Animals and Housing

Adult male wild-type (AB) zebrafish (Danio rerio), 11 months old, bred and held at Instituto Gulbenkian de Ciência (IGC, Oeiras, Portugal) were used. All fish were kept in mixed sex groups in environmentally enriched stock tanks (gravel substrate, artificial plants and rocks), with water temperature at 28 °C and a 14L:10D photoperiod. Water was filtered and monitored for nitrites (<0.2 ppm), nitrates (<50 ppm) and ammonia (0.01–0.1 ppm). Conductivity and pH were maintained at 700 μSm and 7.5 respectively. Fish were fed twice a day with commercial food flakes in the morning and with freshly hatched Artemia salina in the afternoon, except on the day of the experiments. All experiments were performed in accordance with relevant guidelines and regulations, reviewed by the Instituto Gulbenkian de Ciência Ethics Committee and approved by the competent Portuguese authority (Direcção Geral de Alimentação e Veterinária permit 008955).

Behavioural setup

The experimental setup (Fig. 1a,b) consisted of three side-by-side test tanks (13 × 13 × 17 cm each) and three demonstrator tanks (15 × 15 × 17 cm each), one for each experimental treatment. The observation glass side of each test tank was positioned head-to-head to the end glass side of a demonstrator tank. A one-way mirror was placed in-between to prevent interactions between demonstrators and focal fish (bystanders), allowing each bystander full view of the demonstrator fish without itself being seen. A fluorescent light was placed over the demonstrator tanks, creating differential lighting required for the mirror effect. Each test tank had black opaque walls to enhance this effect, with the exception of the transparent glass observation side. This also avoided interference of spurious external visual cues. Each demonstrator tank had white opaque walls, except for the transparent glass end side. All tanks were filled up to a 9 cm water depth. Three B&W mini surveillance cameras (Henelec 300B) with infrared sensitivity (IRs) were placed above each test tank and connected to a laptop (HP Pavilion g6). This allowed a top view video recording of the focal fish and demonstrator fish simultaneously. The setup was placed over an infrared LED (850 nm) custom built lightbox, to increase contrast between the background of the test tanks and the focal fish when video recording, without interfering with their vision, as IR light falls outside zebrafish wavelength sensitivity58. This increased image quality and optimized subsequent video tracking of the fishes’ behaviour. A black curtain separated the setup from the rest of the behavioural room during the experiment and no person was allowed inside during the testing period.

Experimental procedures

A total of 39 focal naïve male zebrafish were used (13 per treatment). Each fish was subjected to a single test corresponding to one of three treatments (Fig. 1c): bystander to male fighting conspecifics (BIC), bystander to non-interacting conspecifics (BNIC) and socially isolated (ISOL). The behavioural setup allowed testing three different bystanders per day. On the day prior to the test, three fish of similar size were randomly removed from the stock tanks and isolated in each test tank overnight. This produced an isolation baseline effect and allowed for setup acclimatization. The order of the treatments attributed to each tank was randomized for each session. To prepare the BIC and BNIC demonstrators, two pairs of unfamiliar zebrafish matched in size, where placed in the corresponding demonstrator tanks. A removable white opaque partition was placed between each pair overnight, allowing chemical but no visual communication. The ISOL treatment was prepared by keeping a demonstrator tank empty, with an opaque partition also placed in the middle to match the other tanks. Removable opaque partitions were additionally placed between each test tank and the one-way mirror, to prevent visual contact between demonstrators and bystanders during the isolation period. The demonstrators were allowed to habituate to the one-way mirror reflection overnight. This avoided interactions with the mirror during the tests. On the following day, at the beginning of each test, the opaque partition that visually separated each test tank from the corresponding demonstrator tank was removed. Each focal fish could then visually observe the corresponding demonstrator tank for 30 minutes. For the BIC treatment, the middle opaque partition separating the demonstrator dyad was also removed simultaneously, prompting demonstrators to fight. For the BNIC treatment, the middle partition remained in place, preventing the two demonstrators to interact. For the ISOL treatment, the middle partition also remained in place. All focal fish behaviours were video recorded for posterior offline behavioural tracking and analysis. Immediately after the test, each focal fish was euthanized with an overdose of tricaine solution (MS222, Pharmaq; 500–1000 mg/L) and sectioning of the spinal cord. Gender was confirmed by dissection of the gonads. Body samples were stored at −80 °C for posterior whole-body hormonal analysis.

