Encephalisation and brain morphology
To standardise dissection and measurement procedures, all were performed
by a single researcher. Heads of trout from half the trial triads
(i.e. 90 individuals), representing all treatment groups, were
removed and fixed for 24 h in 4% buffered (pH 6.9) paraformaldehyde
solution. Brains were removed following the procedure described by
Závorka et al. (2022a) and fixed for a further 24 h in the buffered
paraformaldehyde solution. They were then weighed to the nearest 0.1 mg.
Dorsal and right lateral views of brains were photographed with a Nikon
D50 DSLR camera (Nikon Corporation, Tokyo, Japan) and Sigma 70 mm F2.8
DG Macro lens (Sigma Corporation, Tokyo, Japan). Measurements were made
using Image J 1.48 (Schneider et al. 2021) of the length (L), width (W)
and depth (D) of the whole brain and, independently, of cerebellum,
optic tectum, telencephalon, and olfactory bulb (Fig. 3). Volumes (V)
were calculated for whole brains with a corrected ellipsoid formula. To
temper the systematic overestimation of volume due to deviations of
brain shape from ellipsoid, a correction factor was introduced,
following Pollen et al. (2007), using the slope of the linear regression
of uncorrected brain volume on actual brain mass (i.e. 1.022):
V = (L × W × D) π / (6 × 1.022).
Volumes of brain regions were calculated with the ellipsoid formula
without the correction factor (Pollen et al., 2007). As measures of
relative whole brain mass, residuals of a linear regression of actual
whole brain mass on body mass were used. Similarly, volumes of each
brain region were regressed on whole brain volume, and residuals were
used as relative volumes of each brain region. On eight occasions during
removal, brains were damaged (typically the delicate olfactory bulbs
were severed); these samples were removed from subsequent analyses.