The shape effect of flagella is more important than bottom-heaviness on passive gravitactic orientation in Chlamydomonas reinhardtii.

The way the unicellular, biflagellated, green alga Chlamydomonas orients upward has long been discussed in terms of both mechanics and physiology. In this study, we focus on the mechanics, i.e. the 'passive' mechanisms, of gravitaxis. To rotate the body upwards, cellular asymmetry is critical. Chlamydomonas can be depicted as a nearly spherical cell body with two anterior, symmetric flagella. The present study looks at the question of whether the existence of the flagella significantly affects torque generation in upward reorientation. The 'density asymmetry model' assumes that the cell is spherical and bottom-heavy and that the shape and weight of the flagella are negligible, while the 'shape asymmetry model' considers the shape of the flagella. Both our experimental and simulation results revealed a considerable contribution from shape asymmetry to the upward orientation of Chlamydomonas reinhardtii, which was several times larger than that of density asymmetry. From the experimental results, we also quantified the extent of bottom-heaviness, i.e. the distance between the centers of gravity and the figure when the cell body is assumed to be spherical. Our estimation was approximately 30 nm, only one-third of previous assumptions. These findings indicate the importance of the viscous drag of the flagella to the upward orientation, and thus negative gravitaxis, in Chlamydomonas.

Responses of Bioconvection of Tetrahymena thermophila to Partial Gravity Created by Aircraft Flight.

Bioconvection is a form of collective pattern formation driven by negative gravitaxis of swimming microorganisms. In bioconvection, the interaction between individual swimmers results in self-organization leading to the development of a macroscopic structure typically 100-1000 times greater than an individual microorganism. To gain insight into the role of gravity in this self-organization phenomenon, we investigated the bioconvective behavior of the ciliate Tetrahymena thermophila under short-term partial gravity, i.e., gravitational acceleration < 1 g, achieved by quasiparabolic flight maneuvers of an aircraft. The bioconvective responses of T. thermophila were assessed by observing the collective motion simultaneously in two separate scales, which we call macroscale and microscale, using a newly designed "dual-objective" device with two different magnifications. Microscale analysis revealed that the magnitude of gravikinesis, i.e., active regulation of the propulsive thrust, decreased almost linearly with changes in gravitational acceleration, while gravitactic characteristics, assessed by the distribution of the swimming direction, did not change significantly during partial gravity. Macroscale analysis demonstrated that downward plumes of convection pattern gradually shortened from the lower end, and disappeared under partial gravity. The sustained time of the plumes decreased almost linearly with changes in gravitational acceleration. The response of downward plumes to partial gravity may be attributable to the accumulation of cells into blobs in downward migration, which increases the rate of downward migration enough to exceed the rate of upward movement, which is enhanced due to gravikinesis. This suggests that gravity may act on cells involved in collective pattern formation differently than on free-swimming cells.

Individual Flagellar Waveform Affects Collective Behavior of Chlamydomonas reinhardtii.

Bioconvection is a form of collective motion that occurs spontaneously in the suspension of swimming microorganisms. In a previous study, we quantitatively described the "pattern transition," a phase transition phenomenon that so far has exclusively been observed in bioconvection of the unicellular green alga Chlamydomonas. We suggested that the transition could be induced by changes in the balance between the gravitational and shear-induced torques, both of which act to determine the orientation of the organism in the shear flow. As both of the torques should be affected by the geometry of the Chlamydomonas cell, alteration in the flagellar waveform might change the extent of torque generation by altering overall geometry of the cell. Based on this working hypothesis, we examined bioconvection behavior of two flagellar mutants of Chlamydomonas reinhardtii, ida1 and oda2, making reference to the wild type. Flagella of ida1 beat with an abnormal waveform, while flagella of oda2 show a normal waveform but lower beat frequency. As a result, both mutants had swimming speed of less than 50% of the wild type. ida1 formed bioconvection patterns with smaller spacing than those of wild type and oda2. Two-axis view revealed the periodic movement of the settling blobs of ida1, while oda2 showed qualitatively similar behavior to that of wild type. Unexpectedly, ida1 showed stronger negative gravitaxis than did wild type, while oda2 showed relatively weak gravitaxis. These findings suggest that flagellar waveform, not swimming speed or beat frequency, strongly affect bioconvection behavior in C. reinhardtii.

Swimming ability and flagellar motility of sperm packets of the volvocine green alga Pleodorina starrii.

Eukaryotic flagella collectively form metachronal waves that facilitate the ability to cause flow or swim. Among such flagellated and planktonic swimmers, large volvocine genera such as Eudorina, Pleodorina and Volvox form bundles of small male gametes (sperm) called "sperm packets" for sexual reproduction. Although these sperm packets reportedly have flagella and the ability to swim, previous studies on volvocine motility have focused on asexual forms and the swimming characteristics of sperm packets remain unknown. However, it is important to quantify the motility of sperm packets and sperm in order to gain insights into the significance of motility in the sexual reproduction of planktonic algae. In this study, we quantitatively described the behavior of three flagellated forms of a male strain of Pleodorina starrii-asexual colonies, sperm packets, and single dissociated sperm-with emphasis on comparison of the two multicellular forms. Despite being smaller, sperm packets swam approximately 1.4 times faster than the asexual colonies of the same male strain. Body length was approximately 0.5 times smaller in the sperm packets than in asexual colonies. The flagella from sperm packets and asexual colonies showed asymmetric waveforms, whereas those from dissociated single sperm showed symmetric waveforms, suggesting the presence of a switching mechanism between sperm packets and dissociated sperm. Flagella from sperm packets were approximately 0.5 times shorter and had a beat period approximately twice as long as those from asexual colonies. The flagella of sperm packets were densely distributed over the anterior part of the body, whereas the flagella of asexual colonies were sparse and evenly distributed. The distribution of flagella, but not the number of flagella, appear to illustrate a significant difference in the speeds of sperm packets and asexual colonies. Our findings reveal novel aspects of the regulation of eukaryotic flagella and shed light on the role of flagellar motility in sexual reproduction of planktonic algae.

