Progressive postnatal hearing development limits early parent-offspring vocal communication in the zebra fnnch

Reviewer #1 (Public review):

This work by Antonnen et al. was triggered by claims of auditory-mediated effects on altricial avian embryos which were published without any direct evidence that the relevant parental vocalizations were actually heard. I agree with Anttonen et al. that, based on the available evidence about avian auditory development, those claims are highly speculative and therefore necessitate more direct experimental verification.

Attonen et al. have embarked on a comprehensive series of experiments to

(1) Better characterize acoustically the relevant parental vocalizations (heat whistles; in a separate preprint, not reviewed here)

(2) Characterize the auditory sensitivity of zebra finches at various stages of their posthatching development. Despite the long-standing importance of the zebra finch as a songbird model in neuroethology of learned vocalizations, the auditory development of the species had not been studied so far.

(3) Explore an alternative hypothesis of how the parental vocalizations might be perceived.

The principal method used here is the non-invasive recording of ABR (auditory brainstem response), a standard neurophysiological method in auditory research. The click-evoked ABR provides a quick and objective assessment of basic hearing sensitivity that does not require animal training. Weaknesses of the technique include its limited frequency specificity and low signal-to-noise ratio. The authors are experienced with ABR measurements and well aware of those issues. ABR responses in zebra finches are shown to gradually appear during the first week posthatching and to mature in subsequent weeks, consistent with the auditory development in other altricial bird species studied previously. When matching the acoustic properties of parental heat whistles and auditory sensitivities, hearing of the parental heat whistles by zebra finch hatchlings was convincingly excluded. Although not directly measured, this also convincingly extrapolates to zebra finch embryos. Finally, the authors tested the hypothesis that parental heat whistles could induce perceptible vibrations of the egg and thus stimulate the embryo via a different modality. The method used here was laser doppler vibrometry, an appropriate, state-of-the-art technique that the authors also have proven experience with. The induced vibrations were shown to be several orders of magnitude below known vibrotactile sensitivities in mammals and birds. Thus, although zebra finch vibrotactile thresholds were not obtained directly, the hypothesis of vibrotactile perception of parental heat whistles by zebra finch embryos could also be rejected convincingly.

In summary, even when considering some weaknesses of the techniques (which the authors are aware of), the conclusions of the paper are well supported: Auditory and/or vibration perception of parental heat whistles can be excluded as an explanation for previous reports of developmental programming for high ambient temperatures. As a constructive suggestion towards resolving the apparent paradox, the authors recommend to repeat some of the crucial, previous playback experiments at lower sound levels that better match the natural parental vocalizations.

Reviewer #2 (Public review):

This study by Anttonen, Christensen-Dalsgaard and Elemans describes the development of hearing thresholds in an altricial songbird species, the zebra finch. The results are very clear and along what might have been expected for altricial birds: at hatch (2 days post-hatch), the chicks are functionally deaf. Auditory evoked activity in the form of auditory brainstem responses (ABR) can start to be detected at 4 days post-hatch but only at very loud sound levels. The study also shows that ABR response matures rapidly and reaches adult like properties around 25 days post-hatch. The functional development of the auditory system is also frequency dependent with a low to high frequency time course. All experiments are very well performed. The careful study throughout development and with the use of multiple time-points early in development is important to further ensure that the negative results found right after hatching are not the result of the experimental manipulation. The results themselves could be classified as somewhat descriptive but, as the authors point out, they are particularly relevant and timely. Since 2016, there has been a series of studies published in high profile journals that have presumably showed the importance of prenatal acoustic communication in altricial birds, mostly in zebra finches. This early acoustic communication would serve various adaptive functions. Although acoustic communication between embryos in the egg and parents has been shown in precocial birds (and crocodiles), finding an important function for pre-natal communication in altricial birds came as a surprise. Unfortunately, none of those studies performed a careful assessment of the chicks' hearing abilities. This is done here, and the results are clear: zebra finches at 2 and 6 days post hatch are functionally deaf. Since, it is highly improbably that the hearing in the egg is more developed than at birth, one can only conclude that zebra finches in the egg (or at birth) cannot hear the heat whistles. The paper also ruled out the detection on egg vibrations as an alternative path. The prior literature will have to be corrected, or further studies conducted to solve the discrepancies. For this purpose, the "companion" paper on bioRxiv that studies the bioacoustical properties of heat calls from the same group will be particularly useful. Researchers from different groups will be able to precisely compare their stimuli.

Beyond the quality of the experiments, I also found that the paper was very well written. The introduction was particularly clear and complete (yet concise).

Weaknesses:

My only minor criticism is that you don't discuss potential differences between behavioral audiograms and ABRs. Optimally, one would need to repeat the work of Okanoya and Dooling with your set up and using the same calibration. The ~20dB difference might be real or it might be due to SPL measured with different instruments, at different distances, etc. Either way, you could add a sentence in the discussion that states that even with the 20 dB difference in audiogram heat whistles would not be detected during the early days post-hatch, but that adding a (novel) behavioral assay in young birds could further resolve the issue.

More Minor Points.

(1) As mentioned in the main text, the duration of pips (form pips to bursts) affects the effective bandwidth of the stimulus. I believe that you could give an estimate of this effective bandwidth given what is know from bird auditory filters. I think that this estimate could be useful to compare to the effective bandwidth of the heat-call which you can now also estimate.

(2) Fig 5b. label the green and pink areas as song and heat-call spectrum. Also note that in the legend you say: "Green and red areas display the frequency windows related to the best hearing sensitivity of zebra finches and to heat calls, respectively". I don't think this is what you meant. I agree that 1-4 kHz is the best frequency sensitivity of zebra finches but you probably meant green == "song frequency spectrum" and pink == "heat call spectrum". In either case the figure and the legend need clarification.

(3) Fig 5c. Here also, I would change the song and heat-call labels to "song spectrum", "heat call spectrum". You don't want readers to think you used song and heat calls in these experiments (maybe next time?). For the same reason, maybe in 5a you could add a cartoon of the oscillogram of a frequency sweep next to your speaker.

(4) Methods. In your description of the stimulus, you describe "5ms long tone bursts" but these are the tone pips in the main part of the manuscript. Use the same terms.

Comments on revisions:

In the latest version of this manuscript, Anntonen et al have diligently addressed all the issues raised by the reviewers. As I mentioned in our discussions among reviewers, it is impossible to "prove" a null hypothesis and both methods and experimental design could always be improved. At this stage however, they provide convincing evidence that the sound intensity of heat calls are below the hearing thresholds of zebra finch chicks.

Reviewer #3 (Public review):

Summary

This study aims to contest recent findings that prenatal exposure to natural sounds and anthropogenic noise before hatching affects development and fitness in an altricial songbird. To this aim, it attempts to estimate hearing capacities of zebra finch nestlings. It first uses responses to clicks in nestlings and adults to estimate differences in hearing thresholds but uses experimental parameters that systematically lower nestling responses. It then measures responses to tones but restrict nestling data to a protocol (long tones) that failed in adults; response to tones with a correct protocol (short tones) is repeated in adults only. Thirdly, it includes data on loud airborne sound making eggs vibrate, even though this has no relevance to embryonic vibration perception by direct contact with the incubating parent or in incubators. Lastly, it includes a lengthy discussion on how these results, even though inaccurate or incorrect, would show that zebra finch nestlings and embryos are "functionally deaf". It fails to note that even if the experiment had been performed correctly and still failed to detect an auditory response in 2 day hatchlings - which is unlikely given the above and findings in other songbirds - this would not somehow eliminate the developmental effects of prenatal sounds that have been empirically demonstrated.

