Comparative analysis of zebrafish fear responses to eight different fish species using 3D locomotion tracking assays

Zebrafish (Danio rerio) are widely used in neurobehavioral research due to their translational relevance in studying fear. Eight different fish species and variations were tested to induce fear responses in zebrafish, including one positive control (convict cichlid, Amatitlania nigrofasciata) and negative control (tiger barb, Puntigrus tetrazona) through a shared-environment test. The observation was done in three dimensions (3D) and two dimensions (2D) to assess the impact of dimensionality on the outcome. A single-camera system was used to capture two viewpoints by mirror reflection installed above the fish tank and reconstructed to 3D using F3LA software. Zebrafish showed a similar behavioral response towards Demason's cichlid (Pseudotrophus demasoni) and threadfin acara (Acarichthys heckelii) as they did to A. nigrofasciata, with some minor differences, and a lesser response to green Gymnocorymbus ternetzi, during the shared-environment tests. Meanwhile, presence of B. melanopterus caused zebrafish to have a higher tendency to freeze and display higher entropy, similar to an anxiety-like response. We found no correlation between behavioral response and the body size of the test fishes. However, a correlation was observed when we tested convict cichlids of different ages. Finally, zebrafish color preference was also observed through the use of G. ternetzi with different body colors as test fish, with the zebrafish preferring orange and red G. ternetzi and mostly avoiding green G. ternetzi. We found use of 3D observation superior to 2D observation because several important endpoints are obtainable only from certain viewpoints.