The tropical teleost zebrafish (Danio rerio) is an excellent vertebrate model organism for studying behavioral responses and cognitive functions. Therefore, operations such as precise manipulation of zebrafish, controlled drug exposure, and behavioral monitoring are essential in various biomedical studies. Microfluidics has recently emerged as a potential tool for manipulating zebrafish for various behavioral and neurobiological related studies. In addition, it has been recognized as an alternative technology for overcoming the problems associated with traditional approaches. In this aspect, the current research highlighted the design and development of a novel microfluidic manipulation platform by incorporating an ultrasound transducer which was efficiently used to manipulate zebrafish larvae using the generated acoustics. The developed acoustic microfluidic manipulation platform is purely non-invasive and was operated under various frequencies (200, 600, and 1200 Hz) and signals (sawtooth, square, and triangle) to assess its effects on zebrafish. It was observed that the frequency of sound waves triggered the zebrafish to change the moving path with a characteristic “C-start escape” from the source of the sound. The responses of zebrafish were evaluated by quantifying various motion characteristics such as curvilinear velocity (VCL, mm/s), straight-line velocity (VSL, mm/s), and linearity of forwarding progression (LIN = VSL/VCL, dimensionless). It was observed that VSL was significant (*p < 0.05) in zebrafish (4, 5, and 6 d.p.f.) under sawtooth signal at a frequency of 1200 Hz compared to 200 and 600 Hz. In addition, VCL findings observed in zebrafish (4 and 5 d.p.f.) were 105.19 % and 89.96 % higher than zebrafish (6 d.p.f.). Further, zebrafish larvae were trained according to the simple non-associative learning (stimulus learning) task to test the cognitive performances by significantly manipulating them within the platform’s designated path. In conclusion, the presented work can be extended to manipulate zebrafish into various screening sections (to view dorsal or lateral sections) for employing drug treatment by reducing the time duration significantly in a non-invasive manner. Furthermore, it can serve as a helpful reference framework for future research.

Keywords – microfluidic device, acoustic manipulation, zebrafish, behavioral response, cognitive ability.

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