Strength:

The study is not performed adequately to bring reliable answers, but it addresses the important topic of hearing development in altricial songbirds and the long-held (but untested) assumption that altricial avian embryos cannot hear. More broadly, there is a need to reassess avian auditory perception - and the methodological approaches to measure it - given the accumulating evidence that some bird species respond to high frequency biological sounds beyond their known hearing range.

Weaknesses:

The study presents many experimental flaws that specifically compromise response detection in immature animals in the first experiment, and data from the remaining two experiments are invalid. The revision did not fix any of these issues.

i) Response to clicks, Fig 1: Unlike what the revision claims, the deviations from validated protocols (too rapid stimulation, too few measurements, low temperature, reliance on clicks only) do lead to a potentially large underestimation of nestling hearing sensitivity. Calculation and new data trying to show that these deviations would not matter are wrong or insufficient. Even without strict conventions on ABR methodology, it is striking that every single experimental choice made here i) greatly differs from other avian studies and ii) reduces - rather than maximises - response detection, specifically for low amplitude signals (as in nestlings or near thresholds).

ii) Response to frequency tones, Fig 2: The experiment on frequency sensitivity (long tone burst) failed in adults (positive control) with 0 to only about half of the adults responding (Fig S1), and sensitivity underestimated by 30dB (Fig2). Measures with a failed positive control are invalid, even if the overall pattern of the few data points obtained in nestlings vaguely resemble (but does not match) expectations. That the experiment was repeated in adults with a correct protocol (short tone pips) - but not in nestlings - is highly misleading.

iii) Attempts to validate the protocols above by comparing to published adult values are meaningless (response A.3.1; Fig 5). The one experiment being compared (short tones) was only performed in adults in this study. This cannot validate results obtained by different methodologies in nestlings, for responses to clicks or long tones.

iv) Vibrations: No previous results on the effect of zebra finch heat calls on development rely on the hypothesis or assumption that airborne sound from heat calls makes eggs vibrate. This idea is solely attributable to the authors of the preprint, and is not biologically or experimentally realistic. All speculations from this experiment (Fig3) on vibration perception by embryos or heat call effects are meaningless.

v) Writing and presentation: The text throughout is highly misinformative for non-specialists or any reader not carefully inspecting figures (including in supplementary material) and methods. The confusion is greatly aggravated in the (6-page long) discussion which i) fails to recognise and account for the study shortcomings, and instead ii) greatly overstates the results and what they mean, and iii) misrepresents current knowledge by excluding highly relevant studies showing evidence of early sound perception in embryos and hatchlings, and introducing many errors in the presentation of published papers. This creates an illusion of a strong mismatch between heat-calls and zebra finch sensory capacities, for which the study actually does not provide any evidence for, and which is extremely unlikely to exist.

vi) The revision did not address any of the major flaws of the study outlined above (see detailed assessment below). In particular,
** For point i):
- estimations for the effect of having done too few (400) measurements are wrong - the effect on hearing thresholds cannot be calculated, but would be much greater than 4dB with the expected 37% noise reduction with the standard 1000 sweeps;

- the new data provided does not measure the impact of high stimulus rate, and measures on adults largely underestimate effects on nestling response;

- body temperature, now provided in the revision, is at the lowest extreme for the species, which may increase hearing thresholds;

- tones do elicit a stronger and earlier response than clicks. Whether this is related to stimulus duration does not change the fact that clicks underestimate hearing thresholds, and delay hearing onset by several days.
** For points ii) and iv):
No new data or any valid explanation was provided in the revision. It is still the case that nestling frequency responses were obtained with an experiment where the positive control failed; and the vibrating egg experiment is irrelevant to vibration perception in embryos and any observed effects of heat calls on development.
** The revision greatly lengthens the speculations about heat call perception, based on inaccurate or totally incorrect data.

Conclusion and impact:
i) Overall, the study fails to provide any reliable estimate of zebra finch nestling hearing capacities: it underestimates hearing onsets and thresholds (clicks) and gives no information on nestling frequency sensitivity. It is extremely likely that a better designed ABR experiment, or a more sensitive methodology (e.g. electrophysiology), would have detected a response in 2 day-old nestlings, as in other songbirds. The conclusion of "deafness" in hatchlings (or embryos, which were not tested) is clearly unsupported.
If zebra finch hatchlings were clearly deaf, a well-designed study would have shown this a lot more convincingly.

ii) The data on adults in not new (3 prior studies) - although this study generally underestimates zebra finch high frequency sensitivity. The presentation in relation to heat-calls is flawed: true values of heat-call frequency range and sound levels (>6kHz at 45dB) fall within the adult hearing range.

iii) Without any new evidence, this study does not progress our understanding of heat-call and noise impact on development. Exactly the same issue remains: that heat-call and noise effects on development contradict the general VIEW on hearing ontogeny in altricial birds. But we have learnt nothing from this study on hearing ontogeny or frequency sensitivity. The study provides no reliable neuroscience data to advance the debate.

iv) The largest section of the preprint is a highly speculative discussion, based on erroneous data and wrong interpretations, as well as a misrepresentation of what is known. That these issues would not be recognised is - in my view - of serious concern.

Detailed assessment:

The summary by the authors of my major concerns (R3.A0) is incorrect and misreport many of my statements, without giving any meaningful answers. I will not engage in such discussion.

My actual three major concerns do remain:

(1) Results on Fig 1 overestimate hearing onset and thresholds: All experimental parameters chosen to test responses to clicks are known to lower detection. From their cumulative impact, there is absolutely no doubt that the data in Fig 1 underestimate hearing capacities, and disproportionately so in nestlings compared to adults. Therefore, no quantitative estimate of nestling hearing threshold, absolute or relative (i.e. the estimated "54dB difference"), can be taken from this study. The age of hearing onset based on clicks (4 day old) is also wrong.

(2) All results on Fig 2 are false: 40 to 100% of adults (positive control) failed to respond within their normal hearing range (Fig S1). Data on nestling frequency sensitivity using this methodology (Fig 2) are clearly invalid.

(3) All results on Fig 3 are irrelevant: they assume zebra finch parents are hoovering in front of their nest while heat-calling rather than incubating their eggs, which is nonsense. Any speculation on the role of bone-conduction or vibrotactile stimulation for embryonic heat-call detection is simply unfounded.

The authors' responses to these comments are largely mistaken (see below), and do not change the 3 facts stated above. Overall, the data produced are unreliable, and so are the interpretations and conclusions.
The conclusions that i) heat-calls fall outside of adult hearing range and ii) young nestlings (and embryos) are deaf, are both incorrect. The study provides no actual estimate of nestling hearing and how far off their hearing range heat-calls fall.

(1) Responses to clicks underestimate hearing abilities, specifically in nestlings.

(i) DISPROPORTIONATE EFFECT OF PROTOCOL ON NESTLINGS: As reported in other avian studies (e.g. Brittan Powell et al 2004), because of their immature neural system, nestlings show low amplitude waveforms compared to adults. This occurs even with sound much louder than their hearing threshold. For example here, 8 day old nestlings show waveforms of only low amplitude at 95dB, even though they respond to sound 40dB softer, at 55dB (Fig 1H).