Drastic reorganization of the bioconvection pattern of Chlamydomonas: quantitative analysis of the pattern transition response.

Motile aquatic microorganisms are known to self-organize into bioconvection patterns. The swimming activity of a population of microorganisms leads to the emergence of macroscopic patterns of density under the influence of gravity. Although long-term development of the bioconvection pattern is important in order to elucidate the possible integration of physiological functions of individuals through bioconvection pattern formation, little quantitative investigation has been carried out. In the present paper, we present the first quantitative description of long-term behavior of bioconvection of Chlamydomonas reinhardtii, particularly focusing on the 'pattern transition response'. The pattern transition response is a sudden breakdown of the steady bioconvection pattern followed by re-formation of the pattern with a decreased wavelength. We found three phases in the pattern formation of the bioconvection of C. reinhardtii: onset, steady-state 1 (before the transition) and steady-state 2 (after the transition). In onset, the wavelength of the bioconvection pattern increases with increasing depth, but not in steady-states 1 or 2. By means of the newly developed two-axis view method, we revealed that the population of C. reinhardtii moves toward the bottom of the experimental chamber just before the pattern transition. This indicates that the pattern transition response could be caused by enhancement of the gyrotaxis of C. reinhardtii as a result of the changes in the balance between the gravitactic and gyrotactic torques. We also found that the bioconvection pattern changes in response to the intensity of red-light illumination, to which C. reinhardtii is phototactically insensitive. These facts suggest that the bioconvection pattern has a potential to drastically reorganize its convection structure in response to the physiological processes under the influence of environmental cues.

Gravity-dependent changes in bioconvection of Tetrahymena and Chlamydomonas during parabolic flight: increases in wave number induced by pre- and post-parabola hypergravity.

Bioconvection emerges in a dense suspension of swimming protists as a consequence of their negative-gravitactic upward migration and later settling as a blob of density greater than that of water. Thus, gravity is an important parameter governing bioconvective pattern formation. However, inconsistencies are found in previous studies dealing with the response of bioconvection patterns to increased gravity acceleration (hypergravity); the wave number of the patterns has been reported to decrease during the hypergravity phases of parabolic aircraft flight, while it increases in centrifugal hypergravity. In this paper, we reassess the responses of bioconvection to altered gravity during parabolic flight on the basis of vertical and horizontal observations of the patterns formed by Tetrahymena thermophila and Chlamydomonas reinhardtii. Spatiotemporal analyses of the horizontal patterns revealed an increase in the pattern wave number in both pre- and post-parabola hypergravity. Vertical pattern analysis was generally in line with the horizontal pattern analysis, and further revealed that hypergravity-induced changes preceded at the top layer of the suspensions while microgravity-induced changes appeared to occur from the bottom part of the settling blobs. The responses to altered gravity were rather different between the two sample species: T. thermophila tended to drastically modify its bioconvection patterns in response to changes in gravity level, while the patterns of C. reinhardtii responded to a much lesser extent. This difference can be attributed to the distinct physical and physiological properties of the individual organisms, suggesting a significant contribution of the gyrotactic property to the swimming behavior of some protists.

A Microfluidic Diagnostic Device Capable of Autonomous Sample Mixing and Dispensing for the Simultaneous Genetic Detection of Multiple Plant Viruses.

As an efficient approach to risk management in agriculture, the elimination of losses due to plant diseases and insect pests is one of the most important and urgent technological challenges for improving the crop yield. Therefore, we have developed a polydimethylsiloxane (PDMS)-based microfluidic device for the multiplex genetic diagnosis of plant diseases and pests. It offers unique features, such as rapid detection, portability, simplicity, and the low-cost genetic diagnosis of a wide variety of plant viruses. In this study, to realize such a diagnostic device, we developed a method for the autonomous dispensing of fluid into a microchamber array, which was integrated with a set of three passive stop valves with different burst pressures (referred to as phaseguides) to facilitate precise fluid handling. Additionally, we estimated the mixing efficiencies of several types of passive mixers (referred to as chaotic mixers), which were integrated into a microchannel, through experimental and computational analyses. We first demonstrated the ability of the fabricated diagnostic devices to detect DNA-based plant viruses from an infected tomato crop based on the loop-mediated isothermal amplification (LAMP) method. Moreover, we demonstrated the simultaneous detection of RNA-based plant viruses, which can infect cucurbits, by using the reverse transcription LAMP (RT-LAMP) method. The multiplex RT-LAMP assays revealed that multiple RNA viruses extracted from diseased cucumber leaves were successfully detected within 60 min, without any cross-contamination between reaction microchambers, on our diagnostic device.

Inhomogeneous distribution of Chlamydomonas in a cylindrical container with a bubble plume.

Swimming microalgae show various taxes, such as phototaxis and gravitaxis, which sometimes result in the formation of a cell-rich layer or a patch in a suspension. Despite intensive studies on the effects of shear flow and turbulence on the inhomogeneous distribution of microalgae, the effect of a bubble plume has remained unclear. In this study, we used Chlamydomonas as model microalgae, and investigated the spatial distribution of cells in a cylindrical container with a bubble plume. The results illustrate that cells become inhomogeneously distributed in the suspension due to their motility and photo-responses. A vortical ring distribution was observed below the free surface when the bubble flow rate was sufficiently small. We performed a scaling analysis on the length scale of the vortical ring, which captured the main features of the experimental results. These findings are important in understanding transport phenomena in a microalgae suspension with a bubble plume.