Ship Hull-Fouling Diatoms on Korean Research Vessels Revealed by Morphological and Molecular Methods, and Their Environmental Implications.

Ship biofouling is one of the main vectors for the introduction and global spread of non-indigenous organisms. Diatoms were the early colonizers of ship hulls; however, their community composition on ships is poorly understood. Herein, we investigated the diatom community on the hull samples collected from two Korean research vessels Isabu (IRV) and Onnuri (ORV) on September 2 and November 10, 2021, respectively. IRV showed low cell density (345 cells/cm2) compared to ORV (778 cells/cm2). We morphologically identified more than 15 species of diatoms from the two research vessels (RVs). The microalgae in both RVs were identified as Amphora, Cymbella, Caloneis, Halamphora, Navicula, Nitzschia, and Plagiogramma. Of them, the genus Halamphora was found to be predominant. However, both RVs had a varied dominant species with a significant difference in body size; Halamphora oceanica dominated at IRV, and Halamphora sp. at ORV, respectively. Molecular cloning showed similar results to morphological analysis, in which Halamphora species dominated in both RVs. The hull-attached species were distinct from species found in the water column. These results revealed diatoms communities that are associated with ship hull-fouling at an early stage of biofilm formation. Moreover, ships arriving from different regions could show some variation in species composition on their hull surfaces, with the potential for non-indigenous species introduction.

Mitochondrial and Plastid Genomes of the Monoraphid Diatom Schizostauron trachyderma.

We provide for the first time the complete plastid and mitochondrial genomes of a monoraphid diatom: Schizostauron trachyderma. The mitogenome is 41,957 bp in size and displays two group II introns in the cox1 gene. The 187,029 bp plastid genome features the typical quadripartite architecture of diatom genomes. It contains a group II intron in the petB gene that overlaps the large single-copy and the inverted repeat region. There is also a group IB4 intron encoding a putative LAGLIDADG homing endonuclease in the rnl gene. The multigene phylogenies conducted provide more evidence of the proximity between S. trachyderma and fistula-bearing species of biraphid diatoms.

Teamwork in the viscous oceanic microscale.

Nutrient acquisition is crucial for oceanic microbes, and competitive solutions to solve this challenge have evolved among a range of unicellular protists. However, solitary solutions are not the only approach found in natural populations. A diverse array of oceanic protists form temporary or even long-lasting attachments to other protists and marine aggregates. Do these planktonic consortia provide benefits to their members? Here, we use empirical and modeling approaches to evaluate whether the relationship between a large centric diatom, Coscinodiscus wailesii, and a ciliate epibiont, Pseudovorticella coscinodisci, provides nutrient flux benefits to the host diatom. We find that fluid flows generated by ciliary beating can increase nutrient flux to a diatom cell surface four to 10 times that of a still cell without ciliate epibionts. This cosmopolitan species of diatom does not form consortia in all environments but frequently joins such consortia in nutrient-depleted waters. Our results demonstrate that symbiotic consortia provide a cooperative alternative of comparable or greater magnitude to sinking for enhancement of nutrient acquisition in challenging environments.

Diurnal transcript profiling of the diatom Seminavis robusta reveals adaptations to a benthic lifestyle.

Coastal regions contribute an estimated 20% of annual gross primary production in the oceans, despite occupying only 0.03% of their surface area. Diatoms frequently dominate coastal sediments, where they experience large variations in light regime resulting from the interplay of diurnal and tidal cycles. Here, we report on an extensive diurnal transcript profiling experiment of the motile benthic diatom Seminavis robusta. Nearly 90% (23 328) of expressed protein-coding genes and 66.9% (1124) of expressed long intergenic non-coding RNAs showed significant expression oscillations and are predominantly phasing at night with a periodicity of 24 h. Phylostratigraphic analysis found that rhythmic genes are enriched in highly conserved genes, while diatom-specific genes are predominantly associated with midnight expression. Integration of genetic and physiological cell cycle markers with silica depletion data revealed potential new silica cell wall-associated gene families specific to diatoms. Additionally, we observed 1752 genes with a remarkable semidiurnal (12-h) periodicity, while the expansion of putative circadian transcription factors may reflect adaptations to cope with highly unpredictable external conditions. Taken together, our results provide new insights into the adaptations of diatoms to the benthic environment and serve as a valuable resource for the study of diurnal regulation in photosynthetic eukaryotes.

