DOI: 10.5061/dryad.0k6djhb2s
title: Distance estimation in the Goldfish (Carassius auratus)

description: Neurophysiological advances have given us exciting insights into the
 systems responsible for spatial mapping in mammals. However, we are still
 lacking information on the evolution of these systems and whether the
 underlying mechanisms identified are universal across phyla, or specific
 to the species studied. Here we address these questions by exploring
 whether a species that is evolutionarily distant from mammals can perform
 a task central to mammalian spatial mapping – distance estimation. We
 developed a behavioural paradigm allowing us to test whether goldfish
 (Carassius auratus) can estimate distance and explored the behavioural
 mechanisms that underpin this ability. Fish were trained to swim a set
 distance within a narrow tank covered with striped pattern. After changing
 the background pattern, we found that goldfish use the spatial frequency
 of their visual environment to estimate distance; doubling the spatial
 frequency of the background pattern resulted in a large overestimation of
 the swimming distance. These results provide robust evidence that goldfish
 can accurately estimate distance, and show that they use local optic flow
 to do so. These results provide a compelling basis to utilise goldfish as
 a model system to interrogate the evolution of the mechanisms that
 underpin spatial cognition, from brain to behaviour.
description: Experimental overview. We trained goldfish
 to a target distance in a long and narrow tank and then tested whether
 they could continue to swim the target distance given different optic flow
 information. In training, the fish were exposed to an achromatic striped
 pattern of 2cm and they were given an external cue to indicate when they
 reached the target distance. We then removed the external cue and measured
 if the fish continued to swim the set distance. Finally, we changed the
 optic flow pattern to determine whether the fish would change their
 estimate of distance travelled. Trial videos were then analysed to test
 whether alternative mechanisms, including fin beats and time, could have
 been used for distance estimation. Animal
 husbandry Nine naive goldfish
 (<em>Carassius</em> <em>auratus</em>), sourced
 from a local supplier (The Goldfish Bowl, 118-122 Magdalen Rd, Cowley,
 Oxford OX4 1RQ) were used in experiments. Individuals were housed in 0.35m
 x 0.32m x 0.60m (width x height x length) tanks enriched with 0.5 cm of
 gravel, a terracotta pot, and plastic plants. Because
 <em>C</em>. <em>auratus</em> is a social species,
 individuals were kept in groups of two to three fish. The illumination by
 fluorescent light followed a 12h light /12h dark cycle. Individuals were
 fed twice a day; once in the morning with pellets (<em>Fancy
 Goldfish Sinking Pellets, FishScience</em>) and once in the
 afternoon with spinach or bloodworms to add supplementary nutrients. Tanks
 were cleaned weekly and water quality was maintained at healthy levels for
 this species (pH: 8.2; KH: 7dKH; GH: 8.2; Nitrite: 0ppm; Ammonia, Nitrate:
 &lt; 10ppm). Experimental apparatus
 We used the experimental apparatus built by Karlsson et al.
 (2019). Briefly, fish were trained and tested in an acrylic tank (0.25 m
 high x 0.16 m wide x 1.80 m length, Figure 1) set within a flow-through
 tank. The tank was connected to the home water system to maintain
 consistent water parameters, but the water flow was stopped during
 training and testing sessions. A white and black vertical 2 cm width
 stripe pattern (2 cm pattern) on the floor and walls of the tunnel
 produced optic flow cues. Three additional patterns were used to
 interrogate which optic flow features were used by the goldfish.
 Checker pattern: a 2 cm<sup>2</sup>
 checkerboard pattern with the same optic flow frequency as the training
 pattern was used to evaluate the impact of pattern change on fish distance
 estimation. High-frequency optic flow pattern: a 1cm
 width vertical stripe pattern, used to test the impact of increased optic
 flow on distance estimation. No optic flow pattern: a
 2 cm horizontal stripe pattern used to test the impact of removing optic
 flow cues on distance estimation. To
 control for the use of external visual cues to estimate distance, a
 moveable start area (0.25 m high x 0.16 m wide x 0.20 m length) was placed
 at one of six start positions (10 cm apart). Three start positions (20 cm
 apart) were used to train the fish and three different start positions (20
 cm apart) were used to test them. Therefore, while the target travel
 distance remained constant, the absolute position where the fish must turn
 was different in training and testing. A white partition was placed at the
 end of the tunnel to block external visual stimuli, and a second one was
 placed 20 cm behind the start door. An overhead camera (Point Grey
 GrassHopper 3M- FLIR Machine Vision Cameras), placed 1.05m above the water
 level and connected to a computer displaying individual movement in
 real-time. All training and testing trials were recorded using StreamPix 7
 video capture software (video frame rate = 50 fps).
