Megan E. Winzeler Daniel A. Grear 20240912 spreadsheet https://doi.org/10.5066/P14UVMPL Data in this dataset were collected as a part of a surveillance project for reintroduction of a state endangered species in Indiana. This data was collected in the field by state biologists and sent to the USGS National Wildlife Health Center (NWHC) for testing of four amphibian pathogens, Batrachochytrium dendrobatidis (Bd), Batrachochytrium salamandrivorans (Bs), Ranavirus, and perkinsea. The dataset includes both the field records of the individual amphibians tested and the results for individuals for all four pathogens. These data were collected as preliminary information on donator and recipient sites for the crawfish frog reintroduction program in the state of Indiana. This data could be included in regional studies of the presence or absence of Bd, Bs, Ranavirus, or Perkinsea. 20230711 20240306 ground condition Complete As needed Vanderburgh County, Indiana and Greene County, Indiana -87.8137 -86.6602 39.4616 37.8402 ISO 19115 Topic Category biota None Amphibian disease Batrachochytirum dendrobatidis Batrachochytirum salamandrivorans Ranavirus Perkinsea USGS wildlife disease amphibians fungi viruses polymerase chain reaction USGS Metadata Identifier USGS:667def55d34e29ccacefcbf9 None United States Indiana Vanderburgh County, Indiana Greene County, Indiana None American Bullfrog Small-mouthed Salamander Western Chorus Frog Southern Leopard Frog Rana Kingdom Animalia animals Subkingdom Bilateria triploblasts Infrakingdom Deuterostomia Phylum Chordata chordates Subphylum Vertebrata vertebrates Infraphylum Gnathostomata Superclass Tetrapoda Class Amphibia Amphibians Order Anura Frogs Toads Family Ranidae Genus Lithobates American Water Frogs Species Lithobates catesbeianus American Bullfrog TSN: 775084 Species Lithobates sphenocephalus Southern Leopard Frog TSN: 775116 Genus Rana True Frogs Brown Frogs TSN: 173434 Family Hylidae Hylids New World Tree Frogs Treefrogs Hylid Frogs Subfamily Acridinae Genus Pseudacris Chorus Frogs Spring Peepers Species Pseudacris triseriata Striped Chorus Frog Western Chorus Frog TSN: 173525 Order Caudata Salamanders Family Ambystomatidae Mole Salamanders Genus Ambystoma Mole Salamanders Species Ambystoma texanum Small-mouthed Salamander TSN: 173605 None. Please see 'Distribution Info' for details. None. Users are advised to read the dataset's metadata thoroughly to understand appropriate use and data limitations. Megan Winzeler U.S. Geological Survey mailing and physical

