Vocal panting: a novel thermoregulatory mechanism for enhancing heat tolerance in a desert-adapted bird

Animals thriving in hot deserts rely on extraordinary adaptations and thermoregulatory capacities to cope with heat. Uncovering such adaptations, and how they may be favoured by selection, is essential for predicting climate change impacts. Recently, the arid-adapted zebra finch was discovered to program their offspring’s development for heat, by producing ‘heat-calls’ during incubation in hot conditions. Intriguingly, heat-calls always occur during panting; and, strikingly, avian evaporative cooling mechanisms typically involve vibrating an element of the respiratory tract, which could conceivably produce sound. Therefore, we tested whether heat-call emission results from a particular thermoregulatory mechanism increasing the parent’s heat tolerance. We repeatedly measured resting metabolic rate, evaporative water loss (EWL) and heat tolerance in adult wild-derived captive zebra finches (n = 44) at increasing air temperatures up to 44 °C. We found high within-individual repeatability in thermoregulatory patterns, with heat-calling triggered at an individual-specific stage of panting. As expected for thermoregulatory mechanisms, both silent panting and heat-calling significantly increased EWL. However, only heat-calling resulted in greater heat tolerance, demonstrating that “vocal panting” brings a thermoregulatory benefit to the emitter. Our findings therefore not only improve our understanding of the evolution of passerine thermal adaptations, but also highlight a novel evolutionary precursor for acoustic signals.

. Diagram of the different respirometry values taken throughout a trial (bottom), over a stepped profile of increasing air temperature (T a, top) in zebra finches tested in a flow-through respirometry system with high flow rates. Instantaneously corrected values at the onset of panting (3-min windows before and after, in blue), at the onset of calling (3-min windows before and after, in red), and averaged values over stable air temperature stages (~ 14 min, in green) were used. We also calculated values at the end of the trial (last 3-min window, in purple) to verify a-posteriori the validity of our trial interruption criteria. See supplementary material for details.

Scientific Reports
| (2020) 10:18914 | https://doi.org/10.1038/s41598-020-75909-6 www.nature.com/scientificreports/ or body temperatures for any given individual, (2) heat-calling onset will coincide with an increase in EWL (as also observed at the onset of panting 37 ), and possibly with changes in RMR and (3) ultimately, heat-calling individuals will show higher heat-tolerance (defined as being more likely to reach and tolerate the highest air temperature, without showing severe signs of heat-stress prompting the interruption of the trial), although such heat-tolerance benefit may come at a cost of greater total water loss.

Results
Triggers and repeatability of panting and heat-calling. Heat-calls were emitted during at least one trial by 73% (i.e. 32 out of 44) of the birds. Birds were always panting when they started heat-calling and produced heat-calls within panting bouts as they continued to pant ( Fig. 2A . Overall, the proportion of time each individual spent panting increased gradually across T a stages until reaching its maximum at T a stage = 42 °C (LMM, p < 0.001, Fig. 2B), whereas the proportion of time each individual spent calling remained low and then increased sharply at T a = 42 °C (LMM, p < 0.001, Fig. 2B).
Of the calling birds tested twice, 88% (i.e. 23 out of 26) called during both trials. The T a and T b thresholds for the onset of silent panting and heat-calling were highly repeatable within individuals (respectively for T a and T b , silent panting: ρ = 0.80, p < 0.001 and cor = 0.61, p < 0.001, n = 33 birds; heat-calling: ρ = 0.69, p < 0.0001 and cor = 0.521, p = 0.011, n = 23 birds, Fig. 3A-D), even though trials were conducted two weeks apart. Interestingly, the time elapsed between the onsets of panting and calling (i.e. panting-to-calling delay) was also Panting and heat-calling occurences in wild-derived captive zebra finches. (a) Examples of 1-min windows of three birds (row) in which the individual pants on and off (grey filling) and also calls on and off (red lines) within each panting bout. (b) Proportion of time spent panting (blue dots) and calling (red asterisks) by individual zebra finches exposed to a stepped profile of increasing air temperature. The mean calling (black asterisk) or panting (blue dot) times ± SE are shown (n = 77 trials for 44 birds). Letters indicate significant differences of time spent panting (blue) and calling (red) between each T a stage (p < 0.05) according to post-hoc tests. Overall, both heat-calling and panting were associated with increases in EWL. In contrast, changes in RMR differed between calling and panting, likely because most individuals had not reached their lowest RMR values by the time they started silent panting (at T a = 27 °C and 35 °C), whereas calling onset (at T a = 40 °C and above) occurred after RMR had stabilised. Consistent with this interpretation, we found that when restricting analyses to trials where individuals started silent panting at T a = 40 °C (n = 8 trials for 6 birds), RMR did not change at the onset of panting (LMM: est < 0.01, se = 0.01, p = 0.704).
