DOI: 10.6084/m9.figshare.1002130.v1
title: Data from: Ectoparasites modify escape behaviour, but not performance, in a coral reef fish

description: Parasitized (N = 28) and unparasitized (N = 12) Scolopsis bilineata were collected in February and March 2012 using monofilament barrier nets (10 mm stretched mesh) and silicone hand nets from sites around Lizard Island, Northern Great Barrier Reef, Australia (14° 40’ S; 145° 28’ E). Fish were transported in aerated 20 l Handy Pail buckets (maximum 4 fish per bucket) to the Lizard Island Research Station within 1 h of capture, and transferred with silicone nets to individual holding aquaria (40.0W × 29.0L × 18.0H cm) with a flow-through water system transported directly from the reef. Tanks were kept under a natural light and temperature regime (28 ± 1 °C). All fish were provided with a round PVC shelter for refuge. Fish were fed to satiation once a day with raw prawn and fasted for 24h prior to the experiments to ensure fish were in a standardized (post-absorptive) state that maximizes energy availability for swimming. Holding tanks were siphoned out daily to maintain high water quality. All animals were kept in aquaria for a minimum of three days before performing swim trials to ensure all fish were healthy. Fast-start experiments We tested three groups of fish with different infection statuses: unparasitized (fish total body length BL; 13.2 ± 0.8 cm; mean ± SD, N = 12), parasitized (BL; 11.8 ± 1.1 cm; mean ± SD, N = 16) and parasite-removed (BL; 12.7 ± 1.3 cm; mean ± SD, N = 12). Parasites were removed using forceps 24h before the start of the fast-start experiments by holding the fish in a shallow water bath and gently unhooking the isopod. This procedure took approximately 45s. Fish resumed normal behaviour within 2h of being returned to their holding tanks. Experiments were conducted in rectangular acrylic aquaria (70.0 L × 60W × 35H cm) supported by a wooden stand. Water height was maintained at 12 cm for all trials. This height limited movement of the fish in the vertical plane (Langerhans, 2009). A mirror was inclined at a 45 ° angle below the aquarium to allow filming of the escape responses in 3 dimensions within the same camera frame. Videos where vertical movement occurred were excluded from the analyses (&lt; 2 % of videos). The tank was illuminated by three 150 W spotlights positioned 70 cm above the water. A continuous flow of seawater maintained the water temperature at a constant 28 °C. Prior to escape response trials, fish were transferred from their holding tanks to the experimental arena using silicone nets to prevent mucus loss and fin damage, and left undisturbed for 30 min to acclimate to the arena. Numerous aerial predators, including raptors and pelagic seabirds, are commonly seen feeding on fishes around Lizard Island. Thus we used a mechano-acoustic stimulus, which simulates an aerial attack, to induce escape responses in S. bilineata. We attached a 50 ml PVC container filled with lead weights with a string to a platform 30 cm above the water surface. To ensure that the escape response was not initiated prior to the stimulus’ contact with the water, the stimulus fell inside an opaque 15 cm wide PVC tube positioned 1 cm above the water surface. The time of contact of the stimulus with the water surface was clearly visible in the mirror from below. We filmed the responses at 240 Hz with a high speed digital camera (Exilim EX-FH100, Casio, USA) mounted on a tripod directly facing the aquarium and the mirror. Individuals were stimulated up to three times at 30 minute intervals. The stimulus was dropped when the fish was facing the stimulus at a distance of no more than 10 cm from the bottom of the PVC tube. After the trials, individuals were returned to their holding tanks. In total, we filmed 127 escape responses in 40 fish. Two fish developed a bacterial infection several days following experimentation. These individuals were anesthetized with an overdose of Aqui-S solution and then euthanized in an ice-slurry. No other individuals showed any signs of sickness, and care was taken to prevent the spread of disease by rinsing nets in a freshwater bath prior to handling different individuals. All healthy fish (38 individuals) were released back to their site of collection within 1 week of trials. We used the MtrackJ plugin in the ImageJ v. 1.43 software to analyse the escape sequences. We tracked the two-dimensional coordinates of the fish’s centre of mass (CoM) every 4.2 ms