Fish sampling and data collection
In January 2019 brown trout were captured in Scotland, UK, at Loch Sloy
(56.2632175°N, 4.7707667°W; N = 14; FL = 261±52 mm mean±SD) and Carron
Valley Reservoir (56.0338314°N, 4.1057406°W; N = 14; FL = 227±37 mm)
using 30 m × 1.5 m single-mesh (38 mm) benthic gill nets. Three nets
were deployed in late afternoon and retrieved the following morning.
Loch Sloy has surface area of 1.33 km2 and mean depth
25.5 m. Carron Valley Reservoir has surface area 3.76
km2 and mean depth 9.6 m. Fish community in Loch Sloy
is composed of brown trout, European whitefish Coregonus
lavaretus , and European eel Anguilla Anguilla. Carron Valley
Reservoir contains the same fish species as Loch Sloy complemented by
rainbow trout Oncorhynchus mykiss , three-spined stickleback, and
perch Perca fluviatilis . In October 2019 brown trout were
captured by electrofishing (e-fish, UK) in small tributaries of Loch
Lomond, Scotland, UK (56.0470272°N, 4.5504428°W): Ross Burn (N = 12, FL
= 153±16 mm) and Wood Burn (N = 3, FL = 165±42 mm). Ross Burn has a mean
discharge 0.3 m3.s-1 and length 2.3
km and Wood Burn has a mean discharge 1.1
m3.s-1 and length 3.1 km. Fish
communities in Ross Burn and Wood Burn are the same, composed of brown
trout, brook lamprey Lampetra planeri , European eel, and
occasional Atlantic salmon Salmo salar and European minnowPhoxinus phoxinus . Finally, in January 2020 hatchery
young-of-year brown trout (AE Fishery, Moffat, UK) were transported to
the Scottish Centre for Ecology and the Natural Environment (SCENE).
Hatchery trout were held in unadorned 120 L cylindrical tanks at a
density of 20 fish per tank. Tanks were fed individually with water on a
flow-through system directly from Loch Lomond at natural temperatures
(low of ~3° C in winter to high of ~18°
C in summer). Inflows of ~100 L per hour were angled to
provide a current and an air stone was added to each tank. Lighting
simulated ambient sunlight at the latitude of the facility
(~56° N). Hatchery trout were fed daily to satiation on
commercial salmon pellets containing 31.3 % of fish oil (Ewos Ltd.,
UK). These commercial pellets are a standard diet of hatchery trout and
contain high amount of n-3 LC-PUFA (Heissenberger et al. , 2010).
Hatchery fish were held under these conditions until June 2020, when
brain samples of randomly selected individuals were extracted (N = 15,
FL = 178±22 mm). All fish were killed with an overdose of benzocaine.
Heads of fish were removed and fixed in 4% buffered (pH 6.9)
paraformaldehyde solution. Brains were then dissected out by opening the
skull along the anteroposterior axis and removing muscle tissue and
bones around the brain until the brain could be lifted from the skull.
Dissected brains were stored in 4 % buffered paraformaldehyde until
further procedures were conducted. Brains were photographed with a Canon
EOS 1300D DSLR camera with an EF-S18-55 III lens (Canon) and 13- and
31-mm extension tubes designed for Canon DSLRs (Xit Inc.). For each
dissected brain sample, an image was taken from dorsal, left lateral and
ventral views. Each brain was measured to calculate total volume and the
volumes of the cerebellum, optic tectum, telencephalon, olfactory bulb,
and hypothalamus. Measurements were completed using ImageJ 1.48
(Schneider et al. 2012) and used to calculate volume with the formulas
outlined by Pollen et al. (2007).
We performed geometric morphometric analysis of body shape to confirm
the predominant habitat use of wild caught individuals (see Appendix).
This analysis indicated clear differences in body shape of individuals
caught in lake and stream habitat (Procrustes ANOVA:
F2,64 = 11.6, p = 0.001), which suggests that the
presence of individuals in the sampled habitat (i.e. , lake or
stream) was not coincidental, and it corresponds to their long-term
habitat preference.