2.1 Animal collection and experimental setup
The squid tissues and behavioural data used in this study came from a
previous experiment. Specifically, we used sham-treated squid from the
picrotoxin experiment in Thomas et al. (2021) (Figure 1). As
described in Thomas et al. (2021), male two-toned pygmy squid
(Idiosepius pygmaeus) were collected from the wild and acclimated
in groups at current-day seawater conditions for 1 - 6 days before
transferral to individual treatment tanks set at either current-day
(~400 µatm) or elevated (~1,000 µatm)
CO2 levels, consistent with CO2 levels
projected for 2100 following the representative concentration pathway
RCP8.5 scenario (Bindoff et al., 2019). Experiments were carried
out in four interconnected 8,000 L recirculating seawater systems; two
untreated seawater systems were used for current-day CO2treatments, and two seawater systems were dosed with CO2 using a custom-built pH control system for elevated CO2treatments. The CO2 conditions achieved were current-day
CO2: 407 ± 58 µatm pCO2,
pHT = 8.09 ± 0.10 and elevated CO2:
1,071 ± 71 µatm pCO2, pHT = 7.73
± 0.03 (mean ± SD). Refer to Thomas et al. (2021) for further
details. CO2 variation is common in coastal habitats
(Hannan et al., 2020). However, the coastal waters where we
collected I. pygmaeus show little daily variation in seawater pCO2 levels; average daily range 20.3 ± 8.6 µatm
CO2 (mean ± SD) (Supplementary Text S1 and Figure S1).
Thus, our experimental CO2 levels are ecologically
relevant to the population of I. pygmaeus used.