which is a finding that represents the first evidence of these toxins in strains from Antarctic waters. Toxic phytoplankton in Antarctic waters are still largely unexplored, and their effects on the ecosystem are not well understood. Considering P. subcurvata's prevalence throughout the Southern Ocean, these results highlight the need for further investigations of the harmful properties on the Antarctic phytoplankton community as well as the presence of the toxins in the Antarctic food web, especially in the light of a changing climate.

A case of oral foreign-body granuloma due to diatoms.

Diatoms are photosynthetic algae with a siliceous exoskeleton. Diatoms are utilized by a wide array of industries for applications such as filtration and pest control. Unsubstantiated claims have also propelled their societal reach to trendy oral and topical uses. This case highlights a rare case of an oral granuloma secondary to diatoms. An 80-year-old woman presented with a mobile, firm, asymptomatic submucosal mass on her lower left mandibular vestibular mucosa. Histopathology showed a non-caseating granulomatous reaction to diatoms. Her only verified contact with a diatomaceous earth product was a dental impression using alginate after upper front teeth trauma 5 months before. Although there have been several cases of allergic contact dermatitis attributed to diatoms, there are no reported cases of diatom-induced granuloma formation found in the literature. There are, however, ample data on granulomas initiated by silica. Given the silica-based composition of diatoms, and the broad use of diatoms in industry and alternative medicine, it is unclear why diatom-induced granulomas are not more widely described. This report may alert clinicians to the existence of diatom granulomas and incline them to tailor their history to cover questions about possible exposure when evaluating patients presenting with a localized oral lesion.

Secretion mechanism and adhesive mechanism of diatoms: Direct evidence from the quantitative analysis.

Diatoms are one of the biofouling species attached to the substrate that can cause substrate corrosion, fuel consumption and destruction of the ecological balance. Therefore, the study of single-cellfouling organisms, particularly, the quantitative analysis of extracellular polymeric substances (EPS) is essential for antifouling. Atomic Force Microscope (AFM) was used to quantify three types of diatoms: Nitzschia closterium (N. closterium), Phaeodactylum tricornutum (P. tricornutum) and Halamphora sp. The situation of N. closterium was analyzed multiple times and the results showed that the adhesion value range of N. closterium with nacked chromatophores was three times larger than the mature one. The discovery of the EPS secretion from chromatophore is discussed in this paper, and the proposed mechanism has special implications to study the adhesive protein. Adhesion capabilities of different diatom genera and species were revealed as well. The average adhesion values of N. closterium, P. tricornutum and Halamphora sp. were about 1.7 nN, 3.3 nN and 2.5 nN, respectively, which suggest P. tricornutum could be a better candidate for testing diatom resistance on epoxy materials in the lab. Experimental data and discussions in this paper provide insights for further study of diatoms in the field of antifouling.

Visualization of elemental distributions and local analysis of element-specific chemical states of an Arachnoidiscus sp. frustule using soft X-ray spectromicroscopy.

Using soft X-ray (SX) spectromicroscopy, we show maps of the spatial distribution of constituent elements and local analysis of the density of states (DOS) related to the element-specific chemical states of diatom frustules, which are composed of naturally grown nanostructured hydrogenated amorphous silica. We applied X-ray photoemission electron microscopy (X-PEEM) as well as microprobe X-ray fluorescence (µXRF) analysis to characterize the surfaces of diatom frustules by means of X-ray absorption spectroscopy (XAS) and X-ray emission spectroscopy (XES). We successfully demonstrated that SX spectromicroscopy is able to participate in potential observation tools as a new method to spectroscopically investigate diatom frustules.

Modifying the thickness, pore size, and composition of diatom frustule in Pinnularia sp. with Al3+ ions.