An optimised method for intact nuclei isolation from diatoms.

Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH4F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell ("single nuclei") genomics, transcriptomics and proteomics in different diatom species.

Growth Rate-dependent Cell Death of Diatoms due to Viral Infection and Their Subsequent Coexistence in a Semi-continuous Culture System.

Viral infections are a major factor in diatom cell death. However, the effects of viruses on diatom dynamics remain unclear. Based on laboratory studies, it is hypothesized that virus-induced diatom mortality is dependent on the diatom growth rate. The present study aimed to elucidate the relationship between the diatom growth rate and virus-induced mortality using model systems of the marine planktonic diatom, Chaetoceros tenuissimus and its infectious viruses. We also examined the fate of diatom populations in a semi-continuous dilution culture system, in which host growth rates were controlled at 0.69, 2.08, and 3.47 day-1. Diatom populations gradually decreased following the viral inoculation of each culture system, and virus-induced mortality inversely correlated with the diatom growth rate. Furthermore, the viral burst size was slightly higher in lower growth rate cultures. These results suggested that the host physiological status related to the growth rate affected viral infection and proliferation. Diatom populations were not completely lysed or washed out in any of the dilution systems; they showed steady growth in the presence of infectious viruses. This may be partially explained by defective interference particles from viruses and cell debris. The present results indicate that diatoms in dilution environments maintain their populations, even under viral pressure. Moreover, diatom populations with a low growth rate may partially sustain higher growth populations through nutrient recycling following virus-induced cell death. The results of the present study provide insights into diatom dynamics in natural environments in the presence of infectious viruses.

How marine diatoms cope with metal challenge: Insights from the morphotype-dependent metal tolerance in Phaeodactylum tricornutum.

Metal tolerance in marine diatoms vary between morphotypes, strains, and species due to their long-term adaptations to stochastic environments. The mechanisms underlying this highly variable trait remain a matter of interest in ecotoxicology. In this study, we used several cutting-edge techniques, including a non-invasive micro-test technique, atomic force microscopy, and X-ray photoelectron spectroscopy to examine cadmium (Cd) accumulation and tolerance in the three morphotypes of Phaeodactylum tricornutum. Subcellular Cd distribution, metal transporter expression, and glutathione and phytochelatin activity were also analyzed to characterize the morphology-dependent Cd homeostasis and detoxification. We found that the oval morphotype accumulated more Cd, but was also more Cd tolerant than the other morphotypes. The greater surface binding of Cd to the oval morphotype is attributable to its smaller spherical form, rougher cell surface, and lower surface potential. Moreover, the oval morphotype was less permeable to Cd ions and contained higher phytochelatin and glutathione levels, which explained its higher metal tolerance. Our study offers new explanations for diatom's adaptations to changing environments that may contribute to its evolutionary success.

Characterization of river biofilm responses to the exposure with heavy metals using a novel micro fluorometer biosensor.

