DOI: 10.5061/dryad.qv9s4mwrx
title: High relative humidity and temperature limit disease development and mortality in golden frogs of Panama, Atelopus zeteki, infected with Batrachochytrium dendrobatidis

description: To combat the loss of species due to emerging infectious diseases,
 scientists must incorporate ecological parameters, such as temperature and
 humidity, to understand how the environment affects host–pathogen
 interactions. The fungal disease chytridiomycosis is a compelling case
 study to investigate the role of both temperature and humidity on
 infectious disease, as both the fungal pathogen (Batrachochytrium
 dendrobatidis, Bd) and the host (amphibians) are heavily influenced by
 these abiotic factors. We performed two experiments to investigate the
 importance of relative humidity and temperature on frog immunity
 (production of antimicrobial skin secretions) and disease development in
 captive golden frogs (Atelopus zeteki) of Panama. We found that the
 quantity of skin secretions significantly decreased over time in frogs
 moved from low to medium and high relative humidity treatments. Following
 Bd exposure, frogs in high temperature (26–27 °C) and high relative
 humidity (80–90%) had lower pathogen loads and survived significantly
 longer than frogs kept in all other treatment conditions, including high
 temperature and low relative humidity. These results suggest that high
 relative humidity may be an important, although less understood, mediator
 of Bd infection and the survival of golden frogs. Because the environment
 can drastically alter disease dynamics, understanding how temperature and
 humidity influence chytridiomycosis outcomes in golden frogs may be
 essential for the success of the reintroduction of captive frogs.
description: We obtained Panamanian golden frogs (Atelopus varius) from the Maryland
 zoo as part of the captive breeding program 'Project Golden
 Frog'. For each frog, we measured mass to the nearest 0.01 g, and
 snout-vent length (SVL) to the nearest 0.1 mm using calipers and scales.
 For experiment one, we placed frogs in either high RH (relative humidity),
 medium RH, or low RH conditions. We collected skin swab samples to test
 for Bd presence and infection intensity using standardized swabbing
 techniques. To collect frog skin secretions, we induced half of the frogs
 from each treatment group to secrete their store of secretions from
 cutaneous granular glands by administering a stimulatory injection of 40
 nmol/gram of norepinephrine (NE) below the skin surface (Rollins-Smith et
 al. 2005, Woodhams et al. 2006). The remaining frogs from each RH group
 were given shots of sterile saline as a control. Following a month at each
 humidity treatment, we injected frogs once again with either
 norepinephrine or saline, and then returned them to their humidity
 chambers. Immediately following this second reduction, we exposed frogs to
 the Bd isolate Rio Maria. We measured mass and SVL weekly to calculate
 frog body condition (mass/SVL). We swabbed frogs weekly and used a
 quantitative polymerase chain reaction (qPCR) assay to assess the
 prevalence and intensity of Bd infection in our diagnostic samples. We
 analyzed the quantity of skin secretions collected from each group of
 frogs using a MicroBCA assay using bradykinin to generate a standard
 curve. We assessed the inhibitory effects of these skin secretions
 against Bd in vitro using an MTT assay (Lindauer et al. 2019). For the
 second experiment, we placed frogs in one of the following conditions:
 high RH and high temperature, high RH and low temperature, low RH and high
 temperature, or low RH and low temperature. We exposed half of the frogs
 from each chamber to the Bd isolate Rio Maria. We measured mass and SVL
 weekly to calculate frog body condition (mass/SVL). We swabbed frogs
 weekly and used a quantitative polymerase chain reaction (qPCR) assay to
 assess the prevalence and intensity of Bd infection in our diagnostic
 samples. Data are in .csv files with sheets: ``` 1. Peptide_recovery_data
 ``` Frog_ID: individual frog identification Group: the humidity treatment
 assigned to each frog Protein: the amount of skin secretions collected at
 timepoint 1 in micrograms Protein 2: the amount of skin secretions
 collected at timepoint 2 in micrograms ``` 2. Humidity_Bd_load ```
 Frog_ID: individual frog identification Bd_load: the Bd load (genomic
 equivalents) for each frog at that timepoint. We used a quantitative
 measure of Bd genomic equivalents using a real-time PCR assay with
 universal Bd standards (0.1-100 zoospore equivalents).  Timepoint: the
 week that the Bd load was recorded Date: the calendar date that
 corresponds with each week Group: the humidity treatment assigned to each
 frog Exposure_group: whether the frogs were exposed to Bd or a saline
 control PLUS 1: Adds ‘+1’ to the Bd load column (allows us to take the log
 of the Bd load). Log_GE: the log of the Bd load + 1 for each frog ``` 3.
