Stanek, Ashley E. (ORCID: 0000-0001-5184-2126) Waters-Dynes, Shannon C.(ORCID: 0000-0002-9707-4684) Bowen, Lizabeth (ORCID: 0000-0001-9115-4336) von Biela, Vanessa R. (ORCID: 0000-0002-7139-5981) 20250606 tabular digital data Anchorage, Alaska U.S. Geological Survey, Alaska Science Center Suggested Citation: Stanek, A.E., Waters-Dynes, S.C., Bowen, L., and von Biela, V.R., 2025, Gene transcription in Pacific salmon: U.S. Geological Survey data release, https://doi.org/10.5066/P19M3AYH https://doi.org/10.5066/P19M3AYH This data release contains one table with gene transcription results from muscle samples collected from fish occupying freshwater in various Alaskan watersheds. Heat shock protein 70 (HSP70) results from fish included in this data release are published in a separate data release available at https://doi.org/10.5066/P13ZYKWR. These data were collected to determine the prevalence of heat stress in Pacific salmon migrating in Alaskan freshwater (rivers, streams, and lakes) to spawning grounds. 20190715 20220803 observed In work As needed Alaska -178.4180 -128.6719 71.5805 50.7365 USGS Metadata Identifier USGS:6827a6e6d4be02693eeabdc0 ISO 19115 Topic Category Biota Environment NASA GCMD Earth Science Keyword Thesaurus Animals/vertebrates Fish Freshwater ecosystems USGS CSA Biocomplexity Thesaurus Anadromous species Biological stress Heat tolerance USGS Thesaurus Aquatic biology Fishery resources None Chinook salmon Oncorhynchus tshawytscha Coho salmon Oncorhynchus kisutch USGS Geographic Names Information System (GNIS) Alaska Deshka River None Chinook salmon Oncorhynchus tshawytscha Coho salmon Oncorhynchus kisutch Integrated Taxonomic Information System Unknown on-line database online ITIS-North America Taxonomic details retrieved May 6, 2024 from the Integrated Taxonomic Information System online database https://www.itis.gov https://doi.org/10.5066/F7KH0KBK Fish were identified in the field by skilled observers following Mecklenburg et al. 2002. Kingdom Animalia Subkingdom Bilateria Infrakingdom Deuterostomia Phylum Chordata Subphylum Vertebrata Infraphylum Gnathostomata Superclass Actinopterygii Class Teleostei Superorder Protacanthopterygii Order Salmoniformes Family Salmonidae Subfamily Salmoninae Genus Oncorhynchus Species Oncorhynchus tshawytscha Chinook salmon TSN: 161980 Species Oncorhynchus kisutch Coho salmon TSN: 161977 No access constraints. No use constraints. These data are marked with a Creative Common CC0 1.0 Universal License and are in the public domain. It is requested that this USGS data release be cited for any subsequent publications that reference or utilize these data. Users are advised to read the dataset's metadata thoroughly to understand appropriate use and data limitations. U.S. Geological Survey, Alaska Science Center Mailing and Physical

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Anchorage Alaska 99508 USA 907-786-7000 gs-ak_asc_datamanagers@usgs.gov This work was supported by U.S. Geological Survey Ecosystems Mission Area, U.S. Fish and Wildlife Service, and Alaska Department of Fish and Game. von Biela, V.R. Regish, A.M. Bowen, L. Waters, S. Carey, M.P. Zimmerman, C.E. Gerken, J. Rinella, D. McCormick, S.D. Unknown journal article TBD TBD TBD TBD von Biela, V.R., Regish, A., Bowen, L., Waters, S., Carey, M.P., Zimmerman C.E., Gerken, J., Rinella, D., and McCormick, S.D., in preparation, First evidence of heat stress in subarctic juvenile Pacific salmon. TBD https://doi.org/TBD Stanek, Ashley E. von Biela, Vanessa R. Regish, Amy M. 2025 tabular digital data Stanek, A.E., von Biela, V.R., and Regish, A.M., 2025, Heat shock protein 70 abundance in Pacific Salmon: U.S. Geological Survey data release, https://doi.org/10.5066/P13ZYKWR https://doi.org/10.5066/P13ZYKWR Attribute values were spot checked to identify possible errors. Attribute values fall within expected ranges. Blank cells indicate that no data was collected for that value. Blank cells indicate data were not collected or not provided. Data omissions for particular entities are explained in the Entity and Attribute definitions. Positions determined by hand held GPS; accuracy estimated to be within 3 meters. FIELD (TISSUE SAMPLING): Field collection methods depend on the life stage of the fish collected. For adults, non-lethal muscle biopsies were opportunistically collected at existing adult Pacific salmon monitoring sites, such as weirs, in Alaska. Biopsies were obtained using