Phototactic Algae Research Articles

Effects of oblique irradiation on the onset of phototactic bioconvection

The linear stability of a suspension of phototactic algae is investigated numerically with particular emphasis on the effects of angle of incidence of the illuminating oblique collimated irradiation. The solutions show a transition of the most unstable mode from stationary to overstable state or vice versa for certain parameters at the variation in angle of incidence. Furthermore, a transition from mode 2 to mode 1 instability is observed at the variation of angle of incidence for some parameter values. Oscillatory instabilities are also frequently observed in the rigid upper surface case at the increment in angle of incidence.

Emerging Diversity of Channelrhodopsins and Their Structure-Function Relationships.

Cation and anion channelrhodopsins (CCRs and ACRs, respectively) from phototactic algae have become widely used as genetically encoded molecular tools to control cell membrane potential with light. Recent advances in polynucleotide sequencing, especially in environmental samples, have led to identification of hundreds of channelrhodopsin homologs in many phylogenetic lineages, including non-photosynthetic protists. Only a few CCRs and ACRs have been characterized in detail, but there are indications that ion channel function has evolved within the rhodopsin superfamily by convergent routes. The diversity of channelrhodopsins provides an exceptional platform for the study of structure-function evolution in membrane proteins. Here we review the current state of channelrhodopsin research and outline perspectives for its further development.

Chlamydomonas reinhardtii swimming in the Plateau borders of 2D foams.

Unicellular Chlamydomonas reinhardtii micro-algae cells were inserted into a quasi-2D Hele-Shaw chamber filled with saponin foam. The movement of the algae along the bubble borders was then manipulated and tracked. These self-propelled particles generate flow and stresses in their surrounding matter. In addition, the algae possess the capability of exerting forces that alter bubble boundaries while maintaining an imminent phototactic movement. We find that by controlling the gas fraction of the foam we can change the interaction of the algae and bubbles. Specifically, our data expose three distinct swimming regimes for the algae with respect to the level of confinement due to the Plateau border cross-section: unlimited bulk, transition, and overdamped regimes. At the transition regime we find the speed of the algae to be modeled by a simple force balance equation emerging from the shear inside the Plateau border. Thus, we have shown that it is possible to create an algae-friendly foam while controlling the algae motion. This opens doors to multiple applications where the flow of nutrients, oxygen and recirculation of living organisms is essential.

Probing Channelrhodopsin Electrical Activity in Algal Cell Populations.

Photoelectric recording from populations of phototactic flagellate algae provides a means to study channelrhodopsin functions in vivo. Technical simplicity, versatility, high sensitivity, and reproducibility are the advantages of this assay over recording from individual algal cells by the suction pipette technique. Here we describe the principles and procedures of this assay.

Effects of anisotropic scattering on the onset of phototactic bioconvection with diffuse and collimated irradiation

The ability of many photosynthetic micro-organisms (algae) to detect a localized light source in their environment and move toward (away from) it is known as positive (negative) phototaxis. The phototactic algae absorb the incident light and scatter it isotropically or anisotropically thereafter across the suspension, which is illuminated by both diffuse and collimated irradiation. In this paper, we use the generic model of Panda et al. [“Effects of both diffuse and collimated incident radiation on phototactic bioconvection,” Phys. Fluids 28, 124104 (2016)] for phototaxis and investigate the effects of anisotropic (forward) scattering at the onset of phototactic bioconvection. In the motionless basic state, the up and down swimming caused by positive and negative phototaxis is balanced by diffusion due to randomness in the swimming behavior of algae. The forward scattering improves the underwater light distribution (radiation field) by propagating the light deeper into the algal suspension. At the equilibrium state, the bimodal (respectively, unimodal) base concentration profiles are shifted into the unimodal (respectively, bimodal) ones due to forward scattering for some fixed governing parameters when self-shading (absorption) is insignificant. As a result of forward scattering, for some parameter values, the most unstable solution shifts from mode 1 to mode 2 and a single oscillatory branch bifurcates from the stationary branch or disappears at bioconvective instability when self-shading (absorption) is significant. The dominant bioconvection pattern wavelength at instability is also qualitatively in good agreement with the experimental observations as the forward scattering coefficient is varied.