6006 Schroeder Rd.

Madison WI 53575 USA 608-270-2400 mwinzeler@usgs.gov This project is supported by USGS Amphibian Research Monitoring Initiative. Measures to ensure quality control of this data included instrument calibrations of the qPCR machines, spot checking field data by multiple people, and quality control limitations on the laboratory assays. Values within range. No data have been duplicated or left out. We removed nothing from this dataset. Field collection of swabs were collected on multiple dates during the fall 2023 and early spring 2024 of over-wintering Ranidae tadpoles in Vanderburgh and Greene counties in Indiana. Individual animals were captured by hand, net, or trap and handled while wearing gloves and using clean plastic bags to collect individuals. All handling was conducted with approved Institutional Animal Care and Use Committee with appropriate location permits. We aimed to capture up to 30 individuals at each site for both fall and spring sampling. In the fall, animals were euthanized and whole bodies sent for necropsy and testing for Bd, Bsal, Ranavirus, and Perkinsea. In the spring, we aimed to collect 2 swabs for Bd and Bsal testing, snout-vent length measurements, and age and sex class estimates. Swabs were chilled immediately and then frozen and sent to the U. S. Geological Survey’s National Wildlife Health Center for molecular testing. For Bd and Bs detection, we extracted DNA from swabs as described by Hyatt et al.29 except that 125 μl of PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA) and 100 mg of zirconium/silica beads (Biospec Products, Bartlesville, OK) were used so that the entire swab was immersed. The bead-beating steps were conducted using a FastPrep®-24 homogenizer (MP Biomedicals, Santa Ana, CA). We used a real-time TaqMan polymerase chain reaction (PCR) for detection of Bsal on the extracted DNA as described in Blooi et al. (2013, 2016). We ran reactions on the QuantStudio5 PCR system (Applied Biosystems, Foster City, CA) using QuantiFast Probe RT-PCR mastermix kit with ROX dye (Qiagen, Valencia, CA) and BSA as per the kit instructions. We used five microliters of the PrepMan® solution containing the extracted DNA as template for the PCR. We included a negative extraction control and a standard curve run in duplicate on each PCR plate. The standard curve consisted of five different concentrations of the target sequence for Bsal inserted into plasmids. The concentrations of the standards occurred at ten-fold dilutions ranging from 110–1,100,000 copies (0.5–5,000 fg DNA) per reaction. The threshold for signal detection was set at 5% of the maximum fluorescence of the standards run for that assay. We considered a detection of Bd and Bsal DNA if a detectable signal existed at 40 or fewer PCR cycles and non-detection in all other cases. We calculated the efficiency of each run using standard curve amplification and repeated PCR plates with an efficiency of less than 90% or greater than 110%. To test for Ranavirus we used a magnetic bead-based extraction method (Ambion MagMAX™ 1836, Thermo Fisher Scientific, MA, USA) on the KingFisher™ Flex System (Thermo Fisher Scientific), with sample DNA eluted in a volume of 90 μl. Briefly, we prepped tail clips (~0.5 g) in viral transport media at a concentration of 1:10 respectively, centrifuged at 1,500 g for 25 minutes to clarify and pellet organic material. We followed recommended protocol for the MagMAXTM 1836 before proceeding to quanatitative PCR (qPCR). We ran a qPCR assay following Leung et al. (2017) in a duplex assay with Ranavirus DNA and host nuclear gene (EBF3N). For one reaction, there was 10.25 uL of water, 2.5 uL 10x buffer, 0.5 uL dNTP, 1.25 uL each of forward and reverse Ranavirus primers, 0.625 uL of Ranavirus probe, 1.25 uL of EBF3N forward and reverse primers, 0.625 uL of EBF3N probe, and 0.5 uL Qiagen HotStar Taq DNA polymerase (5u/uL) and 5 uL of sample. All dNTPs, primers, and probes are at 10 uM concentration. The limit of quantification for 1 copy/reaction of Ranavirus and 3 copies/reaction of gDNA was used for diagnostic testing. The qPCR cycling conditions are as follows: 15 minutes at 95C, then 50 cycles of 15 seconds at 95C and 30 seconds at 60C. We included a negative amplification control and a positive amplification standard curve from 1 x 107 to 1 of DNA. If a well did not amplify genomic DNA (gDNA), the ranavirus test was considered invalid. If gDNA amplified, and Ranavirus copy was greater than or equal to 1, the sample is considered positive. Ranavirus copy number less than 1 and greater than 0 is considered equivocal. A negative Ranavirus result was no amplification in the well. We followed the methods described in Isidoro-Ayza et al. 2017 to test the livers for Perkinsea Hyatt A. D. et al. 2007. Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis. Dis. Aquat. Org. 73, 175-192. https://doi.org/10.3354/dao073175 Blooi, M. et al. 2013. Duplex real-time PCR for rapid simultaneous detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in amphibian samples. J. Clin. Microbiol. 51, 4173-4177. https://doi.org/10.1128/jcm.02313-13 Blooi, M. et al. 2016. Correction for Blooi et al., Duplex real-time PCR for rapid simultaneous detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in amphibian samples. J. Clin. Microbiol. 54, 246-246. https://doi.org/10.1128/jcm.02941-15 Waddle, J. H., et al. 2020. Batrachochytrium salamandrivorans (Bsal) not detected in an intensive survey of wild North American amphibians. Scientific Reports 10, 13012. https://doi.org/10.1038/s41598-020-69486-x Leung, T. M. W. et al. 2017. A quantitative-PCR based method to estimate Ranavirus viral load following normalization by reference to an ultraconserved vertebrate target. J Viro Methods 249:147-155. https://doi.org/10.1016/j.jviromet.2017.08.016 Isidoro-Ayza, M. 2017. Pathogenic lineage of Perkinsea associated with mass mortality of frogs across the United States. Scientific Reports 7:10288. DOI:10.1038/s41598-017-10456-1 20240506 2024_Indiana_Amphibian_Pathogen_Surveillance_Swabs.csv Comma Separated Value (CSV) file containing data. Producer Defined Site Name name of the site. should be consistent between repeat visits within and between years Producer Defined Angel Mounds Angel Mounds, Indiana Producer defined Hillenbrand FWA Hillenbrand Fish & Wildlife