Gene Transcription and Heat Shock Protein 70 Abundance in Juvenile Hatchery Reared Coho Salmon and Chinook Salmon during a Manipulative Thermal Experiment, Anchorage, Alaska 2020-2021

Two identical experiments were conducted at the Alaska Department of Fish and Game’s William Jack Hernandez Sport Fish Hatchery in Anchorage, Alaska, USA. The Chinook salmon experiment was conducted in May 2020 and the coho salmon experiment was conducted in May 2021. For each experiment, approximately 150 age-0 (juvenile) individuals were transferred to 15 experimental tanks (75 L). Each tank was randomly assigned one of five treatment temperatures (15, 17, 19, 21, and 23°C), such that each treatment was applied to three tanks. Each tank contained 10 individual fish and one aquarium heater with a temperature controller (Inkbird Tech ITC-306T or ITC-308) that activated the heater when water temperature dropped greater than 0.2°C below the set point. Water temperatures were recorded by a Hobo Tidbit at 5 min intervals. After at least a 1-hour habituation period, water temperatures were warmed at a rate of 2°C per hour. Half of the individuals in each tank were sacrificed after a 12h exposure to the treatment temperature and the other half were sacrificed after 36h. Individuals were dispatched by cranial concussions followed by cervical dislocation. Fork length was measured to the nearest millimeter. Two skeletal muscle tissue samples were removed from above the lateral line using a scalpel and immediately placed in 2ml cryovials and flash frozen in cryoshippers at temperatures below -80°C. One set of samples was sent to the USGS Conte Anadromous Fish Research Laboratory (Turners Falls, MA, USA) for measurement of HSP70 protein concentration. The other set of samples was sent to the USGS Western Ecological Research Center (Davis, CA, USA) for gene transcription analysis.

HSP70 ELIZA: Frozen muscle samples were dissected away from subdermal fat and skin and weighed to the nearest mg, homogenized, and centrifuged. Muscle homogenates were used in a competitive enzyme-linked immunosorbent assay (ELISA), modified from (Faught et al. 2017). Plates were coated with Chinook Salmon HSP (Enzo Life Sciences, USA), washed, blocked with bovine serum albumin buffer, incubated with sample or standard plus primary polyclonal antibody specific to the inducible form of HSP70 (Agrisera, Sweden), washed again, incubated with secondary antibody (goat anti rabbit-HRP Sera Care, USA), and finally developed using a colorimetric substrate 3,3', 5, 5' - tetramethylbenzidine (TMB, Sera Care, USA). HSP70 values based on the standards ranging from 0.6 to 320 ng per mL were calculated with a 4-parameter curve, and final data was corrected by total protein and expressed as ng HSP70 per mg total protein. Measurement of HSP70 protein concentration was conducted at the USGS Conte Anadromous Fish Research Laboratory (Turners Falls, MA, USA).

Gene transcription qPCR: Gene transcription was measured using quantitative real-time polymerase chain reaction (qPCR) assays of mRNA at the USGS Western Ecological Research Center (Davis, CA, USA). Total RNA was extracted from homogenized muscle tissue using the RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, California). To remove contaminating genomic (g)DNA, extracted total RNA was treated with 10 U per µL of RNase-free DNase I (DNase, Amersham Pharmacia Biotech Inc.) at room temperature (20–30°C) for 15 min. The extracted RNA was stored in a –80°C freezer until analyzed (von Biela et al. 2020). A standard cDNA synthesis was performed on 2 µg of RNA template from each Chinook salmon and coho salmon. Reaction conditions included four units reverse transcriptase (Omniscript, Qiagen, Valencia, California), 1 µmol per L random hexamers, 0.5 mmol per L each dNTP, and 10 units RNase inhibitor, in reverse transcription (RT) buffer (Qiagen, Valencia, California). Reactions were incubated for 60 min at 37°C, followed by an enzyme inactivation step of 5 min at 93°C, and then stored at –20°C until further analysis. Briefly, 1 µL of cDNA was added to a mix containing 12.5 µL of QuantiTect Fast SYBR Green Master Mix (5 mmol per L Mg2+; Qiagen, Valencia, California), 0.5 µL each of forward and reverse sequence specific primers with a concentration of 10 µM (von Biela et al., 2020), and 10.5 µL of RNase-free water; total reaction mixture was 25 µL. The primers for heat shock protein for 27, 70, and 90 are specific to the inducible forms of these genes. The reaction mixture cDNA samples for each gene of interest and reference genes were loaded into MicroAmp Fast Optical 96 well reaction plates in duplicate and sealed with optical sealing tape (Applied Biosystems, Foster City, California). Reaction mixtures containing water, but no cDNA, were used as negative controls. Amplifications were conducted on a QuantStudio 3 Real-time Thermal Cycler (Applied Biosystems, Foster City, California), using the QuantStudio 3 software. Reaction conditions were as follows: an initial hold stage of 95°C for 20 s, 40 cycles of 95°C for 1 s, and 60°C for 20 s. The melt curve was 95°C for 1 s, 60°C for 20 s, and 0.3°C per second temperature increase, and then 95°C for 1 s (von Biela et al. 2020). Gene transcription was conducted at the USGS Western Ecological Research Center (Davis, CA, USA).

qPCR data transformation: We transformed the qPCR data according to the 2∧(-CT’) method (Livak and Schmittgen, 2001) as follows. First, we normalized values (threshold crossing (CT) of the reference gene subtracted from the threshold crossing for the gene of interest). We then compared the normalized value of the target gene to the CT of the calibrator sample (the lowest level of transcription for each gene). This gave us normalized transcription values for each gene relative to the maximum observed CT across all samples. We then log-transformed the transcription values, and those log-transformed values served as the basis for all analyses going forward.