Sensitivity of Chlamydomonas reinhardtii to cadmium stress is associated with phototaxis.

Cadmium (Cd) is a common hazardous pollutant to aquatic environments and it easily accumulates in living organisms. The roles of phototactic behavior in Cd tolerance in motile organisms are poorly explored. In this study, two Chlamydomonas reinhardtii strains, a wild type with positive phototaxis (CC125) and a negatively phototactic mutant (agg1), were used to assess the effects of phototaxis on Cd-induced toxicity to algae. Exposure to Cd inhibited the cell growth and photosynthetic activities, reduced the photosynthetic pigment content, and enhanced the intracellular oxidative stress of algae. Well buffered by EDTA in algae medium, the concentrations of Cd causing 50% growth inhibition (EC50) of CC125 and agg1 for 72h of exposure were 55.96 and 77.20 μM L-1, respectively. Photosystem II activities in CC125 were more sensitive to Cd than agg1 at 60 μM L-1 Cd. In addition, agg1 accumulated less intracellular Cd than CC125. The changes of extracellular polymeric substances and intracellular response to Cd stress might be related to the different tolerances of the two algae to Cd. Taken together, phototaxis was demonstrated to be associated with Cd-induced toxicity to C. reinhardtii.

Cytoplasmic extensions of the channelrhodopsins 1 and 2 interacts in Chlamydomonas reinhardtii

Channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2) are directly light-gated ion channels that are being extensively utilized in the field of optogenetics. These proteins are present in the eyespot of a phototactic alga called Chlamydomonas reinhardtii and mediate its motility behavior (phototaxis and photophobic responses). Both ChR1 and ChR2 are two seven transmembrane proteins with N-terminal rhodopsin domain and C-terminal cytoplasmic extensions of unidentified function. Earlier studies have suggested the interaction between ChR1 and ChR2. However, the evidences of physical interaction between ChRs are still lacking. Here, we have dissected the possibility of the direct interaction between the cytoplasmic C-terminus extensions of the ChR1 and ChR2. The immunoprecipitation, co-precipitation, colocalization and the proximity ligation assay showed the interaction between ChR1 and ChR2. In addition, the in vitro GST pull down assay showed that ChR1 and ChR2 interact directly with their C-terminal extensions. Our study leads to the model where C-terminal cytoplasmic extensions of ChR1 and ChR2 could directly interact with each other in Chlamydomonas. In vivo, this interaction could serve to directly regulate the activity or control binding to downstream effectors (Figure 1).

Effects of both diffuse and collimated incident radiation on phototactic bioconvection

Phototaxis denotes swimming towards (positive) or away (negative) from light. The suspension containing phototactic algae is illuminated by both the diffuse and collimated solar radiation. The algae absorb the incident light and scatter it. We use the phototaxis model of Ghorai et al. [“Bioconvection in a suspension of isotropically scattering phototactic algae,” Phys. Fluids 22, 071901 (2010)] and investigate the onset of bioconvection with particular emphasis on the effects of diffuse irradiation. The basic equilibrium state of the bioconvective governing system is defined by assuming that the bulk velocity of the fluid to be zero and the up and down swimming, caused by the positive and negative phototaxis, is balanced with the diffusion. For some values of the parameters, the bimodal steady-state profile transits to a unimodal equilibrium state as the diffuse irradiation is increased. For a small scattering albedo, at the onset of bioconvective instability, this model differs significantly from the up-swimming model of Vincent and Hill [“Bioconvection in a suspension of phototactic algae,” J. Fluid Mech. 327, 343 (1996)], even for small wavelengths. Furthermore, the solutions show a transition of the most unstable mode from the stationary to oscillatory state, and then back to the stationary state again, as the governing parameters are varied. A significant stabilizing effect on suspension has also been observed due to the effects of diffuse irradiation. The effect of the diffuse irradiation on a dominant bioconvection pattern wavelength at instability is also qualitatively in good agreement with the bioconvection experiments.

