Supplementary material from "CO₂-induced ocean acidification does not affect individual or group behaviour in a temperate damselfish"

Open ocean surface CO₂ levels are projected to reach approximately 800 μatm, and ocean pH to decrease by approximately 0.3 units by the year 2100 due to anthropogenic CO₂ emissions and the subsequent process of ocean acidification (OA). When exposed to these CO₂/pH values, several fish species display abnormal behaviour in laboratory tests, an effect proposed to be linked to altered neuronal GABA_A­ receptor function. Juvenile blacksmith (Chromis punctipinnis) are social fish that regularly experience CO₂/pH fluctuations through kelp forest diurnal primary production and upwelling events, so we hypothesized that they might be resilient to OA. Blacksmiths were exposed to control conditions (pH ∼ 7.92; pCO₂ ∼ 540 μatm), constant acidification (pH ∼ 7.72; pCO₂ ∼ 920 μatm) and oscillating acidification (pH ∼ 7.92, pCO₂ ∼ 560 μatm (day), pH ∼ 7.75, pCO₂ ∼ 955 μatm (night)), and caught and tested in two seasons of the year when the ocean temperature was different: winter (16.5 ± 0.1°C) and summer (23.1 ± 0.1°C). Neither constant nor oscillating CO₂-induced acidification affected blacksmith individual light/dark preference, inter-individual distance in a shoal or the shoal's response to a novel object, suggesting that blacksmiths are tolerant to projected future OA conditions. However, blacksmiths tested during the winter demonstrated significantly higher dark preference in the individual light/dark preference test, thus confirming season and/or water temperature as relevant factors to consider in behavioural tests.