In JoVE (1)

Other Publications (17)

Articles by Takashi Toyofuku in JoVE

 JoVE Biology

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

1Department of Biological Sciences, Clemson University, 2Department of Marine Biodiversity Research (BioDive), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 3Advanced Material Research Laboratory (AMRL), Clemson University, 4Swire Institute of Marine Sciences and School of Biological Sciences, The University of Hong Kong


JoVE 55164

Other articles by Takashi Toyofuku on PubMed

Foraminifera Promote Calcification by Elevating Their Intracellular PH

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2009  |  Pubmed ID: 19706891

Surface seawaters are supersaturated with respect to calcite, but high concentrations of magnesium prevent spontaneous nucleation and growth of crystals. Foraminifera are the most widespread group of calcifying organisms and generally produce calcite with a low Mg content, indicating that they actively remove Mg(2+) from vacuolized seawater before calcite precipitation. However, one order of foraminifera has evolved a calcification pathway, by which it produces calcite with a very high Mg content, suggesting that these species do not alter the Mg/Ca ratio of vacuolized seawater considerably. The cellular mechanism that makes it possible to precipitate calcite at high Mg concentrations, however, has remained unknown. Here we demonstrate that they are able to elevate the pH at the site of calcification by at least one unit above seawater pH and, thereby, overcome precipitation-inhibition at ambient Mg concentrations. A similar result was obtained for species that precipitate calcite with a low Mg concentration, suggesting that elevating the pH at the site of calcification is a widespread strategy among foraminifera to promote calcite precipitation. Since the common ancestor of these two groups dates back to the Cambrian, our results would imply that this physiological mechanism has evolved over half a billion years ago. Since foraminifera rely on elevating the intracellular pH for their calcification, our results show that ongoing ocean acidification can result in a decrease of calcite production by these abundant calcifyers.

10 Micrometer-scale SPM Local Oxidation Lithography

Journal of Nanoscience and Nanotechnology. Jul, 2010  |  Pubmed ID: 21128455

10 micrometer-scale scanning probe microscopy (SPM) local oxidation lithography was performed on Si. In order to realize large-scale oxidation, an SPM tip with a contact length of 15 microm was prepared by focused-ion-beam (FIB) etching. The oxidation was carried out in contact mode operation with the contact force ranging from 0.1 to 2.1 microN. The applied bias voltage was 50 V, and scanning speed was varied from 10 to 200 microm/s. The scan length was 15 microm for one cycle. The influence of contact force on the large-scale oxidation was investigated. At high contact force, the Si oxide with good size uniformity was obtained even with high scanning speed. The SPM tip with larger contact length may increase the spatial dimensions of the water meniscus between the SPM tip and sample surface, resulting in the larger dimensions of the fabricated oxide. Furthermore, the throughput of large-scale oxidation reached about 10(3) microm2/s by controlling the scanning speed and contact force of the SPM tip. It is suggested that SPM local oxidation can be upscaled by using a SPM tip with large contact length.

Effects of Hydrogen Sulfide on Bacterial Communities on the Surface of Galatheid Crab, Shinkaia Crosnieri, and in a Bacterial Mat Cultured in Rearing Tanks

Microbes and Environments. 2013  |  Pubmed ID: 23080406

To investigate the effects of H2S on the bacterial consortia on the galatheid crab, Shinkaia crosnieri, crabs of this species were cultivated in the laboratory under two different conditions, with and without hydrogen sulfide feeding. We developed a novel rearing tank system equipped with a feedback controller using a semiconductor sensor for hydrogen sulfide feeding. H2S aqueous concentration was successfully maintained between 5 to 40 µM for 80 d with the exception of brief periods of mechanical issues. According to real-time PCR analysis, the numbers of copies of partial 16S rRNA gene of an episymbiont of the crabs with H2S feeding was three orders of magnitude larger than that without feeding. By phylogenetic analysis of partial 16S rRNA gene, we detected several clones related to symbionts of deep sea organisms in Alphaproteobacteria, Gammaproteobacteria, Epsilonproteobacteria, and Flavobacteria, from a crab with H2S feeding. The symbiont-related clones were grouped into four different groups: Gammaproteobacteria in marine epibiont group I, Sulfurovum-affiliated Epsilonproteobacteria, Osedax mucofloris endosymbiont-affiliated Epsilonproteobacteria, and Flavobacteria closely related to CFB group bacterial epibiont of Rimicaris exoculata. The other phylotypes were related to Roseobacter, and some Flavobacteria, seemed to be free-living psychrophiles. Furthermore, white biofilm occurred on the surface of the rearing tank with H2S feeding. The biofilms contained various phylotypes of Gammaproteobacteria, Epsilonproteobacteria, and Flavobacteria, as determined by phylogenetic analysis. Interestingly, major clones were related to symbionts of Alviniconcha sp. type 2 and to endosymbionts of Osedax mucofloris, in Epsilonproteobacteria.