Diatoms are unicellular photosynthetic algae that produce a silica exoskeleton (frustule) which exposes a highly ordered nano to micro scale morphology. In recent years there has been a growing interest in modifying diatom frustules for technological applications. This is achieved by adding non-essential metals to the growth medium of diatoms which in turn modifies morphology, composition, and resulting properties of the frustule. Here, we investigate the frustule formation in diatom Pinnularia sp., including changes to overall morphology, silica thickness, and composition, in the presence of Al3+ ions at different concentrations. Our results show that in the presence of Al3+ the total silica uptake from the growth medium increases, although a decrease in the growth rate is observed. This leads to a higher inorganic content per diatom resulting in a decreased pore diameter and a thicker frustule as evidenced by electron microscopy. Furthermore, 27Al solid-state NMR, FIB-SEM, and EDS results confirm that Al3+ becomes incorporated into the frustule during the silicification process, thus, improving hydrolysis resistance. This approach may be extended to a broad range of elements and diatom species towards the scalable production of silica materials with tunable hierarchical morphology and chemical composition.

Exopolysaccharides directed embellishment of diatoms triggered on plastics and other marine litter.

In the present study, embellishment or beautification of diatoms on substrates like plastics, polydimethylsiloxane, graphite, glass plate, and titanium dioxide, triggered by exopolysaccharides was examined under laboratory conditions. Exopolysaccharides are secreted mainly by primary colonisers, bacteria, which is succeeded by secondary colonisers i.e. diatoms. Both diatom (Nitzschia sp.4) and bacteria (Bacillus subtilis) were exposed with substrates separately for 30 days. Diatoms adhere on substrates strongly, not only because of surface roughness of different substrates but also the nanoporous architecture of diatoms which enhanced their embellishment. This study attempted to identify the substrates that adhere to diatoms strongly and was mainly analyzed by scanning electron microscope and further the observations are well supported by math work software (MATLAB). The variation of diatom's binding on different substrates is due to the influence of marine litters on diatom population in ocean beds where they undergo slow degradation releasing macro, micro and nanoparticles besides radicals and ions causing cell death. Therefore a proof-of-concept model is developed to successfully deliver a message concerning benefit of using different diatom species.

Evaluation of the toxic response induced by BDE-47 in a marine alga, Phaeodactylum tricornutum, based on photosynthesis-related parameters.

The pollution of polybrominated diphenyl ethers (PBDEs) is becoming a pressing environmental problem in aquatic environments, and its threat to aquatic organism has received much attention. In this study, Phaeodactylum tricornutum was treated with 0.8 and 4 mg L-1 2,2',4,4'-tetrabrominated biphenyl ether (BDE-47), the most toxic PBDEs, for 96 h. BDE-47 inhibited cell growth in a time- and concentration-dependent manner. Observation of cell ultrastructure suggested the damage of the chloroplasts morphology. BDE-47 also decreased the chlorophyll content and the oxygen evolution rate, and altered the performance of photosystems. Transcriptomic analysis revealed differential expression of 62 genes related to photosynthesis in BDE-47 treatments (4 mg L-1) and transcription suppression of 58 genes involved in chlorophyll synthesis, antenna proteins, oxygen evolution, electron transport and downstream carbon fixation, implying potential toxicity targets in cells. Additionally, the levels of reactive oxygen species (ROS) and lipid peroxidation increased under BDE-47 stress and were positively correlated with photosynthesis inhibition. Pretreatment with the ROS scavenger N-acetyl-l-cysteine reduced the extent of inhibition, suggesting that ROS was responsible for these effects. Another experiment with the electron transport chain inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea showed that the generation of ROS was partially blocked, primarily indicating that photosynthetic inhibition induced by BDE-47 contributed to ROS overproduction. Thus, BDE-47 inhibited the photosynthesis by down-regulating the gene expression. This change stimulated ROS production, further leading to chloroplast membrane damage to aggravate this inhibition via a feedback loop. These effects of BDE-47 had adverse outcomes on the entire physiological state and the population growth of the microalgae.

A Widely Distributed Thraustochytrid Parasite of Diatoms Isolated from the Arctic Represents a gen. and sp. nov.

