Fish dissection
A subsample of brown trout (Table 1) at each study site was transported
alive to the department of Biology and Environmental Sciences at the
University of Gothenburg for dissection. Before the dissection, fish
were housed in holding tanks (30 L, 300 × 320 × 340 mm), which provided
shelter structures (rocks and plastic plants) and fresh 12 °C water from
a flow-through filtration system (flow rate: 2 L
min-1). Fish were not fed during this period. All fish
were dissected within 20 hours after capture and to randomize the time
effect, individuals from allopatric and sympatric sites were dissected
in alternating order (with even and odd individuals from allopatry and
sympatry respectively). Before dissection, fish were euthanized by an
overdose of 2-phenoxyethanol (0.5 mL L-1) and body
mass and fork length were measured to the nearest 0.1 g and 1.0 mm
respectively. Sex of individuals was determined by inspection of the
gonads. Heads of fish were removed and fixed in 4% buffered (pH 6.9)
paraformaldehyde solution. Brains were then dissected out as described
in Gonda et al. (2009) by opening the scull along the anteroposterior
axis and removing muscle tissue, nerves and bones until the brain could
be lifted up from the skull and stored in 4% buffered paraformaldehyde
solution until further procedure. Brains were imaged with a digital
camera (EOS 40D with MP-E 65mm lens; Canon Inc., Tokyo, Japan). Images
of brains were taken using the dorsal, left lateral and ventral views to
calculate the total volume and the volume of cerebellum, optic tectum,
telencephalon, olfactory bulb, and hypothalamus. Measurements were
completed using ImageJ 1.48 (Schneider et al. 2021) and used to
calculate volume with the formulas outlined by Pollen et al. (2007).
Brain morphology was assessed by principal component analysis
(Supporting Information S2) yielding two dominant principal components.
PC1 was positively related to the volume of olfactory bulb, and
hypothalamus and negatively related to cerebellum and optic tectum,
while PC2 was positively related to the volume of telencephalon.
Fixing fish brains in paraformaldehyde solution in order to do
morphological measurements prevented us from performing fatty acid
analysis of the brain tissue. Therefore, we have used the muscle tissue
as a proxy of n-3 LC-PUFA content in fish body. Muscles provide a good
proxy for this purpose, because they represent the majority of fish
biomass, and thus muscle metabolic activity and fatty acids content
reflects individuals as a whole (Norin & Malte 2012; Gladyshev et al.
2018). In addition, biochemical composition of muscle tissue of
salmonids has been shown to respond in similar direction to dietary
deprivation of n-3 LC-PUFA as brain tissue, but with higher magnitude
(Závorka et al. 2021). Thus, samples (~1 g of wet mass)
of dorsal and ventral muscle tissue samples were taken from the left
side below the dorsal fin, above and below the lateral line
respectively. Bones and skin residuals were mechanically removed from
the tissue samples, before they were stored on dry ice and subsequently
frozen at -80 °C.