Study species

Acanthochromis polyacanthus and Amphiprion percula are common throughout the Indo-Pacific region. Both species are demersal spawners, laying their eggs within small caves and crevices in the reef matrix. In A. polyacanthus, eggs hatch into small juveniles, with both parents providing care to the eggs and offspring for up to 45 d post-hatching62. In contrast, Am. percula has a relatively short larval phase of approximately 11 d before settling on the reef 63. Both species can be bred and reared in captivity with high success, which has led to their establishment as models for investigating the potential impacts of OA on coral reef fishes25, 27, 31, 50, 55.

Brood-stock and general rearing protocol

Adult A. polyacanthus were collected using hand nets from the Bramble Reef area (site 1: 18°22′S, 146°40′E; site 2: 18°25′S, 146°40′E) of the Great Barrier Reef in July 2015. Fish were transported to an environmentally controlled aquarium research facility at James Cook University (JCU) (Townsville, Australia) where they were housed as breeding pairs in 60 L aquaria at temperature conditions matching the collection location. An existing brood-stock of Am. percula at JCU was used. These pairs had been collected from the Cairns Region of the Great Barrier Reef and housed at JCU for four years. Adult A. polyacanthus and Am. percula pairs were maintained under stable, ambient pCO 2 (~490 µatm). Temperatures were increased at a rate of 0.5 °C per week until the summer breeding temperature of 29 °C was reached in the first week of November 2015. Adult pairs were provided with half a terracotta pot to act as a shelter and spawning site. Aquaria were checked each morning for the presence of newly laid clutches. Pairs were fed ad libitum on commercial fish feed pellets (INVE Aquaculture Nutrition NRD 12/20) once daily outside the breeding season and twice daily during the breeding season (November–May).

Acanthochromis polyacanthus juveniles were fed a combination of freshly hatched Artemia naupli and weaning fish feed (INVE Aquaculture Nutrition Wean-S 200–400 µm) daily for the first four days post hatch (dph). 5–21 dph they were fed daily on the weaning feed and then switched to a small pellet fish feed (INVE Aquaculture Nutrition NRD 5/8) at 22 dph. Rearing of larval Am. percula was performed using methods described by Munday et al.28. Settled juveniles were fed daily on the weaning fish feed.

Experimental design

Experiment one was carried out at the aquarium research facility at JCU. For details on the experimental system refer to Supporting Information. Fish were reared at two stable (480 and 1000 µatm) and two cycling (1000 ± 300 and 1000 ± 500 µatm) CO 2 treatments (Table 1 and Figure S3). The stable 1000 µatm pCO 2 treatment represented the open ocean projection for the end of this century, typically used in many OA experiments11. The cycling pCO 2 treatments matched levels that have been observed in some tidal lagoons38. Diel pCO 2 fluctuations of between ±50–150 µatm are more typical in other reef areas40, 41, however, the magnitude of fluctuations seen in tidal lagoons today may become more common in other reef areas by the year 2100, as a amplification in diel pCO 2 fluctuations is predicted to occur over this time period5. Mean values for seawater parameters in experiment one are presented in Table 1.

Table 1 Seawater parameters for experiment one. Values are means ± 1 SD for daily average, minimum, maximum and range of pH T and pCO 2 . Full size table

Experiment two was carried at the National Sea Simulator (SeaSim) facility at the Australian Institute of Marine Science (AIMS) (Cape Cleveland, Australia). Fish were reared at three stable (460, 750 and 1000 µatm) and two cycling (750 ± 300 and 1000 ± 300 µatm) CO 2 treatments (Table 2 and Figure S4). For details on the experimental system refer to Supplementary Information. Previous experiments indicate that behavioural abnormalities are first evident at around 700 µatm CO 2 , although the magnitude of effect is often not as large as observed at higher CO 2 levels27, 28, 31. Therefore, the inclusion of the 750 and 750 ± 300 µatm CO 2 treatments enabled us to determine how diel pCO 2 cycles may affect the onset threshold of behavioural abnormalities. Mean values for seawater parameters in experiment two are presented in Table 2.

Table 2 Seawater parameters for experiment two. Values are means ± 1 SD for daily average, minimum, maximum and range of pCO 2 . Full size table

A similar protocol was employed in both experiments. Three offspring clutches were used per species, each from a different parental pair. In experiment one, A. polyacanthus and Am. percula clutches were transferred to the experimental system and split between pCO 2 treatments in duplicate tanks (12–15 A. polyacanthus per tank and 10 Am. percula per tank) at 1 and 12 dph respectively. In experiment two, offspring clutches were transferred to the experimental system and split between pCO 2 treatments in duplicate tanks (one tank per line; 15 A. polyacanthus per tank and 13–15 Am. percula per tank) at 14 and 12 dph respectively. A. polyacanthus clutches were transferred at 14 dph in experiment two, compared with 1 dph in experiment one, due to logistical reasons.

Behavioural lateralization trials on A. polyacanthus were performed 40–42 dph, which equated to approximately six and four weeks of exposure to pCO 2 treatments in experiments one and two respectively. Predator cue trials on Am. percula were performed 18–20 dph, which equated to approximately 1 week of exposure to pCO 2 treatments in both experiments. All behavioural trials were performed between 09:00 and 17:00. Fish were gently transferred to the behavioural arenas using a glass beaker to minimise handling stress. Fish from each pCO 2 treatment were tested at random times throughout the day to account for any possible time of day effects in the fluctuating treatments. Each fish was tested once, being placed in an isolation chamber within their experimental tank after a trial for the rest of the day. Research was carried out under approval of the James Cook University animal ethics committee (permit: A2210) and according to the University’s animal ethics guidelines.