Clustering instability of focused swimmers

One of the hallmarks of active matter is its rich nonlinear dynamics and instabilities. Recent numerical simulations of phototactic algae showed that a thin jet of swimmers, obtained from hydrodynamic focusing inside a Poiseuille flow, was unstable to longitudinal perturbations with swimmers dynamically clustering (Jibuti L. et al., Phys. Rev. E, 90, (2014) 063019). As a simple starting point to understand these instabilities, we consider in this paper an initially homogeneous one-dimensional line of aligned swimmers moving along the same direction, and characterise its instability using both a continuum framework and a discrete approach. In both cases, we show that hydrodynamic interactions between the swimmers lead to instabilities in density for which we compute the growth rate analytically. Lines of pusher-type swimmers are predicted to remain stable while lines of pullers (such as flagellated algae) are predicted to always be unstable.

Penetrative phototactic bioconvection in a two-dimensional non-scattering suspension

Phototaxis is a directed swimming response dependent upon the light intensity sensed by microorganisms. Positive (negative) phototaxis denotes the motion directed towards (away from) the source of light. In this paper, we simulate numerically penetrative phototactic bioconvection in a non-scattering suspension of phototactic algae in the non-linear regime. The suspension is confined by a stress-free top boundary, and rigid bottom and lateral boundaries. The algae receive light from the source directly above it and thus they swim vertically upward by neglecting the effects of scattering. We use the phototaxis model proposed by Vincent and Hill [“Bioconvection in a suspension of phototactic algae,” J. Fluid Mech. 327, 343 (1996)] to investigate the onset of bioconvection in two dimensions using the stream function-vorticity formulation. The critical conditions from the onset of bioconvection are used to study the effects of different governing parameters on the structure and stability of the obtained solutions. The resulting bioconvective patterns differ qualitatively from those found by Ghorai and Hill [“Penetrative phototactic bioconvection,” Phys. Fluids 17, 074101 (2005)] at some particular swimming velocity of microorganisms due to rigid lateral walls. A significant stabilizing effect on suspension has been also observed due to lateral rigid walls for some governing parameters.

306 走光性藻類ボルボックスを駆動源とする光制御型マイクロシステムの開発(OS3-2 MEMSと先進材料(2),OS3 MEMSと先進材料)

306 走光性藻類ボルボックスを駆動源とする光制御型マイクロシステムの開発(OS3-2 MEMSと先進材料(2),OS3 MEMSと先進材料)

Characterization of a Highly Efficient Blue-shifted Channelrhodopsin from the Marine Alga Platymonas subcordiformis

Rhodopsin photosensors of phototactic algae act as light-gated cation channels when expressed in animal cells. These proteins (channelrhodopsins) are extensively used for millisecond scale photocontrol of cellular functions (optogenetics). We report characterization of PsChR, one of the phototaxis receptors in the alga Platymonas (Tetraselmis) subcordiformis. PsChR exhibited ∼3-fold higher unitary conductance and greater relative permeability for Na(+) ions, as compared with the most frequently used channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Photocurrents generated by PsChR in HEK293 cells showed lesser inactivation and faster peak recovery than those by CrChR2. Their maximal spectral sensitivity was at 445 nm, making PsChR the most blue-shifted channelrhodopsin so far identified. The λmax of detergent-purified PsChR was 437 nm at neutral pH and exhibited red shifts (pKa values at 6.6 and 3.8) upon acidification. The purified pigment undergoes a photocycle with a prominent red-shifted intermediate whose formation and decay kinetics match the kinetics of channel opening and closing. The rise and decay of an M-like intermediate prior to formation of this putative conductive state were faster than in CrChR2. PsChR mediated sufficient light-induced membrane depolarization in cultured hippocampal neurons to trigger reliable repetitive spiking at the upper threshold frequency of the neurons. At low frequencies spiking probability decreases less with PsChR than with CrChR2 because of the faster recovery of the former. Its blue-shifted absorption enables optogenetics at wavelengths even below 400 nm. A combination of characteristics makes PsChR important for further research on structure-function relationships in ChRs and potentially useful for optogenetics, especially for combinatorial applications when short wavelength excitation is required.