A unicellular, heterotrophic, eukaryotic parasite was isolated from nearshore Arctic marine sediment in association with the diatom Pleurosigma sp. The parasite possessed ectoplasmic threads that could penetrate diatom frustules. Healthy and reproducing Pleurosigma cultures would begin to collapse within a week following the introduction of this parasite. The parasite (2-10 µm diameter) could reproduce epibiotically with biflagellate zoospores, as well as binary division inside and outside the diatom host. While the parasite grew, diatom intracellular content disappeared. Evaluation of electron micrographs from co-cultures revealed the presence of hollow tubular processes and amorphic cells that could transcend the diatom frustule, generally at the girdle band, as well as typical thraustochytrid ultrastructure, such as the presence of bothrosomes. After nucleotide extraction, amplification, and cloning, database queries of DNA revealed closest molecular affinity to environmental thraustochytrid clone sequences. Testing of phylogenetic hypotheses consistently grouped this unknown parasite within the Thraustochytriidae on a distinct branch within the environmental sequence clade Lab19. Reclassification of Arctic high-throughput sequencing data, with appended reference datasets that included this diatom parasite, indicated that the majority of thraustochytrid sequences, previously binned as unclassifiable stramenopiles, are allied to this new isolate. Based on the combined information acquired from electron microscopy, life history, and phylogenetic testing, this unknown isolate is described as a novel species and genus.

Morphology, phylogeny, and molecular dating in Plagiogrammaceae family focused on Plagiogramma-Dimeregramma complex (Urneidophycidae, Bacillariophyceae).

Although previous phylogenetic analyses suggested that the araphid diatom family Plagiogrammaceae is monophyletic, there is still not a clear understanding of relationships among the genera, and the taxonomy of several genera--Dimeregramma and Plagiogramma--remains questionable in light of paraphyly for both genera using molecular and morphological data. We have expanded the available DNA for molecular work for dozens of plagiogrammacean clones and analyzed 29 morphological characters from plagiogrammarian taxa and closely related genera, to increase understanding of the evolutionary history and systematics of the family and re-evaluate the current taxonomical classification of plagiogrammacean genera. The addition of more taxa and more data confirm the results from previous molecular phylogenies: most plagiogrammacean genera are monophyletic, except for Dimeregramma and Plagiogramma. Interestingly, the morphological analysis resolves only Talaroneis and Glyphodesmis as monophyletic. Given these results, we feel there is limited support for retaining Dimeregramma and Plagiogramma as distinct genera, and formally propose amending Plagiogramma and transferring six Dimeregramma species. As the Plagiogrammaceae is also one of the first-diverging clades of pennate diatoms, we also used these molecular data to estimate the age of the family, based on multiple calibration points derived from fossil taxa within or close to the Plagiogrammaceae. The results indicated that the Plagiogrammaceae evolved more than 114 million year ago and its diversification appears to correspond to a time of climate cooling. Additionally, we described a new monotypic genus (Coccinelloidea) with one new species C. gracilis, and five new species within established genera, e.g. Plagiogramma marginalis, Plagiogramma harenae, Plagiogramma porcipellis, Neofragilaria montgomeryii and Psammogramma anacarae.

Structural features of the diatom photosystem II-light-harvesting antenna complex.

In photosynthesis, light energy is captured by pigments bound to light-harvesting antenna proteins (LHC) that then transfer the energy to the photosystem (PS) cores to initiate photochemical reactions. The LHC proteins surround the PS cores to form PS-LHC supercomplexes. In order to adapt to a wide range of light environments, photosynthetic organisms have developed a large variety of pigments and antenna proteins to utilize the light energy efficiently under different environments. Diatoms are a group of important eukaryotic algae and possess fucoxanthin (Fx) chlorophyll a/c proteins (FCP) as antenna which have exceptional capabilities of harvesting blue-green light under water and dissipate excess energy under strong light conditions. We have solved the structure of a PSII-FCPII supercomplex from a centric diatom Chaetoceros gracilis by cryo-electron microscopy, and also the structure of an isolated FCP dimer from a pennate diatom Phaeodactylum tricornutum by X-ray crystallography at a high resolution. These results revealed the oligomerization states of FCPs distinctly different from those of LHCII found in the green lineage organisms, the detailed binding patterns of Chl c and Fxs, a huge pigment network, and extensive protein-protein, pigment-protein, and pigment-pigment interactions within the PSII-FCPII supercomplex. These results therefore provide a solid structural basis for examining the detailed mechanisms of the highly efficient energy transfer and quenching processes in diatoms.

