Stanek, Ashley E. (ORCID: 0000-0001-5184-2126) von Biela, Vanessa R. (ORCID: 0000-0002-7139-5981) Regish, Amy M. (ORCID: 0000-0003-4747-4265) 20250325 tabular digital data Heat Shock Protein 70 Abundance in Pacific Salmon ver 3.0, January 2026 Anchorage, Alaska U.S. Geological Survey, Alaska Science Center Suggested Citation: Stanek, A.E., von Biela, V.R., and Regish, A.M., 2025, Heat shock protein 70 abundance in Pacific Salmon (ver 3.0, January 2026): U.S. Geological Survey data release, https://doi.org/10.5066/P13ZYKWR https://doi.org/10.5066/P13ZYKWR This data release contains one table with heat shock protein 70 (HSP70) abundance measurements of Pacific salmon (Oncorhynchus spp.) muscle samples, collected from fish occupying freshwater in various Alaskan watersheds. Water temperatures associated with a sampled fish are also included in the dataset, when available. Gene transcription results from fish included in this data release are published in a separate data release; https://doi.org/10.5066/P19M3AYH. 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. HSP70 is a heat stress biomarker that increases with protein damage in the cells associated with exposure to warm water temperatures. Heat stress is a life-threatening condition when warm temperatures are prolonged (a week or more) and/or extreme (>21°C), although individuals can recover when the duration of exposure to warm temperatures is short (e.g., hours) and temperatures are only moderately warm. HSP70 values are also associated with most recent water temperature readings, when water temperature data is available from a collaborator. HSP70 increases are not exclusively related to warm water temperatures, so the inclusion of water temperature data provides for indirect validation that HSP70 increases are related to water temperatures, and confirms HSP70 as a biomarker of heat stress for each species and stock of Pacific salmon studied. HSP70 protein is elevated in tissues in response to thermal stress where it aids in the repair of heat damaged proteins and helps to maintain cellular function. HSP70 has been used as a cellular biomarker of thermal stress in fishes (von Biela et al. 2020, 2023, Mohanty et al. 2018) and is thought to be a physiological mechanism for thermal acclimation (Narum et al. 2013). In the short term HSP70 helps return animals to homeostatic norms, however under chronic or severe heat stress, whole-animal changes in performance and survival can result from heat stress. 20190715 20240910 observed In work As needed Alaska, USA, and Yukon, Canada -162.883000 -135.041434 66.573701 60.296085 USGS Metadata Identifier USGS:66e9a2dfd34e0606a9dba178 ISO 19115 Topic Category Biota Environment NASA GCMD Earth Science Keyword Thesaurus Animals/vertebrates Fish USGS CSA Biocomplexity Thesaurus Anadromous species Heat shock proteins Biological stress Heat tolerance USGS Thesaurus Aquatic biology Fishery resources NIH MeSH Thesaurus Enzyme-Linked Immunosorbent Assay USGS Geographic Names Information System (GNIS) Alaska Yukon River Kuskokwim River Norton Sound George River Kwethluk River Shaktoolik River Unalakleet River Aniak River Eagle Crooked Creek The Rapids Fort Yukon Pilot Station Deshka River NGA GEOnet Names Server Canada Yukon Whitehorse None Chinook salmon Oncorhynchus tshawytscha Coho salmon Oncorhynchus kisutch Integrated Taxonomic Information System Unknown online database online ITIS-North America Taxonomic details retrieved January 21, 2026 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 and National Fish and Wildlife Foundation. Project partners include: Bering Sea Fishermen's Association, Native Village of Napaimute, Alaska Department of Fish and Game, Kuskokwim River Intertribal Fish Commission, Norton Sound Economic Development Corporation, U.S. Fish and Wildlife Service, and subsistence fishers. von Biela, V.R. Regish, A.M. McCormick, S.D. Spaeder, J. Whitworth, K. Leon, J. Gillikin, D. Liller, Z. Ivanoff, R. Bell, J. Larson, S.D. Carey, M.P. Zimmerman, C.E. 2025 journal article Canadian Journal of Fisheries and Aquatic Sciences 83 Online Canadian Science Publishing von Biela, V.R., Regish, A.M., McCormick, S.D., Spaeder, J., Whitworth, K., Leon, J., Gillikin, D., Liller, Z., Ivanoff, R., Bell, J., Larson, S.D., Carey, M.P., and Zimmerman C.E., 2025, Migration water temperature and heat stress assessments in Western Alaska Chinook salmon overlapping the 2019 heatwave. Canadian Journal of Fisheries and Aquatic Sciences 83, https://doi.org/10.1139/cjfas-2025-0109 https://doi.org/10.1139/cjfas-2025-0109 Lee, M.L. von Biela, V.R. McCormick, S.D. Regish, A.M. Seits, A. Westley, P.A.H. Unknown journal article Transactions of the American Fisheries