Motility patterns of Euglena species in confining microfluidic geometries.

Protists rely on motility to explore their environment, enabling them to access resources such as nutrients and light, while avoiding unfavorable conditions and predators. To advance the current understanding of how motility and morphological adaptations modulate spatial navigation and interactions with confining physical environments, this work analysed the motility of E. gracilis and E. acus, which share a similar overall body plan but differ in flagellum length and pellicle configuration. Cell motility was quantified under progressively increasing confinement in microfluidic devices comprising quasi-open spaces (0D), single-wall interactions (1D), and two-wall confinement (2D), including corners and channels with varying geometries and complexities. The motility of Euglenids was characterized using single-cell swimming trajectories, wall-scattering dynamics, trapping time, switching of propulsion mode, swimming velocity, and quantification of successful navigation through structured environments. Five distinct wall-scattering behaviors were observed in low-confinement conditions. Both species mainly escaped geometrical confinements through collision-mediated scattering. E. gracilis exhibited larger average deflection angles and greater variability in its trajectory angles. In 2D corner confinement, E. gracilis presented extended trapping times and larger deflection angles, consistent with an increased interaction with walls. Under strong lateral confinement (channel widths approximately 1-2× the cell diameter), both species shifted from flagellar swimming to friction-assisted back-and-forth motion accompanied by pellicle-driven deformation (metaboly). These transitions in motility behavior were geometry-dependent and species-specific. E. acus exhibited higher metaboly incidence in highly tortuous channels, whereas E. gracilis relied more on flagellar propulsion and back-and-forth motility. Upon exiting channels with quasi straight channels (lower tortuosity), E. gracilis adopted curvilinear trajectories, while E. acus presented straighter trajectories. Our results suggest that E. gracilis did not show significant conditioning to confining environments, while E. acus presented a 'directional memory'-like movement, with trajectories in open spaces strongly influenced by the geometry of the preceding channel. These observations are consistent with the hypothesis that morphological traits, such as flagellum length and pellicle organization, modulate the observed differences in behavioral motility. These findings also provide a framework for understanding how morphological features may influence navigation in structured microenvironments and inform the design of microfluidic systems for applications such as motility-based cell sorting, photo-microbioreactors, and biocomputation.