Yoshihiro Mogami

Theoretical and Experimental Dissection of Gravity-Dependent Mechanical Orientation in Gravitactic Microorganisms

Mechanisms of gravitactic behaviors of aquatic microorganisms were investigated in terms of their mechanical basis of gravity-dependent orientation. Two mechanical mechanisms have been considered as possible sources of the orientation torque generated on the inert body. One results from the differential density within an organism (the gravity-buoyancy model) and the other from the geometrical asymmetry of an organism (the drag-gravity model). We first introduced a simple theory that distinguishes between these models by measuring sedimentation of immobilized organisms in a medium of higher density than that of the origanisms. Ni2+-immobilized cells of Paramecium caudatum oriented downwards while floating upwards in the Percoll-containing hyper-density medium but oriented upwards while sinking in the hypo-density control medium. This means that the orientation of Paramecium is mechanically biased by the torque generated mainly due to the anterior location of the reaction center of hydrodynamic stress relative to those of buoyancy and gravity; thus the torque results from the geometrical fore-aft asymmetry and is described by the drag-gravity model. The same mechanical property was demonstrated in gastrula larvae of the sea urchin by observing the orientation during sedimentation of the KCN-immobilized larvae in media of different density: like the paramecia, the gastrulae oriented upwards in hypo-density medium and downwards in hyper-density medium. Immobilized pluteus larvae, however, oriented upwards regardless of the density of the medium. This indicates that the orientation of the pluteus is biased by the torque generated mainly due to the posterior location of the reaction center of gravity relative to those of buoyancy and hydrodynamic stress; thus the torque results from the fore-aft asymmetry of the density distribution and is described by the gravity-buoyancy model. These observations indicate that, during development, sea urchin larvae change the mechanical mechanism for the gravitactic orientation. Evidence presented in the present paper demonstrates a definite relationship between the morphology and the gravitactic behavior of microorganisms.

Sperm-activating peptide induces asymmetric flagellar bending in sea urchin sperm.

Abstract Speract, a sperm-activating peptide (SAP) from sea urchin eggs, induces various sperm responses including a transient increase in the intracellular Ca2+ concentration. However, it has not been clarified how speract modulates sperm motility and whether it functions as a chemoattractant. To confirm the effect of speract on sperm motility, we observed the flagellar bending response to speract in sperm of Hemicentrotus pulcherrimus, in experiments using caged speract and a lighting system for a microscope newly developed with a power LED. We found that speract induces increases in curvature of swimming paths and changes flagellar bending shape to asymmetric. These facts show that speract directly regulates flagellar motility, and suggest that speract-induced increases in intracellular Ca2+ concentration play an actual role in regulation of the flagellar movement.

Bioconvective pattern formation of Tetrahymena under altered gravity

SUMMARY Bioconvection is a result of the negative gravitactic behavior of microorganisms. When the top-heavy density gradient generated by gravitaxis grows sufficiently large, an overturning convection occurs leading to a formation of characteristic patterns, which involve highly concentrated aggregation of cells into extended two-dimensional structures. Although gravity is a crucial factor, few experiments have been done with reference to gravity as an experimental variable. In order to gain an insight into the hydrodynamic as well as biological dependence of the convective motion on gravity, we investigated changes in bioconvective patterns of Tetrahymena under altered gravity acceleration generated by a long-arm centrifuge. Bioconvective patterns recorded of three different cell strains (T. pyriformis, T. thermophila and its behavioral mutant, TNR) were analyzed quantitatively using space-time plot and Fourier analysis. For example, under subcritical conditions, when T. pyriformis (1.0×106 cells ml-1) was placed in a 2 mm-deep chamber, no spatial pattern was observed at 1 g. When the suspension was centrifuged, however, patterns began to appear as acceleration increased over a critical value (1.5 g), and then remained steady. The formation was reversible, i.e. the patterns disappeared again as acceleration decreased. Under supracritical conditions, i.e. when a suspension of the same density was placed in a 4 mm-deep chamber, a steady state pattern was formed at 1 g. The pattern spacing in the steady state was observed to decrease stepwise in response to step increases in acceleration. Fourier analysis demonstrated that for TNR the mean wave number changed almost simultaneously with step changes in acceleration, whereas the responses were less sharp in the wild-type strains. This may suggest that the locomotor phenotype of the cell, such as its avoiding response ability, has a crucial role in bioconvective pattern formation. These findings are discussed in relation to former theoretical studies.

Peptide-induced hyperactivation-like vigorous flagellar movement in starfish sperm

Asterosap, a sperm-activating peptide (SAP) from the starfish egg jelly coat, is diffusible and controls a cGMP-signalling pathway in starfish sperm in the same manner as resact, a potent chemoattracting SAP in sea urchins. This fact suggests that asterosap may serve as a chemoattractant like resact at concentrations with appropriate gradients. Since asterosap is one of three egg jelly components, which in concert induce the acrosome reaction, it is still worthwhile to evaluate how asterosap modulates sperm motility prior to this reaction. We analysed the flagellar movement of sperm of the starfish Aphelasterias japonica in artificial seawater (ASW) containing the asterosap isoform P15 at 1 micromol l(-1). We found that sperm swim straighter with more symmetrical flagellar movement in P15 than in ASW, but without any significant difference in the flagellar beat frequency and the swimming velocity. The flagellar movement is, however, dramatically different between sperm firmly attached to the solid surface by the head in P15 and those attached in ASW: in P15 the flagellum bends to a greater extent, with higher curvature and with higher shear angle up to a right angle to the flagellar wave axis, and beats at an increased frequency. The vigorous flagellar movement of sperm, which can be activated when sperm are placed in high-load circumstances just as entering into a jelly layer, may increase propulsive forces and hydrodynamic resistances, allowing sperm to undergo the acrosome reaction as effectively as possible.

