Imaging and Quantitative Analysis on the Etching of Diatom Frustules via Digital Holographic Microscopy.

Frustules, whose length spans from a few micrometers to more than a hundred micrometers, have been the subject of various modifications to improve their physical properties because of their complex porous silica structure. However, three-dimensional measurements of these changes can be challenging because of the complex 3D architecture and limitations of known methods. In this study, we present a new method that applies digital holographic microscopy (DHM) to analyze controlled etched frustules and observe real-time degradation of frustules at the single-cell level. Frustules obtained from Craspedostauros sp. diatoms were etched in 1 N NaOH for 5 min at 25 and 60 °C, respectively, and the frustule's valve was analyzed using DHM. DHM uses a combination of holography and tomography to reconstruct a 3D refractive index image of the frustule. Measurements of the width, volume, and surface area are achieved. Results showed that at 60 °C of etching, a significant difference with the unetched frustule was observed for all measurements but with high fluctuation values. Finally, real-time observation of the degradation of the frustule is observed when immersed in a high concentration of NaOH. This is the first time the real-time etching of the frustule is observed at the single-cell level. This research provides an easy estimation of the 3D measurements of frustules that may provide new fundamental information and applications.

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 500 µg/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.

Correction to "Fabrication of a Floatable Micron-Sized Enzyme Device Using Diatom Frustules".

[This corrects the article DOI: 10.1021/acsomega.3c02104.].

Fabrication of a Floatable Micron-Sized Enzyme Device Using Diatom Frustules.

Immobilization of enzymes has been widely reported due to their reusability, thermal stability, better storage abilities, and so on. However, there are still problems that immobilized enzymes do not have free movements to react to substrates during enzyme reactions and their enzyme activity becomes weak. Moreover, when only the porosity of support materials is focused, some problems such as enzyme distortion can negatively affect the enzyme activity. Being a solution to these problems, a new function "floatability" of enzyme devices has been discussed. A "floatable" micron-sized enzyme device was fabricated to enhance the free movements of immobilized enzymes. Diatom frustules, natural nanoporous biosilica, were used to attach papain enzyme molecules. The floatability of the frustules, evaluated by macroscopic and microscopic methods, was significantly better than that of four other SiO2 materials, such as diatomaceous earth (DE), which have been widely used to fabricate micron-sized enzyme devices. The frustules were fully suspended at 30 °C for 1 h without stirring, although they settled at room temperature. When enzyme assays were performed at room temperature, 37, and 60 °C with or without external stirring, the proposed frustule device showed the highest enzyme activity under all conditions among papain devices similarly prepared using other SiO2 materials. It was confirmed by the free papain experiments that the frustule device was active enough for enzyme reactions. Our data indicated that the high floatability of the reusable frustule device, and its large surface area, is effective in maximizing enzyme activity due to the high probability to react to substrates.

Attachment of DNA-Wrapped Single-Walled Carbon Nanotubes (SWNTs) for a Micron-Sized Biosensor.

We fabricated a micron-sized biodevice based on the near-infrared photoluminescence (PL) response of single-walled carbon nanotubes (SWNTs). Various biosensors using the unique optical responses of SWNTs have been proposed by many research groups. Most of these employed either colloidal suspensions of dispersed SWNTs or SWNT films on flat surfaces, such as electrodes. In this study, we attached DNA-wrapped SWNTs (DNA-SWNTs) to frustule (micron-sized nanoporous biosilica) surfaces, which were purified from cultured isolated diatoms. After the injection of an oxidant and a reductant, the SWNTs on the frustules showed prominent PL responses. This suggests that the biodevice functions as a micron-sized redox sensor. Frustules can be easily suspended in aqueous solutions because of their porous structures and can easily be collected as pellets by low-speed centrifugation. Thus, the removal of unbound SWNTs and the recovery of the fabricated DNA-SWNT frustules for reuse were achieved by gentle centrifugation. Our proposal for micron-sized SWNT biodevices would be helpful for various biological applications.

An Efficient Method of Observing Diatom Frustules via Digital Holographic Microscopy.

