Gene Transcription and Heat Shock Protein 70 Abundance Measured in Muscle Tissue of Chinook Salmon, Yukon River Watershed, Alaska, 2016–2018

Muscle biopsy samples were collected from 477 live Chinook salmon during their spawning migration in the Yukon River watershed during summer 2016 and 2017 and 22 Chinook salmon from a controlled temperature experiment conducted near Pilot Station, AK, in 2018. The timing of sample collection varied by sites to generally align with the peak of spawning migration at each location. Collection locations were annual management assessment projects or subsistence fishing efforts. Chinook salmon were sampled in Alaska at three locations on the main-stem Yukon River and three tributaries. Main-stem Yukon River locations were near the mouth at Emmonak (ADF&G test fishery), subsistence fish wheels in the middle portion of the main-stem Yukon River (Rapids Fish Wheel operated by Stan Zuray and others), and just before the Canadian border at Eagle (ADF&G test fishery). Upstream migration in the main-stem occurs primarily in June and July. Tributary collections occurred at weirs in the East Fork Andreafsky and Gisasa rivers (U.S. Fish and Wildlife Service) and electrofishing in the Chena and Salcha rivers (ADF&G). Fish collected in the Chena and Salcha rivers were grouped together for analysis because both are components of the Tanana River population with similar migration routes and temperature histories. Sampling at all tributary locations occurred primarily in July.

A subset of the samples included were from 22 Chinook salmon held in a controlled temperature experiment conducted near Pilot Station, AK, in 2018. Briefly, up to three individual Chinook salmon were acquired daily from the ADF&G test fishery across nine consecutive days (n = 27; n = 5 individuals either died or did not acclimate to the experiment and no data are included here for those individuals) and randomly assigned to one of three tanks: control (river ambient; ~15 °C), 18 °C, or 21 °C. Treatment tanks were 587 L polyethylene stock tanks with a liquid propane fueled heater to raise temperature, electric aquarium heaters to maintain temperature, circulation pumps to prevent thermal stratification, aerators to supplement dissolved oxygen, and temperature loggers to record water temperature every five minutes. Each experimental run lasted approximately six hours with a minimum of four hours at the treatment temperature. Prior experiments indicate that four hours is sufficient to e;ocotgene transcription and protein responses to heat stress (Buckley et al. 2006). The rate of temperature rise to treatment temperatures was 3.71 ± 1.31 °C (mean ± SD) with a mean hold temperature of 18.0 °C in the low heat stress treatment (range = 17.3 – 18.6 °C) and 20.9 °C in the high heat stress treatment (range = 19.8 – 22.0 °C). Individuals in the control group were held at a near consistent ambient water temperature until the completion of the heat treatments each day. Details of the heating and control system used for these experiments can be found in Donnelly et al. (2020)

All samples were immediately placed in microcentrifuge tubes and then into a liquid nitrogen dry shipper. All tissue samples were stored at –70 °C or below until laboratory work was conducted to assess the gene transcription and protein abundance.

Gene transcription was measured using quantitative real-time polymerase chain reaction (qPCR) assays of mRNA at the U.S. Geological Survey Western Ecological Research Center in Davis, CA, USA. Total RNA was extracted from ground muscle tissue using the RNeasy Lipid Tissue Mini Kit (Qiagen; www.qiagen.com). To remove contaminating genomic (g)DNA, extracted total RNA was treated with 10 U μl−1 of RNase-free DNase I (DNase, Amersham Pharmacia Biotech Inc.; www.apbiotech.com) at room temperature (20-30°C) for 15 min. The extracted RNA was stored in a -80 °C freezer until analyzed.