This characteristic of nestling waveforms means that:
- nestling responses are harder to detect,
- nestling responses are more easily attenuated below the detection criteria (>2 S/N),
- a low amplitude response in nestlings at a given sound level does not predict that softer sounds will not be perceived by the animal.

Any deviation in protocol that attenuates waveform amplitude will therefore disproportionately affect nestling thresholds (and artificially lead to the "54dB" estimate).
Even without universal standards (resp R3.A1.1), knowing this, the protocol should be adjusted to maximise response detection. This study does exactly the opposite.

(ii) INSUFFICIENT MEASUREMENTS: using only 400 sweeps, rather than the typical 1000 sweeps, reduces response detection, especially in nestlings.

- The claim that using 1000 sweeps would only decrease the hearing threshold by 4dB (response A3.2; ms L530) is false:
- Based on the square root relationship mentioned by the authors, using 1000 averages instead of 400 would improve the signal-to-noise ratio by 37%, which would allow detecting many small amplitude waves which are currently hidden in the abnormally high noise.
- Lowering the noise floor level by 4dB does not mean that the hearing threshold would only decrease by 4dB. The improvement in hearing threshold would be much greater, especially in nestlings.

(iii) UNSUITABLY HIGH STIMULATION RATE: the new data on pairs of clicks greatly underestimate the attenuation by high stimulation rates, and so do adult measurements compared to nestlings'.
- the click rate used in this study (25 clicks per second) is 5 to 25 times faster than most previous studies in young birds (e.g. Saunders et al 1973, 1974: 1 stim/sec or less; Katayama 1985: 3.3 clicks/sec; Brittan-Powell et al 2004: 4 stim/sec), and 6 times faster than zebra finch heat-calls.
- responses to pairs of clicks (new data in Fig S3; L 543-552, response A3.3) does not measure the response dampening caused by high stimulation rates. Response attenuation (adaptation) after a single click is much weaker than after a train of 400 consecutive clicks (or even just 30). This paired-click measure ignores the cumulative attenuation observed with many consecutive stimuli. Paired-click paradigm is used to measure immediate refractoriness for other purposes (e.g. temporal resolution, diagnosis tool) but does not replicate high stimulation rates.
It is unclear why the authors chose to use this weak approximation rather than simply replicating the measurements at the same slow rate as in other avian studies. This would have given a straightforward answer, comparable to previous studies that have demonstrated the detrimental impact of fast rate on response strength by directly comparing responses to different stimulation rates (e.g. Saunders et al 1973; Brittan-Powell et al 2004).
- Adults are less sensitive to high stimulation rates than immature individuals (e.g. Saunders et al 1973; Khayutin 1985; Brittan-Powell et al 2004 and 6 references cited therein). Effects of fast rate in adults (new data in Fig S3) therefore largely underestimate effects on nestlings (not measured), and the high stimulation rate used in this study increases the relative difference between nestlings and adults.
- Given the 2 points above, it is incorrect to conclude from this new data that the high stimulation rate used had "no effect on ABR amplitude or thresholds" (responses A3.3 L551). Instead, given that 40ms (i.e. interval for 25 clicks/ sec) is at the limit of what adults can handle after a single click, this new data does confirm that the stimulation rate is indeed too high and underestimates hearing in nestlings (and adults to a lesser extent).

(iv) LOW BODY TEMPERATURE can reduce ABR response.
- The average body temperature (39.5C) now provided in the revised manuscript (response A2) is at the lowest extreme of the range for zebra finches. The normal average body temperature for zebra finches is 41C at low ambient temperature, rising to 43-44C at high ambient temperatures (when heat-calls would be produced). This value of 41C is consistent across many studies in wild and domestic zebra finches (e.g. Wojciechowski et al 2020 [avr 41C at 23C]; Udino and Mariette 2022 [avg 41, min=40C at 32C]; Bech and Midtgard 1981 [avr ~41C]; Pessato et al 2022 avg=41C at 27C]). The only study reporting a body temperature average as low as 39C (Cooper et al 2020) was indeed under hypothermic conditions, obtained in adult zebra finches under extreme fasting conditions (17hrs of food deprivation), at ambient temperature below thermoneutrality, during the night (i.e. during "nocturnal hypothermia", when bird body temperature is normally lower).
Therefore 39.5C does corresponds to hypothermic conditions for most individuals. While this body temperature remains considerably higher than that used experimentally to supress hearing response, using a below-normal body temperature, added to other factors, may lower wave amplitude and therefore increase hearing threshold estimates.

(v) CLICKS UNDERESTIMATE HEARING ONSETS
- My statement that, compared to tones, clicks elicit a smaller response, at a later age, is correct (Saunders et al 1973; Brittan-Powell et al 2004).
- It does not matter whether this is due to differences in stimulation duration (response R3.A2.1), since clicks are always much shorter than tones. What matters is that hearing onset based on responses to clicks has been found to underestimate the earliest age at which an auditory response can be obtained by several days (Saunders et al 1973).
- Without any valid nestling data for tones (see below), this study, based on clicks only, does not provide the true age of hearing onset.
- The suggestion of using 170dB clicks (response R3.A2.1) is very odd. The correct approach would have been to use a correct protocol for tones in nestlings, rather than solely correcting it for adults (see below).

(vi) CONFUSING INTERPRETATION OF WAVE AMPLITUDE AND LATENCY
- the text implies throughout that nestlings lacking an adult-like amplitude and latency is a sign of poor hearing (e.g. L122 "ABR wave I gradually reaches maturity 25 days after hatching").
- It should be clarified that this is a characteristic of immature systems but does mean these nestlings do not hear. For example, this characteristic persists even in 10d old nestlings which have similar hearing thresholds to adults.

(2). Results on nestling frequency sensitivity are invalid. Using long (25ms) tones with subdermal electrodes is incorrect, and not used by anyone other than the authors. The failure to repeat the experiment with a correct protocol (5ms tones) in nestlings is misleading:

(i) When 60% of 4-day old chicks respond to clicks (Fig 1), they are described as "functionally deaf" (L78, L261). By contrast, when 0% to 60% of adults respond to tones in the core of their hearing range (Fig S1 for data shown in Fig 2), this is merely presented as a methodological limitation (text added L186-197), and the authors still proceed to presenting results on nestlings with a method that failed in adults (positive control).

(ii) When the positive control fails, the experiment is failed. This principle applies in Neuroscience as in any scientific field.

(iii) That the experiment repeated in adults with the standard and correct short tone protocol (5ms) gave normal results, is not a validation. Instead, it proves the point that 25ms is unsuitable (regardless of whether that is due to rising time, L399). This correction does NOTHING to fix results in nestlings, which were ONLY done with the unsuitable 25ms tones.

(iv) The mixture of adult data with 5 and 25ms tones, instead, creates confusion, because differences in protocols between nestlings and adults are blurred in the text. The abnormally small proportion of adults responding (0 to 60%) with 25ms tones is only shown in supplementary material, and attenuated in the main text (L186: "about half").

(v) The claim (response R3.A1.2) that long 25ms tones is commonly used with the ABR methodology used here is false:
** ALL (but 1) avian studies using tones of 20ms or longer (cited by myself or by the authors in their reply R3.A1.2) used a different ABR methodology, with electrodes implanted through the skull. The only exception, using 25ms tones with subdermal electrodes (as here), is a study by the authors themselves.
** All other avian studies with subdermal electrodes used short tones, of 5ms or less (e.g. Brittan-Powell et al 2002, 2004; Henry & Lucas 2008), as in the corrected adult experiment.
** My initial comment already specified this difference in electrode placement.
** The results here (failing in adults) unquestionably show that the method of long tones with subdermal electrodes does not work, and the literature shows that no one else uses it.

