All procedures were approved by Deakin University Animal Ethics Committee (G06-2017), the Animal Ethics Committee of the University of Pretoria (protocol EC048-18) and the Research and Scientific Ethics Committee of the South African National Biodiversity Institute (P18/36). All experiments were performed in accordance with Australian guidelines and regulations for the use of animals in research. This study was conducted in compliance with the ARRIVE guidelines (https://arriveguidelines.org).
Experimental acoustic treatments and housing
Experimental birds were wild-derived zebra finches from an acoustic playback experiment previously presented in Mariette and Buchanan31. At laying (Feb–March 2014), eggs were collected from outdoor aviaries (Deakin University, Geelong, Australia), replaced by dummy eggs and placed in an artificial incubator at 37.5 °C and 60% relative humidity. After nine days, whole clutches were randomly assigned to one of two acoustic playback groups: treatment eggs were exposed to heat-calls (aka “incubation calls”) and controls to adult contact calls (i.e. tet calls), whilst both groups were also exposed to common nest-specific calls (i.e. whine calls) to ensure normal acoustic stimulation. Playbacks had 20 min of heat-calls or tet calls per 1h15, separated by silence and whine calls, and played from 9:30 a.m. to 6:30 p.m.31. To avoid any differences in incubation conditions, eggs and sound cards were swapped daily between incubators. After hatching, nestlings were reared in mixed or single-group broods, in the same outdoor aviaries (see Supplementary Material).
At adulthood (March–April 2018), we tested 34 experimental birds (16 females and 18 males; 15 treatment and 19 control birds) at the end of their fourth summer. From February 2018, birds were moved to indoor cages for acclimation, at least 27 days before experimental trials, at a constant room temperature of 25 °C and day-night cycle of 12 h:12 h, and supplied with ad libitum finch seed mix, grit, cucumber and water. After three days, we implanted a temperature-sensitive passive integrated transponder (PIT) tag (Biomark, Boise ID, USA) subcutaneously into the bird’s flank. Subcutaneous PIT tags reduce the risk of injuries and generally yield Tb values similar to those obtained using intraperitoneally-injected tags in small birds such as the zebra finch62,63.
Experimental heat exposure protocol
All birds were tested twice. Each individual’s second trial occurred on a different day than the first, with an average of 16 days between the two trials, but each bird was tested in the morning for one trial (~ 10:30 a.m.) and in the afternoon (~ 2:50 p.m.) for the other, in random order. On average, trials lasted 125 min (range: 93–151 min). The predicted mean digesta retention time for a 12 g bird is ~ 50 min64. Hence, to ensure birds were post-absorptive, they were fasted (but with ad-libitum water) for two hours before each trial, within auditory and visual contact of conspecifics. Birds were then weighed to measure the initial mass (massinit ± 0.01 g), before being placed individually in the metabolic chamber within a temperature-controlled cabinet. There were no significant difference in massinit between heat-call (12.04 ± 0.18 g) and control individuals (12.03 ± 0.15 g; t (60) = − 0.059, p = 0.953).
During each trial, Ta in the metabolic chamber was gradually increased in a succession of “stages”. Trials started with Ta = 27 °C for 25 min or 45 min (for the first or second trial respectively), then Ta = 35 °C for 30 min (i.e. thermoneutrality54, followed by 20-min stages in succession at Ta = 40, 42 and 44 °C. Temperature transition took 1 (for 2 °C) to 6 min (for 8 °C increments).
To “complete the trial”, individuals had to be able to remain in the chamber for 20 min at Ta = 44 °C. Bird behaviour in the chamber was monitored using two infrared video cameras by an experimenter (AP) blind to playback treatments. The trial was terminated early if the bird showed sustained escape behaviour, or reached a thermal endpoint (e.g., loss of balance or severe hyperthermia with Tb > 45 °C16,52). Immediately after trial termination or completion, birds were taken out of the chamber and exposed to room temperature. They were then weighed (massend), given water on their bill, and transferred to the holding room at 25 °C in an individual cage with ad libitum seeds and water. After one hour, birds were weighed again (mass1h). No bird died during the trials.