sterile, 6mm, biopsy punches (Integra, USA) without the use of anesthesia. The biopsy was taken dorsally (above the lateral line of the fish) to sample white muscle tissue. To sample juveniles, individuals were euthanized with an overdose of AQUI-S (AQII-S, New Zealand) and a dorsal muscle sample was dissected. After collection, muscle samples were stored in dry cryoshippers at ultra cold temperatures (-70°C or colder) while in the field and during shipping. Upon arrival at the USGS Alaska Science Center from the field, samples were stored at -80°C until analyzed. Unknown LABORATORY (GENE TRANSCRIPTION): 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). Unknown LABORATORY (qPCR DATA NORMALIZATION AND TRANSFORMATION): To generate a measure of the relative amount of transcription, we first normalized values to the threshold crossing (CT) by subtracting the CT of the reference gene, rpL8, from the CT of the gene of interest. A change in normalized value of two is approximately equivalent to a four-fold change in the amount of the transcript. These delta CT values are related to the number of PCR cycles, so lower values indicate higher levels of mRNA (with the maximum value of 36 indicating gene transcripts are at or below detection limits) and thus are not intuitive. Therefore, we then transposed the values by subtracting delta CT from 40 so that a higher value indicates more mRNA transcript. The resulting values did not meet the statistical assumption of normality so a log10 transformation was applied. This data release includes both the normalized qPCR values (delta CT values) (i.e. delta CT values) in attributes labeled with the gene name, while the attributes with the gene name followed by “tt” are the transposed and transformed qPCR values. Unknown LITERATURE CITED: Mecklenburg, C.W., Mecklenburg, T.A., Thorsteinson, L.K., 2002. Fishes of Alaska. American Fisheries Society, Bethesda, Maryland. 1116 p. von Biela, V.R., Bowen, L., McCormick, S.D., Carey, M.P., Donnelly, D.S., Waters, S., Regish, A.M., Laske, S.M., Brown, R.J., Larson, S., Zuray, S., Zimmerman, C.E., 2020. Evidence of prevalent heat stress in Yukon River Chinook salmon. Canadian Journal of Fisheries and Aquatic Sciences 77: 1878–1892. https://doi.org/10.1139/cjfas-2020-0209 Unknown Spatial information in this data set includes named locations and geographic coordinates in decimal degrees. Point 0.00001 0.00001 Decimal degrees World Geodetic System of 1984 (WGS84) World Geodetic System of 1984 (WGS84) 6378137 298.257223563 genes_heatStress_pacificSalmon.csv Table containing gene transcription values for Pacific salmon sampled in Alaska, along with sampling date and location, and length measurements. Presented in a Comma Separated Value (CSV) formatted table. Author defined FishID FishID is a concatenation of CHIN for Chinook salmon or COHO for coho salmon, a two-letter code for each collection location, and a 3-digit number for each individual fish sampled. Note that for some juvenile fish from the Deshka River, 'FishIDs' do not follow the same naming convention. In these cases, samples were put into the incorrect vial in the field, thus the 'FishID' reflects the species and/or year on the vial label and does not necessarily reflect the sample. The correct species and date of collection is included in the species and date attributes. Samples that do not follow the naming convention are: 2022-COHODR020, 2022-COHODR021, 2022-COHODR022, 2022-COHODR023, 2022-COHODR024, 2022-COHODR072, COHODR068, COHODR069, COHODR070, COHODR071. Where conflicts between naming convention and other data columns occur, the information in the data columns for species and year are correct. Author defined The unique identifier for individual fish sampled. Species Common name of the fish species sampled. Author defined Chinook Salmon A Chinook salmon (Oncorhynchus tshawytscha) was sampled. Author defined Coho Salmon A coho salmon (Oncorhynchus kisutch) was sampled. Author defined Date Date of the fish sample collection. Author defined 2019-07-15 2023-08-03 Date (YYYY-MM-DD) Collector The name of the project partner that collected the fish muscle sample that was analyzed. Author defined The name of the organization that collected the fish muscle sample that was analyzed. Location The name of the river or site where each fish was sampled, approximate coordinates for the sampling location are included in the 'Latitude' and 'Longitude' attributes