Progression of Flagellar Stages during Artificially Delayed Motility Initiation in Sea Urchin Sperm

Abstract Transition from immotile to motile flagella may involve a series of states, in which some of regulatory mechanisms underlying normal flagellar movement are working with others being still suppressed. To address ourselves to the study of starting transients of flagella, we analyzed flagellar movement of sea urchin sperm whose motility initiation had been retarded in an experimental solution, so that we could capture the instance at which individual spermatozoa began their flagellar beating. Initially straight and immotile flagella began to shiver at low amplitude, then propagated exclusively the principal bend (P bend), and finally started stable flagellar beating. The site of generation of the P bend in the P-bend propagating stage varied in position in the basal region up to 10 μm from the base, indicating that the ability of autonomous bend generation is not exclusively possessed by the very basal region but can be unmasked throughout a wider region when the reverse bend (R bend) is suppressed. The rate of change in the shear angle, the curvature of the R bend and the frequency and regularity of beating substantially increased upon transition from P-bend propagating to full-beating, while the propagation velocity of bends remained unchanged. These findings indicate that artificially delayed motility initiation may accompany sequential modification of the motile system and that mechanisms underlying flagellar motility can be analyzed separately under experimentally retarded conditions.

Responses to Hypergravity in Proliferation of Paramecium tetraurelia

Abstract It has been reported that Paramecium proliferates faster when cultured under microgravity in orbit, and slower when cultured under hypergravity. This shows that the proliferation rate of Paramecium affected by gravity. The effect of gravity on Paramecium proliferation has been argued to be direct in a paper with an axenic culture under hypergravity. To clear up uncertainties with regard to the effect of gravity, Paramecium tetraurelia was cultured axenically under hypergravity (20 × g) and the time course of the proliferation was investigated quantitatively by a new non-invasive method, laser-beam optical slice, for measuring the cell density. This method includes optical slicing a part of the culture and computer-aided counting of cells in the sliced volume. The effects of hypergravity were assessed by comparing the kinetic parameters of proliferation that were obtained through a numerical analysis based on the logistic growth equation. Cells grown under 20 × g conditions had a significantly lower proliferation rate, and had a lower population density at the stationary phase. The lowered proliferation rate continued as long as cells were exposed to hypergravity (> one month). Hypergravity reduced the cell size of Paramecium. The long and short axes of the cell became shorter at 20 × g than those of control cells, which indicates a decrease in volume of the cell grown under hypergravity and is consistent with the reported increase in cell volume under microgravity. The reduced proliferation rate implies changes in biological time defined by fission age. In fact the length of autogamy immaturity decreased by measure of clock time, whereas it remained unchanged by measure of fission age.

Effect of gravity field on the nonequilibrium/nonlinear chemical oscillation reactions

Biological systems have evolved for a long time under the normal gravity. The Belousov-Zhabotinsky (BZ) reaction is a nonlinear chemical system far from the equilibrium that may be considered as a simplified chemical model of the biological systems so as to study the effect of gravity. The reaction solution is comprised of bromate in sulfuric acid as an oxidizing agent, 1,4-cyclohexanedione as an organic substrate, and ferroin as a metal catalyst. Chemical waves in the BZ reaction-diffusion system are visualized as blue and red patterns of ferriin and ferroin, respectively. After an improvement to the tubular reaction vessels in the experimental setup, the traveling velocity of chemical waves in aqueous solutions was measured in time series under normal gravity, microgravity, hyper-gravity, and normal gravity using the free-fall facility of JAMIC (Japan Microgravity Center), Hokkaido, Japan. Chemical patterns were collected as image data via CCD camera and analyzed by the software of NIH image after digitization. The estimated traveling velocity increased with increasing gravity as expected. It was clear experimentally that the traveling velocity of target patterns in reaction diffusion system was influenced by the effect of convection and correlated closely with the gravity field.

Developmental changes in Ca2+ sensitivity of sea-urchin embryo cilia

Abstract 1. 1. The swimming velocity and direction of sea-urchin embryos were directly measured by multiple-exposure photography. 2. 2. An increase in the Ca2+ concentration of the medium decreased the velocity in the stages from blastula through pluteus. 3. 3. The calcium ionophore A23187 added in the medium enhanced this Ca2+ effect throughout all stages, and further significantly increased the number of embryos that stopped swimming and the ratio of backward vs forward, only when added after a critical stage, where spontaneous backward swimming began to occur. 4. 4. These findings suggest that the regulatory mechanism of ciliary activity changes in its sensitivity to Ca2+ during development.

Behavior of Japanese tree frogs under microgravity on MIR and in parabolic flight.

Japanese tree frogs (Hyla japonica) were flown to the space station MIR and spent eight days in orbit during December, 1990. Under microgravity, their postures and behaviors were observed and recorded. On the MIR, floating frogs stretched four legs out, bent their bodies backward and expanded their abdomens. Frogs on a surface often bent their neck backward and walked backwards. This behavior was observed on parabolic flights and resembles the retching behavior of sick frogs on land--a possible indicator of motion sickness. Observations on MIR were carried out twice to investigate the frogs adaptation to space. The frequency of failure in landing after a jump decreased in the second observation period. After the frogs returned to earth, readaptation processes were observed. The frogs behaved normally as early as 2.5 hours after landing.

Three‐dimensional recording and measurement of swimming paths of micro‐organisms with two synchronized monochrome cameras

Instrumentation is described that permits analysis of the three‐dimensional (3‐D) swimming paths of micro‐organisms. The signals from two monochrome cameras for the front and side views of micro‐organisms are encoded into a composite signal for a video tape recorder (VTR). The signals decoded from the VTR or those directly from the cameras are processed by computer.