Thermoregulatory measurements and data processing
We used an open flow-through respirometry system to measure CO2 production and EWL, following Whitfield et al.52 and as commonly used to assess avian thermoregulation in the heat19,53,60. Dry air was pushed into a 1.5-L plastic metabolic chamber, maintained at low humidity levels (< 0.72 kPa in excurrent air) by regulating the flow rate (range: 1–3.5 L.min-1) with a mass flow controller. Air was subsampled and pulled into H2O (RH-300, Sable Systems) and CO2 analysers (CA-10, Sable Systems). Details of the respirometry system and calibration procedures are in the Supplementary Material.
Following Whitfield et al.52, in Expedata, for each Ta stage, we selected the 1-min window with lowest and least variable CO2 and H2O values, after ≥ 10 min (or ≥ 5 min at Ta = 42–44 °C) of stable Ta. We calculated MR and EWL using equations 9.5 and 9.6 from Lighton65, assuming a respiratory exchange ratio (RER) of 0.71 for fasted individuals66. Using a RER of 0.83 (i.e. metabolism of approximately equal mix of lipids and carbohydrates60 did not change any result. We computed relative water economy (RWE) as the ratio of metabolic water production (MWP; calculated from rates of CO2 production) to EWL2,59; and the evaporative cooling capacity as the ratio of EHL (calculated from EWL) to MHP (approximated by MR, see Supplementary Material)67. Body temperature was recorded every 10 s using a PIT tag reader, and averaged Tb calculated for the 1-min sampling window, accounting for 99% equilibrium time68 (6.9 min and 2 min for flow rates of 1 L min−1 and 3.5 L min−1, respectively).
Bird behaviour was monitored every 30 s and activity scored as: 0 = resting or sleeping, 1 = looking around while sitting mostly still, 2 = moving with no or small displacement by stepping, 3 = displacement usually by hopping, 4 = hopping repeatedly or jumping, 5 = sustained escape behaviour, jumping continuously. At each Ta stage, we averaged the activity (i) over the first 3 min at stable air temperature (activitystage) to test for inter-individual differences in activity levels under standard conditions, and (ii) over the 10 min prior and during measurement windows (activitymeas) to account for current and carry-over effects of activity on metabolism (after equilibrium time68). Importantly, as per52, only data from calm birds were retained in analyses of thermoregulatory variables (i.e. here, activity ≤ 3 during the 1-min measurement, as well as the preceding 10 min).
We calculated mass loss over the trial (i.e. massinit-massend) as a proxy for total water loss (including through defecation, as faeces contain 80% water54) and mass recovery post-trial as the percentage of mass loss regained after 1 h (i.e. [(mass1h − massend)/(massinit − massend)] * 100).
Data analyses
All analyses were performed using R (v3.6.1) in RStudio (v1.1.1335). The total data set corresponded to 67 trials (n = 34 birds). One trial (out of 68) could not be used because the flow rate was set incorrectly. As data were restricted to calm birds, and some trials had to be terminated before reaching Ta = 44 °C, analyses were conducted on data from all 67 trials at Ta = 27, 35 and 40 °C, but 55 trials at Ta = 42 °C and 28 trials at Ta = 44 °C (Fig. 3). As on rare occasions the PIT tag angle or position prevented its detection by the antenna, sample sizes for Tb are n = 66 at Ta = 27 °C and n = 65 at Ta = 35 °C. For every model, predictors were centered and scaled and residuals checked for normality and homoscedasticity.
In all models (apart from segmented analyses), we tested for effects of prenatal playback, massinit, time-of-day (AM or PM), trial number (1st or 2nd trial) and sex as fixed factors, together with the interaction between playback and time-of-day, and with individual ID as a random factor. Non-significant interactions (p < 0.05) were not retained (full models are presented in Supplementary Material Tables S1–S8).