(vi) When the aim of the study is to contest effects of high frequency sounds (L39-45), it is odd to choose a protocol specifically directed at testing responses to very low frequencies (responses A3.1, R3A.0; L398-403) and that compromises all results.

(vii) That the shape of the few data points obtained in nestlings with long tones would broadly resemble expectations (responses A3.1, R3A.0) is not a validation: See ii.
- The shape is not even correct:
** Why aren't 4 and 6 day old nestlings responding to any frequency when they were responding to clicks (in spite of poor detection conditions for clicks)?
*** Why are they not responding, when 2 day old flycatchers respond to tones from 1 to 4kHz at 45dB, and 0.5 to 5kHz at 60dB (Aleksandrov and Dmitrieva 1992, Korneeva et al 2006)?

(viii) That only relative measures between nestlings and adults matter (resp R3.A4; L404) is incorrect. Measures that are qualitatively (see vii) and quantitatively (see i) wrong cannot be compared.
If these data with long tones were indeed acceptable, why repeat the experiment in adults with a correct protocol, even before this flaw was pointed to during the review?

(ix) The unusually low number of measurements (400 instead of 1000; L530) will have reduced detectability of low amplitude responses, in nestlings and near thresholds, here as in the click experiment (see above). The claim that this would only increase threshold by 4dB (responses A3.2, L530) is wrong (see above).

(x) There is no explanation as to why only half of the 6-day old nestlings (n=5-6) were tested with tones. Would having 10 in this group shown a response at 6-day old?

(xi) Even the correct 5ms tone protocol in adults overestimated thresholds at higher frequencies (>3kHz) by up to 30dB compared to other published estimates (Fig 5). If inter-population differences among domestic zebra finches (L 393; Fig 5 legend; resp R3.A4) were enough to cause 30dB differences, results on domestic zebra finches here should not be extrapolated to those on wild-derived Australian zebra finches documenting developmental effects of heat calls.

The authors give no other elements than the above in their responses that could demonstrate the validity of their nestling frequency data.

Based on other studies, we can expect zebra finch hearing to develop sensitivity to high frequencies after that to middle frequencies. But pretending that this study provides any evidence towards this is wrong. There is no valid data.

(3) The experiment using loud sounds to make eggs vibrate is biologically and experimentally meaningless. The claim that these measurements would rule out vibration perception in embryos is totally unfounded. The preprint is creating the illusion of having ruled-out a mechanism that they have not tested.

(i) zebra finches are not hovering in front of their nest when heat-calling. They are in physical contact with the eggs while incubating, with vibrations expected to travel directly through solids from adults to eggs, without attenuation.

(ii) the claim that previous studies on heat-call developmental impact rely on the assumption of airborne sound making egg vibrate (response A3.5, R3.A8) is incorrect. This is conceptually wrong and there is no indication of this in any paper.

(iii) vibration perception in embryos in birds and other taxa (e.g. amphibian, reptiles and insects) rely on direct contact with the source, not on loud airborne sounds shaking eggs. Beyond any consideration on heat-calls, claiming that this experiment could give any indication of vibration perception in embryos is nonsense.

The authors give no information in their responses that could demonstrate the validity of this experiment.

(4) Interpretation and discussion

All other songbird studies, individually and collectively, show much greater hearing sensitivity in nestlings that what this study is trying to suggest (e.g Khayutin 1985; Korneeva et al 2006; Aleksandrov and Dmitrieva 1992; Rivera et al 2018; Platzen and Magrath 2004; Haff & Magrath 2012). The only way the authors can reach their conclusion is by:
- presenting data that greatly underestimate hearing sensitivity in zebra finches (see sections 1 and 2) and overstating them, and;
- misrepresenting current knowledge by excluding relevant papers and being unclear about what the literature shows.
This study tries to impose the idea that zebra finch do not detect any sound before day 4-6 post-hatch, and have extremely rudimental hearing until day 8-10 post-hatch. By contrast, other studies show very young hatchlings respond to sound 1 to 3 days after hatching (first age tested) and show quite sophisticated, and totally functional, responses to relevant sounds at 5 days old.

This is not to say that hearing does not continue to improve post-hatch in birds, or that sensitivity to mid frequencies post-hatch would not precede that to high frequencies. But statements throughout this study are so exaggerated and/or wrong that nothing can be learnt. It is undeniable that this study fails to provide the useful and balanced assessment needed to establish were true heat-calls actually sit relative to zebra finch adult, nestling and embryonic hearing range.

It is literally impossible to correct every wrong statement in the discussion and responses to reviewers. I focus here on some examples related the claims of "nestling deafness" or of heat-calls being outside of adult zebra finch hearing range, as well as inaccuracies leading to an apparent match with current literature.

(i) MISALIGNMENT WITH OTHER STUDIES AND MISREPORTING

*** Neurological evidence

- Highly relevant evidence on response to sound in zebra finch embryos (Rivera et al
2018) is totally excluded.
Excluding this study on the basis that it used a different methodology that does not directly quantifying auditory sensitivity (resp R3.A6a) is a poor justification. Evidence, even indirect or imperfect, should be brought to the attention of the readers.

- This applies to many other studies cited in my first review and arbitrarily excluded here. If one wants to conclude on "deafness", all evidence, even indirect, should be considered. Excluding non-ABR studies means the conclusion cannot be extended beyond flat ABR traces.

- Other studies show much greater sensitivity that what the text describes:

* Flycatcher hatchlings at 2-3d post hatch (first age tested), respond across a wide range of frequencies (0.3 to 5kHz), at low to moderate sound levels (45-65dB)
(Aleksandrov and Dmitrieva 1992, Korneeva et al 2006).

* Stating that these studies in flycatchers "likely yield lower thresholds" (L364) is an astonishing understatement. Thresholds in Aleksandrov and Dmitrieva (1992) at 2-3 day old were 35 dB lower than those here at 4 day old with clicks, and 60 to 80dB lower than those with tones of 1-2kHz at 8 day old.

* Claims that "sensitivity improves rapidly postnatally, with ~40 dB threshold decreases (L365)" is also misreporting these studies' findings. Thresholds decreased by 25dB consistently at 9 out of 11 frequencies tested from 0.3 to 8kHz (Aleksandrov and Dmitrieva, 1992). A difference of 40dB was only found at 5-6kHz (Aleksandrov and Dmitrieva, 1992). Likewise, improvement also varied from 25 to 40dB in Korneeva 2006. These do not average to "~40 dB".

* Even birds developing 4 times slower than songbirds (budgerigars) show a response at 5 day old. My statement that Brittan-Powell 2004 shows an auditory response at 5d old is correct. Re-response R3.A2.2: Fig 1B shows one example for ONE individual. All other figures based on multiple individuals shows at least some individuals responded at 5-6 days old at frequencies less than 4Khz (Fig 1C, 4 and 5).

- Many inaccuracies on avian hearing remain uncorrected in the revision.
* e.g. L 329: extrapolating high frequency hearing (>6khZ) from precocial species is incorrect because even adult chicken and ducks are not sensitive to high frequencies.
The authors imply elsewhere that species of songbirds cannot be compared (resp A4, R3.A6), but make extrapolations from species that are far more remote, phylogenetically, developmentally and ecologically than other songbirds.