Herein, we propose a convenient method to enable pretreatment of target objects using digital holographic microscopy (DHM). As a test sample, we used diatom frustules (Nitzschia sp.) as the target objects. In the generally used sample preparation method, the frustule suspension is added dropwise onto a glass substrate or into a glass chamber. While our work confirms good observation of purified frustules using the typical sample preparation method, we also demonstrate a new procedure to observe unseparated structures of frustules prepared by baking them on a mica surface. The baked frustules on the mica surface were transferred to a glass chamber with 1% sodium dodecyl sulfate solution. In this manner, the unseparated structures of the diatom frustules were clearly observed. Furthermore, metal-coated frustules prepared by sputtering onto them on a mica surface were also clearly observed using the same procedure. Our method can be applied for the observation of any target object that is pretreated on a solid surface. We expect our proposed method to be a basis for establishing DHM techniques for microscopic observations of biomaterials.

Genome evolution of a nonparasitic secondary heterotroph, the diatom Nitzschia putrida.

Secondary loss of photosynthesis is observed across almost all plastid-bearing branches of the eukaryotic tree of life. However, genome-based insights into the transition from a phototroph into a secondary heterotroph have so far only been revealed for parasitic species. Free-living organisms can yield unique insights into the evolutionary consequence of the loss of photosynthesis, as the parasitic lifestyle requires specific adaptations to host environments. Here, we report on the diploid genome of the free-living diatom Nitzschia putrida (35 Mbp), a nonphotosynthetic osmotroph whose photosynthetic relatives contribute ca. 40% of net oceanic primary production. Comparative analyses with photosynthetic diatoms and heterotrophic algae with parasitic lifestyle revealed that a combination of gene loss, the accumulation of genes involved in organic carbon degradation, a unique secretome, and the rapid divergence of conserved gene families involved in cell wall and extracellular metabolism appear to have facilitated the lifestyle of a free-living secondary heterotroph.

Sinking of Four Species of Living Diatom Cells Directly Observed by a "Tumbled" Optical Microscope.

The study of the sinking phenomenon of diatom cells, which have a slightly larger specific gravity (~1.3) compared to that of water, is an important research topic for understanding photosynthetic efficiency. In this study, we successfully demonstrated the observation of the sinking behaviors of four different species of diatom using a homemade "tumbled" optical microscope. A homemade 1 mm3 microchamber was employed to decrease the effects of convection currents. In the microchamber, diatom cells were basically settled in a linear manner without floating, although some of the cells were rotated during their sinking. Sinking speeds of the four species of diatom cells, Nitzschia sp., Pheodactylum tricornutum, Navicula sp., and Odontella aurita, were 0.81 ± 5.56, 3.03 ± 10.17, 3.29 ± 7.39, and 11.22 ± 21.42 µm/s, respectively, based on the automatic tracking analysis of the centroids of each cell. Manual analysis of a vector between two longitudinal ends of the cells (two-point analysis) was effective for quantitatively characterizing the rotation phenomenon; therefore, angles and angular velocities of rotating cells were well determined as a function of time. The effects of the cell shapes on sinking velocity could be explained by simulation analysis using the modified Stokes' law proposed by Miklasz et al.

Comparative Gene Analysis Focused on Silica Cell Wall Formation: Identification of Diatom-Specific SET Domain Protein Methyltransferases.

Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.

Corrigendum to "Label-free imaging and analysis of subcellular parts of a living diatom cylindrotheca sp. using optical diffraction tomography" [MethodsX: 7 (2020) 100889].

[This corrects the article DOI: 10.1016/j.mex.2020.100889.].

Label-free imaging and analysis of subcellular parts of a living diatom cylindrotheca sp. using optical diffraction tomography.

Optical diffraction tomography is an emerging label-free microscopic technique with its capability of label-free, quantitative, and rapid imaging of biological samples. In this work, we present the imaging and analysis of a living diatom Cylindrotheca sp. in seawater without using any pretreatment such as fluorescence staining. The 3D refractive index (RI) of a living diatom cell was measured, to which quantitative image analysis was perform to investigate subcellular parts of the diatom. Each part of the cell was well distinguished as RI values and distributions. From the analysis, RI values of frustules, protoplasm, vacuole, and chloroplast were estimated to be in the range of 1.352-1.388, 1.363-1.381, 1.388-1.395, and 1.403-1.436, respectively. Our results suggest that the present method will be a powerful tool not only for observing diatom cells but also for studying various cells and mesoscopic materials.•Subcellular parts of a living diatom cell was well visualized by digital holographic microscope.•Subcellular parts could be identified as differences of refractive indexes.•The observation was achieved without any pre-treatment of the living cell.