Heat tolerance and body mass variation
The effect of prenatal playback on heat tolerance was assessed using two proxies as response variables: the maximum Ta reached (Max Ta = 42 or 44 °C, n = 67), and whether or not individuals reaching Ta = 44 °C (n = 53) completed the trial (i.e., spent 20 min at Ta = 44 °C). We fitted generalized linear mixed-effects models (GLMMs, glmer function from lme4 R package) with a binomial error distribution and the fixed and random effects described above.
The effects on individual total water loss during the trial and subsequent body water replenishment in the following hour were investigated using two LMMs with predictors as described above and either mass loss (n = 67), or post-trial mass recovery (n = 66, as one individual was not weighed after 1 h), as response variables.
Variation in activity throughout the trial
First, considering all birds, we tested how activity varied as a function of increasing Ta. We defined the inflection point for activitystage, for Ta ≥ 35 °C, using a Davies test and the function segmented from the segmented R package69. We then fitted linear mixed models (LMMs, lmer function from the lme4 R package) above the inflection point, with prenatal playback, massinit, trial number, time-of-day, sex and the interaction between prenatal playback and recorded Ta as fixed effects, and trial nested within individual ID as random effects. Given the interaction was significant (see “Results”), we computed separate regression lines for treatment and control birds. Second, we tested for differences between prenatal playback groups on activity, separately at each Ta stage where thermoregulatory values were investigated: at the max Ta reached, Ta = 35 °C and Ta = 27 °C. We used LMMs, with activitystage (i.e. activity in first 3 min at stable Ta) or activitymeas (i.e. activity in the 10 min before and during metabolic measurements; square root transformed) as a response variable. Analyses on activitystage were performed on all birds (except n = 12 when the stage lasted < 3 min at stable Ta before trial interruption), to test for overall playback effects (i.e. n = 55 at max Ta reached, n = 67 otherwise). Analyses on activitymeas however were restricted to calm birds only (i.e. activity scores ≤ 3), to match thermoregulatory analyses (i.e. n = 32 at max Ta and n = 67 otherwise). We used the same fixed and random factors as described above for all statistical analyses, in addition to Ta (= 42 or 44 °C) for analyses at the max Ta reached only.
To establish if there were any bias in trial termination criteria between playback groups (even though the observer was blind to treatment), we tested for differences in activity level during the last 3 min at Ta = 42 °C for birds reaching Ta = 44 °C, (i.e. activity42-end, n = 53 trials). We fitted a LMM with predictors and random effect as described above. Activity42-end was square root transformed to meet linear model assumptions.
Thermoregulatory responses above thermoneutral zone
To investigate individual overall thermoregulatory response to heat, we first defined the upper limit of thermoneutrality (i.e. increase in MR) and inflection points for other variables (EWL, Tb, RWE, EHL/MHP) for Ta ≥ 35 °C, using a Davies test and segmented function, as described above for activitystage. This was then again followed by LMMs above the respective inflection points, with predictors, random effect and interaction as above.
To examine responses at the most extreme Ta stage reached (i.e. Max Ta = 42 or 44 °C, n = 32 trials with measurements on calm individuals), we fitted LMMs on MR, EWL, Tb, RWE and EHL/MHP, with predictors as described above. We included Max Ta (42 or 44 °C) as an additional fixed factor, and activitymeas as a covariate and in interaction with playback, to account for potential activity effects on thermoregulatory values.
Thermoregulatory response at mild temperatures
We examined the effect of playback on each thermoregulatory value (MR, EWL, Tb, RWE and EHL/MHP) (i) at thermoneutrality (Ta = 35 °C, n = 67 trials) and (ii) at mild Ta (Ta = 27 °C, n = 67 trials) using LMMs, with predictors as described above and activitymeas and its interaction with playback.
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