Microorganisms can change their mode of locomotion by responding to various stimuli, such as chemicals (Berg, 1975), gravity (Häder et al., 2005), and light (Häder and Iseki, 2017). These behavioral responses are called taxes (Pedley and Kessler, 1992); these responses cause a macroscopic phenomenon called bioconvection. Wager (1911) reported net-like patterns that appeared within dense Euglena suspensions in various containers and demonstrated that variations in aggregation occurred when stimulated by external light targeted vertically down from the top. Platt (1961) considered the reasons for the onset of bioconvection patterns observed in various microorganisms; consequently, these bioconvections were not due to Rayleigh–Bénard thermal convections. The container's depth and the suspension's average cell density were determined to be important for the formation of this bioconvection. Additionally, Williams and Bees (2011) showed the time evolution of pattern formation of Chlamydomonas bioconvections in dense suspensions. Chlamydomonas bioconvection occurs due to three taxes (phototaxis, gyrotaxis, and gravitaxis) which are not completely independent. They showed variation in this time evolution depending on the intensity of light treatment from above and below the suspensions. Recently, Ramamonjy et al. (2022) demonstrated the detailed interaction between phototaxis and bioconvection in Chlamydomonas.

This study focused on the bioconvection that was induced in a Euglena suspension due to negative phototaxis. Euglena is a well-known unicellular flagellate with a body length of approximately 50 μm and spindle-like shape. This flagellate moves forward by moving a single anterior flagellum (puller-type swimmer) (Ogawa et al., 2017; Rossi et al., 2017). Euglena is a photosynthetic microorganism with positive and negative phototaxis (Diehn, 1973). Euglena moves toward a weak light source (positive phototaxis) and away from a strong light source (negative phototaxis). When a strong light from below illuminates the diluted suspension of Euglena, it moves toward the top of the suspension due to the corresponding negative phototaxis. As a result, the local cell density of the microorganisms near the top of the suspension becomes higher. As the specific gravity of Euglena is greater than one, it causes an unstable stratification of mass density, where the top layer is heavier than bottom layer. In such a situation, Rayleigh–Taylor instability occurs, where a downward flow occurs from the top to the bottom of the suspension. In this process, Euglena is swept to the bottom layer by the downward flow. Additionally, simultaneous migration to the upper layer of suspension is maintained via the Euglena-dependent negative phototaxis. The balance between the microorganisms' upward and downward movements generates a macroscopic convective flow (bioconvection) in the suspension, as illustrated in Figure 1A.

Recent studies demonstrated that spatially-localized bioconvection can occur within dilute suspensions of Euglena (Suematsu et al., 2011; Shoji et al., 2014). Suematsu et al. (2011) revealed that bioconvection cells appeared when a Euglena suspension was illuminated by a strong light from below and spatially localized in a thin cylindrical container. Overall, the pattern of the localized bioconvection cells depends on the average cell density and container thickness. When the average cell density increases, the number of convection cells increases. However, the size of the bioconvection cells does not change, and there is no considerable change in the distance between the convection cells. This phenomenon differs from Rayleigh–Bénard thermal convection, in which the convection cells cover the entire container. Shoji et al. (2014) investigated the existence and behavioral characteristics of the bioconvection unit consisting of two convection rolls in an annular container with periodicity in a circumferential direction. They demonstrated a two-dimensional-like unit that maintained its structure and moved in a circumferential direction along the side wall. When two bioconvection units exist within a container, they can either merge with time or maintain a constant distance between them. The stable non-equilibrium states of the bioconvection unit in an annular container are similar to thermal convection in binary fluid mixtures (Bensimon et al., 1990; Watanabe et al., 2011, 2012). Nonetheless, the laws governing the motion of bioconvection units remain unknown. No prior study has investigated an isolated, single, three-dimensional bioconvection cell in a container such as a petri dish. Therefore, whether such a bioconvection cell can maintain its structure without other convection cells surrounding it is unclear.

