A cost-effective method to assess light/dark preference and activity in zebrafish larvae


March 11, 2026

The light-dark test is a widely used behavioral assay for studying light/dark preferences in zebrafish (Danio rerio) and rodents. Currently, options for conducting this experiment in larval zebrafish are limited and expensive, restricting its accessibility. Here, we present a simple, cost-effective strategy that can be implemented in any laboratory environment. This approach uses infrared video-tracking technology to monitor zebrafish larvae during alternating light and dark phases, allowing for continuous monitoring of larval behavior without the need for sophisticated or expensive equipment. We evaluated two different protocols (40-min habituation in dark followed by 20-min habituation in light; and 30-min habituation in the light) at two developmental stages, specifically 7-days post-fertilization (dpf) and 14 dpf. After the habituation period, larvae were exposed to multiple cycles of light and darkness, enabling the assessment of locomotor activity and light/dark preferences. Behavioral parameters such as distance traveled and movement activity during light versus dark phases were measured using freely available open-source software, and data were analyzed using custom-written Python codes. The results demonstrated erratic movements of the larvae during the first protocol, while typical larval behaviors in the light and dark were observed during the protocol using 30-min habituation, highlighting the importance of an appropriate habituation phase when conducting the LDT. This method can be used to investigate behavioral activity in larval zebrafish, retaining high experimental rigor while significantly reducing costs, making it accessible to a broader range of zebrafish research laboratories.