Detailed methods from Ocean acidification effects on fish hearing

Humans are rapidly changing the marine environment through a multitude of effects, including increased greenhouse gas emissions resulting in warmer and acidified oceans. Elevated CO₂ conditions can cause sensory deficits and altered behaviours in marine organisms, either directly by affecting end organ sensitivity or due to likely alterations in brain chemistry. Previous studies show that auditory-associated behaviours of larval and juveniles fishes can be affected by elevated CO₂ (1000 µatm). Here, using auditory evoked potentials (AEP) and micro-computer tomography (microCt) we show that raising juvenile snapper, Chrysophyrs auratus, under predicted future CO₂ conditions resulted in significant changes to their hearing ability. Specifically, snapper raised under elevated CO₂ conditions had a significant decrease in low frequency (less than 200 Hz) hearing sensitivity. MicroCt demonstrated that these elevated CO₂ snapper had sacculus otolith's that were significantly larger and had fluctuating asymmetry, which likely explains the difference in hearing sensitivity. We suggest that elevated CO₂ conditions have a dual effect on hearing, directly effecting the sensitivity of the hearing end organs and altering previously described hearing induced behaviours. This is the first time that predicted future CO₂ conditions have been empirically linked through modification of auditory anatomy to changes in fish hearing ability. Given the widespread and well-documented impact of elevated CO₂ on fish auditory anatomy, predictions of how fish life-history functions dependent on hearing may respond to climate change may need to be reassessed.