Attachment and Detachment of Living Microorganisms Using a Potential-controlled Electrode

Marine Biotechnology (New York, N.Y.). Aug, 2013  |  Pubmed ID: 23420537

We developed an electrical modulation method for attachment and detachment of microorganisms. Living microorganisms suspended in non-nutritive media such as PBS⁻ and artificial seawater were attracted by and selectively attached to indium tin oxide (ITO)/glass electrode regions to which a negative potential was applied. The microorganisms suspended in LB medium and glucose solution were not attracted to the ITO electrode. Dead microorganisms were not attracted to the ITO electrode. The living microorganisms were retrieved after detachment from the ITO electrode by application of a high-frequency triangular wave potential. When we applied this method to separate microorganisms from deep-sea sediment, bacteria belonging to 19 phyla and 23 classes were collected without undesirable high molecular weight contaminants such as humic acids. At the phylum and class level, respectively, 95 and 87 % of the phylotypes among electrically retrieved bacteria were common to the gene clones from the direct sediment DNA extraction. This technique is a novel useful method to prepare bacterial cells in a single population or a community for metagenomic analyses.

Hadal Disturbance in the Japan Trench Induced by the 2011 Tohoku-Oki Earthquake

Scientific Reports. 2013  |  Pubmed ID: 23715086

In situ video observations and sediment core samplings were performed at two hadal sites in the Japan Trench on July, 2011, four months after the Tohoku-Oki earthquake. Video recordings documented dense nepheloid layers extending ~30-50 m above the sea bed. At the trench axis, benthic macrofauna was absent and dead organisms along with turbid downslope current were observed. The top 31 cm of sediment in the trench axis revealed three recent depositions events characterized by elevated (137)Cs levels and alternating sediment densities. At 4.9 km seaward from the trench axis, little deposition was observed but the surface sediment contained (134)Cs from the Fukushima Dai-ichi nuclear disaster. We argue that diatom blooms observed by remote sensing facilitated rapid deposition of (134)Cs to hadal environment and the aftershocks induced successive sediment disturbances and maintained dense nepheloid layers in the trench even four months after the mainshock.

Exclusive Localization of Carbonic Anhydrase in Bacteriocytes of the Deep-sea Clam Calyptogena Okutanii with Thioautotrophic Symbiotic Bacteria

The Journal of Experimental Biology. Dec, 2013  |  Pubmed ID: 24031050

Deep-sea Calyptogena clams harbor thioautotrophic intracellular symbiotic bacteria in their gill epithelial cells. The symbiont fixes CO2 to synthesize organic compounds. Carbonic anhydrase (CA) from the host catalyzes the reaction CO2 + H2O ↔ HCO3(-) + H(+), and is assumed to facilitate inorganic carbon (Ci) uptake and transport to the symbiont. However, the localization of CA in gill tissue remains unknown. We therefore analyzed mRNA sequences, proteins and CA activity in Calyptogena okutanii using expression sequence tag, SDS-PAGE and LC-MS/MS. We found that acetazolamide-sensitive soluble CA was abundantly expressed in the gill tissue of C. okutanii, and the enzyme was purified by affinity chromatography. Mouse monoclonal antibodies against the CA of C. okutanii were used in western blot analysis and immunofluorescence staining of the gill tissues of C. okutanii, which showed that CA was exclusively localized in the symbiont-harboring cells (bacteriocytes) in gill epithelial cells. Western blot analysis and measurement of activity showed that CA was abundantly (26-72% of total soluble protein) detected in the gill tissues of not only Calyptogena clams but also deep-sea Bathymodiolus mussels that harbor thioautotrophic or methanotrophic symbiotic bacteria, but was not detected in a non-symbiotic mussel, Mytilus sp. The present study showed that CA is abundant in the gill tissues of deep-sea symbiotic bivalves and specifically localizes in the cytoplasm of bacteriocytes of C. okutanii. This indicates that the Ci supply process to symbionts in the vacuole (symbiosome) in bacteriocytes is essential for symbiosis.

