Sumihiro Koyama

Pressure-stat aquarium system designed for capturing and maintaining deep-sea organisms

Abstract A novel pressure-stat aquarium system has been developed for the study of living deep-sea multicellular organisms under high hydrostatic pressure. The system is designed for operation by submersibles and captures deep-sea organisms with a suction servomotor. The system can maintain an inner pressure of 20 MPa (1 bar ≈0.1 MPa ) even when the submersible surfaces from the deepest sea bottom. The system can control the pressure range up to 20 MPa within a fluctuation of ±0.1∼0.2 MPa by exchanging seawater. A specimen of the deep-sea fish Ebinania brephocephala collected by trawl net was kept alive under pressure in the pressure-stat aquarium system for 64 days. Oxygen consumption rate of a deep-sea fish E. brephocephala (total length 12.9 cm , wet weight 61.1 g ) was calculated as 0.10 μl O 2 / mg wet weight/h. Based on the calculation, two deep-sea fishes of the same size could be kept alive in the system. We also succeeded in maintaining the deep-sea fish Zoarcidae sp. captured by the submersible Shinkai 2000 at a depth of 1171 m in Sagami Bay, Japan.

Tissue culture of the deep-sea bivalve Calyptogena soyoae

Abstract Tissue culture for the deep-sea clam Calyptogena soyoae (C. soyoae) has been examined. Mantle tissue was cultured in Dulbeccos modified Eagle medium that was prepared using artificial seawater supplemented with fetal bovine serum (FBS) and the body fluid of C. soyoae. The mantle cells were viable in culture for at least 13 days at 4°C and atmospheric pressure on a polylysine-coated dish, although no cells attached in the body fluid-free culture medium. It was found that mantle cells synthesized DNA and seemed to proliferate under atmospheric conditions.

Brevundimonas denitrificans sp. nov., a denitrifying bacterium isolated from deep subseafloor sediment

A novel Gram-stain-negative, aerobic, heterotrophic, stalked and capsulated bacterium with potential denitrification ability, designated strain TAR-002(T), was isolated from deep seafloor sediment in Japan. Colonies lacked lustre, and were viscous and translucent white. The ranges of temperature, pH and salt concentration for growth were 8-30 °C, pH 6.0-10.0 and 1-3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain TAR-002(T) belongs to the genus Brevundimonas of the class Alphaproteobacteria. Levels of similarity between the 16S rRNA gene sequence of strain TAR-002(T) and those of the type strains of species of the genus Brevundimonas were 93.5-98.9%; the most closely related species was Brevundimonas basaltis. In DNA-DNA hybridization assays between strain TAR-002(T) and its phylogenetic neighbours, Brevundimonas lenta DS-18(T), B. basaltis J22(T), Brevundimonas subvibrioides ATCC 15264(T) and Brevundimonas alba DSM 4736(T), mean hybridization levels were 6.4-27.7%. The G+C content of strain TAR-002(T) was 70.3 mol%. Q-10 was the major respiratory isoprenoid quinone. The major fatty acids were C(18:1)ω7c and C(16:0), and the presence of 1,2-di-O-acyl-3-O-[D-glucopyranosyl-(1 → 4)-α-D-glucopyranuronosyl]glycerol (DGL) indicates the affiliation of strain TAR-002(T) with the genus Brevundimonas. On the basis of biological characteristics and 16S rRNA gene sequence comparisons, strain TAR-002(T) is considered to represent a novel species of the genus Brevundimonas, for which the name Brevundimonas denitrificans sp. nov. is proposed; the type strain is TAR-002(T) ( =NBRC 110107(T) =CECT 8537(T)).

Survival of Deep-Sea Shrimp (Alvinocaris sp.) During Decompression and Larval Hatching at Atmospheric Pressure

We report successful larval hatching of deep-sea shrimp after decompression to atmospheric pressure. Three specimens of deep-sea shrimp were collected from an ocean depth of 1157 m at cold-seep sites off Hatsushima Island in Sagami Bay, Japan, using a pressure-stat aquarium system. Phylogenetic analysis of Alvinocaris sp. based on cytochrome c oxidase subunit gene sequences confirmed that these species were a member of the genus Alvinocaris. All 3 specimens survived to reach atmospheric pressure conditions after stepwise 63-day decompression. Two of the specimens contained eggs, which hatched after 10 and 16 days, respectively, of full decompression. Although no molting of the shrimp larvae was observed during 74 days of rearing under atmospheric pressure, the larvae developed conventional dark-adapted eyes after 15 days.