Effect of panting or heat-calling on heat tolerance and water loss. Birds in 75% of the trials reached T a = 44 °C, whereas other trials had to be interrupted at T a = 42 °C because the bird showed signs of severe heat stress (e.g. loss of balance or rapid increase of T b ). Among trials reaching T a = 44 °C, only 27% were completed, with the individual tolerating T a = 44 °C for 20 min. Importantly, the likelihood of completing a trial was highly repeatable within individuals (R = 0.97, p < 0.001, n = 33 birds). Furthermore, the birds completing the trial had significantly lower RMR end , EWL end and T b-end values in the last 3 min of their trial than those that had to be interrupted, confirming the validity of our interruption criteria (see methods). www.nature.com/scientificreports/ Critically, in support of our prediction, trial outcome was significantly related to the proportion of time spent calling: birds completing trials had called for longer (GLMM: est = 3.67, se = 1.69, p = 0.030, n = 77 trials for 44 birds, Fig. 5A). By contrast, the proportion of time spent panting had no effect on trial completion (GLMM: est = 1.28, se = 1.72, p = 0.458).
Nonetheless, the thermoregulatory benefit of vocal panting came at a cost of greater water loss, as indicated by individuals' fractional mass loss during the trial in relation to the cumulative effects of panting and calling. Indeed, calling birds had significantly higher mass loss than non-calling birds (LMM; est = 0.16, se = 0.07,   Fig. 5B).

Discussion
As predicted, we found that heat-calling (i) occurred at a predictable stage of panting, (ii) triggered changes in EWL, and (iii) importantly, was associated with a higher likelihood of tolerating acute heat exposure in adult wild-derived captive zebra finches. Our data therefore reveal a novel mechanism for thermoregulation that increased individual's heat tolerance, supporting the hypothesis that vocal panting may offset the inherent inefficiency of panting, as an evaporative cooling pathway in a small arid-zone passerine. Furthermore, we provide the first evidence for consistent individual differences in thermoregulatory strategies, with heat-call production, as well as silent panting, being highly repeatable traits within individuals. Overall, our study makes a significant step towards a greater understanding of the selection forces that may act on thermoregulatory capacities in a desert avian species. Such advances are particularly crucial at a time when desert avian communities are highly threatened by unmitigated climate warming [8][9][10][11] . Furthermore, our findings highlight that respiratory movements for thermoregulatory purposes may represent a novel evolutionary precursor of acoustic signals.
Vocal panting as a strategy to improve heat tolerance. Individuals showed notable differences in heat tolerance, where some individuals showed signs of distress at T a = 42 °C, whereas others tolerated T a = 44 °C for up to 20 min. Importantly, individuals' capacities to tolerate the highest T a and complete the trial increased with the proportion of time they spent heat-calling (Fig. 5A), whereas panting duration had no such effect. Nonetheless, EWL increased at the onset of vocal panting compared to silent panting, and calling birds lost more mass than non-calling birds, indicative of elevated water loss throughout the trial (Fig. 5B). Taken together, our findings therefore demonstrate that vocal panting-or the thermoregulatory mechanism associated with heatcalling-improves heat tolerance by increasing evaporative water loss. Interestingly, the onset of heat-calling in our experiment at T b ~ 43 °C coincides with the T b at which zebra finches have classically been described to reach hyperthermia 41 . At moderately high air temperatures, avian T b increases to maximise passive heat loss through non-evaporative avenues and thus save water 31,42 . A previous classic study postulated that, above T b = 43 °C www.nature.com/scientificreports/ nonetheless, zebra finches would favour water loss over detrimental effects of hyperthermia 41 . Our results suggest that vocal panting may correspond to such change in thermoregulation strategy predicted by that study 41 , to increase evaporative water loss when individuals are no longer able to maintain a viable T b via silent panting.