Behavioural tracking

All focal fish behaviours were tracked from a top-down view perspective, using a custom made tracking software (see https://github.com/joseaccruz/fishtracker) developed in Python (pythonTM). For each behavioural video, a 2D region (arena) was defined for tracking (Fig. 1b). The arena’s position and size took into account the camera’s perspective distortion caused by the water depth and comprised the inner area of the bystander tank (12 × 12 cm) including the stimulus observation side, where the lighting contrast between the white background and the fish was high. It excluded the black outer walls sidelines where contrast was low. The fish were tracked at a 29 fps rate. The tracking software determined and extracted into data files, the pixel coordinates of the head, centroid and tail for each frame (see Fig. 1d and Supplementary Video 1). This allowed determination of position and orientation (Fig. 1e) of the fish every 1/29 s. It also identified and counted all frames in which the fish was not detected. This only occurred at surface level, alongside the tank’s black outer walls (on average 4% of the total time). After tracking, the head, centroid and tail coordinates were projected over the video (see Fig. 2a) allowing the manual inspection of the tracking quality and an easy early detection of possible tracking errors. https://github.com/joseaccruz/fishtracker

Behavioural data analysis

All tracked data files were imported to MATLAB (MathWorks ®) and behavioural parameters were determined using a custom-made script. A region of interest (ROI) with 12 × 3 cm (25% of the tank) corresponding to the width of the tank and the mean body length of an adult zebrafish, was defined in the area of the arena closest to the observation glass (Fig. 1c). The focal fish was considered in the ROI when its centroid point was inside that region. The following behavioural parameters were determined and analysed at an individual and group level for each treatment: total time spent at each position in the arena, total time at each direction, percentage of time spent in the ROI, mean speed in the ROI, total distance covered in the arena, mean preferred orientation in the arena and directional focus. The determination of the total distance covered by each fish took into account an estimation of the distance covered when the fish could not be tracked, by considering it proportional to the total distance covered when detected. To determine the mean resultant vector for the fish, each orientation taken during the 30 minutes test was first transformed to a unit vector . Where α i was the angle formed by the fish’s centroid-to-head axis direction relative to the horizontal axis in each frame. The mean resultant vector was thus defined as the mean of all frames’ unit vectors taken by the fish during the testing period and calculated by . The mean resultant vector’s length , a measure of directional focus and inversely related to the angular standard deviation, was calculated by the mean vector’s norm and ranged from 0 to 1. The closer (Fig. 1e) is to one, the more concentrated the n orientations are around the mean direction. The projection of onto the stimulus tank’s direction (180°) was determined by , where α (Fig. 1e) is the angle formed by the mean resultant vector r. This allowed measurement of the mean directional focus of each fish relative to the stimulus direction, using a linear scale ranging from 1 to −1. Positive values indicate directionality towards the stimulus, negative values away from it and null values no directionality. For each treatment, the group mean resultant vector , correspondent mean angle and length , was determined by the grand mean of all focal fishes’ mean resultant vectors , weighted by their individual lengths R.

The temporal dynamics and correlation of the BIC vs. ISOL treatments for the mean time spent in ROI and , was calculated in 30 seconds bins (Fig. 3).

Video playbacks-experimental setup and procedures

The original experimental setup was adapted by replacing the demonstrator tanks (Fig. 4a) with a 10-inch, 1024 × 768 LCD tablet, positioned adjoining the end glass side of a removable bystander tank. A camera was placed above the tank for a top-down view video recording and later tracking of the focal fish. The same lighting conditions were maintained to match the previous experimental settings. In this experiment, the number of bystander focal fish was increased to 23 per treatment. Each focal fish was subjected to a single 30 minutes test corresponding to one of four new treatments: (1) bystander to a video of fighting conspecifics (BVIC) comprising a pre-resolution, resolution and post-resolution stage31; (2) bystander to a video of fighting dots (BVID), where the original fight video was manipulated by replacing the fighting fish by circles (dots), while maintaining the same original movements (see Supplementary Video 2); (3) bystander to a video of non-interacting conspecifics (BVNIC); and (4) observing a video of an empty tank (VISOL), as control for the stimuli and any possible effects of the screen itself. Each video presented was previously recorded with a digital video camera at a 25 fps and 720 × 576 pixel resolution, using the same conditions and settings of the previous experiment. The videos were displayed on the tablet using real size images.