A standard cDNA synthesis was performed on 2 μg of RNA template from each salmon. Reaction conditions included 4 units reverse transcriptase (Omniscript, Qiagen, Valencia, CA), 1 μM random hexamers, 0.5 mM each dNTP, and 10 units RNase inhibitor, in RT buffer (Qiagen, Valencia, CA). Reactions were incubated for 60 min at 37 °C, followed by an enzyme inactivation step of 5 minutes 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 [5mM Mg2+] (Qiagen, Valencia, CA), 0.5 μl each of forward and reverse sequence specific primers (Table 2), and 10.5 μl of RNase-free water; total reaction mixture was 25 μl. 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, CA). 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, CA), 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/s temperature increase, and then 95 °C for 1 s. We analyzed qPCR data using normalized values calculated as the threshold cycle (CT) of the reference gene subtracted from the CT of the gene of interest where CT is the amplification cycle that allows for detection. Because samples with inherently higher numbers of transcripts require fewer amplification cycles for detection, lower normalized values indicate that more transcripts are present. A change in normalized value of 2 is approximately equivalent to a 4-fold change in the amount of the transcript.

HSP70 protein abundance was analyzed at the U.S. Geological Survey S.O. Conte Anadromous Fish Research Laboratory, Turners Falls, MA, USA. Muscle from the dermal punch was separated from subdermal fat and skin and weighed to the nearest milligram. All tissues were thawed and homogenized with a Kontes Pestle Pellet handheld homogenizer (Thermo Fisher Scientific, Hampton, NH, USA) in 10 volumes of SEID (150 mM sucrose, 10 mM EDTA and 50 mM imidazole, pH 7.3 plus 0.1% deoxycholic acid). Homogenates were centrifuged at 3000 x g for 7 min at 4 °C. A portion of the resulting supernatant was immediately diluted with an equal volume of 2 × Laemmli buffer, heated for 15 min at 65 °C and stored at -80 °C. A small volume of supernatant was used to determine total protein concentration in quadruplicate using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Hampton, NH, USA). Thawed samples were run on a 7.5% SDS-PAGE gel along with Precision Plus protein standards at 5 µg in a reference lane (Bio-Rad Laboratories, Hercules, CA, USA). Dilution titration for tissue homogenates was completed to establish the range of linearity. A total of 10 µg of muscle protein was loaded per sample. Two lanes were reserved on each gel for a standard consistent tissue preparation as reference to control for blot-to-blot variation and to allow for comparison across all treatments and locations. Following electrophoresis, proteins were transferred to Immobilon polyvinylidene difluoride (PVDF) transfer membrane (Millipore, Bedford, MA, USA) at 30 V overnight in 25 mMTris, 192 mM glycine buffer, pH 8.3. Equal loading was verified by reversible total protein staining with Ponceau S. Samples with unequal loading or alternate banding patterns were removed from analysis. PVDF membranes were blocked with 5% non-fat dry milk in PBST (phosphate buffered saline plus 0.1% Triton X-100) for 1 h at room temperature, rinsed in PBST, and exposed to primary polyclonal antibody specific for the inducible form of salmonid HSP70 (AS05061; Agrisera, Vannas, Sweden) at 1:25,000 dilution in PBST with 5% non-fat dry milk for 1 h at room temperature. After rinsing in PBST, blots were exposed to goat anti-rabbit IgG conjugated to horseradish peroxidase diluted 1:10 000 in PBST and 5% non-fat dry milk for 1 h at room temperature. Blots were washed in PBST and incubated for 1 min in a 1:1 mixture of enhanced chemiluminescent (ECL) solution A (396 µMcoumaric acid, 2.5 µM luminol, 100 mM Tris, pH 8.5) and ECL B (0.018% H2O2, 100 mM Tris, pH 8.5), and then digitally imaged and quantified (Syngene PXi, GeneTools, Frederick, MD, USA). All blots were normalized to the internal standard consistent tissue preparation and are represented as a ratio to the mean standard value that we refer to as HSP70 relative abundance.

Heat stress was inferred through differences between fish held in the control and elevated temperature treatments that allow for high classification accuracy (correct classification > 75%) for both the gene transcript levels and HSP70 protein abundance. Following separate statistical analysis for the two biomarkers, heat stress classifications based on gene transcription and HSP70 protein were considered jointly with indications of heat stress in both biomarkers interpreted as more severe stress (Lund et al. 2002; Lewis et al. 2016).