Society TBD TBD TBD Lee, M.L., von Biela, V.R., McCormick, S.D., Regish, A.M., Seitz, A., Westley, P.A.H., in review, Factors beyond temperature influence heat stress responses of adult Chinook Salmon in Southcentral Alaska. Transactions of the American Fisheries Society. TBD https://doi.org/TBD Stanek, A.E. Waters, S. Bowen, L. von Biela, V.R. Unknown data release online USGS Alaska Science Center Stanek, A.E., Waters, S., Bowen, L., von Biela, V.R., in review, Gene transcription in Pacific salmon: U.S. Geological Survey data release, https://doi.org/10.5066/P19M3AYH https://doi.org/10.5066/P19M3AYH 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): Non-lethal muscle biopsies were opportunistically collected at existing adult Pacific salmon monitoring sites, such as test fisheries or weirs, in Alaska and at a hatchery in Whitehorse, Yukon, Canada. Biopsies were obtained from adults using sterile, 6mm, biopsy punches (Integra, USA). To sample juveniles, a dorsal muscle sample was dissected from lethally sampled fish. 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 FIELD (WATER TEMPERATURES): Water temperatures (Celsius) were recorded in the field by study partners. Summarized values used in data analysis are included in this data release; details regarding water temperature values can be found in the associated interpretative publication listed in the associated data table "heatStress_pacificSalmon.csv". A citation for publicly available data or general information on the data collector, location, and sampling interval of water temperature values is provided. Summary methods are briefly described in the attribute descriptions. Unknown LABORATORY (HSP70 ELISA): Frozen muscle samples were dissected away from subdermal fat and skin and weighed to the nearest milligram, 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 (Oncorhynchus tshawytscha) 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). The ELISA has a lower detection limit of 0.3 ng per mL such that any instance of values <0.3 ng per mL were replaced with 0.3 ng per mL. 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. Measurements of HSP70 protein concentration were conducted at the USGS Conte Anadromous Fish Research Laboratory (Turners Falls, MA, USA). HSP70 values can be categorized as elevated and reflective of heat stress or normal and unstressed using a threshold of >2 ng HSP70 per mg total protein (von Biela et al. 2023). This heat stress threshold was developed using a heat stress experiment with juvenile Chinook salmon (n=141) where individuals were held at five different temperature treatments (15, 17, 19, 21, and 23°C) for 12 or 36 hours and identified that cooler treatments resulted in HSP70 values ≤2 ng HSP70 per mg total protein in 96% of individuals held in water temperatures of 15–19°C and >2 ng HSP70 per mg total protein in 100% of individuals held at 21 and 23°C (von Biela et al. 2023). Unknown LITERATURE CITED: Faught E., Henrickson L., Vijayan M.M., 2017. Plasma exosomes are enriched in Hsp70 and modulated by stress and cortisol in rainbow trout. Journal of Endocrinology 232(2): 237–246. doi:10.1530/JOE-16-0427 Mecklenburg, C.W., Mecklenburg, T.A., Thorsteinson, L.K., 2002. Fishes of Alaska. American Fisheries Society, Bethesda, Maryland. 1116 p. Mohanty, B.P., Mahanty, A., Mitra, T., Parija, S.C., Mohanty, S., 2018. Heat Shock Proteins in Stress in Teleosts. In: Asea, A., Kaur, P. (eds) Regulation of Heat Shock Protein Responses. Heat Shock Proteins, vol 13. Springer, Cham. https://doi.org/10.1007/978-3-319-74715-6_4 Narum, S.R., Campbell, N.R., Meyer, K.A., Miller, M.R. and Hardy, R.W., 2013. Thermal adaptation and acclimation of ectotherms from differing aquatic climates. Molecular Ecology 22: 3090-3097. https://doi.org/10.1111/mec.12240 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 von Biela, V.R., Regish, A.M., Bowen, L., Stanek, A.E., Waters, S., Carey, M.P., Zimmerman, C.E., Gerken, J., Rinella, D., McCormick, S.D., 2023. Differential heat shock protein responses in two species of Pacific salmon and their utility in identifying heat stress. Conservation Physiology 11(1): coad092; doi:10.1093/conphys/coad092 Unknown Spatial information in this data set include named locations and geographic coordinates in decimal degrees. Point 0.000001 0.000001 Decimal degrees World Geodetic System of 1984 (WGS84) World Geodetic System of 1984 (WGS84) 6378137 298.257223563 HSP70_pacificSalmon.csv Table containing HSP70 measurements for Pacific salmon sampled in Alaska and Yukon, Canada, along with sampling date and location, and length measurements. Summarized water temperature values used in data analysis are included, when applicable. Presented