Unique observation method of temperature dependence of diatom floating by direct microscope.

Diatoms are one of the earth's major oxygen producers. For that reason, studying the floating phenomena of living diatom cells in water is an important research subject. Efficiency of photosynthesis of diatom cells may be heavily affected by their floating behavior. In our previous research, we devised a 'tumbled' microscope, a device created by tilting an inverted microscope (CKX53, OLYMPUS) by 90 degrees, due to which allowed observation with a sample stage perpendicular to the ground. When we observed a Petri dish filled with diatom cell suspension, the floating behavior of diatom cells were well visualized. Cyclotella meneghiniana was isolated and subcultured in bold modified basal freshwater nutrient solution liquid medium (B5282-500ML, Sigma-Aldrich) at 18 °C. Before the microscopic observation, cell suspension was cultured for two weeks after the final subculture. Observation was performed at room temperature, 30 °C, and 40 °C with a temperature sensor in the center of the chamber (inside). Observations were started as soon as the sample was installed. In a typical image obtained using the tumbled microscope, the diatom cells were found to move from the top to the bottom. In order to analyze floating velocity and trajectory, observation was continued for 35 min at room temperature, 30 °C, and 40 °C. Tracking analysis was carried out using the two-dimensional motion image measurement software Move-tr/2D. The average speed of 100 cells was 7.0 ± 4.3 µm/s at room temperature, 85.6 ± 31.9 µm/s at 30 °C and 470.1 ± 279.8 µm/s at 40 °C. In this study, we devised the unique observation to visualize the temperature dependence of diatom cells.

Single cell analysis of sinking diatoms studied using a homemade 'tumbled' optical microscope system.

Diatoms are one of the major photosynthetic planktons. Here, we studied movements of aqueous suspensions of diatoms using a home-made 'tumbled' optical microscope system. The usual inverted optical microscope was reoriented using a homemade microscope stand so that the vertical sample stage contacted the surface. To observe the intrinsic sinking phenomenon of individual Navicula sp. cells, which have slender bodies, a homemade microchamber (1 mm3) was employed. Most of the cells uniformly sunk with a velocity of 2 to 14 µm/s. Automatic and manual two points trajectory analyses were carried out. The manual analysis was able to assess the rotation of cells. The novel methods provide new information about cell movements in aqueous systems that could not be obtained using conventional methods.

Differences in dynamic behavior of single diatom cells caused by changing wavelengths.

We investigate a motion of diatom cells stimulated by a halogen lamp irradiation. Diatom cells are single-celled organisms which have chloroplast. Chloroplast contains photosynthetic pigment which absorbs blue light (wave length of the light is 400 nm-450 nm) and red one (650 nm-700 nm). Light intensity of the halogen lamp is fixed about 500 Lx during the experiment. We used colored films to cut the blue or red light and observed motion of diatom cells by using the optical microscope. We found that the speed of diatom cells decreases when the colored film is inserted, and it increases after ejecting the film. It is noted that the light intensity is constant during the experiment, which means that we change wave length of the irradiated light. Our results show that the average speed of diatom cells is influenced by not the light intensity but the wave length of the light.

A Non-photosynthetic Diatom Reveals Early Steps of Reductive Evolution in Plastids.

Nonphotosynthetic plastids retain important biological functions and are indispensable for cell viability. However, the detailed processes underlying the loss of plastidal functions other than photosynthesis remain to be fully understood. In this study, we used transcriptomics, subcellular localization, and phylogenetic analyses to characterize the biochemical complexity of the nonphotosynthetic plastids of the apochlorotic diatom Nitzschia sp. NIES-3581. We found that these plastids have lost isopentenyl pyrophosphate biosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase-based carbon fixation but have retained various proteins for other metabolic pathways, including amino acid biosynthesis, and a portion of the Calvin-Benson cycle comprised only of glycolysis/gluconeogenesis and the reductive pentose phosphate pathway (rPPP). While most genes for plastid proteins involved in these reactions appear to be phylogenetically related to plastid-targeted proteins found in photosynthetic relatives, we also identified a gene that most likely originated from a cytosolic protein gene. Based on organellar metabolic reconstructions of Nitzschia sp. NIES-3581 and the presence/absence of plastid sugar phosphate transporters, we propose that plastid proteins for glycolysis, gluconeogenesis, and rPPP are retained even after the loss of photosynthesis because they feed indispensable substrates to the amino acid biosynthesis pathways of the plastid. Given the correlated retention of the enzymes for plastid glycolysis, gluconeogenesis, and rPPP and those for plastid amino acid biosynthesis pathways in distantly related nonphotosynthetic plastids and cyanobacteria, we suggest that this substrate-level link with plastid amino acid biosynthesis is a key constraint against loss of the plastid glycolysis/gluconeogenesis and rPPP proteins in multiple independent lineages of nonphotosynthetic algae/plants.