Determination of Extremely High Pressure Tolerance of Brine Shrimp Larvae by Using a New Pressure Chamber System

Zoological Science. Nov, 2013  |  Pubmed ID: 24224473

Hydrostatic pressure is the only one of a range of environmental parameters (water temperature, salinity, light availability, and so on) that increases in proportion with depth. Pressure tolerance is therefore essential to understand the foundation of populations and current diversity of faunal compositions at various depths. In the present study, we used a newly developed pressure chamber system to examine changes in larval activity of the salt-lake crustacean, Artemia franciscana, in response to a range of hydrostatic pressures. We showed that A. franciscana larvae were able to survive for a short period at pressures of ≤ 60 MPa (approximately equal to the pressure of 6000 m deep). At a pressure of > 20 MPa, larval motor ability was suppressed, but not lost. Meanwhile, at a pressure of > 40 MPa, some of the larval motor ability was lost without recovery after decompression. For all experiments, discordance of movement and timing between right and left appendages, was observed at pressures of > 20 MPa. Our results indicate that the limit of pressure for sustaining active behavior of A. franciscana larvae is ∼20 MPa, whereas the limit of pressure for survival is within the range 30-60 MPa. Thus, members of the genus Artemia possess the ability to resist a higher range of pressures than their natural habitat depth. Our findings demonstrated an example of an organism capable of invading deeper environment in terms of physical pressure tolerance, and indicate the need and importance of pressure study as an experimental method.

Lateral Transfer of Eukaryotic Ribosomal RNA Genes: an Emerging Concern for Molecular Ecology of Microbial Eukaryotes

The ISME Journal. Jul, 2014  |  Pubmed ID: 24451210

Ribosomal RNA (rRNA) genes are widely utilized in depicting organismal diversity and distribution in a wide range of environments. Although a few cases of lateral transfer of rRNA genes between closely related prokaryotes have been reported, it remains to be reported from eukaryotes. Here, we report the first case of lateral transfer of eukaryotic rRNA genes. Two distinct sequences of the 18S rRNA gene were detected from a clonal culture of the stramenopile, Ciliophrys infusionum. One was clearly derived from Ciliophrys, but the other gene originated from a perkinsid alveolate. Genome-walking analyses revealed that this alveolate-type rRNA gene is immediately adjacent to two protein-coding genes (ubc12 and usp39), and the origin of both genes was shown to be a stramenopile (that is, Ciliophrys) in our phylogenetic analyses. These findings indicate that the alveolate-type rRNA gene is encoded on the Ciliophrys genome and that eukaryotic rRNA genes can be transferred laterally.

Unexpected Biotic Resilience on the Japanese Seafloor Caused by the 2011 Tōhoku-Oki Tsunami

Scientific Reports. Dec, 2014  |  Pubmed ID: 25515588

On March 11(th), 2011 the Mw 9.0 2011 Tōhoku-Oki earthquake resulted in a tsunami which caused major devastation in coastal areas. Along the Japanese NE coast, tsunami waves reached maximum run-ups of 40 m, and travelled kilometers inland. Whereas devastation was clearly visible on land, underwater impact is much more difficult to assess. Here, we report unexpected results obtained during a research cruise targeting the seafloor off Shimokita (NE Japan), shortly (five months) after the disaster. The geography of the studied area is characterized by smooth coastline and a gradually descending shelf slope. Although high-energy tsunami waves caused major sediment reworking in shallow-water environments, investigated shelf ecosystems were characterized by surprisingly high benthic diversity and showed no evidence of mass mortality. Conversely, just beyond the shelf break, the benthic ecosystem was dominated by a low-diversity, opportunistic fauna indicating ongoing colonization of massive sand-bed deposits.

Whole Genome Amplification Approach Reveals Novel Polyhydroxyalkanoate Synthases (PhaCs) from Japan Trench and Nankai Trough Seawater

BMC Microbiology. Dec, 2014  |  Pubmed ID: 25539583

Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host.