in a Comma Separated Value (CSV) formatted table. Author defined FishID Unique identifier for individual fish sampled. For samples collected at Rampart Rapids and Pilot station, the FishID is a concatenation of RARA or PIST, respectively, followed by the year and a three-digit number for each individual fish. For samples from all other locations the FishID is Aa concatenation of CHIN for Chinook salmon or COHO for coho salmon, a two-letter code for each experiment location, and a three digit number for each individual fish sampled. Note that for some juvenile fish from the Deshka River, FishIDs do not follow the 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: 2021-COHODR020, 2021-COHODR021, 2021-COHODR023, 2021-COHODR024, 2021-COHODR072, 2021-COHODR073, 2021-COHODR074, 2021-COHODR075, 2022-COHODR020, 2022-COHODR021, 2022-COHODR022, 2022-COHODR023, 2022-COHODR024, 2022-COHODR072, COHODR068, COHODR069, COHODR070, COHODR071. 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 sample collection. Author defined 2019-07-15 2024-09-10 Date (YYYY-MM-DD) Region The watershed or general region where the fish was sampled. Author defined The watershed or general region where the fish was sampled. Collector The name of the project partner that collected the fish muscle sample that was analyzed. Author defined The name of the project partner 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 (decimal degrees) of the location where the sampled fish was captured. Author defined 60.296085 66.573701 Decimal degrees (WGS84) Longitude Longitude (decimal degrees) of the location where the sampled fish was captured. Author defined -162.883000 -135.041434 Decimal degrees (WGS84) LengthMetric The type of measurement 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; measured to the nearest millimeter. The type of measurement is included in the 'LengthMetric' column. 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 24 995 millimeters HSP70 HSP70 protein abundance is determined by enzyme-linked immunosorbent assay (ELISA) and calculated by comparison of observed values to a 4-parameter standard curve generated from known concentrations of purified HSP70. These values are normalized to the total amount of muscle protein assayed. The resulting units are defined as nanograms HSP70 per milligrams of total protein. Author defined 0.01 118.15 nanograms (ng) of HSP70 protein per milligram (mg) of total protein TempMetric The method used to summarize water temperature values included in the 'TempC' attribute. Blank cells indicate that a temperature value is not associated with this sample. Author defined Maximum three-day water temperature Value in the 'TempC' attribute is the estimated maximum water temperature to which each fish was exposed, given their sampling date and location. The value is calculated based on the maximum water temperature recorded in a three-day window that includes the date of fish capture and two days prior. The source of temperature values which were summarized, and the location of measurements used in the calculation is included in the 'TempSource' attribute. Author defined TempC Summarized water temperature value at a location and time period that is relevant to the HSP70 value measured in the fish muscle. The method used to calculate the summarized value reported here is described in the 'TempMetric' attribute. Blank cells indicate that water temperatures were not measured. Author defined 12.57 21.008 degrees Celsius TempSource The source of the water temperature data. If temperature data is publicly available, a citation is included, otherwise the name of the project that collected the temperature data and the recording interval is included. Blank cells indicate that the temperature source was not recorded. Author defined The source of the water temperature data; either a citation or the name of the project that collected the temperature data and the recording interval in hours. InterpretativeCitation The citation abbreviation of the interpretative product where this data was analyzed. Full citation details can be found in the cross-reference section of this metadata record. Blank cells indicate that the data are not associated with a particular product. Author defined The citation abbreviation of the interpretative product where this data was analyzed. Flag This column contains additional information of which users must be aware. Blank cells indicate that no additional information needs to be shared. Author defined This column contains additional information of which users must be aware. 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/P13ZYKWR None 20260121 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