Behavioral Dynamics of Zebrafish Under Hydrodynamic Stimuli Induced by Magnetic Microactuator in Microfluidics

Behavioral investigation in zebrafish is essential for understanding adaptive responses, where learning represents a key process influenced by external stimuli. The applied stimulus plays a critical role in shaping such responses, therefore making physiologically relevant stimulation strategies important. Hydrodynamic stimuli represent one such modality, providing a natural and non-invasive means of activating mechanosensory responses in aquatic organisms, thereby enabling behavioral manipulation in microfluidic environments. To address this, a microfluidic assay was developed to generate controlled hydrodynamic environments by employing multiple S-shaped magnetic microactuators (SMMAs). Further, motions of these SMMAs were independently controlled to produce spatiotemporally varying vortical flow fields, enabling flow-induced transportation of zebrafish larvae. Flow dynamics were characterized by employing micro-particle image velocimetry (µPIV). Compared to the control condition, transportation time under microactuator-assisted guidance was significantly reduced, with a maximum improvement of 94.3% observed for a representative target zone. Building on this validated transport capability, training-dependent behavioral adaptation was quantified using latency under repeated hydrodynamic-training, where a reduction of 82.7% was achieved. Post-training assessment further demonstrated short-term retention of the acquired behavioral response followed by progressive extinction. These findings demonstrate that the proposed paradigm serves as a foundational behavioral assay leveraging hydrodynamic cues for studying adaptive responses in microfluidics.