Cytologic and Genetic Characteristics of Endobiotic Bacteria and Kleptoplasts of Virgulinella Fragilis (Foraminifera)

The Journal of Eukaryotic Microbiology. Jul-Aug, 2015  |  Pubmed ID: 25510528

The benthic foraminifer Virgulinella fragilis Grindell and Collen 1976 has multiple putative symbioses with both bacterial and kleptoplast endobionts, possibly aiding its survival in environments from dysoxia (5-45 μmol-O2 /L) to microxia (0-5 μmol-O2 /L) and in the dark. To clarify the origin and function of V. fragilis endobionts, we used genetic analyses and transmission electron microscope observations. Virgulinella fragilis retained δ-proteobacteria concentrated at its cell periphery just beneath the cell membranes. Unlike another foraminifer Stainforthia spp., which retains many bacterial species, V. fragilis has a less variable bacterial community. This suggests that V. fragilis maintains a specific intracellular bacterial flora. Unlike the endobiotic bacteria, V. fragilis klepto-plasts originated from various diatom species and are found in the interior cytoplasm. We found evidence of both retention and digestion of kleptoplasts, and of fragmentation of the kleptoplastid outer membrane that likely facilitates transport of kleptoplastid products to the host. Accumulations of mitochondria were observed encircling endobiotic bacteria. It is likely that the bacteria use host organic material for carbon oxidation. The mitochondria may use oxygen available around the δ-proteobacteria and synthesize adenosine triphosphate, perhaps for sulfide oxidation.

Involvement of Flocculin in Negative Potential-applied ITO Electrode Adhesion of Yeast Cells

FEMS Yeast Research. Sep, 2015  |  Pubmed ID: 26187908

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between -0.2 and -0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.

Deep-sea Whale Fall Fauna from the Atlantic Resembles That of the Pacific Ocean

Scientific Reports. Feb, 2016  |  Pubmed ID: 26907101

Whale carcasses create remarkable habitats in the deep-sea by producing concentrated sources of organic matter for a food-deprived biota as well as places of evolutionary novelty and biodiversity. Although many of the faunal patterns on whale falls have already been described, the biogeography of these communities is still poorly known especially from basins other than the NE Pacific Ocean. The present work describes the community composition of the deepest natural whale carcass described to date found at 4204 m depth on Southwest Atlantic Ocean with manned submersible Shinkai 6500. This is the first record of a natural whale fall in the deep Atlantic Ocean. The skeleton belonged to an Antarctic Minke whale composed of only nine caudal vertebrae, whose degradation state suggests it was on the bottom for 5-10 years. The fauna consisted mainly of galatheid crabs, a new species of the snail Rubyspira and polychaete worms, including a new Osedax species. Most of the 41 species found in the carcass are new to science, with several genera shared with NE Pacific whale falls and vent and seep ecosystems. This similarity suggests the whale-fall fauna is widespread and has dispersed in a stepping stone fashion, deeply influencing its evolutionary history.

Intracellular Isotope Localization in Ammonia Sp. (Foraminifera) of Oxygen-Depleted Environments: Results of Nitrate and Sulfate Labeling Experiments

Frontiers in Microbiology. 2016  |  Pubmed ID: 26925038

Some benthic foraminiferal species are reportedly capable of nitrate storage and denitrification, however, little is known about nitrate incorporation and subsequent utilization of nitrate within their cell. In this study, we investigated where and how much (15)N or (34)S were assimilated into foraminiferal cells or possible endobionts after incubation with isotopically labeled nitrate and sulfate in dysoxic or anoxic conditions. After 2 weeks of incubation, foraminiferal specimens were fixed and prepared for Transmission Electron Microscopy (TEM) and correlative nanometer-scale secondary ion mass spectrometry (NanoSIMS) analyses. TEM observations revealed that there were characteristic ultrastructural features typically near the cell periphery in the youngest two or three chambers of the foraminifera exposed to anoxic conditions. These structures, which are electron dense and ~200-500 nm in diameter and co-occurred with possible endobionts, were labeled with (15)N originated from (15)N-labeled nitrate under anoxia and were labeled with both (15)N and (34)S under dysoxia. The labeling with (15)N was more apparent in specimens from the dysoxic incubation, suggesting higher foraminiferal activity or increased availability of the label during exposure to oxygen depletion than to anoxia. Our results suggest that the electron dense bodies in Ammonia sp. play a significant role in nitrate incorporation and/or subsequent nitrogen assimilation during exposure to dysoxic to anoxic conditions.