Extensive Cryptic Diversity in the Terrestrial Diatom Pinnularia borealis (Bacillariophyceae).

With the increasing application of molecular techniques for diatom species discovery and identification, it is important both from a taxonomic as well as an ecological and applied perspective, to understand in which groups morphological species delimitation is congruent with molecular approaches, or needs reconsideration. Moreover, such studies can improve our understanding of morphological trait evolution in this important group of microalgae. In this study, we used morphometric analysis on light microscopy (LM) micrographs in SHERPA, detailed scanning electron microscopy (SEM), and cytological observations in LM to examine 70 clones belonging to eight distinct molecular lineages of the cosmopolitan terrestrial diatom Pinnularia borealis. Due to high within-lineage variation, no conclusive morphological separation in LM nor SEM could be detected. Morphological stasis due to the "low-morphology" problem or stabilizing selection, as well as parallel/convergent evolution, phenotypic plasticity and structural inheritance are discussed as potential drivers for the observations. Altogether, P. borealis is truly cryptic, in contrast to the majority of other diatom species complexes which turned out to be pseudo-cryptic following detailed morphological analysis.

A new widespread subclass of carbonic anhydrase in marine phytoplankton.

Most aquatic photoautotrophs depend on CO2-concentrating mechanisms (CCMs) to maintain productivity at ambient concentrations of CO2, and carbonic anhydrase (CA) plays a key role in these processes. Here we present different lines of evidence showing that the protein LCIP63, identified in the marine diatom Thalassiosira pseudonana, is a CA. However, sequence analysis showed that it has a low identity with any known CA and therefore belongs to a new subclass that we designate as iota-CA. Moreover, LCIP63 unusually prefers Mn2+ to Zn2+ as a cofactor, which is potentially of ecological relevance since Mn2+ is more abundant than Zn2+ in the ocean. LCIP63 is located in the chloroplast and only expressed at low concentrations of CO2. When overexpressed using biolistic transformation, the rate of photosynthesis at limiting concentrations of dissolved inorganic carbon increased, confirming its role in the CCM. LCIP63 homologs are present in the five other sequenced diatoms and in other algae, bacteria, and archaea. Thus LCIP63 is phylogenetically widespread but overlooked. Analysis of the Tara Oceans database confirmed this and showed that LCIP63 is widely distributed in marine environments and is therefore likely to play an important role in global biogeochemical carbon cycling.

ROS changes are responsible for tributyl phosphate (TBP)-induced toxicity in the alga Phaeodactylum tricornutum.

As a newly emerging environmental contaminant, tributyl phosphate (TBP) is an additive flame retardant of high production volume that is frequently detected in biota and the environment. Despite evidence that TBP is a potential threat to marine organisms, ecotoxicology data for TBP in marine organisms at low trophic levels are scarce. In this study, the acute toxicological effect of TBP on the marine phytoplankton Phaeodactylum tricornutum was thoroughly investigated, and the possible mechanism was explored. The results showed that TBP at concentrations ≥0.2 mg L-1 significantly inhibited P. tricornutum growth in a clear dose-response manner, with 72-h EC10, EC20, EC50 and EC90 values of 0.067, 0.101, 0.219 and 0.716 mg L-1, respectively. Algal cells treated with TBP exhibited distorted shapes, ruptured cell membranes and damaged organelles, especially mitochondria. Additionally, apoptosis was triggered, followed by a decrease in mitochondrial membrane potential, indicating that cellular damage occurred during exposure. Although the activities of two antioxidant enzymes, superoxide peroxidase and catalase, were upregulated by TBP at 1.2 mg L-1, excess reactive oxygen species (ROS) and malondialdehyde still accumulated in algal cells after exposure, suggesting that the cells experienced oxidative stress. Moreover, both growth inhibition and apoptosis were positively correlated with ROS levels and were ameliorated by pretreatment with the ROS scavenger N-acetyl-l-cysteine. Taken together, the results indicate that TBP exposure leads to growth inhibition and cellular damage in P. tricornutum, and a ROS-mediated pathway might contribute to these observed toxicological effects.