*** Behavioural evidence

- contradicts this study findings:

* When correctly cited and described, the literature, does not support the authors' statement that nestling behavioural response "typically emerges between ~5-10 days post-hatch" (L358). It emerges earlier, at an unknown age, including potentially from hatch (present at 1.5day) for innate responses (see below).

* Results in other songbirds are not consistent with this study finding that a "response to loud click stimuli is first detectable at 4-8 days post hatch" (L230). Instead they show nestling hearing capacities described here are abnormally poor.

* Therefore, the conclusion that "The timeline of behavioural studies closely matches the onset and maturation of ABR responses observed here in zebra finches" (L360) is wrong.

- shows a very early response (1-2day post hatch), with no known onset:

* songbird behavioural response to sound, with parental alarm call suppressing begging, has been demonstrated in nestlings as young as 1.5 or 3 days old (Khayutin 1985, Korneeva et al 2006, Aleksandrov and Dmitrieva 1992). None of these results are mentioned in the revision when discussing behavioural evidence (L357-360).

* Instead, they exclusively mention studies that started testing nestlings at 5-6 days old, or much later (e.g. 17 day old: Suzuki 2011; 14 day old: Barati & McDonald 2017).

* ALL of these studies (cited or not) demonstrated a significant response of nestlings to calls on the first age tested. NONE tested the onset of this response.

* the one study looking at progression across 3 ages, at 5, 8 and 11 day old shows parental alarm calls suppress nestling calling at day 5 as much as later on, with no effect of age (Platzen and Magrath 2004). The authors failed to acknowledge this in their response (resp A4) or revision (L359).

* the claim that Haff & Magrath (2012) showed nestlings did not respond at 5-6 day old but did at 10-11 days (resp A4) is wrong. At 5 day old, they responded to their own species alarm calls, as well as to another similar sounding species and to the sound of predators themselves (Table2 in Haff & Magrath 2012; as in Platzen and Magrath 2004).

- learning, not just hearing, improves nestling response with age:

* Haff & Magrath (2012) showed that by 10-11 day old, nestlings had learnt to also respond to heterospecifics, demonstrating that learning improves nestling response with age.

* the intensity of the response to low frequency sound improved more with age than that to high frequency calls. If improvement were related to hearing limitations, the opposite would be expected (Haff & Magrath (2012).

- nestlings discriminate complex calls, including at high frequency

* By 5-6 day old, nestlings can already discriminate several different sounds indicative of danger, among the complex natural acoustic background (Haff & Magrath 2012).

* nestling do not respond indiscriminately to any calls, but only to relevant sounds that specifically present a threat to them (Haff and Magrath 2012; Magrath et al 2006).

* the idea that nestlings only distinguish "low frequency broadband cues" (L362) is inaccurate (resp R3.A6). Nestlings respond to scrubwren chip calls and fairywren alarm calls that are narrowband calls with the fundamental at 8 and 10 kHz respectively (Platzen and Magrath 2004; Haff and Magrath 2012).

* These studies indeed "do not imply mature auditory sensitivity" (L362), they show that auditory maturity is not needed to show a perfectly functional response to biologically meaningful sounds in a natural context.

- Overall, every other songbird species tested shows greater hearing sensitivity than that proclaimed here for zebra finches. It is very unlikely that zebra finches would be such an outlier.

(ii) INCORRECT CONCLUSION ON DEAFNESS

Deafness of young zebra finch nestlings cannot be demonstrated because:

- the study has no valid response to tones in nestlings to establish the age of hearing onset.

- responses to clicks are greatly underestimated (see 1). Had correct, more sensitive, protocol parameters been used, it is very likely that:
* Most 2 day old nestlings would have responded to clicks, instead of 60% of 4 day old nestlings.
* Thresholds would be lower, especially in nestlings.
The >54dB difference between nestlings and adults based on an assumed 95dB threshold in 2d old nestlings is wrong.

- based on data on other songbird nestlings, a difference of 25dB would be more realistic (at frequencies of 1-2 kHz, comparable to clicks, Aleksandrov and Dmitrieva 1992, Korneeva et al 2006). This greatly contrasts with the >54dB estimate here. While the authors qualify their estimate as "conservative" (legend Fig4, L293), it is actually greatly overestimated.

- Even assuming the data in this study were correct, the interpretation is erroneous. Concluding "deafness of young nestlings" is incorrect when 60% of 4 day old nestlings respond to clicks at 80dB. This is especially wrong given that the ABR method systematically overestimates thresholds by 20-40dB.

- That ABR is used in humans to diagnose deafness (L263) does not make ABR the most suitable method for birds, when evidence shows that other methods (e.g. electrophysiology or behaviour) are more accurate. Hearing screening in humans can only use non-invasive methods, and has additional criteria than accuracy (price, ease, etc).

- The discussion fails to acknowledge the implications of the limitations of the study (detailed above). For example, talking in broad terms of differences in protocols (L392-395), does not tell readers that this study, because of the parameters chosen, led to an underestimation of zebra finch hearing capacities.
The discussion instead greatly overstates what the study shows (e.g. L229-232, 261, 277-278, 288, 332, 340, etc).

(iii) INCORRECT CONCLUSION THAT HEAT-CALL FALL OUTSIDE THE ADULT HEARING RANGE.

- heat-calls fall outside the adult hearing range solely because of the authors' decision to:
* restrict heat-call frequency range to the authors' own estimation (7-10kHz) on 4 birds instead of using published values that caused developmental impact (e.g. Katsis et al: 6-10kHz), or were produced in vitro (5.9kHz; Anttonnen et al 2025);

* lowering heat call sound level (34dB at 10 cm) to measurements on 4 isolated birds under unknown temperature conditions for an unknown amount of time, when the same paper gives values of 43.4 dB at 1 m (range: 30.7-53.2) in standard in vitro conditions.

* As soon as EITHER of these two values is corrected, the statement that heat calls are outside zebra finch adult hearing range is false.

* In addition, assuming constant heat-call sound level across contexts is unreasonable. That zebra finch can produce inspiratory syllable very similar to heat calls and during inspiration at 65dB during song (Goller & Dalley 2001) argues against the assumption that heat-calls are always soft.

- Other species also show perception, not just production (resp R3.A10a), of calls above their known hearing range (10-20kHz; e.g. Duque et al 2020). The zebra finch is not the only case in birds of mismatch between signals perceived and known hearing range.

Reviewer #4 (Public review):

Reviewing Editor:

There is a virtuous circle between behavior and neuroscience. Sometimes neuroscience identifies a signal that no behavioral study alone could discern: place cells in rats, replayed song sequences in sleeping birds, compass-like signals in the central complex of the fruit fly. Sometimes behavioral observation is what inspires neuroscience: precise measurements of short and long latency reflexes implicate different neural pathways, and directed escape responses in fish are so fast that they require specialized and lateralized escape circuits. And sometimes a behavioral claim requires an animal's sensory system to possess a capacity nobody had documented. A prime example is bat echolocation: Spallanzani inferred in 1793 that bats navigate by hearing, but the claim was dismissed for over a century because the signal he proposed could not be detected. It was vindicated only when Griffin and Galambos measured the ultrasonic emissions directly in 1940, and the loop closed fully when Suga and colleagues found cortical neurons in the mustached bat tuned to precisely the echo delays and Doppler shifts the behavior required.