Penetrative phototactic bioconvection in an isotropic scattering suspension

Phototaxis is a directed swimming response dependent upon the light intensity sensed by micro-organisms. Positive (negative) phototaxis denotes the motion directed towards (away from) the source of light. Using the phototaxis model of Ghorai, Panda, and Hill [“Bioconvection in a suspension of isotropically scattering phototactic algae,” Phys. Fluids 22, 071901 (2010)]10.1063/1.3457163, we investigate two-dimensional phototactic bioconvection in an absorbing and isotropic scattering suspension in the nonlinear regime. The suspension is confined by a rigid bottom boundary, and stress-free top and lateral boundaries. The governing equations for phototactic bioconvection consist of Navier–Stokes equations for an incompressible fluid coupled with a conservation equation for micro-organisms and the radiative transfer equation for light transport. The governing system is solved efficiently using a semi-implicit second-order accurate conservative finite-difference method. The radiative transfer equation is solved by the finite volume method using a suitable step scheme. The resulting bioconvective patterns differ qualitatively from those found by Ghorai and Hill [“Penetrative phototactic bioconvection,” Phys. Fluids 17, 074101 (2005)]10.1063/1.1947807 at a higher critical wavelength due to the effects of scattering. The solutions show transition from steady state to periodic oscillations as the governing parameters are varied. Also, we notice the accumulation of micro-organisms in two horizontal layers at two different depths via their mean swimming orientation profile for some governing parameters at a higher scattering albedo.

Bioconvection in an anisotropic scattering suspension of phototactic algae

Phototaxis is a directed swimming response dependent upon the light intensity sensed by microorganisms. Positive phototaxis denotes motion directed towards the source of light and negative phototaxis is motion directed away from it. In this paper, we investigate the onset of bioconvection in a suspension of anisotropic scattering phototactic algae illuminated by collimated radiation at the top. The basic state of the system is defined by the zero fluid flow and the up and down swimming, caused by the positive and negative phototaxis, is balanced by the diffusion. A comprehensive numerical study of the linear stability is presented with particular emphasis on the forward scattering effect. The onset of bioconvection occurs either via a stationary mode or an oscillatory mode. The transition from a stationary mode to an oscillatory mode or vice versa has been observed as the anisotropic coefficient is varied for certain parameter values.

2D measurement of ion currents associated to the signal transduction of the phototactic alga Chlamydomonas reinhardtii

Our objective was to develop a simple procedure for the detection of light-induced ion currents of photomotile cells in two dimensions. The novel technique was based on the light gradient method (LGM), and the model object was Chlamydomonas reinhardtii, a phototactic unicellular alga, ideal for such experiments. The conventional LGM cuvette was modified such that the electrode pair could be rotated around the sample and pick up the electric signals from arbitrary directions. The experiments were performed with and without the application of an auxiliary light beam preorienting the motile cells. The analysis of the detected traces revealed two main vectorial components of the signal by the help of singular value decomposition (SVD), in concert with previous experimental findings and theoretical considerations suggesting different origins of the “fast” and “slow” components of the photoelectric response of Chlamydomonas and Haematococcus cells. Using plausible assumptions, our method allowed a quantitative analysis of the signal, assigning size and direction to the two vectorial components. The method allows a rapid and accurate way to measure electric signals of photomotive cells in 2D, and particularly to test the physiological activity and in vivo-kinetics of site-directed mutants of ChR1 or ChR2, providing novel photo-electrophysiological methods with important quantitative information.

A tale of three taxes: photo-gyro-gravitactic bioconvection

The term bioconvection encapsulates the intricate patterns in concentration, due to hydrodynamic instabilities, that may arise in suspensions of non-neutrally buoyant, biased swimming microorganisms. The directional bias may be due to light (phototaxis), gravity (gravitaxis), a combination of viscous and gravitational torques (gyrotaxis) or other taxes. The aim of this study is to quantify experimentally the wavelength of the initial pattern to form from an initially well-mixed suspension of unicellular, swimming green algae as a function of concentration and illumination. As this is the first such study, it is necessary to develop a robust and meticulous methodology to achieve this end. The phototactic, gyrotactic and gravitactic alga Chlamydomonas augustae was employed, with various red or white light intensities from above or below, as the three not altogether separable taxes were probed. Whilst bioconvection was found to be unresponsive to changes in red light, intriguing trends were found for pattern wavelength as a function of white light intensity, depending critically on the orientation of the illumination. These trends are explored to help unravel the mechanisms. Furthermore, comparisons are made with theoretical predictions of initial wavelengths from a recent model of photo-gyrotaxis, encouragingly revealing good qualitative agreement.