of this entity. Author defined The name of the river or site where each fish was sampled. Latitude Latitude of the location where the sampled fish was captured. Author defined 61.78522 62.33378 Decimal degrees (WGS84) Longitude Longitude of the location where the sampled fish was captured. Author defined -150.5849 -150.3474 Decimal degrees (WGS84) LengthMetric The type of measurement for values recorded in the mmLength attribute. Measurements for adult fish (>150mm) were made from the mid-eye to the fork (MEF) of the tail. Fork length was measured for juvenile fish (<150mm) from the tip of the nose to the fork of the tail. Blank cells indicate that the length of the fish at capture was not recorded. Author defined Mid-eye to the fork (MEF) length Fish were measured from mid-eye to the fork of the tail. Author defined Fork length Fish were measured from the tip of the nose to the fork of the tail Author defined mmLength The length of the fish at capture. Blank cells indicate that the length of the fish at capture was not recorded. Author defined 24 870 millimeters Gata3 A measurement of the relative amount of transcript for the Th2-specific transcription factor (Gata3) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined 2.9722 16.5170 normalized qPCR values (delta CT values) (delta CT values) AHR A measurement of the relative amount of transcript for the Aryl hydrocarbon receptor (AHR) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined 3.2639 10.9707 normalized qPCR values (delta CT values) (delta CT values) HSP27 A measurement of the relative amount of transcript for the heat shock protein 27 (HSP27) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined -1.7616 7.9125 normalized qPCR values (delta CT values) (delta CT values) HSP90 A measurement of the relative amount of transcript for the heat shock protein 90 (HSP90) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined -5.7625 5.3223 normalized qPCR values (delta CT values) (delta CT values) IFNa A measurement of the relative amount of transcript for the Th1-specific interferon alpha, type I (IFNa) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined 1.7723 12.2332 normalized qPCR values (delta CT values) (delta CT values) IFNg2 A measurement of the relative amount of transcript for the interferon gamma 2 (IFNg2) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined 5.2982 19.1341 normalized qPCR values (delta CT values) Leptin A measurement of the relative amount of transcript for the Leptin gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined 5.2982 19.7926 normalized qPCR values (delta CT values) MT_A A measurement of the relative amount of transcript for the Metallothionein A (MT-A) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined -6.5258 5.1666 normalized qPCR values (delta CT values) MX1 A measurement of the relative amount of transcript for the Orthomyxovirus resistance gene 1 (MX1) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined 5.2982 17.2465 normalized qPCR values (delta CT values) New_CYP1A A measurement of the relative amount of transcript for the hydrocarbon-inducible cytochrome P540 1A (CYP1A) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined 1.9021 13.7170 normalized qPCR values (delta CT values) HSP70 A measurement of the relative amount of transcript for the heat shock protein 70 (HSP70) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined -2.2040 4.2299 normalized qPCR values (delta CT values) SOD A measurement of the relative amount of transcript for the Cyp1A Superoxide dismutase (SOD) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined -0.4523 5.7248 normalized qPCR values (delta CT values) tbx21 A measurement of the relative amount of transcript for the Th1-specific Tbox transcription factor (tbx21) gene; measured by quantitative real-time polymerase chain reaction (qPCR) and normalized to the reference gene as described in the methodology section of this data release. Author defined 4.855 13.188 normalized qPCR values (delta CT values) Gata3tt Value resulting from the transformation of normalized qPCR values (delta CT values) of the Th2-specific transcription factor (Gata3) gene following methods described in the methodology section of this data release. Author defined 1.37 1.57 transposed and transformed qPCR values AHRtt Value resulting from the transformation of normalized qPCR values (delta CT values) of the Aryl hydrocarbon receptor (AHR) gene