River biofilms are a suitable indicator of toxic stress in aquatic ecosystems commonly exposed to various anthropogenic pollutants from industrial, domestic, and agricultural sources. Among these pollutants, heavy metals are of particular concern as they are known to interfere with various physiological processes of river biofilm, directly or indirectly related to photosynthetic performance. Nevertheless, only limited toxicological data are available on the mechanisms and toxicodynamics of heavy metals in biofilms. Pulse Amplitude Modulated (PAM) fluorometry is a rapid, non-disruptive, well-established technique to monitor toxic responses on photosynthetic performance, fluorescence-kinetics, and changes in yield in other non-photochemical processes. In this study, a new micro-PAM-sensor was tested to assess potential acute and chronic effects of heavy metals in river biofilm. Toxicity values across the three parameters considered in this study (photosynthetic yield YII, non-photochemical quenching NPQ, and basal fluorescence F0) were comparable, as determined EC50 were within one order of magnitude (EC50 ∼1-10 mg L-1). However, the stimulation of NPQ was more clearly associated with early acute effects, especially in illuminated samples, while depression of YII and F0 were more prevalent in chronic tests. These results have implications for the development of functional indicators for the biomonitoring of aquatic health, in particular for the use of river biofilm as a bioindicator of water quality. In conclusion, the approach proposed seems promising to characterize and monitor the exposure and impact of heavy metals on river periphyton communities. Furthermore, this study provides a fast, highly sensitive, inexpensive, and accurate laboratory method to test effects of pollutants on complex periphyton communities that can also give insights regarding the probable toxicological mechanisms of heavy metals on photosynthetic performance in the river biofilm.

Nucleotide substitution rates of diatom plastid encoded protein genes are positively correlated with genome architecture.

Diatoms are the largest group of heterokont algae with more than 100,000 species. As one of the single-celled photosynthetic organisms that inhabit marine, aquatic and terrestrial ecosystems, diatoms contribute ~ 45% of global primary production. Despite their ubiquity and environmental significance, very few diatom plastid genomes (plastomes) have been sequenced and studied. This study explored patterns of nucleotide substitution rates of diatom plastids across the entire suite of plastome protein-coding genes for 40 taxa representing the major clades. The highest substitution rate was lineage-specific within the araphid 2 taxon Astrosyne radiata and radial 2 taxon Proboscia sp. Rate heterogeneity was also evident in different functional classes and individual genes. Similar to land plants, proteins genes involved in photosynthetic metabolism have lower synonymous and nonsynonymous substitutions rates than those involved in transcription and translation. Significant positive correlations were identified between substitution rates and measures of genomic rearrangements, including indels and inversions, which is a similar result to what was found in legume plants. This work advances the understanding of the molecular evolution of diatom plastomes and provides a foundation for future studies.

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.

Optimized mRuby3 is a Suitable Fluorescent Protein for in vivo Co-localization Studies with GFP in the Diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum is an ecologically and evolutionarily relevant microalga that has developed into an important model for molecular biological studies on organisms with complex plastids. The diatom is particularly suitable for in vivo protein localization analyses via fluorescence microscopy in which the green fluorescent protein (GFP) and its derivatives are dominantly used. Whereas GFP fluorescence emission is usually measured between 500 and 520nm in confocal microscopy, the autofluorescence of the P. tricornutum plastid is detected above 625nm. Here we established the fluorescent protein mRuby3 as tag for efficient in vivo protein localization studies by expressing a codon-optimized gene in P. tricornutum. mRuby3 was directed to seven different subcellular localizations by means of full-length marker protein or N-/C-terminal targeting signal fusions; its emission was detected efficiently between 580 and 605nm, being unequivocally distinguishable from the plastid autofluorescence in vivo. Moreover, mRuby3 proved to be highly suitable for co-localization experiments using confocal laser scanning microscopy in which mRuby3 fusion proteins were expressed in parallel with GFP-tagged proteins. Our results show the potential of mRuby3 for its application in studying protein targeting and localization in P. tricornutum, particularly underlining its compatibility with GFP and the plastid autofluorescence in signal detection.

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.

Testing eco-evolutionary predictions using fossil data: Phyletic evolution following ecological opportunity.