Bioconvection in a suspension of isotropically scattering phototactic algae

Phototaxis is a directed swimming response toward a light source sensed by micro-organisms. Positive phototaxis represents swimming toward the source of light intensity and negative phototaxis is the swimming away from it. In this paper we develop a new model for phototaxis that incorporates the effects of absorption and scattering by the micro-organisms. This model is then used to analyze the linear stability of a suspension of phototactic algae illuminated by a collimated radiation at the top. A comprehensive numerical study of the linear stability is presented with particular emphasis on the scattering effect. As a result of scattering, for some parameter values, the micro-organisms accumulate in two horizontal layers at different depths in the basic equilibrium state. Examples of oscillatory instabilities are also found.

Two Open States with Progressive Proton Selectivities in the Branched Channelrhodopsin-2 Photocycle

Channelrhodopsins are light-gated ion channels that mediate vision in phototactic green algae like Chlamydomonas. In neurosciences, channelrhodopsins are widely used to light-trigger action potentials in transfected cells. All known channelrhodopsins preferentially conduct H+. Previous studies have indicated the existence of an early and a late conducting state within the channelrhodopsin photocycle. Here, we show that for channelrhodopsin-2 expressed in Xenopus oocytes and HEK cells, the two open states have different ion selectivities that cause changes in the channelrhodopsin-2 reversal voltage during a light pulse. An enzyme kinetic algorithm was applied to convert the reversal voltages in various ionic conditions to conductance ratios for H+ and divalent cations (Ca2+ and/or Mg2+), as compared to monovalent cations (Na+ and/or K+). Compared to monovalent cation conductance, the H+ conductance, α, is ∼3 × 106 and the divalent cation conductance, β, is ∼0.01 in the early conducting state. In the stationary mixture of the early and late states, α is larger and β smaller, both by a factor of ∼2. The results suggest that the ionic basis of light perception in Chlamydomonas is relatively nonspecific in the beginning of a light pulse but becomes more selective for protons during longer light exposures.

Photoresponses of the purple nonsulfur bacteria Rhodospirillum centenum and Rhodobacter sphaeroides.

We have measured the photoresponse of two purple nonsulfur bacteria, Rhodobacter sphaeroides and Rhodospirillum centenum, under defined conditions in a light beam propagating at 90 degrees to the optical axis of the microscope. This beam presented cells with a steep gradient of intensity perpendicular to the direction of propagation and a shallow gradient in the direction of light propagation. R. centenum, a species that reverses to change direction, accumulated in the light beam, as expected for a "scotophobic" response, while R. sphaeroides, which stops rather than reverses, accumulated outside the light beam. We also compared the behavior of liquid-grown R. centenum, which swims by using a single polar flagellum, to that of surface-grown R. centenum, which swarms over agar by using many lateral flagella and has been shown to move as colonies toward specific wavelengths of light. When suspended in liquid medium, both liquid- and surface-grown R. centenum showed similar responses to the light gradient. In all cases, free-swimming cells responded to the steep gradient of intensity but not to the shallow gradient, indicating they cannot sense the direction of light propagation but only its intensity. In a control experiment, the known phototactic alga Chlamydamonas reinhardtii was shown to swim in the direction of light propagation.

Electric currents evoked by double-flash excitation of the phototactic alga Chlamydomonas reinhardtii

The electric responses that accompany photosensory transduction in Chlamydomonas reinhardtii were measured using a recently developed suspension method. Double-laser-flash excitations were applied to acquire more information on the nature of the photocycle of the retina containing protein in the eye spot, and on the nature of the different components of the photoelectric signals (‘photoreceptor potential’ and ‘regenerative response’). It was found that blue light, as a second illumination following the driving flash with a wavelength of 500 nm, did not affect the membrane-coupled electric events. A characteristic turnover time of nearly 1 s was established for these processes with a second flash of wavelength 500 nm. The results are discussed in terms of their relevance to the photocycle of the retinal protein and to the signal transduction mechanism.