In this manuscript we are faced with a fascinating and contemporary instance of this important dialogue between behavior and neurophysiology. Several high profile papers have reported that incubating zebra finch parents produce high frequency "heat calls" when ambient temperatures rise, and that playback of these calls to eggs during late incubation alters offspring growth, begging behavior, thermal preference, and reproductive success in adulthood. That function requires that the embryo be able to sense, presumably to hear, the heat calls. This is textbook ecology and often cited as the prime example of adult behavior affecting the development of their unborn (or unhatched) offspring.

Yet the auditory capacity of very young zebra finches had never been carefully examined. This paper does exactly that, using auditory brainstem responses, a method that detects synchronous volleys of afferent input through low levels of the auditory system. ABRs are not perfect and may miss very subtle or sparsely represented signals, but they are a time-tested way to assess hearing capacity and the development of a system.

Here, the authors show, convincingly in my view and in the views of Reviewers 1 and 2, that 2 DPH hatchlings have no detectable ABR to a 95 dB SPL broadband click, and that sensitivity then rises progressively across the first two postnatal weeks. This is the most consequential result, since it bears directly on whether an auditory route to heat call perception is feasible at all. A second finding is that ABR wave I amplitude continues to mature until 20 to 25 days post hatch, coinciding with the onset of sensory song learning, just as young finches need to form a template of an adult male song to copy.

The key question relevant to mediating this review process is: If a two-day-old hatchling shows no detectable auditory brainstem response, over 400 averaged sweeps, to a 95 dB SPL broadband click, in 14/14 animals tested, then how could that same animal, two days earlier and inside an egg, have been sensitive to a far weaker stimulus of roughly 33 dB SPL at 6.8 kHz?

Below I set out the considerations that influenced my judgment as I handled these reviews.

Strengths:

The developmental series is the strength of the design. Seven ages, with multiple early time points, means the negative result at 2 DPH is not a lone flat trace but sits at one end of a graded and internally consistent trajectory. Body temperature was monitored and held within {plus minus}0.5{degree sign}C, and differed by no more than 0.6{degree sign}C across age groups. Every stimulus parameter was applied identically at every age, so the developmental comparison is a within-method one. The vibrometry experiment addresses the most obvious alternative modality with an appropriate and state-of-the-art technique.

The key findings related to the development of the ABR response and, presumably, the hearing capacity. At two days post hatch, the authors presented broadband clicks of 20 µs duration at 95 dB SPL peak equivalent, a stimulus carrying energy across the full spectrum including the 6.8 kHz of the heat whistle. Clicks were delivered at 25 Hz with a 40 ms inter-stimulus interval, 400 presentations per condition with alternating polarity, and responses were scored both by an automated signal-to-noise criterion and by independent visual inspection. No response was observed in any of the 14 animals tested. Two days later, under the same protocol, eight of thirteen animals responded, with a mean threshold of 80.6 {plus minus} 1.6 dB SPL and wave I latencies of roughly 4.0 to 4.7 ms against 1.8 to 2.5 ms in adults. These responses were small, broad, and slow, the signature of an immature auditory system. By 6 DPH nine of ten animals responded, and from 8 DPH onward every animal did at every age tested. Adult thresholds settle at 40.6 {plus minus} 4.0 dB SPL, more than 54 dB below the 2 DPH value. The 4 DPH data show the preparation resolves weak, desynchronized responses under exactly the parameters used at 2 DPH, and that sensitivity emerges on a trajectory consistent with auditory development in other birds and in mammals.

On Reviewer 3's methodological objections:

Reviewer 3 raised a series of objections to the recording parameters: the stimulus rate is faster than in comparable avian developmental studies, 400 sweeps is fewer than the conventional 1000, body temperature sits at the low end of the reported range for the species, and clicks lag tones in developmental onset. Each of these describes a mechanism that would reduce the amplitude of an evoked response relative to some ideal stimulus. But in aggregate my take was that none of these would completely abolish a response that is detectable two days later. That distinction is the crux of my reading and others may disagree. A response reduced by 70 percent is still a response, and the question at 2 DPH is not why the trace was small but why there was no trace at all over 400 averages in all 14 animals tested.

Reviewer 3's concerns are difficult to translate into threshold shifts, and on the narrow point that amplitude reductions do not map cleanly onto decibels, the difficulty that the reviewer faced in converting stimulus concerns to impact on dB threshold is well taken. But the figures supplied are themselves bounded: a 37 percent noise reduction from additional sweeps, a 70 percent amplitude reduction from stimulus rate in the youngest birds, a four day shift in apparent onset from using clicks rather than tones. These are large effects. They are not unbounded ones. And because the decibel is a logarithmic unit, in which every 20 dB corresponds to a tenfold change in sound pressure, the gap they are being asked to explain, exceeding 54 dB, amounts to a difference of more than 500-fold.

The 4 DPH data bear directly on this, because every parameter at issue was applied identically at that age. The same 25 Hz rate, the same 400 sweeps, the same temperature, the same click stimulus resolved small, dispersed, long latency responses in 8 of 13 animals two days later. These are precisely the weak and desynchronized responses an immature auditory system is expected to produce, and precisely what the objections predict should have been lost. Whatever theoretically possible deficits in stimulus design, they did not prevent detection of a marginal response in animals two days older, and no mechanism has been proposed by which their cost would fall from total suppression at 2 DPH to negligible at 4 DPH.

A 95 dB SPL broadband click carries energy at every frequency including 6.8 kHz and exceeds the level at which heat whistles arrive at 10 cm by more than 60 dB, before any attenuation by shell or by an incubating parent. Even a substantial underestimate of hatchling sensitivity leaves that gap open, and at 6.8 kHz it is a broadband stimulus being compared against a narrowband signal in the frequency region that matures last, in this dataset and in every developmental series reported.

Sensory systems as matched filters:

A second consideration: sensory systems as matched filters (survival-critical sensory responses are typically associated with expanded sensory sensitivity for them). Failure to detect an auditory response is not proof of functional deafness. An evoked potential measures synchronized population activity, so sparse discharge below the threshold for ABR sensitivity (a population response) can never be excluded. This is the crux of Reviewer 3's concerns.

But this second consideration makes the difficulty of finding any evidence of hearing or vibrotactile responsiveness in these animals more compelling. Rüdiger Wehner, working on desert ant navigation, noted that sensory systems are matched filters. They are tuned to the narrow slice of the world that matters for survival and reproduction and discard the rest. From the tuning of an ant's polarization channel you can infer what it navigates by, and from the work of Capranica we know that the tuning of a frog's inner ear relates to the sound of conspecific frogs. If acoustic or vibrotactile processing of heat calls were genuinely critical to the embryo, natural selection would have built the outsized sensitivity to receive it. Instead there is a greater than 54 dB deficit and a frequency range where nothing is detectable at all. I interpret this as evidence that evolution did not build auditory sensitivity into two-day-old hatchlings, which suggests functional deafness in the embryo.

One might invert this argument and object that the filter in the embryo is precisely for the heat whistle itself, so that neither a broadband click nor a 25 ms tone burst, the only two stimuli presented to 2 DPH animals, is the right probe. Feature detectors that respond to a specific signal while ignoring stimuli of far greater energy are common: cricket AN2 neurons fire to bat-like pulse intervals and stay quiet to loud broadband noise, and anuran midbrain neurons are interval-tuned and silent to spectrally matched noise exceeding the call in level. Embryonic sensory systems also carry transient specializations that later disappear, and the heat whistle is unusually specifiable, narrowband near 6.8 kHz and delivered in rhythmic trains. A detector tuned to that rhythm would not have been engaged by any stimulus used here, since the actual call was never played to any animal at any age.

