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The adaptation to a strong light is one of the essential characteristics of green algae, yet lacking relatively the information about the photophobic responses of Eukaryotic microalgae. We investigated the photophobic step-up responses of Euglena gracilis over a time course of several hours with alternated repetition of blue-light pulse illumination and spatially patterned blue-light illumination. Four distinctive photophobic motions in response to strong blue light were identified in a trace image analysis, namely on-site rotation, running and tumbling, continuous circular swimming, and unaffected straightforward swimming. The cells cultured in autotrophic conditions under weak light showed mainly the on-site rotation response at the beginning of blue-light illumination, but they acquired more blue-light tolerant responses of running and tumbling, circular swimming, or straightforward swimming. The efficiency of escaping from a blue-light illuminated area improved markedly with the development of these photophobic motions. Time constant of 3.0 h was deduced for the evolution of photophobic responses of E. gracilis. The nutrient-rich metabolic status of the cells resulting from photosynthesis during the experiments, i.e., the accumulation of photosynthesized nutrient products in balance between formation and consumption, was the main factor responsible for the development of photophobic responses. The reduction-oxidation status in and around E. gracilis cells did not affect their photophobic responses significantly, unlike the case of photophobic responses and phototaxis of Chlamydomonas reinhardtii cells. This study shows that the evolution of photophobic motion type of E. gracilis is dominated mainly by the nutrient metabolic status of the cells. The fact suggests that the nutrient-rich cells have a higher threshold for switching the flagellar motion from straightforward swimming to rotation under a strong light.

We found that the transient freezing behavior in photophobic responses of Euglena gracilis is a good indicator of the metabolic status of the cells. The transient blue light photophobic responses of E. gracilis cells were investigated on-chip using a new measurement, 'trace momentum' (TM), to evaluate their swimming activity quantitatively in real time. When blue light of intensity >30 mW cm(-2) was repeatedly switched on and off, a large negative spike in the TM was observed at the onset of the 'blue-light-off' phase. Single-cell trace analysis at a blue light intensity of 40 mW cm(-2) showed that 48% (on average, n = 15) of tumbling Euglena cells ceased activity ('freezing') for 2-30 s at the onset of blue-light-off before commencing forward motion in a straight line (termed 'straightforward swimming'), while 45% smoothly commenced straightforward swimming without delay. The proportion of freezing Euglena cells depended on the blue light intensity (only 20% at 20 mW cm(-2)). When the cells were stimulated by four blue light pulses at the higher intensity, without pre-exposure, the transient freezing behavior was more prominent but, on repeating the stimuli after an 80 min interval in red light, the same cells did not freeze. This shows that the metabolism of the cells had changed to anti-freezing during the interval. The relationship between the interval time with/without light irradiation and the blue light adaptation was elucidated experimentally. The origin of the freezing behavior is considered to be a shortage of a metabolic substance that promotes smooth switching of flagellum movement from in situ rotation mode to a straightforward swimming mode., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We examined two-dimensional (2D) optical feedback control of phototaxis flagellate Euglena cells confined in closed-type microfluidic channels (microaquariums), and demonstrated that the 2D optical feedback enables the control of the density and position of Euglena cells in microaquariums externally, flexibly, and dynamically. Using three types of feedback algorithms, the density of Euglena cells in a specified area can be controlled arbitrarily and dynamically, and more than 70% of the cells can be concentrated into a specified area. Separation of photo-sensitive/insensitive Euglena cells was also demonstrated. Moreover, Euglena-based neuro-computing has been achieved, where 16 imaginary neurons were defined as Euglena-activity levels in 16 individual areas in microaquariums. The study proves that 2D optical feedback control of photoreactive flagellate microbes is promising for microbial biology studies as well as applications such as microbe-based particle transportation in microfluidic channels or separation of photo-sensitive/insensitive microbes.

Abstract In this study, we developed a microfluidic system to elucidate the behavioral response of Euglena gracilis cells to simultaneous competing optical and chemical stimuli. The system illuminated a nonuniform blue light of 0.5–18 mW/cm2 on cells confined in a 2D microchamber to induce a photophobic response (step-up photo-shock). After cells accumulated in areas of weak blue light, a CO 2 gradient (0%–100%) was generated in the microchamber to induce counter chemotaxis (aerotaxis). E. gracilis cells showed negative chemotaxis for areas of higher CO 2 concentrations rather than strong blue light, suggesting that CO 2 chemotaxis is dominant over blue light photophobicity. We also examined phototaxis instead of photo-shock responses, using in-plane uniform blue light illumination in a counter direction to the CO 2 gradient. The cells exhibited both CO 2 chemotaxis and blue light phototaxis, and swam back and forth randomly. In both cases, some sensitive cells accumulated in niche areas, primarily with CO 2 concentration < 25% and secondarily with a weaker blue light intensity (further away from the blue light source). The results indicate that competing chemotactic and photophobic or phototactic stimuli induced superimposed responses, although E. gracilis was more strongly affected by CO 2 chemotaxis than by blue light. We propose that the two independent chemotaxis and photoresponse signal-transduction pathways are merged into one flagellar control mechanism. Moreover, a small proportion of cells were resistant to CO 2 or blue light, showing the diversity in cell metabolic status. Highlights • Development of a microfluidic system, where competing optical and chemical stimuli are simultaneously imposed to microalgae cells confined in a microchamber • Video observation of superimposed behavior of Euglena gracilis , with a non-absolute priority of CO 2 chemotaxis to photo-shock or phototaxis • Existence of diversity of cell characteristics involved in chemotaxis/phototaxis, originating from differences in metabolic status of the cells [ABSTRACT FROM AUTHOR]