Long-term Cultivation of the Deep-Sea Clam Calyptogena Okutanii: Changes in the Abundance of Chemoautotrophic Symbiont, Elemental Sulfur, and Mucus

The Biological Bulletin. Jun, 2016  |  Pubmed ID: 27365420

Survival of deep-sea Calyptogena clams depends on organic carbon produced by symbiotic, sulfur-oxidizing, autotrophic bacteria present in the epithelial cells of the gill. To understand the mechanism underlying this symbiosis, the development of a long-term cultivation system is essential. We cultivated specimens of Calyptogena okutanii in an artificial chemosynthetic aquarium with a hydrogen sulfide (H2S) supply system provided by the sulfate reduction of dog food buried in the sediment. We studied morphological and histochemical changes in the clams' gills by immunohistochemical and energy-dispersive X-ray analyses. The freshly collected clams contained a high amount of elemental sulfur in the gill epithelial cells, as well as densely packed symbiotic bacteria. Neither elemental sulfur nor symbiotic bacteria was detected in any other organs except the ovaries, where symbiotic bacteria, but not sulfur, was detected. The longest survival of an individual clam in this aquarium was 151 days. In the 3 clams dissected on Days 57 and 91 of the experiment, no elemental sulfur was detected in the gills. The symbiotic bacteria content had significantly decreased by Day 57, and was absent by Day 91. For comparison, we also studied the deep-sea mussel Bathymodiolus septemdierum, which harbors a phylogenetically close, sulfur-oxidizing, symbiotic bacterium with similar sulfur oxidation pathways. Sulfur particles were not detected, even in the gills of the freshly collected mussels. We discuss the importance of the proportion of available H2S and oxygen to the bivalves for elemental sulfur accumulation. Storage of nontoxic elemental sulfur, an energy source, seems to be an adaptive strategy of C. okutanii.

Cytological Observations of the Large Symbiotic Foraminifer Amphisorus Kudakajimensis Using Calcein Acetoxymethyl Ester

PloS One. 2016  |  Pubmed ID: 27812157

Large benthic foraminifera are unicellular calcifying reef organisms that can form symbiotic relationships with a range of different microalgae. However, the cellular functions, such as symbiosis and calcification, and other aspects of cellular physiology in large benthic foraminifera are not fully understood. Amphisorus kudakajimensis was used as a model to determine the detailed cellular characteristics of large benthic foraminifera. We used calcein acetoxymethyl ester (calcein AM) as a fluorescent indicator for live confocal imaging. We demonstrated that calcein AM is a useful fluorescent indicator to stain the fine network of reticulopodia and the cytoplasm in living A. kudakajimensis. We showed that at least two types of reticulopodia exist in A. kudakajimensis: the straight bundle of reticulopodia that spreads from the aperture and the fine reticulopodia along the surface of the aperture and chamber walls. The cytoplasm in outer chambers was highly branched and contained a few dinoflagellates. In contrast, the inner chamberlets contained condensed cytoplasm and many dinoflagellates, suggesting that the cytoplasm of A. kudakajimensis performs different functions based on its location within the large test. Our confocal detailed image analysis provides real-time cellular morphology and cell physiology of living foraminifera.

Proton Pumping Accompanies Calcification in Foraminifera

Nature Communications. Jan, 2017  |  Pubmed ID: 28128216

Ongoing ocean acidification is widely reported to reduce the ability of calcifying marine organisms to produce their shells and skeletons. Whereas increased dissolution due to acidification is a largely inorganic process, strong organismal control over biomineralization influences calcification and hence complicates predicting the response of marine calcifyers. Here we show that calcification is driven by rapid transformation of bicarbonate into carbonate inside the cytoplasm, achieved by active outward proton pumping. Moreover, this proton flux is maintained over a wide range of pCO2 levels. We furthermore show that a V-type H(+) ATPase is responsible for the proton flux and thereby calcification. External transformation of bicarbonate into CO2 due to the proton pumping implies that biomineralization does not rely on availability of carbonate ions, but total dissolved CO2 may not reduce calcification, thereby potentially maintaining the current global marine carbonate production.

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