Repeatability in thermoregulation mechanisms within individuals.
To the best of our knowledge, our study is the first to show the repeatability of thermoregulatory mechanisms and heat tolerance within individuals. We showed that individual heat tolerance, as measured by their capacity to reach and withstand the air temperature stage of T a = 44 °C, was remarkably repeatable between two trials conducted 2 weeks apart. In addition, in agreement with findings on basal metabolic rate 43,44 , our analyses reveal that T a and T b thresholds triggering panting, as well as heat-calling, were highly consistent within individuals. Furthermore, we showed each individual triggered heat-calling at a specific instant after silent panting had started, with the panting-to-calling delay varying up to eightfold between individuals but being extremely repeatable within individuals. This further suggests that heat-calling is tightly linked to thermoregulation, and specific to the individual. These findings also indicate that, under controlled conditions, individuals consistently differ in how they use thermoregulatory mechanisms to withstand a heat challenge. This points to the occurrence of individual-specific thermoregulatory strategies within populations, even though individual thermoregulatory capacities may vary plastically across seasons or acclimation conditions 38,45,46 . Importantly, to limit sources of inter-individual variation at adulthood, all individuals tested in this study were of similar age (4 year-old) and thermal experience (always held together in the same outdoor aviaries). However, it is currently not clear whether the inter-individual differences we found in adult thermoregulation strategies and the production of heat-calls were due to differences in genotypes, or induced by developmental plasticity in early life. In particular, future studies may establish whether prenatal exposure to heat-calls and/or high temperatures during development favours the production of heat-calls at adulthood or improves individuals' thermoregulation capacities. Nonetheless, our findings that thermoregulatory behaviour consistently vary between individuals open the possibility for selection to act on this trait 47,48 .
The evolution of vocal panting. Our findings reveal the existence of a novel modification of panting in a passerine species, which improves thermotolerance. It is unlikely that vocal panting is a phenomenon that uniquely evolved in zebra finches. For example, it is noteworthy that other Australian passerines such as fairy wren species, breeding in hot climates, produce similar fast, high-pitch calls during incubation, even though their possible association with heat (or thermoregulation) has not been investigated 49,50 . We predict that vocal panting may also occur in other small-sized heat-adapted passerines, where the reliance on facultative hyperthermia is common 31 . Furthermore, given that vocal panting is costly in terms of water loss, it may be more likely to occur in drinking species than non-drinking species 51,52 . Therefore, examining the occurrence of heat-calling across passerines species occupying hot environments may bring new insights on the resilience of desert avian communities to climate warming. In addition, our study provides evidence that a vocal signal can be associated with a thermoregulatory mechanism. Whilst this may seem surprising, there is evidence for a similar phenomenon occurring with visual signals. Indeed, some visual cues, such as feather erection or skin flushing (i.e. redness of highly vascularised skin patch), that were primarily associated with a thermoregulation function, have then evolved to become visual signals during social displays in some species [53][54][55] . In our study, consistent with previous findings in wild and captive zebra finches 26,27 , we showed that heat-calls, starting at T a ~ 41 °C and T b ~ 43 °C, can reliably signal hot environmental conditions and the emitter's hyperthermic state to embryos (or possibly to conspecifics). Therefore, it is plausible that heat-calls could have evolved from a cue initially associated with a thermoregulatory mechanism into a signal directed at embryos, which increases offspring fitness. Such a hypothesis is consistent with the evidence showing that heat-calling occurs in multiple contexts but is most common towards the end of incubation, when embryos might be able to perceive that signal 26,28 .