Fossil sequences provide observations of phenotypes within a lineage over time and represent essential data for increasing our understanding of phyletic evolution beyond microevolutionary timescales. I investigate if fossil time series of the diatom Stephanodiscus niagarae/yellowstonensis follow evolutionary dynamics compatible with hypotheses for how the adaptive landscape changes when a population enters a new environment. The lineage-which has a remarkably detailed stratigraphic record-invaded Yellowstone Lake immediately after recession of ice from the basin 14,000 years ago. Several phyletic models portraying different types of evolutionary dynamics-both compatible and not compatible with changes in the adaptive landscape following ecological opportunity-were fitted to the fossil times-series of S. niagarae/yellowstonensis. Different models best describe the three analyzed traits. Two of the models (a new model of decelerated evolution and an Ornstein-Uhlenbeck model) capture trait dynamics compatible with an event of ecological opportunity, whereas the third model (random walk) does not. Entering a new environment may accordingly affect trait dynamics for thousands of years, but the effects can vary across phenotypes. However, tests of model adequacy reveal shortcomings in all three models explaining the trait dynamics, suggesting model development is needed to more fully understand the phyletic evolution in S. niagarae/yellowstonensis.

High-resolution label-free 3D mapping of extracellular pH of single living cells.

Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.

Distribution of aerophilous diatom communities associated with terrestrial green macroalgae in the South Shetland Islands, Maritime Antarctica.

The establishment of diatom communities depends on environmental factors such as the type of substrate and geographic conditions that influence the dispersal processes of these organisms. The main goal of this study was to evaluate the similarity between diatom communities associated with the macroalgae Prasiola crispa (Lightfoot) Kützing in relation to spatial distance from six sampled sites located in the South Shetland Islands, Maritime Antarctica. The diatom flora associated with Prasiola crispa was represented by 23 species distributed in 15 genera. Pinnularia australoschoenfelderi Zidarova, Kopalová & Van de Vijver, Luticola austroatlantica Van de Vijver, Kopalová, S.A.Spaulding & Esposito, Luticola amoena Van der Vijver, Kopalová, Zidarova & Levkov, Pinnularia austroshetlandica (Carlson) Cleve-Euler and Psammothidium papilio (D.E. Kellogg et al.) Kopalová & Zidarova were the most abundant species in our samples, together they represented 68% of the total number of individuals collected. There was great similarity and abundance of the diatom communites among the sampled points, which resulted in the absence of a linear relationship pattern with distance between sampling points. We conclude that distance was not a factor of differentiation of Antarctic diatom communities associated with terrestrial green macroalgae. This suggests that Antarctic environments may have unique characteristics with homogeneous abiotic factors, at least in relation to this substrate.

Unmasking "The Eldest Son of The Father of Protozoology": Charles King.

In 1703 two articles appeared in the Transactions of the Royal Society, authored by an unnamed gentleman. The articles, with deference to Leeuwenhoeck, described recent observations made with a microscope. Clifford Dobell, in his biography of Leeuwenhoeck, remarked at length on the extraordinary quality of the illustrations and descriptions of "animalcules". He declared the anonymous author to be the scion and master draughtsman of Leeuwenhoeck's followers. Still today, one of the illustrations is credited with being the first unambiguous depiction of a diatom. Here I present evidence that the anonymous author was Charles King of Staffordshire and evidence of his talent. John Hill is often credited for the first naming and illustrating Paramecium and other ciliates in his 1752 book, but it has been claimed repeatedly that he copied the anonymous 1703 illustrations without attribution. Here, the illustrations from 1703 and 1752 are given, and casual examination suffices to show not only that the illustrations were copied, but also that the 1703 illustrations (and text descriptions) of Charles King are of a far higher quality than those of John Hill. Although very little is known about Charles King, he deserves recognition as a pioneer of protistology.

Metabolic Innovations Underpinning the Origin and Diversification of the Diatom Chloroplast.

