Social preference test

The social preference test was adapted from Crawley’s preference for social novelty test for mice⁹¹ and similar tests in zebrafish^46,92. We used a transparent plastic tank divided in five compartments: a central area (13 × 19 cm) surrounded by two smaller compartments (6.5 × 9 cm) on either side. The walls between the central and the side compartments contained 1 mm holes to permit movement of water. A single focal fish was placed in the central area and allowed to interact with a stimulus fish placed into the side compartments. The central arena was divided conceptually into four equal size sections (Fig. 3a), and the time the focal fish spent in each area was recorded. We performed two experiments using this setup:

This test consisted of two consecutive 5 min recordings. In the first session (interaction 1), an unfamiliar female WT (stranger 1) was placed into one of the small compartments and the focal fish was placed into the central arena. In the second session (interaction 2), a second unfamiliar female WT (stranger 2) was placed in the compartment diagonally-opposite to stranger 1. The choice of compartment to use in each test was randomised. The focal fish was recorded for another 5 min. In interaction 1 we compared the time spent in the central quadrant closest to stranger 1 with the time spent in the central quadrant closest to the empty compartment diagonally opposite. In interaction 2 we compared the time spent near stranger 1 with the time spent near stranger 2. We used females as stimulus fish since they have been found to attract both male and female zebrafish, whereas males induce different responses in males and females⁹³. We used 16 WT focal fish (8 males, 8 females; size: 3.37 ± 0.16 cm) and 16 ednraa^−/− focal fish (8 males, 8 females; size: 3.43 ± 0.03 cm). We used different stimulus fish for each interaction (size: 3.36 ± 0.07 cm).

In this test we placed an unfamiliar female WT (WT stranger) in one compartment and an unfamiliar female mutant (ednraa^−/− stranger) in the compartment diagonally-opposite. We assessed the preference of WT and ednraa^−/− focal fish (both male and female) when stranger fish of each genotype were presented simultaneously in a 5 min recording. The time spent in the proximity of each stranger was measured. We used 16 WT focal fish (8 males, 8 females; size: 3.39 ± 0.13 cm) and 16 ednraa^−/− focal fish (8 males, 8 females; size: 3.41 ± 0.06 cm). We used different stimulus fish for each focal fish (WT size: 3.40 ± 0.11 cm; ednraa^−/− size: 3.38 ± 0.14 cm).