Highlights • Bio-inspired neurocomputing with software noise oscillators that mimic the photophobic behaviors of microalgae cells of Euglena gracilis. • Advanced study from Euglena -cell-based neurocomputing we published in Appl. Soft Comput. [K. Ozasa et al., Appl. Soft Comput., 13 (2013) 527–538]. • Taking the spontaneous behaviors and the survival strategies of the real living cells into neurocomputing. • Dynamic transition among best or 2nd best solutions in a 16-city TSP combinatorial optimization. • Focusing on the elucidation of the origin of the relatively good performance in Euglena -inspired neurocomputing. Abstract We developed a bio-inspired neurocomputing approach based on our earlier biological neurocomputer, which leverages the survival strategies of living micro-algae cells ( Euglena gracilis ) to soft computing. Instead of using the real living cells, the bio-inspired neurocomputing in this study (namely, Euglena -inspired neurocomputing) mimics the photophobic responses of the cells using photo-responsive (PR) noise oscillators. The PR noise oscillators play the role of neurons during computation and their output signals are autonomously changed both by noise generation and firing of the neuron. The Euglena -inspired neurocomputing achieved a high performance in searching for multiple solutions continuously and autonomously for a combinatorial optimization problem, 16-city TSP as instance. We analyzed the temporal evolution of the computation and its dependence on the parameter set of the PR noise oscillators and identified the source of the high performance as the trade-off between noise amplitude and the reduction ratio of the oscillators. We next introduced two specific survival strategies observed in the real Euglena cells to PR noise oscillators, and elucidated their positive effects on the performance. The Euglena -inspired neurocomputing examined in this study can be used to address dynamically changing optimization problems, since the computation tracks changes in the imposed conditions by autonomous and non-converged searching for the solutions. [ABSTRACT FROM AUTHOR]

We improved our previous micro-device containing motile micro-algae ( Euglena gracilis ) cells for toxicity monitoring, by introducing micro-trenches between a micro-aquarium and two surrounding channels. Target test solution was introduced from the bypass channel to the micro-aquarium through micro-trenches, with a pulse injection and continuous diffusion. Although the chemical gradient formed in the micro-aquarium was not permanently sustainable but only temporal for a couple of 10 min, the device was simple, easy-to-use, and cost-effective to evaluate the toxicity of the test solution. The cells showed their chemotaxis to the temporal chemical gradients, resulting in the deviated distribution of the cells in the micro-aquarium, whereas metabolic disturbing effects of toxic substances induced the unusual motion of the cells. [ABSTRACT FROM AUTHOR]

Artificial linking of two isolated culture dishes is a fascinating means of investigating interactions among multiple groups of microbes or fungi. We examined artificial interaction between two isolated dishes containing Euglena cells, which are photophobic to strong blue light. The spatial distribution of swimming Euglena cells in two micro-aquariums in the dishes was evaluated as a set of new measures: the trace momentums (TMs). The blue light patterns next irradiated onto each dish were deduced from the set of TMs using digital or analogue feedback algorithms. In the digital feedback experiment, one of two different pattern-formation rules was imposed on each feedback system. The resultant cell distribution patterns satisfied the two rules with an AND operation, showing that cooperative interaction was realized in the interlink feedback. In the analogue experiment, two dishes A and B were interlinked by a feedback algorithm that illuminated dish A (B) with blue light of intensity proportional to the cell distribution in dish B (A). In this case, a distribution pattern and its reverse were autonomously formed in the two dishes. The autonomous formation of a pair of reversal patterns reflects a type of habitat separation realized by competitive interaction through the interlink feedback. According to this study, interlink feedback between two or more separate culture dishes enables artificial interactions between isolated microbial groups, and autonomous cellular distribution patterns will be achieved by correlating various microbial species, despite environmental and spatial scale incompatibilities. The optical interlink feedback is also useful for enhancing the performance of Euglena-based soft biocomputing. [ABSTRACT FROM AUTHOR]

