Data from Chapter 4 of the PhD thesis: Thermal thresholds in the amphibian disease chytridiomycosis.

Thermoregulation in frogs may be a normal part of day-to-day physiology or a behavioral fever response to infection. Regardless of the reason, thermoregulatory behaviors may cause frogs to experience daily spikes in temperature, which can help frogs avoid, tolerate, or clear infections by cold-tolerant parasites. Infections by the fungus Batrachochytrium dendrobatidis (Bd) alter internal homeostasis and water balance and may therefore alter frogs’ ability to tolerate elevated temperatures, which could discourage protective thermoregulatory behaviors. We tested for effects of Bd infection on host thermal tolerance in the model frog species Litoria spenceri.

We first inoculated frogs with Bd and acclimated them to four temperature treatments. Two temperature treatments represented high elevation conditions and two temperature treatments represented low elevation conditions. The two high elevation treatments were (1) a daily rectangular wave with trough at 15°C for 20 h per day and crest at 26°C for four hours per day (hereafter high elevation heat pulse) and (2) a constant 15°C control treatment (hereafter high elevation constant). Our two low elevation treatments were (1) a daily rectangular wave with trough at 18°C for 20 hours per day and crest at 29°C for four hours per day (hereafter low elevation heat pulse) and (2) a constant 18°C control treatment (hereafter low elevation constant).

To ensure infection, we inoculated frogs with Bd on two consecutive days. On each day, we prepared a zoospore suspension of 1 x 106 zoospores per ml. We inoculated frogs at room temperature on three consecutive days. To inoculate, we placed each frog into an individual 70-ml plastic container and added 3 ml of zoospore inoculant or sham inoculant (enough to cover the bottom of the container) to each container using a syringe. We left frogs in inoculant baths for eight hours per day. To ensure regular contact of frogs with the inoculant, we monitored frogs every 15 minutes during each inoculation period. If a frog had climbed out of the inoculant onto the wall of the container, we gently tilted the container to bathe the frog in the inoculant. After each inoculation period, we returned frogs with their inoculant to individual permanent enclosures comprising 70 x 120 x 170 mm plastic containers lined with tap water-saturated paper towel. We allocated frogs in their individual enclosures to temperature-controlled chambers on the day after the last inoculation. To monitor Bd infection status and intensity, we swabbed frogs upon delivery from the captive breeding facility and every eight days thereafter and determined the number of Bd zoospore genome equivalents (ZGE) per swab with a real-time quantitative PCR protocol. Frogs were acclimated to the temperature treatments for a minimum of 36 days.

To measure thermal tolerance, we placed individual frogs into a perforated container containing a suspended thermocouple. Each frog was brought to room temperature in its permanent enclosure and then transferred to the perforated container and placed in a temperature-controlled chamber programmed to increase from room temperature at a rate of ~1°C per minute. We used two measures of thermal tolerance: onset of spasms and loss of righting ability. Onset of spasms was the temperature at which frogs began displaying erratic movements such as increased jumping and leg twitches. After onset of spasms, at each 1°C increase in chamber temperature, we quickly opened the chamber, gently moved the container until the frog jumped, and closed the chamber. Loss of righting ability was the temperature at which animals were unable to right themselves for three seconds after this manipulation. To minimize stress to the frogs, we elected to record the ambient (i.e., thermocouple) temperature at each behavioral indicator of thermal tolerance for each frog. Frogs were then immediately placed in room-temperature water to recuperate. To convert ambient temperatures to frog body temperatures, we later exposed four haphazardly selected L. spenceri of average sizes to the same program of gradually increasing temperature in the same chamber. For each frog, we recorded body temperature at 25°C, 30°C, 35°C, and 40°C ambient temperature. We measured body temperature with a non-contact infrared thermometer (OS425-LS, Omega Engineering Ltd, Irlam, Manchester, UK; emissivity 0.95). We then modeled the relationship between ambient and body temperatures using linear regression and used this analysis to convert ambient temperatures to body temperatures for all experimental frogs.

This dataset shows all data from this experiment. Column headings are explained below.

Species = frog species tested
Date = date tested
Day = day of experiment
Frog = individual frog identifier
Mass = frog mass in grams
Sul = frog snout-urostyle length in mm
Treat = acclimation temperature treatment
Inc = temperature-controlled chamber identifier
Type = control (uninfected) or treatment (infected)
Status = disease status (positive or negative)
Discomf = ambient temperature at onset of spasms
Discomf_body = frog body temperature at onset of spasms
Ctmax = ambient temperature at loss of righting response
Ctmax_body = frog body temperature at loss of righting response
Load = Bd zoospore equivalents detected on swab
Log_load = Log10-transformed Bd zoospore equivalents detected on swab