Nonetheless, the mechanisms for heat-call production and its associated evaporative cooling benefits remain to be established. For example, contrary to anecdotal evidence on a single individual zebra finch suggesting that respiration rate might increase during heat-call production 28,56 , we found no supporting evidence for a rapid increase in RMR at calling onset. Moreover, the mode of heat-call production requires further investigation. For example, a possible mechanism underlying the increase in EWL and thus thermoregulatory benefits of vocal panting is that, similarly to gular flutter during panting 19 , heat-calling increases the area for evaporative water loss by moving air across parts of the respiratory tract that are not usually involved in panting. However, we currently do not know where the sound originates in the respiratory tract and which element might be vibrating, although the involvement of the medial and lateral labia, which are known to vibrate to produce sound 57 , are worth investigating. Overall, establishing the mechanical pathway for sound production during heat-calling may give insights into its associated thermoregulatory benefits.

Conclusion
Our study reveals the intriguing phenomenon of vocal panting, whereby a vocalisation can be associated with a thermoregulatory mechanism which improves heat tolerance. This, together with the adaptive developmental effects of heat-call exposure on zebra finch embryos 26 , add to the remarkable adaptations displayed by species exposed to extreme environmental conditions. Whilst more investigation is needed to understand the underlying mechanisms of vocal panting, as well as heat-call occurrence among avian taxa, our findings suggest that, beside panting, some passerine species are likely to show additional thermoregulatory mechanisms for heat tolerance, perhaps analogous to lingual flutter in parrots 24,25 . Overall, our study highlights the importance of understanding how thermoregulatory mechanisms vary within species, in order to predict selection on heat tolerance and how species may fare under climate change. Indeed, future climate projections have shown that the increase in www.nature.com/scientificreports/ chronic hot weather will threaten the persistence of bird populations inhabiting arid environments, by dramatically increasing breeding failure 10 . In addition, the risk of lethal dehydration and hyperthermia is predicted to increase dramatically in these environments for small passerines species, including the zebra finch 8,9 . Therefore, as our study hints to how much remains to be discovered, understanding avian thermoregulatory mechanisms and their implications for adaption to hot climates is more important than ever.

Materials and methods
Study species and housing. Between March and June 2018, 44 adult wild-derived zebra finches (26 males and 18 females) were transferred from outdoor aviaries to indoor cages (4-5 same-sex individuals per cage; 1 × 0.42 × 0.42 m) at Deakin University (Geelong, Australia) for acclimation to a constant temperature of 25 °C and photoperiod of 12L:12D. Birds were supplied with ad libitum finch seed mix, grit, cucumber and water. After three days, a temperature-sensitive passive integrated transponder (PIT) tag (Biomark, Boise, USA) was inserted subcutaneously onto the bird's flank, which have been demonstrated to give similar T b to those implanted intraperitoneally in zebra finches 58 . Physiological measurements took place after 31 days (range 27-36 days) of acclimation for the first trial (with the exception of a female after 96 days) and after 47 days (range 42-51 days) of acclimation for the second trial, with an average of 16 days (range 6-24 days) between the two trials.