following methods described in the methodology section of this data release. Author defined 1.46 1.57 transposed and transformed qPCR values HSP27tt Value resulting from the transformation of normalized qPCR values (delta CT values) of the Aryl hydrocarbon receptor (AHR) gene following methods described in the methodology section of this data release. Author defined 1.51 1.62 transposed and transformed qPCR values HSP90tt Value resulting from the transformation of normalized qPCR values (delta CT values) of the heat shock protein 90 (HSP90) gene following methods described in the methodology section of this data release. Author defined 1.54 1.66 transposed and transformed qPCR values IFNatt Value resulting from the transformation of normalized qPCR values (delta CT values) of the Th1-specific interferon alpha, type I (IFNa) gene following methods described in the methodology section of this data release. Author defined 1.44 1.58 transposed and transformed qPCR values IFNg2tt Value resulting from the transformation of normalized qPCR values (delta CT values) of the interferon gamma 2 (IFNg2) gene following methods described in the methodology section of this data release. Author defined 1.32 1.54 transposed and transformed qPCR values Leptintt Value resulting from the transformation of normalized qPCR values (delta CT values) of the Leptin gene following methods described in the methodology section of this data release. Author defined 1.31 1.54 transposed and transformed qPCR values MT_Att Value resulting from the transformation of normalized qPCR values (delta CT values) of the Metallothionein A (MT-A) gene following methods described in the methodology section of this data release. Author defined 1.54 1.67 transposed and transformed qPCR values MX1tt Value resulting from the transformation of normalized qPCR values (delta CT values) of the Orthomyxovirus resistance gene 1 (MX1) gene following methods described in the methodology section of this data release. Author defined 1.36 1.54 transposed and transformed qPCR values New_CYP1Att Value resulting from the transformation of normalized qPCR values (delta CT values) of the hydrocarbon-inducible cytochrome P540 1A (CYP1A) gene following methods described in the methodology section of this data release. Author defined 1.42 1.58 transposed and transformed qPCR values HSP70tt Value resulting from the transformation of normalized qPCR values (delta CT values) of the heat shock protein 70 (HSP70) gene following methods described in the methodology section of this data release. Author defined 1.55 1.63 transposed and transformed qPCR values SODtt Value resulting from the transformation of normalized qPCR values (delta CT values) of the Cyp1A Superoxide dismutase (SOD) gene following methods described in the methodology section of this data release. Author defined 1.53 1.61 transposed and transformed qPCR values tbx21tt Value resulting from the transformation of normalized qPCR values (delta CT values) of the Th1-specific Tbox transcription factor (tbx21) gene following methods described in the methodology section of this data release. Author defined 1.43 1.55 transposed and transformed qPCR values U.S. Geological Survey USGS ScienceBase Team Mailing and Physical

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Denver Colorado 80225 USA 1-888-275-8747 sciencebase@usgs.gov The U.S. Geological Survey, Alaska Science Center is the authoritative source of these data, distributed by ScienceBase (a USGS Trusted Digital Repository). Unless otherwise stated, all data, metadata and related materials are considered to satisfy the quality standards relative to the purpose for which the data were collected. Although these data and associated metadata have been reviewed for accuracy and completeness and approved for release by the U.S. Geological Survey, no warranty expressed or implied is made regarding the display or utility of the data for other purposes or on all computer systems, nor shall the act of distribution constitute any such warranty. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. CSV Tabular data in CSV format; FGDC metadata in XML and HTML formats. https://doi.org/10.5066/P19M3AYH None 20250606 U.S. Geological Survey, Alaska Science Center Mailing and Physical

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Anchorage Alaska 99508 USA 907-786-7000 gs-ak_asc_datamanagers@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999