Heating experiment and temperature measurements
Nests were heated starting on the day after hatch (day 1 of the nestling stage) until the first hatched nesting was 18 days old. The treatment was suspended at this age to provide a consistent amount of heat to all our samples within and among nests, as all birds fledged at days 19 or 20 of age. A Kapton flexible heating device (Omega Engineering KHA-404(10)-P), powered by a DC regulated Powertech power supply (HW1200R-12), was connected to a Vemer digital heat regulator (HT NIPT-1P3A VM628500) set at 40 ± 0.2°C, and to a digital time switch (Vemer MICRO-D) that automatically started and ended the daily treatment. The heat output was regulated on the base of the nest temperature recorded by a Vemer VE122800 double injection IP68 probe placed in the nest. Whenever the air temperature in the nest was higher than the set point (40°C) the circuit was automatically turned off. Vice versa when nest temperature was lower, power was activated and heating resumed. This temperature value was chosen to replicate daily maximal Ta conditions recorded in the natural habitat for this species during a substantial part of their breeding season (Griffithet al. 2016). Also, 40 °C represents an ideal compromise between challenging the nestlings to maintain homeostasis (Wojciechowskiet al. 2021), and yet moderating exposure to harmful temperatures. The heat treatment was applied for six hours a day from 9:00 until 15:00. In this way we simulated the natural pattern of heat waves experienced in the field, while also allowing parental care and measurements to be taken by us before and after this time interval. Control nests were exposed to the same type of disturbance as heated nests with the difference that sham devices substituted batteries, thermostats, heaters and wires.
Two distinct temperature values were measured every 24 seconds at two separate positions inside each nest box between day one and day eight post-hatch. One probe was suspended 3 cm below the box roof to record ambient temperature Ta. A second probe was placed on the bottom of the nest cup in contact with the nestlings to record brood temperature Tbr. Both probes were wired to a Gemini Tinytag Plus 2 data logger. Temperature was recorded only during this first ectothermic half of the post-natal period because measurements of later treatment effects on Tbr become unreliable due to the increased mobility of nestlings at older ages that often leads them to cluster in corners. Temperature traces were analysed and processed using Tinytag Explorer (ver. 4.7) and during the experiment both were significantly higher in treatment (Ta=37.3 ± 1.33°C; Tbr=35.4 ± 1.84°C) compared to control boxes (Ta=21.5 ± 1.84°C, t1,11 = 19.99, p < 0.001; Tbr=33.7 ± 1.33°C, t1,11 = 2.49, p = 0.01) (See Ton et al. 2021a for further details).