: Of all the eukaryotic algal groups, diatoms make the most substantial contributions to photosynthesis in the contemporary ocean. Understanding the biological innovations that have occurred in the diatom chloroplast may provide us with explanations to the ecological success of this lineage and clues as to how best to exploit the biology of these organisms for biotechnology. In this paper, we use multi-species transcriptome datasets to compare chloroplast metabolism pathways in diatoms to other algal lineages. We identify possible diatom-specific innovations in chloroplast metabolism, including the completion of tocopherol synthesis via a chloroplast-targeted tocopherol cyclase, a complete chloroplast ornithine cycle, and chloroplast-targeted proteins involved in iron acquisition and CO2 concentration not shared between diatoms and their closest relatives in the stramenopiles. We additionally present a detailed investigation of the chloroplast metabolism of the oil-producing diatom Fistuliferasolaris, which is of industrial interest for biofuel production. These include modified amino acid and pyruvate hub metabolism that might enhance acetyl-coA production for chloroplast lipid biosynthesis and the presence of a chloroplast-localised squalene synthesis pathway unknown in other diatoms. Our data provides valuable insights into the biological adaptations underpinning an ecologically critical lineage, and how chloroplast metabolism can change even at a species level in extant algae.

Collective sinking promotes selective cell pairing in planktonic pennate diatoms.

Finding a partner in an inherently unsteady 3-dimensional system, such as the planktonic marine environment, is a difficult task for nonswimming organisms with poor control over their orientation. We experimentally investigate the process of cell pairing in pennate marine diatoms and present field evidence of its occurrence in the ocean. We describe the mechanism as a 3-step process in which pennate diatoms (i) vertically reorient while sinking from surface turbulent waters to a more stable environment (i.e., under the seasonal pycnocline), (ii) segregate from incompatible partners (e.g., dead or different sized cells), and (iii) pair with other partners as a result of the hydrodynamic instabilities generated by collective cell sinking. This is, eminently, a cell abundance-dependent process, therefore being more effective when population sinking is synchronized. We suggest that this selective process, enabling matching of size-compatible healthy partners, could be fundamental in understanding sexual reproduction in pennate diatoms.

Can diatom girdle band pores act as a hydrodynamic viral defense mechanism?

Diatoms are microalgae encased in highly structured and regular frustules of porous silica. A long-standing biological question has been the function of these frustules, with hypotheses ranging from them acting as photonic light absorbers to being particle filters. While it has been observed that the girdle band pores of the frustule of Coscinodiscus sp. resemble those of a hydrodynamic drift ratchet, we show using scaling arguments and numerical simulations that they cannot act as effective drift ratchets. Instead, we present evidence that frustules are semi-active filters. We propose that frustule pores simultaneously repel viruses while promoting uptake of ionic nutrients via a recirculating, electroosmotic dead-end pore flow, a new mechanism of "hydrodynamic immunity".

Contribution of frustules and mucilage trails to the mobility of diatom Navicula sp.

The secreted mucilage trails of the diatom Navicula sp. in the process of motility were studied by scanning electron microscopy (SEM), transmission electron microscope (TEM), atomic force microscopy (AFM) and Raman spectra etc. Contrary to previous studies, force measurement was taken directly on the mucilage trails of live cells using the method of in situ force mapping by AFM. The retraction force curve presented an increased tip-substrate peak and a small saw-tooth pattern tip-mucilage peak. Especially, same measurements on various substrates with different surface energy revealed that the mucilage trails actually functioned as a medium increasing the adhesive force between the diatom and substrates, which is crucial to diatom's adhesion and locomotion. In addition, the mechanical properties of mucilage trails were quite different from mucilage strands in the maximum adhesive force and the maximum polymer extension length. Raman spectra indicated the difference in compositions that both of the two kinds of mucilages had proteins and polysaccharide, but the mucilage strands contained some other components with C=O, -CH2- and -CH3 asymmetric and symmetric stretches. This research hammers out more precise information about mucilage trails which would be useful in terms of diatom motility and biofouling prevention.