 Training We used an operant training
 paradigm with a food reward to train the fish to swim to a target distance
 of 0.70 m. For each training session, the start area was randomly assigned
 to one of the three training start positions. During the first stage of
 training, bloodworms were placed along the bottom of the tank to encourage
 the fish to swim through. A transparent acrylic barrier was placed 70 cm
 apart from the door to stop the fish from swimming further than the target
 distance. Bloodworms were then spaced throughout the tunnel to provide
 motivation for exploration. The number of bloodworms gradually decreased
 throughout training until only one bloodworm was placed at the barrier and
 one at the start position. Gradually, the barrier was replaced by a 5 cm
 partition, then a 0.5 cm stick, and finally the physical barrier was
 removed from the tank altogether. Those intermediate steps were necessary
 as they provided a physical cue indicating that the target distance was
 reached but also allowed the fish to get around it, swim further than the
 target distance and explore the tank. If the fish swam further than the
 target distance, no food reward was provided when it came back to the
 start position. The experimenter stood 1 m apart from the experimental
 tank, observing the fish movements on the computer screen. The
 experimenter was not visible to the fish. The experimenter waved at the
 fish when it reached the target distance (with the 5 cm partition, 0.5 cm
 stick, and without a physical barrier) to provide a cue for turning. At
 the final stage of training, the fish had to swim and turn when the
 experimenter waved above the experimental tank and then come back directly
 to the start position to receive their food reward. If the fish returned
 to the start position before reaching the target distance or explored the
 tunnel further away, no food reward was given. The training was completed
 once a fish reached the target distance in 80% of trials (i.e., swim out
 to the wave and come back directly to the start area) over three
 consecutive sessions. Training sessions lasted for 10 minutes or until 10
 trials were completed. Fish were trained twice a day, once in the morning
 and once in the afternoon, five days a week.
 Testing During testing, three start
 positions (different from the three training start positions) were
 randomly assigned and 15 trials per fish were completed at each start
 position. As a result, 45 distance estimation tests were recorded for each
 fish. For each session, fish first performed six to seven training trials
 with the experimenter providing a turning cue. The training start position
 was then moved to the test start position and the fish were tested for
 three to four trials with no turning cues. A food reward was given in all
 test trials regardless of the distance travelled. To test alternative
 optic flow patterns, fish were returned to their home tank after training
 while the experimenter changed the optic flow pattern. Fish were then
 moved back into the experimental tank and tested for a further three to
 four trials. A total of 45 distance estimation tests were also recorded
 for each optic flow pattern. A few exceptions led to
 discarding a trial: (i) if the fish reached the very end of the
 experimental tank or if it only got its body length out of the start
 position before turning. (ii) If the fish turned multiple times in the
 tunnel before returning to the start position. (iii) If the fish showed
 erratic swimming movement indicating stress for the individual. In this
 final case, the trial was discarded, the session was ended and the fish
 returned to its home tank and monitored. Six of the nine fish were tested
 with the four different patterns. The power achieved to test the effect of
 the change of optic flow pattern on distance travelled with six
 individuals was above the 80% threshold criteria (power for 1000
 iterations of the generalised linear mixed model= 94,50%, confident
 interval<sub>95%</sub> [92.90; 95.83], R package
 <em>simr</em>, Green et al., 2016). Data
 collection The videos were recorded using StreamPix7
 software. To measure the goldfish distance estimate for each test trial,
 frames were extracted from video recordings at the point when the fish
 exited the start area and when it turned into the experimental tank. The
 pixel coordinates of the fish mouth were then manually recorded for those
 two events using custom video tracking software (Matlab version R2022a,
 MathWorks Inc.). The difference in pixel coordinate between the exit of
 the start area and the turn position was then converted into a distance
 estimate measured in centimetres (ratio 1 pixel = 0.0664 cm). The absolute
 turn position was measured using the coordinate of the turn frame
 converted in cm.   The time taken to turn in seconds
 was measured using the number of frames elapsed between the exit of the
 start area and the turn position. The frame rate (50 fps) was used to
 convert the number of frames to seconds. The number of caudal fin beats
 for each test trial was manually counted by the experimenter using
 StreamPix software that allowed frame-by-frame video inspection.
 To determine if the goldfish used optic flow to estimate
 distance, we compared the average turning distance and the absolute turn
 position obtained with the 2cm checker pattern (same density optic flow),
 the 1cm vertical stripes pattern (high-frequency optic flow), and the
 horizontal stripes pattern (no optic flow).