starting 21 ms (5 frames) before and ending 84 ms (20 frames) after the onset of the fish’s first movement. The CoM was visually estimated at a proportional distance from the tip of the head corresponding to approximately 29% of an individual’s total length. The following escape performance metrics were measured: response latency (time between stimulus onset and fish response in ms), size- adjusted cumulative distance travelled (D in fish total body lengths; BL), size-adjusted maximum escape speed (Umax in BL s-1) and maximum acceleration (Amax in m s-2). Distance–time variables (D, Umax, Amax) were evaluated within a fixed time period of 58 ms (14 frames), corresponding to the approximate mean duration of stages 1 and 2 of the escape response across all treatments. A five-point moving quadratic polynomial regression was used to obtain smoothed values of speed and acceleration, the first and second derivatives of distance. For each fish, the best value (e.g., highest Umax or shortest latency) of each escape performance variable across the three stimulus presentations were chosen for analysis (see Domenici, 2011; Marras et al., 2011). Flight initiation distance We estimated FID in S. bilineata from the lagoon and adjacent reefs in front of the Lizard Island Research Station on calm weather days from July-August 2013. Water depth varied between 2 and 3 m at these sites with a visibility of approximately 15 m. We used snorkelers as the stimulus for flight initiation. Many studies of FID in terrestrial and aquatic systems have used humans as a stimulus to elicit flight. Recent studies found FID estimates to be relatively robust to variation among observers regardless of experience or, in aquatic systems, whether observations were made on snorkel or SCUBA. Two snorkelers swam around the reef in search of S. bilineata. Only solitary, adult individuals that were foraging or moving slowly over the reef in an open area where they could be approached directly were targeted. Individuals less than 1.5 m from branching corals or other shelter were not approached to avoid the confounding effects of distance to a refuge on FID. Similarly, trials were abandoned if individuals began swimming in any direction at a consistent speed before the observer initiated their approach. Once a suitable individual was spotted, we recorded fish infection status (parasitized or unparasitized, hereafter referred to as treatment) and total length (± 1 cm; actual error). Before data collection, all observers practiced estimating fish length underwater using model fish and objects of various sizes until they reached a precision of ± 1 cm. One snorkeler positioned themselves in direct line of view of the fish at a distance of approximately 5 m. The other snorkeler positioned themselves off to the side to avoid obstructing the trial. The first snorkeler duck-dived under the water until they were close to the substrate (within 1 m), and visually relocated the individual. The snorkeler then approached the focal fish at a quick but steady swimming speed, holding two weights marked with flagging tape beside their head, which was assumed to be the onset of the stimulus. When the fish began to flee (i.e. first began to turn away from the approaching snorkeler), the snorkeler dropped one weight where they were, and took a visual landmark of where the fish had been, which was marked with the second weight. The two snorkelers then measured the horizontal distance between the two landmarks with a tape measure to the nearest 1 cm (FID). The observers also scored the strength of an individual’s reaction, or flight intensity, on a scale from 0 – 4 as follows: 0: no response (i.e. fish did not move in response to the snorkeler), 1: fish ceased previous activities (i.e. foraging) and moved a short distance away, but did not leave the immediate area, 2: fish changed directions and began a slow displacement away from the area, 3: fish changed directions and fled the area at a fast, but constant speed, 4: fish initiated an escape response characterized by a “C-start” unsteady burst behaviour. Increasing intensity was assumed to be related to more energetically costly forms of locomotion, and therefore provided an estimate of the costs of flight. Recent studies suggest that S. bilineata are strongly site attached and rarely travel far from their small territories during the day. Therefore, to avoid pseudoreplication, we never sampled two similarly sized fish with the same infection status within 25 m.