Overall, we aimed to understand the localized bioconvection of Euglena and demonstrate the existence of an isolated, single, three-dimensional bioconvection cell, which was termed a “bioconvection spot.” In this study, we established a “position-control” experiment method to generate a Euglena-dependent bioconvection spot in a container. In addition, we established that this bioconvection spot can exist alone, without a wall nearby, and maintain its corresponding structure via the position-control technique. Recently, Arrieta et al. (2019) reported a control method for the bioconvection plumes of Chlamydomonas. They controlled the local cell density of a dense Chlamydomonas suspension by LED illumination using optical fiber. Alternatively, time-varying non-uniform light fields were used in this study. Ogawa et al. (2016) observed the cell numbers of a Euglena suspension in a capillary laid horizontally, which had two regions illuminated with different light intensities from below. The cell number in each region was biased depending on the differences in the average intensity and the gap between the two light intensities. This indicated that Euglena exhibits lateral movements in response to light. By utilizing this response and exposing the suspension to a controlled light environment with bright and dark regions that changed temporally, we manipulated the local cell density of the Euglena suspension. As a result, the bioconvection spot would emerge at a specific location within the dilute suspension where the local cell density of Euglena reaches a high level, which was mediated by this indirect manipulation with light.

Euglena gracilis strain Z (Figure 1B) was pre-cultured using Koren–Hunter medium with continuous light illumination. The cells were inoculated into a 2g/L HYPONeX aqueous solution with periodic light illumination at room temperature (20±1°C). The light period (4.27 μmol/m²/s, 306 lx) was conducted between 7 a.m. to 10 p.m. (15 h light/9 h dark cycle). The average cell densities of the suspension were prepared by diluting the suspension extracted from the cell culture with a HYPONeX aqueous solution of the same concentration. The suspension was left in the darkroom for an hour before placing it on our experimental device. We conducted all experiments from 1 p.m. to 7 p.m. Figure 1C shows a schematic of the experimental setup used in this study. We used a projector (PicoCube X, Felicross Co. Ltd.) as the light source, and varying light conditions were projected onto a screen made of white polystyrene sheet (thickness: 0.2 mm). The light environment corresponded to a flip drawing controlled by an in-house Python program with library Pygame. The Euglena suspension was placed in a cylindrical acrylic container (40 mm in diameter and 5mm in thickness) with a top cover made of glass. This container was placed on the screen and observed from above using a digital single-lens reflex camera (Z 7II & NIKKOR Z 50 mm f/1.8 S, Nikon).

Euglena migrates away from strong light when the environment suddenly changes (Ogawa et al., 2016; Tsang et al., 2018). By exposing the Euglena suspension to an artificial, non-uniform light environment, we manipulated the local cell density of the Euglena by utilizing this movements. As shown in Figure 1C, the artificial light environment had two regions, (I) and (II), with different light colors and brightness. Region (II) incorporated a darker color than that of region (I). The circle radius for region (II) was initially 20 mm (covering the whole region of the cylindrical container). The radius of this region decreased linearly with time until this region vanished after a specific designated time. The light intensity of the projected pattern fluctuated (50 Hz) due to refreshing the image. To obtain an average light brightness for each region, we measured the light 240 times using a spectrometer (MK350S Premium, UPRtek) with an exposure time of 500 μs. Figure 1D shows the profiles of photon flux density (PFD) for wavelengths from 380 nm to 780 nm. Black-solid and red-dashed lines represent the averaged PFD profiles of the light conditions that were projected in white and red colors, respectively. Gray and bright-red lines indicated the PFD profiles obtained using each measurement. The net light brightness, an integrated PFD with respect to wavelength, of the white and red regions was 55.4 μmol/m²/s (3500 lx) and 15.6 μmol/m²/s (695 lx), respectively.