But feature selectivity is typically computed centrally, downstream of peripheral transduction, and ABR wave I reflects auditory nerve output, below any circuit that could implement pattern selectivity. A central detector still requires afferent input to operate on, and it is that input which seems to be absent here. It's conceivable that the cochlea itself exhibits some very special tuning to the heat whistle and implements some kind of yet-to-be discovered surround suppression at the periphery, such that a click would not be able to activate the hair cells in the heat whistle's frequency range. But 2 DPH animals were also tested directly with narrowband 6 and 8 kHz stimuli at 95 dB SPL, bracketing the heat whistle frequency, also without response. The surviving version of the objection therefore requires a pathway with too few fibers to generate a far-field response, tuned to a temporal structure nobody tested, in an animal whose auditory nerve shows no signal at some 60 dB above the natural signal level.

Caveats:

The paper comes with some caveats, which the authors note in their discussion. First, no embryo was tested. The embryonic claim is an extrapolation from hatchlings. I regard it as a reasonable one, since sound must additionally traverse the shell and since auditory systems gain rather than lose function as development proceeds, but it is an extrapolation nonetheless. Second, frequency-specific thresholds in nestlings were not obtained with a full stimulus set. The low to high developmental sequence therefore rests on a conservative stimulus. Pip data in nestlings would be a valuable addition to the record. Finally, the study offers no pre-neural measure, such as cochlear microphonics or otoacoustic emissions. Such a measure would distinguish a cochlea that is not transducing from one that transduces without synchronized output, and would address the strongest form of the objection raised in review.

Summary:

Absence of evidence is not evidence of absence, and some future study could in principle identify single neuron responses to heat calls in an embryo. The evidence in this paper makes that outcome unlikely in my assessment. It is worth noting what the precedent actually supports. Prenatal hearing is real and well documented in precocial birds: mallard embryos deprived of exposure to their own calls fail to recognize the maternal assembly call after hatching, and chickens show evoked responses well before hatching. But those animals hatch at a developmental stage altricial songbirds do not reach until days later, and the calls involved carry their energy below 3 kHz, the frequency region that comes online first. The claim at issue here requires sensitivity at 6.8 kHz, the region that matures last, in an animal at a far earlier stage. Something may still be happening to the embryo during heat calls, but whatever the mechanism, it is probably not the embryo listening. Behavioral claims that lack strong precedent, such as hearing through an eggshell, need to sit in a virtuous circle with neurophysiological investigation, and this study is what that looks like from the physiological side.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

This work by Antonnen et al. was triggered by claims of auditory-mediated effects on altricial avian embryos, which were published without any direct evidence that the relevant parental vocalizations were actually heard. I agree with Anttonen et al. that, based on the available evidence about avian auditory development, those claims are highly speculative and therefore necessitate more direct experimental verification.

Attonen et al. have embarked on a comprehensive series of experiments to:

(1) Better characterize acoustically the relevant parental vocalizations (heat whistles; in a separate preprint, not reviewed here)

(2) Characterize the auditory sensitivity of zebra finches at various stages of their posthatching development. Despite the long-standing importance of the zebra finch as a songbird model in neuroethology of learned vocalizations, the auditory development of the species has not been studied so far.

(3) Explore an alternative hypothesis of how the parental vocalizations might be perceived.

The principal method used here is the non-invasive recording of ABR (auditory brainstem response), a standard neurophysiological method in auditory research. The click-evoked ABR provides a quick and objective assessment of basic hearing sensitivity that does not require animal training. Weaknesses of the technique include its limited frequency specificity and low signal-to-noise ratio. The authors are experienced with ABR measurements and well aware of those issues. ABR responses in zebra finches are shown to gradually appear during the first week posthatching and to mature in subsequent weeks, consistent with the auditory development in other altricial bird species studied previously. When matching the acoustic properties of parental heat whistles and auditory sensitivities, hearing of the parental heat whistles by zebra finch hatchlings was convincingly excluded. Although not directly measured, this also convincingly extrapolates to zebra finch embryos. Finally, the authors tested the hypothesis that parental heat whistles could induce perceptible vibrations of the egg and thus stimulate the embryo via a different modality. The method used here was laser doppler vibrometry, an appropriate, state-of-the-art technique that the authors also have proven experience with. The induced vibrations were shown to be several orders of magnitude below known vibrotactile sensitivities in mammals and birds. Thus, although zebra finch vibrotactile thresholds were not obtained directly, the hypothesis of vibrotactile perception of parental heat whistles by zebra finch embryos could also be rejected convincingly.

In summary, even when considering some weaknesses of the techniques (which the authors are aware of), the conclusions of the paper are well supported: Auditory and/or vibration perception of parental heat whistles can be excluded as an explanation for previous reports of developmental programming for high ambient temperatures. As a constructive suggestion towards resolving the apparent paradox, the authors recommend repeating some of the crucial, previous playback experiments at lower sound levels that better match the natural parental vocalizations.

(R1. A1) We thank the reviewer for their time and effort to thoroughly review our paper and for the positive comments on our manuscript. In the revised manuscript we have addressed the concerns that you have raised in the Joint recommendations above (Pages 1-4).

Reviewer #2 (Public review):

This study by Anttonen, Christensen-Dalsgaard, and Elemans describes the development of hearing thresholds in an altricial songbird species, the zebra finch. The results are very clear and along what might have been expected for altricial birds: at hatch (2 days post-hatch), the chicks are functionally deaf. Auditory evoked activity in the form of auditory brainstem responses (ABR) can start to be detected at 4 days post-hatch, but only at very loud sound levels. The study also shows that ABR response matures rapidly and reaches adult-like properties around 25 days post-hatch. The functional development of the auditory system is also frequency dependent, with a low-to-high frequency time course. All experiments are very well performed. The careful study throughout development and with the use of multiple time-points early in development is important to further ensure that the negative results found right after hatching are not the result of the experimental manipulation. The results themselves could be classified as somewhat descriptive, but, as the authors point out, they are particularly relevant and timely. Since 2016, there have been a series of studies published in high-profile journals that have presumably shown the importance of prenatal acoustic communication in altricial birds, mostly in zebra finches. This early acoustic communication would serve various adaptive functions. Although acoustic communication between embryos in the egg and parents has been shown in precocial birds (and crocodiles), finding an important function for prenatal communication in altricial birds came as a surprise. Unfortunately, none of those studies performed a careful assessment of the chicks' hearing abilities. This is done here, and the results are clear: zebra finches at 2 and 6 days post-hatch are functionally deaf. Since it is highly improbable that the hearing in the egg is more developed than at birth, one can only conclude that zebra finches in the egg (or at birth) cannot hear the heat whistles. The paper also ruled out the detection on egg vibrations as an alternative path. The prior literature will have to be corrected, or further studies conducted to solve the discrepancies. For this purpose, the "companion" paper on bioRxiv that studies the bioacoustical properties of heat calls from the same group will be particularly useful. Researchers from different groups will be able to precisely compare their stimuli.

Beyond the quality of the experiments, I also found that the paper was very well written. The introduction was particularly clear and complete (yet concise).

