DOI: 10.5061/dryad.6wwpzgn5q
title: Data for: Testing the population-level effects of stress-induced susceptibility in the ranavirus-wood frog system

description: In this study we tracked experimentally-induced epidemics of ranavirus
 infections in 96 populations of wood frog (Lithobates sylvaticus) larvae
 (in addition to 32 control populations) raised in mesocosms with low or
 high salinity (well water, 109-177uS/cm; or well water + road salt
 1900-2000uS/cm) crossed factorially with ambient or elevated (ambient +
 3°C) water temperature. Epidemics were initiated by the introduction of
 two ranavirus-infected larvae (waterbath exposed to 2.5 × 104
 plaque-forming units/mL of a local ranavirus isolate for the prior 24h)
 into each of the 96 virus mesocosms or two uninfected larvae in the 32
 control mesocosms. Mesocosms were checked daily for carcasses and
 metamorphosing individuals (=front limb emergence), which were removed and
 frozen for later analyses. Recovered carcasses and a subset of
 metamorphosing individuals were then screened for ranavirus infection with
 a quantitative realtime PCR assay. 
description: This data set consists of the identity (SampleID, mesocosm
 [Tank]), treatment information, date of collection (Mortality_Date or date
 of metamorphosis [Meta_Date]), and infection status (PCR positive or
 negative [Pos], mean ranavirus gene copy number [MeanCopy] if positive)
 for each individual animal recovered during the daily checks of the
 experimentally-induced ranavirus epidemics. More details are available in
 the README file 
description: # Data for: Environmental stressors do not lead to more severe ranavirus
 epidemics
 [https://doi.org/10.5061/dryad.6wwpzgn5q](https://doi.org/10.5061/dryad.6wwpzgn5q) This data set consists of the identity, treatment information, date of collection, and infection status for each individual animal recovered during the daily checks of experimentally-induced ranavirus epidemics. Epidemics were initiated with the introduction of two ranavirus-infected larvae in each of 96 populations of wood frog (*Lithobates sylvaticus*) larvae (or the addition of two uninfected larvae in each of the 32 control populations) raised in mesocosms with low or high salinity (well water, 109-177uS/cm; or well water + road salt 1900-2000uS/cm) crossed factorially with ambient or elevated (ambient + 3°C) water temperature. Mesocosms were checked daily for carcasses and metamorphosing individuals (=front limb emergence), which were removed and frozen for later analyses. Recovered carcasses and a subset of metamorphosing individuals were then screened for ranavirus infection with a quantitative realtime PCR assay.  Small pieces (≤ 2 mm-square) of liver, interrenal gland, and intestines, as available, were dissected from the frozen carcasses and DNA was extracted using Qiagen DNeasy blood and tissue kits, following the manufacturer’s recommendations. All instruments were disinfected in a 50% solution of commercial bleach for ≥ 1 min and then rinsed in fresh water prevent cross contamination. Pieces of liver from uninfected bullfrogs (Lithobates catesbeianus) or chicken were extracted with each set of samples as negative extraction controls. Extracted DNA was then run in triplicate 20 µL quantitative real time PCR reactions targeting the viral major capsid protein (MCP) gene (Stilwellet al. 2018). Each 96 well PCR plate included a serial dilution of known number of copies of a gBlock synthetic oligonucleotide (Integrated DNA Technologies, Coralville, IA, USA) containing the target sequence to serve as a standard and no-template controls to detect contamination in the PCR setup. Samples with ambiguous results (e.g., one of three wells showing amplification) were re-run. We used a threshold of 5 copies to separate positive from negative samples. Stilwell, N. K., R. J. Whittington, P. M. Hick, J. A. Becker, E. Ariel, S. van Beurden, N. Vendramin, N. J. Olesen, and T. B. Waltzek. 2018. Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses. Diseases of Aquatic Organisms 128:105-116. ### Description of the data and file structure Each row represents one animal collected during the experiment. Missing data and entries that are "not applicable" are represented with "NA". Dates are provided in the POSIXct date-time format, but no time information is included. Column are as follows: SampleID is the unique identifier of each recovered animal. * Date is the date of collection * Tank is the unique identifier of the mesocosm in which the animal was housed. * Mortality is a logical value indicating whether the animal was found dead (=TRUE) or alive (FALSE) * Meta is a logical value indicating whether the animal was found with its front legs having emerged (TRUE) or not (FALSE) * Stage is a categorical variable describing whether the animal was found as a larvae (=Larva) or metamorph (Meta; with front legs visible) * Mortality_Date is the date when the animal died (usually the same as Date of collection), if it was found dead, or NA otherwise. * Meta_Date is the date when the animal was found with its front legs visible, or NA otherwise. * Meta_Euth_Date is the date that an animal that was collected as a live metamorph was subsequently euthanized, or NA otherwise. * Pos is a categorical variable describing whether a sample was considered positive (Pos) or negative (Neg) for ranavirus DNA, or NA if that information is not available. * MeanCopy is the estimated number of copies of the target ranavirus gene averaged across the three replicate wells or reactions of the quantitative realtime PCR. * Salinity is a categorical variable indicating whether the water in the mescocosm was high (High salt) or low (Low salt) salinity. * Temperature is a categorical variable indicating whether the water temperature was Ambient or Elevated (Ambient + 3°C) * Treatment is a categorical variable that includes the salinity and temperature variables. * Pathogen is a categorical variable that indicates whether the mesocosm had virus-exposed (Virus) or mock-exposed (Control) larvae introduced. * Date_Exposure is the date when the virus- or mock-exposed larvae were added. * DaysPI is the number of days since Date_Exposure the animal was collected. Not that there were two larvae that were collected dead two days prior to the introduction of the virus- or mock-exposed larvae. ### Code/Software R is required to run "MesocosmEpidemic2023Analysis.R" The script was created using version 4.3.2. The script includes the libraries required, loading data, creating data summaries, statistical analyses, and figure generation. Annotations are provided throughout to explain the code.