Behavioural assays

Behavioural lateralization trials

Behavioural lateralization (i.e., favoring the left or right side during behavioural activities) is an expression of brain functional asymmetry and a strong determinant of fish behaviour. Lateralized individuals show higher performance in cognitive tasks64, schooling behaviour65 and escape reactivity66. Lateralization in juvenile A. polyacanthus was determined using a detour test in a two-way T-maze using methods similar to those described by31. The two-way T-maze consisted of an experimental arena (60 cm × 30 cm × 20 cm), with a runway in the middle (25 cm × 2 cm, length × width), and at both ends of the runway (2 cm ahead of the runway) an opaque barrier (12 cm × 12 cm × 1 cm) was positioned perpendicular to the runway. The maze was filled to a depth of 4 cm with the respective treatment water of the fish being tested, being changed after each trial. A single fish was placed at one end of the T-maze and given a 3 min habituation period, during which time it could explore the apparatus. At the end of the habituation period the fish was gently guided into the runway using a plastic rod with the observer standing directly behind the fish (the plastic rod was never placed closer than approximately twice the body length of the fish). At this point to minimise human interference affecting direction turned the observer slowly stepped back from the maze and the fish was allowed to swim to the end of the runway. In instances when a fish did not swim to the end, encouragement was provided by gently moving the plastic rod around at the beginning of the runway. Direction choice was recorded as the first direction turned when the fish exited the runway. Ten consecutive runs were recorded per fish. Twenty fish from each clutch (ten per tank) were tested per CO 2 treatment. To account for any possible asymmetry in the maze, turns were recorded alternately on the two ends of the runway. Turning preference (i.e. bias in left or right turns) at the population level was assessed using the relative lateralization index (L R , from −100 to +100, indicating complete preference for left and right turning, respectively) according to the following formula: L R = [(Turn to the right − Turn to the left)/(Turn to the right + Turn to the left)] * 100. The strength of lateralization (irrespective of its direction) was also assessed at the individual-level using the absolute lateralization index L A (ranging from 0 (an individual that turned in equal proportion to the right and to the left) to 100 (an individual that turned right or left on all 10 trials)). Lateralization trials in experiment two were performed with the observer blinded to the experimental treatments.

Predator cue trials

The ability to detect and elicit appropriate antipredator behaviour is critical for survival, especially in early life-stages that experience a greater predation threat67. The response of juvenile Am. percula to a predator cue was tested in a two-channel choice flume using methods similar to those described by29. The flume combination was predator cue water versus untreated water. Water at the same pCO 2 level from two different sources (9 L buckets) was gravity fed into the choice flume, which was divided down half of its length. A constant flow rate of 100 ml min−1 was maintained and monitored using a flow meter and dye test after every water change. Water was changed after each trial. Fish were tested under the mean pCO 2 level of their respective treatments (i.e. fish reared under both 1000 and 1000 ± 300 µatm were tested at 1000 µatm), due to the logistical difficulties involved in manipulating predator cue water pH across a daily cycle. While this resulted in fish from cycling treatments experiencing a change in pCO 2 between experimental and test water, recent work has shown this has no effect on the response of Am. percula to a predator cue at far greater changes than experienced in this study29. For each trial, a single test fish was placed in the centre of the downstream end of the choice flume and given a 2 min acclimation period. The position of the fish was then recorded every five seconds for a total of 2 min. A rest period of 4 min followed, during which time the water sources were switched to eliminate potential side preferences. The position of the fish was then once again recorded every five seconds for a total of 2 min. Fish were not disturbed during the trial. Temperatures during the trials were kept within 1 °C of the temperature in the rearing tanks. Eight fish from each clutch were tested per pCO 2 treatment (4 per tank). Predator cues were obtained from three common coral-cod, Cephalopholis miniatus, as described by ref. 29. Response to predator cue was assessed as the percentage of time spent in the cue water. In experiment one, the control fish from one clutch exhibited no response to the predator cue (i.e. did not avoid the predator cue) and so this clutch was excluded from data analysis.

Statistical analyses

The effects of pCO 2 treatment on absolute lateralization (L A ), relative lateralization (L R ) and percentage time spent in cue water were tested using mixed-effects logistic regressions68. Models for L A data from experiment one and predator cue data from experiments one and two were over dispersed and so were re-run using a penalised quasi-likelihood. In all models, parental pair and tank were included as random factors, with tank nested within parental pair. Pairwise comparisons were performed using Tukey’s post hoc tests. To determine if a treatment group demonstrated a turning direction preference Pearson’s Chi-square tests were used, where we expected a 50:50 ratio for left/right turning preference. Finally, differences in the relative frequency distribution of L R between treatments were tested using Kolmogorov-smirnov tests. Mixed-effects logistic regressions with and without penalised quasi-likelihood were conducted in R version 3.3.269 using the lme470 and MASS71 packages respectively. Pairwise comparisons were conducted using the multcomp72 package. Pearson’s Chi-square tests were performed using Minitab 17.

Data availability

The datasets generated during and analysed during the current study are available from the corresponding author on request or via the Tropical Research Data Hub (doi:10.4225/28/5923bfed71f8d).