The following image analysis was performed to quantify the relationship between Euglena-rich regions observed in a container and the average cell density of the suspensions. We extracted the Euglena-rich regions inside a container from the images of a whole region using an in-house Python program with library Pillow. We converted the images taken after region (II) vanished to 8bit-grayscale images; we then inverted the grayscale of these images. We eliminated the regions with bubbles in the images. Additionally, we set an image intensity threshold to 145 (57% of a maximum intensity on 8-bit grayscale) to extract the regions corresponding to the green haze. For all images, we utilized the same threshold value; therefore, the values in each image smaller than this threshold were considered noise and replaced with zero. We calculated the average image intensity of the converted images by dividing the sum of the image intensity of each pixel by the total number of pixels inside the container. This average image intensity was used to represent the extent of pattern formation considering each degree of aggregation of the bioconvection cells induced within the container.

The occurrence of a bioconvection spot in Euglena suspension and the external light environment in this study may or may not exist in natural environment. Our experimental setups involved artificial environments designed to demonstrate that a bioconvection spot can maintain its structure alone. Further, they are used to test the behavior of the bioconvection spot, by which we can analyze the characteristics of collective motion. We call such a well-designed environment “diorama environment”. We expect Euglena's potential ability or adaptability that may be hidden in a complex natural environment can emerge using the diorama environment. This study could contribute to understanding microorganisms' behavioral characteristics and fluid dynamics. In an annular container, the behavior of Euglena bioconvection resembles that of thermal convection in binary fluid; nonetheless, their convection mechanisms are different. Overall, our position-control technique can be used for more controlled experiments, leading to an essential understanding of the dynamics of spatially localized structures.

Bioconvection did not always occur for the suspension with approximately 1 × 10⁵ cells/mL in a uniformly lit environment. This implies that 1 × 10⁵ cells/mL is close to the critical cell density required for bioconvection. We presumed the initial uniform distribution of cell density; however, the slight difference in initial distributions may contribute to the occurrence of bioconvection. In a uniformly lit environment, experiments for non-uniform initial distributions are difficult to conduct because of distribution control. To observe the induction of a bioconvection spot in a container, it is essential to perform position-control experiments, which manipulate the local cell density of the suspension.

We demonstrated the linear relationship between the average image intensity of bioconvection observed and the average cell density of the corresponding suspensions (Figure 3F). One factor for the linear relationship is that the number of spots increases as the suspension of average cell density increases (Figures 3B–D). However, the average image intensities differed in the suspensions of 0.37 × 10⁵ cells/mL (Figure 3A) and 0.79 × 10⁵ cells/mL (Figure 3B), nonetheless the number of a bioconvection spot formed was one in both suspensions. Figures 4B, C show that the spot observed in Figure 3B radiated more pulses than that in Figure 3A. These indicate that the spot in Figure 3B contains more microorganisms than that in Figure 3A. In addition, an upper limit on the number of cells in which a bioconvection spot can be constructed exists, because several bioconvection spots emerged in the higher average cell density suspension (Figures 3C, D). Considering the lower limit (the critical density for bioconvection), we suggest that the effective cell number range exists to maintain the bioconvection spot structure.

We established position-control experiments and determined that a bioconvection spot can exist independently and radiate pulses of local cell density. Moreover, we showed that the behavior of pulses changes depending on the average cell density of the suspension used. Our new experimental setup is expected to understand the localized bioconvection of Euglena and to reveal the behavioral characteristics of an isolated single three-dimensional bioconvection cell (bioconvection spot). For example, by using this setup, we can investigate the competition between the spots in events such as collision and coexistence. This can be conducted by generating two controlled bioconvection spots in a container that approach each other. In future studies, we will investigate the factors involved in the structural changes of bioconvection spots and aim to elucidate the mechanism of these radiated pulses and the laws of motion that govern them.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

HY, NS, and MI conceptualized the research and designed the experimental setup. SI cultivated and provided Euglena. HY, TK, and KM conducted preliminary experiments to establish the experiments. HY performed the experiments, analyzed the data, and drafted the manuscript. All authors confirmed the manuscript and contributed to the revision. All authors contributed to the article and approved the submitted version.