 Environmental_con_Bd_load ``` Frog_ID: individual frog identification
 Humidity_group: the humidity and temperature treatment that each frog was
 assigned Exposure: whether the frog was exposed to Bd or was a control
 frog. Timepoint: The week that the Bd load was recorded Date: The calendar
 date that corresponds with each week Bd_load: the Bd load (genomic
 equivalents) for each frog at that timepoint. We used a quantitative
 measure of Bd genomic equivalents using a real-time PCR assay with
 universal Bd standards (0.1-100 zoospore equivalents).  Plus_1: the Bd
 load column plus 1 (allows us to take the log of Bd load) Log_GE: the log
 of the Bd load +1 for each frog ``` 4. Humidity_survival ``` Frog_ID:
 individual frog identification DOD: the calendar date that each frog died
 (DOD: date of death) Group: the humidity treatment group that each frog
 was placed in Days survived: the number of days each frog survived ``` 5.
 Environmental_con_survival ``` Frog_ID: individual frog identification
 DOD: the calendar date that each frog died (DOD: date of death) Group: the
 humidity and temperature group that each frog was placed in Days survived:
 the number of days each frog survived Week: the number of weeks each frog
 survived ``` 6. Inhibitory_effectiveness ``` Frog_ID: individual frog
 identification Group: the humidity treatment group of each frog
 Timepoint_1: the inhibitory effect of peptides against Bd from frogs taken
 at timepoint 1. Inhibitory effectiveness of the peptides is based on their
 ability to limit Bd growth. The amount of Bd present after addition of the
 peptides is measured by optical density (OD 570 nm).  Timepoint_2: the
 inhibitory effect of peptides against Bd from frogs taken at timepoint 2.
 Inhibitory effectiveness of peptides is based on their ability to limit Bd
 growth. The amount of Bd present after addition of the peptides is
 measured by optical density (OD 570 nm).  ``` 7. Humidity_Body_condition
 ``` Frog_ID: individual frog identification Treatment: The humidity group
 (high, medium, low) and exposure status (Bd exposed or control) of each
 frog Time: The week that the body condition was recorded Date: The
 calendar date corresponding to each week Mass: The mass of each frog at
 each timepoint measured in grams (g) SVL: The SVL (Snout-Vent-Length) of
 each frog at each timepoint measured in millimeters (mm) Body condition:
 The mass divided by the SVL for each frog at each timepoint ``` 8.
 Environmental_con_body_con ``` Frog_ID: individual frog identification
 Treatment: The humidity/temperature group and exposure status (Bd exposed
 or control) of each frog. Time: The week that body condition was recorded
 Date: The calendar date corresponding to each week Mass: The mass of each
 frog at each timepoint measured in grams (g) SVL: The SVL
 (Snout-Vent-Length) of each frog at each timepoint measured in millimeters
 (mm) Body condition: The mass divided by the SVL for each frog at each
 timepoint ``` 9. Panama_env_data ``` Date Time: The date and time that the
 temperature and humidity was recorded Ttemp: The temperature (degrees
 Celsius) taken at the first field station TRH: The relative humidity taken
 at the first field station Btemp: The temperature (degrees Celsius) taken
 at the second field station BRH: The RH taken at the second field station
 ``` 10. Lab_temp_hum ``` Date: The date and time that temperature and
 humidity data were collected Chamber 1 Temp, °F: The temperature from
 chamber 1 (High RH High Temp) Chamber 1 RH, %: The Relative humidity from
 chamber 1 (High RH High Temp) Chamber 2 Temp, °F: The temperature from
 chamber 2 (High RH Low Temp) Chamber 2 RH, %: The relative humidity from
 chamber 2 (High RH Low Temp) Chamber 3 Temp, °F: The temperature from
 chamber 3 (Low RH High Temp) Chamber 3 RH, %: The relative humidity from
 chamber 3 (Low RH High Temp) Room Temp, °F: The temperature from the room
 (Low RH Low Temp) Room RH, %: The relative humidity from the room (Low RH
 Low Temp)