2.1 Sample collection, group assignment, and tissue harvesting
All samples of westslope cutthroat trout were sampled on a single day in May 2019 at the Montana Fish, Wildlife, & Parks Sekokini Springs hatchery in West Glacier, MT. We collected 120 samples, which were divided in three treatment groups as follows (Supporting Information Table S1): group 1 = net-sampling (n = 40); group 2 = electrofishing (n = 40); group 3 = electrofishing, tissue harvested from fish 5 minutes after death by pithing (see below, tissues from group 1 and 2 fish were harvested immediately) (n = 40). All the fish were fry from the same breeding stock and were offspring from parents from Danaher Creek (MT). Before sampling for tissue harvesting, fish were netted as needed from the raceway and transferred to holding buckets with hatchery system water. In order to simulate field work conditions, each raceway collection was at least 10 minutes apart. Independent of the sampling method, fish were sampled five at a time to reduce the interval between sampling and tissue harvesting. Each fish was then captured by net from one of the holding buckets or shocked with a backpack electrofishing unit set to 150 volts with a standard pulse for a duration of 3 seconds and then netted. Length and weight data were collected for each fish. Fish were then euthanized by pithing and immediately 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 euthanasia by pithing they were placed in a separate holding bucket of water for 5 minutes before tissue harvesting.
Electrofishing, which consists of a backpack mounted electrofishing unit that applies an electrical current in the water to momentarily stun the fish, is one of the most common fisheries sampling techniques. This technique may cause the fish to express genes in response to the electric current, and may affect individual fish and tissue types differently, increasing variation among biological replicates. An alternative to electrofishing is dip netting. While nets may potentially result in a lower effect on gene expression and lower risk of inadvertently killing both target and non-target organisms, it is more laborious and time consuming in the field where circumstances may not allow for long sampling periods or aquatic systems may have obstacles that prevent effective capture with nets (e.g. fallen tree limbs and rocks). Capturing fish by dip netting may still influence gene expression through stress, as the fish tries to escape being captured.
Processing times of fish and tissues may depend on which tissue needs to be harvested and how many fish are captured at the same time. Therefore, time between capture and euthanasia and duration of tissue collection were recorded for each individual. Average time in the bucket was around 2 minutes before euthanasia and average time of tissue collection was around 3 minutes (data available upon request). 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 DNA 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 G. I. 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 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.