Experimental protocol. Experimental trials were conducted during the day, when the birds would naturally experience higher temperatures. Thirty-three birds were tested twice, once in the morning (start 10:30 am ± 13 min) and once in the afternoon (start 2:50 pm ± 16 min) in random order. An additional 11 birds were tested only once due to time constraints. To maximise the likelihood of birds being post-absorptive during measurements, birds were fasted for two hours before the trial 59 with ad libitum water in an individual cage (32 × 50 × 33 cm), in auditory and visual contact with others in the holding room. Birds were then weighed (g ± 0.01, Sartorini scale) before being placed in the metabolic chamber. During measurements, air temperature was initially maintained at 27 °C for 25 min (or 45 min for the second trial), then 35 °C for 30 min (i.e. thermoneutral temperature 41 ), followed by 20 min stages at 40 °C, 42 °C and 44 °C. The 27 °C stage for the second trial was longer to ensure that patterns of panting or calling were directly linked to temperature, rather than time spent in the chamber. After 10 min at T a = 27 °C, we started a playback of birds in the holding room to lower stress due to social isolation as commonly done in behavioural studies 60,61 . We used an open flow-through respirometry system to measure individual CO 2 production and EWL as previously described 32 (see supplementary material for details). Trials were considered 'complete' when the individual remained in the chamber for 20 min at T a = 44 °C. The trial was stopped before this if the bird showed a persistent escape behaviour (attempting to fly, pecking container), a loss of balance, an abrupt drop in the metabolic traces or a high T b (> 45 °C), as 32 . The experimenter was blind to heat-call production or to results from the previous trial. To independently confirm the validity of our interruption criteria, and that birds completing the trial were less severely heat-stressed than those in interrupted trials, we tested for differences in thermoregulatory values at the end of the trial (last 3-min, Fig. 1) between birds completing the trial and those that did not, using Linear Mixed Models (LMM) (one per thermoregulatory variable) with individual as a random factor, and trial completion (yes/no), mass and sex as fixed effects. Because some birds did not reach T a = 44 °C, we had n = 7 observations at T a = 42 °C and n = 25 observations at T a = 44 °C for final values. Therefore, we run these analyses using data for all birds (n = 32 trials for 24 birds) or only for birds that reached T a = 44 °C (n = 25 trials for 18 birds). In agreement with our decision criteria, birds in completed trials had lower thermoregulatory end values than those that had to be interrupted. Specifically, completing birds had a significantly lower RMR end , EWL end and T b-end than birds interrupted at T a = 44 °C (respectively, p < 0.001, p = 0.007, p = 0.031, n = 25), and a significantly lower RMR end (LMM: est = − 0.10, se = 0.03, p = 0.005, n = 32) and marginally lower T b-end (LMM: est = − 0.47, se = 0.25, p − 0.067, n = 32) than birds interrupted at either T a = 42 °C or 44 °C.
After the trial, birds were weighed and given water (by depositing drops of water on their bill). All birds were subsequently monitored to ensure they recovered from the experiment and had access to ad libitum water and food in their home cage.
All procedures were approved by Deakin University Animal Ethics Committee (G06-2017). All experiments were performed in accordance with Australian guidelines and regulations for the use of animals in research.
Behavioural monitoring. Behavioural monitoring consisted of a 5-s scan every 30 s using two infrared video cameras (mini CCD camera with IR, Signet) placed into the cabinet. We scored the activity (0 = resting, 1 = looking around, 2 = moving, 3 = hopping, 4 = agitated, 5 = very agitated), as well as panting behaviour (i.e. opening of the bill 62 ). We considered as the onset of panting when the individual was panting for at least three consecutives scans. Proportion of time spent panting was computed over each air temperature stage and for the total trial length.
Acoustic recording. Heat-call production was recorded using a microphone (Seinnheiser MKE 2P, Germany) inserted through the metabolic chamber lid and connected to an external recorder (H6 handy recorder, Zoom). Audio recordings were analysed visually and audibly in Adobe Audition CC (Creative Cloud 2018) by two researchers who were blind to the respirometry values, behavioural data and air temperatures. The onset of calling corresponded to the time when the bird initiated the first heat-call sequence; and the duration of each sequence was noted as previously described 27 . We calculated the proportion of time spent vocal panting over each T a stage and for the total trial duration. We also defined "calling" as a categorical variable with 1 corresponding to birds calling for a total duration of at least 20 s and 0 for birds not calling, or calling for less than 20 s. www.nature.com/scientificreports/ This threshold corresponded to a gap in the histogram of calling duration and allowed us to consider ambiguous individuals calling for less than 20 s as non-calling birds.