On the day prior to the test, fish of similar size were randomly removed from the stock tanks and isolated in each bystander test tank overnight, next to the experimental setup. This produced an isolation baseline effect and allowed for setup lighting acclimatization. Removable white opaque partitions were placed on the observation glass side of each test tank to prevent visual contact with the outside. On the following day, prior to the beginning of each test, a test tank with an isolated focal fish inside was placed in the setup (with the opaque partition still in place), positioned in front of the tablet screen and allowed to habituate for 30 min. At the beginning of the test, the video started playing on the screen and the opaque partition was immediately removed. Each focal fish could then visually observe a video for 30 min. The order of the video treatments was randomized for each session. All focal fishes’ behaviours were video recorded for posterior offline behavioural tracking and analysis. Immediately after the test, each focal fish was euthanized. All samples were stored at −80 °C for posterior analysis.

Manipulation and activity analysis of the fighting conspecifics video

Replacement of the fighting fish by dots was achieved firstly by tracking and extracting both fighters’ centroid coordinates, size, colour and contrast for each frame, using a custom-made tracking software. Two circles with the mean area, colour and contrast of the original fish were then placed at the corresponding centroid positions, over the fish-subtracted background images of the tank. This allowed exact replication of the fighters’ movement, while eliminating their form features (see Supplementary Movie 2). As a measure of activity on screen throughout the 30 min video fight, the mean speed of the fighting dyad was calculated in 30 s bins (Fig. 5a) using the fighters’ tracked data. This allowed profiling the temporal dynamics of the fight’s level of activity and posterior correlation analysis with the bystanders’ mean time spent in the ROI, when observing the video fight.

Hormonal analysis

Cortisol whole-body (WB) levels were measured for each focal fish. For the hormone extraction, the collected WB samples, kept at −80 °C, were first measured in body weight and length for normalization purposes. Each sample was partially thawed, weighed and dissected on ice into smaller parts for efficient homogenization. 500 μl of EIA Buffer (from Cayman EIA kit) were added and vortexed for 3 s. The samples were then transferred to extraction glass tubes and homogenized using a mechanical homogenizer (IKA Labortechnik) for 30 s on ice. The homogenization rotor blade was washed with additional 500 μl of ice-cold EIA buffer and collected in the glass tube containing the homogenate. Samples were sonicated for 30 s on ice, added 3 ml of diethyl ether, vortexed, stirred for 10 min in the orbital shaker and then centrifuged at 2000 rpm (4 °C) for 15 min. Following centrifugation, samples were frozen at −80 °C for 15 min and the organic layer (containing the hormones) was removed from each sample and placed in a separate test tube. Ether was evaporated with a speed vacuum centrifuge (Speedvac Savant SC 1101) equipped with a cryotrap. Samples were reconstituted in 1 ml of EIA buffer after evaporation and kept at −20 °C until analysis. Cortisol levels were assayed using enzyme immunoassay (EIA) kits from Cayman Chemical Company (#500360) following the manufacturer’s instructions. In the cases where samples were too concentrated, dilutions were performed and measurements repeated. For the first experiment, the intra-assay coefficient of variation was 3.20% and inter-assay coefficient of variation was 8.79%. For the video experiment, the intra-assay coefficient of variation was 5.10% and inter-assay coefficient of variation was 2.80%.

Statistics

Behavioural and hormonal results were represented as mean ± SEM unless stated otherwise. Statistical significance was considered for P < 0.05. For the behavioural parameters’ comparisons between treatments, one-way ANOVAs were performed when normality and homogeneity of variances (Levene’s test) was verified, followed by post-hoc Tukey HSD tests or contrasts for specific planned comparisons. When normality was verified but not homogeneity of variances, Welch’s ANOVAs were used, followed by Games-Howell post-hoc tests. When normality was not verified, non-parametric Kruskall-Wallis ANOVAs were used. Cortisol concentrations were first ln transformed to meet the assumption of a normal distribution. Deviation from uniformity of the fishes’ individual mean orientations distribution was tested using the non-parametric Moore’s Modified Rayleigh test, for each treatment. The angles of the group mean resultant vectors were represented as mean, 95% C.I. when directionality was significant. Correlations were performed using a non-parametric Spearman rank correlation. All analyses were performed using MATLAB R2012b (MathWorks) with the CircStat toolbox59, STATISTICA 12 (StatsoftInc), SPSS Statistics 22 (IBM) and Oriana 4 (Kovach Computing Services).