Electrically modulated attachment and detachment of animal cells cultured on an optically transparent patterning electrode.

The purpose of this study was to develop the modulation methods for the attachment and detachment of specifically positioned adhesive animal cells cultured on an electrode surface with the application of a weak electrical potential. A patterned indium tin oxide (ITO) optically transparent working electrode was placed on the bottom of a chamber slide with a counter-(Pt) and reference (Ag/AgCl) electrode. The ITO patterning was formed by a reticulate ITO region and arrayed square glass regions of varying size. Using the 3-electrode culture system, the author succeeded in modulation of the attachment and detachment of animal cells on the working electrode surface. Animal cells suspended in serum or sera containing medium were drawn to and attached on a reticulate ITO electrode region to which a +0.4-V vs. Ag/AgCl-positive potential was applied. Meanwhile, the cells were successfully placed on the square glass regions by -0.3-V vs. Ag/AgCl-negative potential application. Animal cells were detached not only from the ITO electrode but also from the square glass regions after the application of a ±10-mV vs. Ag/AgCl, 9-MHz [corrected] rectangular wave potential in PBS(-) for 30-60 min. Rectangular wave potential-induced cell detachment is almost completely noncytotoxic, and no statistical differences between trypsinization and the high frequency wave potential application were observed in HeLa cell growth. The electrical modulation of the specifically positioned cell attachment and detachment techniques holds potential for novel optical microscopic cell sorting analysis in lab-on-chip devices.

Post-transcriptional regulation of immunomodulatory cytokines production in human skin fibroblasts by intense mechanical stresses

We found that extremely high hydrostatic pressure stresses induced a variety of cytokines production in normal human dermal fibroblasts. Normal human dermal fibroblasts were found to survive and were active in producing interleukin (IL) -6, -8, and monocyte chemoattractant protein-1 (MCP-1) under extremely high hydrostatic pressure, up to 70 MPa (=690.8 atm=713.8 kgf/cm2). 70 MPa pressure application extremely enhanced IL-6 and IL-8 secretions (about 130 folds) without transcriptional enhancement. Although induction of IL-1alpha, IL-1beta, and IL-12 mRNAs was appeared under high hydrostatic pressure condition, no translation of IL-1alpha, IL-1beta, and IL-12 proteins was found. Extracellular accumulation of constitutively produced MCP-1 was down regulated in the pressure-applied fibroblasts in the absence of transcriptional repression. These results indicated that hydrostatic pressure stresses triggered post-transcriptional regulation mechanisms that modulated the cytokines production.

Vertical body orientation by a snipe eel (Nemichthyidae, Anguilliformes) in the deep mesopelagic zone along the West Mariana Ridge

A nemichthyid eel apparently of the genus Avocettina was observed and video recorded during the day from a submersible operating above the deep western slope of the West Mariana Ridge at a depth of about 800 m. The eel was oriented vertically, head-upward, with its long-thin body held rigidly straight. It remained motionless for more than 1 min except for small constant oscillations of the tip of the tail. It showed no reaction to the presence of the approaching submersible until contacting it. This body position has been seen in nemichthyid eels previously by submersibles in the Atlantic, Pacific and Indian oceans. It is postulated to be a prey or predator detection tactic that optimizes the use of the visual or lateral line senses. It may also be an efficient way to maintain a position or to move slowly through the water column without disrupting sensory function.

Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells.

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.

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

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.

Physical Modulation of Cellular Information Networks

In this chapter, we reviewed the effects of physical stimulation including electrical potential, hydrostatic pressure, electromagnetic field, shear stress, and heat shock application on cellular functions such as proliferation, differentiation, gene expression, and protein production. Prospects for future investigation are also discussed.