Respirometry data processing. We calculated whole animal RMR, EWL using equations previously described 63 . Our birds being post-absorptive, we assumed a respiratory exchange ratio of 0.71 32,64 . We computed thermoregulatory values for each individual at three time points: at the onset of panting, at the onset of calling and at the end of the trial (Fig. 1, see supplementary material for details). We also computed the mean values for each T a stage above the thermoneutral zone of the zebra finch (T a ≥ 40 °C, Fig. 1 shown as mean ± standard error. The total data set was 77 trials for 44 birds, including 33 birds tested twice. Nonetheless, we excluded values when individuals were agitated (activity > 3) for the analyses with thermoregulatory variables (RMR and EWL), as previously described 32 , and sample sizes were smaller in some analyses since not all birds called (see "Results"). For every model, predictors were normalised and residuals were checked for normality and homoscedasticity. Results from the full models are shown.
Triggers and repeatability of panting and heat-calling. To understand in which conditions heat-calling occurs, we examined the effect of T a on the proportion of time spent calling and panting at each air temperature stage using separate LMMs (lme function from the nlme R package) with individual ID and trial number as random effects and T a as a fixed effect (n = 370 observations for 77 trials and 44 birds), followed by post-hoc pairwise Turkey comparisons test (emmeans function from the emmeans R package).. To investigate the link between panting and calling, we calculated the time delay between the onset of each behaviour (panting-to-calling delay). Within-individual repeatability in panting-to-calling delay between the two trials was assessed using a Pearson correlation test (n = 23 birds). In addition, within-individual repeatability in T a and T b thresholds triggering panting (n = 33 birds) and calling (n = 23 birds) between the two trials was assessed using either a Pearson or Spearman correlation test, depending on whether the data were normally distributed.
Thermoregulatory changes associated with panting and heat-calling. We first compared the instantaneous changes in thermoregulatory variables at the onset of panting and calling in separate models. We investigated the effect of the sampling window (3-min before/after the onset, Fig. 1) on RMR and EWL using LMMs with trial number nested in individual ID as a random factor, and mass, sex and T a-std as fixed effects (lme function from the nlme R package). T a-std corresponds to T a centred on the median T a stage for the focal behaviour (i.e. coded as 0 at T a = 35 °C for panting and T a = 42 °C for calling), with increasing or decreasing temperatures equally spaced (one increment) from the centred temperature (e.g. for calling T a = 35 °C coded − 2, T a = 40 °C coded − 1, T a = 44 °C coded 1). Because we could not obtain values when the behaviour onset occurred during baseline measurements, the sample sizes were n = 32 trials (for 24birds) for panting onset and n = 29 trials (for 23 birds) for calling onset.
Then, to corroborate these short-term (i.e. 3-min) changes at panting and calling onsets, and to circumvent the issue of onsets occurring during baseline measurements, we assessed the effect of the proportion of time spent calling and panting per T a stages on the average RMR Ta and EWL Ta values during these T a (ca. 14 min, see above , Fig. 1), for T a above the thermoneutral zone. We fitted LMMs with the proportion of time spent panting or calling as predictors in separate models, individual ID and trial number as random effects and sex, mass and T a-std as fixed effects using lme function (n = 140 observations for 77 trials and 44 birds).
Effect of panting or heat-calling on heat tolerance and water loss. To determine whether panting or heat-calling improve heat tolerance (i.e. the ability to withstand high T a without showing severe signs of heat-stress such as loss of balance or rapid increase of T b ), we evaluated potential predictors of successful trial completion (i.e. remained calm with T b < 46 °C for 20 min at T a = 44 °C). Within-individual repeatability of trial completion probability was assessed using Generalized Linear Mixed Models (GLMMs) based repeatability estimation for binary data using rptR R package 65 . To test for the effect of the proportion of time spent panting or calling on the trial completion we fitted separate GLMMs with a binomial error distribution and individual as a random factor (n = 77 trials for 44 birds), and mass, sex and trial number as fixed effects.
To investigate the potential costs of panting and heat-calling on total water loss, we tested, in the same LMM model, the effects of the air temperature stage at which bird started panting (T a-panting ) and calling occurrence (0/1) on the fractional mass loss (n = 77 trials for 44 birds). We used calling occurrence rather than the proportion of time spent calling, to allow retaining non-calling birds in the analysis. We also included mass, sex and trial completion (yes/no) as fixed effects, and individual as a random factor (lmer function).