Effects of elevated CO₂ on predator avoidance behaviour by reef fishes is not altered by experimental test water

Pioneering studies into the effects of elevated CO₂ on the behaviour of reef fishes often tested high-CO₂ reared fish using control water in the test arena. While subsequent studies using rearing treatment water (control or high CO₂) in the test arena have confirmed the effects of high CO₂ on a range of reef fish behaviours, a further investigation into the use of different test water in the experimental arena is warranted. Here, we used a fully factorial design to test the effect of rearing treatment water (control or high CO₂) and experimental test water (control or high CO₂) on antipredator responses of larval reef fishes. We tested antipredator behaviour in larval clownfish Amphiprion percula and ambon damselfish Pomacentrus amboinensis, two species that have been used in previous high CO2 experiments. Specifically, we tested if: (1) using control or high CO₂ water in a two channel flume influenced the response of larval clownfish to predator odour; and (2) using control or high CO₂ water in the test arena influenced the escape response of larval damselfish to a startle stimulus. Finally, (3) because the effects of high CO₂ on fish behaviour appear to be caused by altered function of the GABA-A neurotransmitter we tested if antipredator behaviours were restored in clownfish treated with a GABA antagonist (gabazine) in high CO₂ water. Larval clownfish reared from hatching in control water (496 µatm) strongly avoided predator cue whereas larval clownfish reared from hatching in high CO₂ (1,022 µatm) were attracted to the predator cue, as has been reported in previous studies. There was no effect on fish responses of using either control or high CO₂ water in the flume. Larval damselfish reared for four days in high CO₂ (1,051 µatm) exhibited a slower response to a startle stimulus and slower escape speed compared with fish reared in control conditions (464 µatm). There was no effect of test water on escape responses. Treatment of high-CO₂ reared clownfish with 4 mg l^-1 gabazine in high CO₂ seawater restored the normal response to predator odour, as has been previously reported with fish tested in control water. Our results show that using control water in the experimental trials did not influence the results of previous studies on antipredator behaviour of reef fishes and also supports the results of novel experiments conducted in natural reef habitat at ambient CO₂ levels.