Abstract: We report on neurocomputing performed with real Euglena cells confined in micro-aquariums, on which two-dimensional optical feedback is applied using the Hopfield–Tank algorithm. Trace momentum, an index of swimming activity of Euglena cells, is used as the input/output signal for neurons in the neurocomputation. Feedback as blue-light illumination results in temporal changes in trace momentum according to the photophobic reactions of Euglena. Combinatorial optimization for a four-city traveling salesman problem is achieved with a high occupation ratio of the best solutions. Two characteristics of Euglena-based neurocomputing desirable for combinatorial optimization are elucidated: (1) attaining one of the best solutions to the problem, and (2) searching for a number of solutions via dynamic transition between the best solutions. Mechanisms responsible for the two characteristics are analyzed in terms of network energy, photoreaction ratio, and dynamics/statistics of Euglena movements. The spontaneous fluctuation in input/output signals and reduction in photoreaction ratio were found to be key factors in producing characteristic (1), while the photo-insensitive Euglena cells or the accidental evacuation of cells from non-illuminated areas causes characteristic (2). Furthermore, we show that the photophobic reactions of Euglena involves various survival strategies such as adaptation to blue-light or awakening from dormancy, which can extend the performance of Euglena-based neurocomputing toward deadlock avoidance or program-less adaptation. Finally, two approaches for achieving a high-speed Euglena-inspired Si-based computation are described. [Copyright &y& Elsevier]

Cell division of unicellular microalgae is a fascinating process of proliferation, at which whole organelles are regenerated and distributed to two new lives. We performed dynamic live cell imaging of Euglena gracilis using optical microscopy to elucidate the mechanisms involved in the regulation of the eyespot and flagellum during cell division and distribution of the organelles into the two daughter cells. Single cells of the wild type (WT) and colorless SM-ZK cells were confined in a microfluidic device, and the appearance of the eyespot (stigma) and emergent flagellum was tracked in sequential video-recorded images obtained by automatic cell tracking and focusing. We examined 12 SM-ZK and 10 WT cells and deduced that the eyespot diminished in size and disappeared at an early stage of cell division and remained undetected for 26–97 min (62 min on average, 22 min in deviation). Subsequently, two small eyespots appeared and were distributed into the two daughter cells. Additionally, the emergent flagellum gradually shortened to zero-length, and two flagella emerged from the anterior ends of the daughter cells. Our observation revealed that the eyespot and flagellum of E. gracilis are degraded once in the cell division, and the carotenoids in the eyespot are also decomposed. Subsequently, the two eyespots/flagella are regenerated for distribution into daughter cells. As a logical conclusion, the two daughter cells generated from a single cell division possess the equivalent organelles and each E. gracilis cell has eternal or non-finite life span. The two newly regenerated eyespot and flagellum grow at different rates and mature at different timings in the two daughter cells, resulting in diverse cell characteristics in E. gracilis. [ABSTRACT FROM AUTHOR]

Gravitaxis is one of the most important issues in the growth of microalgae in the water column; it determines how easily cells receive sunlight with a comfortable intensity that is below the damaging threshold. We quantitatively investigated and analyzed the gravitaxis and cell multiplication of Euglena gracilis using vertically placed microchambers containing a single cell. A temporal change in gravitaxis and cell multiplication was observed after transferring the cells to fresh culture medium for 9 days. We performed 29 individual experiments with 2.5 mm × 2.5 mm × 0.1 mm square microchambers and found that the cells showed positive, negative, and moderate gravitaxis in 8, 7, and 14 cases, respectively, after transferring to fresh culture medium. A common trend was observed for the temporal change in gravitaxis for the eight initially positive gravitaxis cases. The cells with initially positive gravitaxis showed a higher rate of cell multiplication than those with initially negative gravitaxis. We also discussed the gravitaxis mechanism of E. gracilis from the observed trend of gravitaxis change and swimming traces. In addition, bioconvection in a larger and thicker chamber was investigated at a millimeter scale and visualized. [ABSTRACT FROM AUTHOR]

Abstract: Microbe-based neural network computing, where the reaction of microbial cells to external stimuli is incorporated in the function of virtual neurons, has high potential for developing soft computing based on the survival strategies of the microbe. To utilize reaction-threshold diversity among the cells, we examined analog feedback in Euglena-based neurocomputing by solving a simple combinatorial optimization problem. The analog feedback was performed by blue light illumination to Euglena cells, where the intensity of the blue light was controlled using the Hopfield-Tank algorithm with a sigmoid function. The solution patterns obtained with analog feedback had greater variations than those with digital feedback, implying that the solution-search capability of Euglena-based neurocomputing is enhanced by analog feedback. Moreover, the solutions obtained with analog feedback comprised one stable core-motive selection and additional flexible selections, which are associated with hesitation shown by humans when faced with a frustrated task. The study shows that using analog feedback in Euglena-based neurocomputing is promising in terms of incorporating the diversity of photoreactions of Euglena cells to enhance the solution-search capability for combinatorial optimization problems and to utilize the adaptive reaction of Euglena cells. [Copyright &y& Elsevier]