Gymnoxanthella radiolariae gen. et sp. nov. (Dinophyceae), a dinoflagellate symbiont from solitary polycystine radiolarians.

The symbiotic dinoflagellate Gymnoxanthella radiolariae T. Yuasa et T. Horiguchi gen. et sp. nov. isolated from polycystine radiolarians is described herein based on light, scanning and transmission electron microscopy as well as molecular phylogenetic analyses of SSU and LSU rDNA sequences. Motile cells of G. radiolariae were obtained in culture, and appeared to be unarmored. The cells were 9.1-11.4 µm long and 5.7-9.4 µm wide, and oval to elongate oval in the ventral view. They possessed an counterclockwise horseshoe-shaped apical groove, a nuclear envelope with vesicular chambers, cingulum displacement with one cingulum width, and the nuclear fibrous connective; all of these are characteristics of Gymnodinium sensu stricto (Gymnodinium s.s.). Molecular phylogenetic analyses also indicated that G. radiolariae belongs to the clade of Gymnodinium s.s. However, in our molecular phylogenetic trees, G. radiolariae was distantly related to Gymnodinium fuscum, the type species of Gymnodinium. Based on the consistent morphological, genetic, and ecological divergence of our species with the other genera and species of Gymnodinium s.s., we considered it justified to erect a new, separate genus and species G. radiolariae gen. et sp. nov. As for the peridinioid symbiont of radiolarians, Brandtodinium has been erected as a new genus instead of Zooxanthella, but the name Zooxanthella is still valid. Brandtodinium is a junior synonym of Zooxanthella. Our results suggest that at least two dinoflagellate symbiont species, peridinioid Zooxanthella nutricula and gymnodinioid G. radiolariae, exist in radiolarians, and that they may have been mixed and reported as "Z. nutricula" since the 19th century.

Proposal of a Twin Arginine Translocator System-Mediated Constraint against Loss of ATP Synthase Genes from Nonphotosynthetic Plastid Genomes. [Corrected].

Organisms with nonphotosynthetic plastids often retain genomes; their gene contents provide clues as to the functions of these organelles. Yet the functional roles of some retained genes-such as those coding for ATP synthase-remain mysterious. In this study, we report the complete plastid genome and transcriptome data of a nonphotosynthetic diatom and propose that its ATP synthase genes may function in ATP hydrolysis to maintain a proton gradient between thylakoids and stroma, required by the twin arginine translocator (Tat) system for translocation of particular proteins into thylakoids. Given the correlated retention of ATP synthase genes and genes for the Tat system in distantly related nonphotosynthetic plastids, we suggest that this Tat-related role for ATP synthase was a key constraint during parallel loss of photosynthesis in multiple independent lineages of algae/plants.

Single cell analysis using a glass microchamber for studying movement fluctuations of Navicula pavillardii and Seminavis robusta diatom cells.

We propose a new technique for quantitative trajectory analysis of gliding phenomenon of Navicula pavillardii (NP) and Seminavis robusta (SR) diatom cells by single cell observation using a glass microchamber in this short technical note. Two-dimensional trajectory analysis of cell movements was used to determine the angular velocity, velocity, and migration distances of the diatom movement. Based on the trajectory analysis, we found that asymmetrically shaped SR had a larger angular velocity with large fluctuations compared to symmetrically shaped NP, although the velocity of SR was less than that of NP. It suggests that lateral frictional force in a culture medium is an important factor for diatom movements. Our results revealed that the single cell observation using a glass microchamber is effective on quantitative analysis of angular velocity of diatom gliding.