2.1 Sample collection, group assignment, and tissue harvesting
All samples of westslope cutthroat trout were collected on a single day
in May 2019 at the Montana Fish, Wildlife, & Parks Sekokini Springs
hatchery in West Glacier, MT (USA). We collected 30 fish divided in
three treatment groups (10 fish per group), each with 4 tissues for a
total of 120 samples as follows (Tables 1 and 2 and available on Dryad):
group 1 = net-sampling with immediate tissue harvest (n samples = 40);
group 2 = electrofishing with immediate tissue harvest (n samples = 40);
group 3 = electrofishing, tissue harvested from fish 5 minutes after
death by pithing (see below, n samples = 40). All fish were fry (1 year
old; fish were non-sexually mature as they cannot be sexed until 2-4
years old) from the same breeding stock and were offspring (F1) from
wild parents from Danaher Creek (MT). Average size of fish was 108 mm
+/- 11 and average weight was 10g +/- 3.
Fish were gently netted five at the time from the raceway into holding
buckets containing hatchery system water. Fish were then either captured
from the bucket by net or electrofished backpack electrofishing unit set
to 150 volts with a standard pulse for a duration of 3 seconds. Fish
were then euthanized by pithing and processed for tissue harvesting,
except for group 3. Fish from group 3 were sampled in the same way as
fish from group 2, except that after pithing they were placed in a
separate holding bucket of water for 5 minutes before tissue harvesting
to test for the influence of delayed tissue harvesting. Time between
capture and euthanasia and duration of tissue collection were recorded
for each individual. Average time in the bucket was approximately 2
minutes before euthanasia and average time of tissue collection after
pithing was approximately 3 minutes, except for group 3, for which
tissue harvesting began 2-3 min after the 5 min from pithing. Length and
weight data were collected for each fish. Sample information, including
times of tissue harvest after euthanasia for each sample can be found in
Table S1 on Dryad).
Tissue removal was performed using single use scalpels on a nylon
cutting board. Tissue samples from each fish were collected in the
following order: blood, dorsal muscle, liver, and gills. We first
collected the blood immediately before euthanasia as coagulated blood
may affect RNA quality (Chiari and Galtier, 2011). To obtain the blood
sample, the tail was removed by a diagonal cut made through the caudal
peduncle from dorsally anterior of the anal fin to ventrally posterior
of the anal fin to avoid intersecting the gastrointestinal tract. Slight
pressure was applied to the body of the fish and blood was allowed to
drip out of the cut directly into the 2 mL tube. Muscle tissue was
sliced into smaller pieces to allow penetration of the preservative
(Gayral et al., 2011). Sampling tools and the cutting board were
thoroughly cleaned with 10% bleach first and then purified water
between fish to avoid sample and tissue contamination. Tissue samples
were placed in 2 mL sterile tubes filled with RNAlater (Qiagen) for
preservation. Tubes were left at room temperature overnight and then
stored at -80C (or in dry ice for transportation) until the RNA
extraction was carried out. All sampling was carried out according to
the Institutional Animal Care and Use Committee (IACUC) approved permit
#AUP 007-19GLFLBS-062819 to GL.