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  • Subjects were 59 adult wild-type (AB) zebrafish, of mixed gender, raised in the laboratory from a line originally obtained from the University of Oregon breeding facility. Subjects were housed in an Aquaneering table-top housing rack, with a recirculating filtration system using mechanical, biological, and chemical filtration. The subjects were housed in groups of 20, in 10 L system tanks. Because each subject was run in a single session, individuals were removed from the group of naïve fish, and then returned to a separate, identical tank containing experienced fish. The temperature of the tanks was held at 25°C, and the room was maintained on a 14/10 light/dark cycle. Subjects were fed 1–2 times daily on a mixed diet of live brine shrimp, freeze-dried brine shrimp, and Tetra-Min® flake food. The housing conditions and protocols were approved by the University of San Diego IACUC. The split-depth tank was a rectangular glass aquarium (20×15×20 cm; length×width×depth), like that described in Blaser & Goldsteinholm [26], [27]. In the ‘split’ configuration, one side of the tank was set to a depth of 10 cm using a plexiglas partition, and the other side set to a depth of 15 cm. In the ‘shallow’ configuration, both sides of the tank were set to a depth of 5 cm. In the ‘deep’ configuration, both sides of the tank were set to a depth of 15 cm. In all cases, gravel substrate was placed on a floor 5 cm below the plexiglas partition on each side. The sides of the tank were either left uncovered (transparent), covered in black paper (black), covered in white paper (white), or covered in black on one side, and white on the other. Figure 1 illustrates the apparatus for each configuration tested. The experimental setup was lit from above such that the inside of the apparatus ranged from 400 lux (in the black condition) to 600 lux (in the transparent condition). A video camera located approximately 1 m above the tank was used to monitor the location and activity of the fish. The video fed directly to a desktop computer which used Noldus Ethovision® to track the swim-patterns of the fish. The video-tracking data were then used to determine relevant measures of behavior including location in the tank (duration in each side, distance to the outer walls) and locomotor behavior (path length, immobility, shuttling). The animals were divided into five groups of 11–13 fish. Three of the groups (Depth Preference Groups – DP) were tested for depth preference while being confined to a single wall color (Black, White, or Transparent). All of these animals were tested in the split-tank configuration, with the walls either uncovered (transparent), or completely covered in black or in white. The other two groups (Color Preference Groups – CP) were tested for color preference while being confined to a single depth (Shallow or Deep). All of these animals were tested in a black/white tank, with the depth set to either 5 cm (Shallow), or 15 cm (Deep). Each subject was observed individually in a single session lasting 15 minutes. Subjects were gently netted from their home tank and placed into the center of the experimental tank. Recording began immediately, and continued for the entire 15 minutes of the test. After the test was complete, animals were returned to a separate home tank housing experienced individuals. For the DP groups, the primary dependent measure was Duration in Shallow, which was defined as the duration of each 1-minute interval that the animal spent on the shallow side of the tank in seconds. For the CP groups, the primary dependent measure was Duration in White, which was defined as the duration of each 1-minute interval that the animal spent on the white side of the tank in seconds. Additional dependent measures included the Distance from Walls (average distance of the animal from the nearest outer wall), Shuttling (total number of center-crosses in each minute), Path Length (total swim path length of the subject in each 1-min interval), and Immobility (movement rate of <1 cm/sec). Separate analyses were used for the CP and DP groups. For the CP groups, behaviors were analyzed using a 2×2×15 (Depth: Deep, Shallow×Side: Black, White×1-min Interval) repeated-measures Analysis of Variance (ANOVA), with Depth as a between-subjects measure and Side and Interval as within-subjects measures. For the DP groups, behaviors were analyzed using a 3×2×15 (Color: Black, White, Transparent×Side: Deep, Shallow×1-min Interval) repeated-measures ANOVA, with Color as a between-subjects measure and Side and Interval as within-subjects measures. The duration in shallow/white and shuttling behaviors were analyzed using only data from the less-preferred side, since the scores for one side are not statistically independent of the scores for the other side. A single-sample t-test was used to confirm that DP animals exhibited a significant preference for the deep side, and that CP animals exhibited a significant preference for the black side. Tukey's HSD was used for posts-hoc analysis as needed.
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