Area, Indiana Producer defined Date date that the sampling occurred. Format = YYYY-MM-DD. Range = 2020-02-22 - ongoing Producer Defined 7/11/2023 3/6/2024 Swab Code 10 digit numeric code assigned to each unique sample Producer Defined 2303000193 2400400246 Species Code 4-letter code to designate species from which the sample was collected. Values = SpeciesCode field in sppCode table Producer Defined unRA Unidentified Ranid Frog (Rana sp.) Producer defined PTRI Western Chorus Frog (Pseudacris triseriata) Producer defined AMTE Small-mouthed Salamander (Ambystoma texanum) Producer defined RASP Southern Leopard Frog (Rana sphenocephalus) Producer defined RACA American Bullfrong (Rana catesbeiana) Producer defined Sex Sex of the individual from which the sample was collected. M = male, F = female, U = unknown, NA = not recorded Producer Defined U Unknown Producer defined M Male Producer defined F Female Producer defined Age Age of the individual from which the sample was collected. A = adult, J = juvenile, L = larval, SA = subadult, NA = not recorded Producer Defined L Larval Producer defined A Adult Producer defined J Juvenile Producer defined SVL units = mm. Snout Vent Length of the individual from which the sample was collected. NA = not recorded Producer Defined NA Not Available Producer defined 21 79 Aquatic or Terrestrial Capture environment. Values = Aquatic, Terrestrial, Arboreal, and NA=not recorded Producer Defined A Aquatic Producer defined Lesions Yes or No, were any skin lesions or abnormalities noted. Describe in notes. Values = Yes, No, Blank = not recorded, NA = not recorded Producer Defined NA Not recorded Producer defined N No Producer defined Comments Notes or comments about the sample Producer Defined << empty cell >> No comments available Producer defined Notes or comments about the sample State Location of the state of animal capture Producer Defined Indiana Indiana Producer defined County Location of the county of animal capture Producer Defined Vanderburgh County Producer defined Greene County Producer defined 2024_Indiana_Amphibian_Pathogen_Surveillance_Results.csv Comma Separated Value (CSV) file containing data. Producer Defined Site Name name of the site. should be consistent between repeat visits within and between years Producer Defined Hillenbrand FWA Hillenbrand Fish & Wildlife Area, Indiana Producer defined Angel Mounds Angel Mounds, Indiana Producer defined Date date that the sampling occurred. Format = YYYY-MM-DD. Producer Defined 7/11/2023 3/6/2024 Swab Code Identifier for the individual animal that the result was generated for. May appear multiple times when there are multiple results for an individual. Producer Defined 2303000193 2400400246 SampleID Identifier for the sample collected from the individual to produce a result. Producer Defined 2302500016 2400400246 SampleType type of sample NA= sample type not recorded. Values = Skin Swab, Skin Swab (Frozen or Chilled), NA, Tissue, Environmental Sample Producer Defined Liver Liver Producer defined Swab Skin swab Producer defined Tail Tail Producer defined Bd Detection of Bd target DNA was detected in qPCR reaction. Values = detected: amplification detected a PCR cycle < 40 at 5% of max flourescence and subsequent signal followed a true amplification curve. Not detected = no signal detected; No Amp = signal detected, but due to signal noise and not amplificaiton of the target; equivocal = signal detected during last cycle with likely amplification, but reaction cycles stopped before true amplification could be determined. Producer Defined NA No Data Producer defined Negative Negative result Producer defined Equivocal signal detected during last cycle with likely amplification, but reaction cycles stopped before true amplification could be determined Producer defined Positive Positive result Producer defined Bsal Detection of Bsal target DNA was detected in qPCR reaction. Values = detected: amplification detected a PCR cycle < 40 at 5% of max flourescence and subsequent signal followed a true amplification curve. Not detected = no signal detected; No Amp = signal detected, but due to signal noise and not amplification of the target; equivocal = signal detected during last cycle with likely amplification, but reaction cycles stopped before true amplification could be determined. Producer Defined NA No Data Producer defined Negative Negative result Producer defined No Amp signal detected, but due to signal noise and not amplification of the target Producer defined Ranavirus result of Ranavirus qPCR test; Values = non-detection: no signal detected, invalid: genomic DNA sample was not positive, positive: amplification detected on a PCR cycle < 40 cycles and subsequent signal followed a true amplification curve Producer Defined NA No Data Producer defined Invalid genomic DNA sample was not positive Producer defined Negative negative result Producer defined genomic DNA result of genomic DNA qPCR test; Values = non-detection: no signal detected, detection: numeric Ct value detected genomic DNA presence Producer Defined NA No Data Producer defined numeric Ct value detected genomic DNA presence Perkinsea result of perkinsea PCR test; Values = non-detection: no presence of a band in PCR test, detection: band present on PCR test and sequencing matched Perkinsea Producer Defined NA No Data Producer defined result of perkinsea PCR test; Values = non-detection: no presence of a band in PCR test, detection: band present on PCR test and sequencing matched Perkinsea Comments Notes or comments about the sample Producer Defined << empty cell >> No Data Producer defined Notes or comments about the sample GS ScienceBase U.S. Geological Survey mailing address

Denver Federal Center, Building 810, Mail Stop 302

Denver CO 80225 United States 1-888-275-8747 sciencebase@usgs.gov 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 (USGS), no warranty expressed or implied is made regarding the display or utility of the data for other purposes, nor on all computer systems, nor shall the act of distribution constitute any such warranty. Digital Data https://doi.org/10.5066/P14UVMPL None 20240912 Megan Winzeler U.S. Geological Survey mailing and physical

6006 Schroeder Rd.

Madison WI 53575 USA 608-270-2400 mwinzeler@usgs.gov FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata FGDC-STD-001.1-1999