Weaknesses:

My only minor criticism is that the authors do not discuss potential differences between behavioral audiograms and ABRs. Optimally, one would need to repeat the work of Okanoya and Dooling with your setup and using the same calibration. The ~20dB difference might be real, or it might be due to SPL measured with different instruments, at different distances, etc. Either way, you could add a sentence in the discussion that states that even with the 20 dB difference in audiogram heat whistles would not be detected during the early days post-hatch. But adding a (novel) behavioral assay in young birds could further resolve the issue.

(R2. A0) We thank the reviewer for their time and effort to thoroughly review our paper, and for the positive comments on our manuscript.

In our revision, we have added a new figure (Fig 5) and three new paragraphs (Lines 387-422) in the discussion to compare all published ABR and behavioral audiograms and the differences between and among these datasets. Our adult data is consistent with the reported findings from four other labs despite differences in stimulus design, setups and genetic background of the animals, providing strong support for our findings.

Furthermore we have more clearly presented our argument why we think juvenile and embryos cannot detect heat whistles. For clarification, we have added a new figure (Fig 4) and four new paragraphs (Lines 271-355) in the discussion.

We agree with the reviewer that more data is needed on the development of hearing in songbirds and zebra finches especially; both anatomical data and functional, such as innervation. We emphasize this need in our discussion (Lines 352-355 and Lines 405-411).

More Minor Points:

(1) As mentioned in the main text, the duration of pips (from pips to bursts) affects the effective bandwidth of the stimulus. I believe that the authors could give an estimate of this effective bandwidth, given what is known from bird auditory filters. I think that this estimate could be useful to compare to the effective bandwidth of the heat-call, which can now also be estimated.

(R2. A1) Please see answer A3.4 under Joint Recommendations.

(2) Figure 5b. Label the green and pink areas as song and heat-call spectrum. Also note that in the legend the authors say: "Green and red areas display the frequency windows related to the best hearing sensitivity of zebra finches and to heat calls, respectively". I don't think this is what they meant. I agree that 1-4 kHz is the best frequency sensitivity of zebra finches, but they probably meant green == "song frequency spectrum" and pink == "heat call spectrum". In either case, the figure and the legend need clarification.

(R2. A2) We thank the reviewer for pointing out these issues. We have changed the figure and legend accordingly. In the meantime, we published a paper measuring the in vivo source levels of the heat whistles (Anttonen et al., Current Biology 2025), and we have carefully gone through this manuscript to incorporate those findings and adjust the text accordingly. We have therefore adjusted the analysis in Fig 3B to correct for the narrower frequency distribution of the heat whistles.

(3) Figure 5c. Here also, I would change the song and heat-call labels to "song spectrum", "heat call spectrum". The authors would not want readers to think that they used song and heat calls in these experiments (maybe next time?). For the same reason, maybe in 5a you could add a cartoon of the oscillogram of a frequency sweep next to your speaker.

(R2. A3) We thank the reviewer for pointing out these issues We mention the frequency sweep in the legend for panel A, but decided against including this in the figure to prevent too much clutter. We have changed the figure = legend to (new text underlined):

Legend Fig 3. “A Setup used to measure sound-induced vibrations of eggs. A 94 dB, 0.25-to-10 kHz frequency sweep was played at the eggs to determine the vibration transfer function.”

(4) Methods. In the description of the stimulus, the authors describe "5ms long tone bursts", but these are the tone pips in the main part of the manuscript. Use the same terms.

(R2. A4) Thank you for catching this, we have changed this into “5ms long tone pips".

Reviewer #3 (Public review):

Summary

Following recent findings that exposure to natural sounds and anthropogenic noise before hatching affects development and fitness in an altricial songbird, this study attempts to estimate the hearing capacities of zebra finch nestlings and the perception of high frequencies in that species. It also tries to estimate whether airborne sound can make zebra finch eggs vibrate, although this is not relevant to the question.

Strength

That prenatal sounds can affect the development of altricial birds clearly challenges the long-held assumption that altricial avian embryos cannot hear. However, there is currently no data to support that expectation. Investigating the development of hearing in songbirds is therefore important, even though technically challenging. More broadly, there is accumulating evidence that some bird species use sounds beyond their known hearing range (especially towards high frequencies), which also calls for a reassessment of avian auditory perception.

Weaknesses

Rather than following validated protocols, the study presents many experimental flaws and two major methodological mistakes (see below), which invalidate all results on responses to frequencyspecific tones in nestlings and those on vibration transmission to eggs, as well as largely underestimating hearing sensitivity. Accordingly, the study fails to detect a response in the majority of individuals tested with tones, including adults, and the results are overall inconsistent with previous studies in songbirds. The text throughout the preprint is also highly inaccurate, often presenting only part of the evidence or misrepresenting previous findings (both qualitatively and quantitatively; some examples are given below), which alters the conclusions.

Conclusion and impact

The conclusion from this study is not supported by the evidence. Even if the experiment had been performed correctly, there are well-recognised limitations and challenges of the method that likely explain the lack of response. The preprint fails to acknowledge that the method is well-known for largely underestimating hearing threshold (by 20-40dB in animals) and that it may not be suitable for a 1-gram hatchling. Unlike what is claimed throughout, including in the title, the failure to detect hearing sensitivity in this study does not invalidate all previous findings documenting the impacts of prenatal sound and noise on songbird development. The limitations of the approach and of this study are a much more parsimonious explanation. The incorrect results and interpretations, and the flawed representation of current knowledge, mean that this preprint regrettably creates more confusion than it advances the field.

(R3. A0) We thank the reviewer for their detailed and critical assessment. We agree that establishing auditory sensitivity in very young altricial birds is technically challenging and that careful interpretation of ABR data is essential. We also appreciate the reviewer’s recognition of the importance of obtaining direct physiological data on auditory development.

However, we respectfully disagree with the reviewer’s central claim that our methodology is flawed or that our conclusions are unsupported. Many of the concerns raised reflect misunderstandings of ABR methodology, selective interpretation of the literature, or assumptions that are not supported by empirical evidence. Below, we address the main points in turn.

As also stated in our Provisional response, the reviewer’s critique can be distilled into four main arguments:

(1) ABR cannot be reliably measured in very small animals.

(2) Our stimulus design (especially 25 ms tone bursts) invalidates frequency-specific results.

(3) ABR thresholds should be corrected to behavioral thresholds, which would alter conclusions.

(4) Our findings are inconsistent with prior studies in songbirds.

We address each of these below before responding point-by-point.

(1) Suitability of ABR in small animals.

Reviewer claim: ABR may not be suitable for very small hatchlings.

This claim is not supported by existing evidence. ABR measures summed neural activity, and signal amplitude depends in part on the distance between neural tissue and recording electrodes. In smaller animals, this distance is reduced, which can increase signal amplitude and improve signal-to-noise ratio.

Consistent with this, ABR has been successfully recorded in animals substantially smaller than zebra finch hatchlings, including zebrafish (Jørgensen et al., 2012), 10 mm froglets (Goutte et al., 2017) and 5 mm salamanders (Capshaw et al., 2020). It is in fact much more surprising the technique still provides robust signals even in extremely large animals such as Minke whales, where the distance between electrodes and brain is on the decimeter scale (Houser et al., 2024). We have extensive experience of recording ABRs in such small systems.

Thus, there is no principled reason why ABR would be an invalid method to study auditory sensitivity in zebra finch hatchlings.

(2) Stimulus design and tone duration

Reviewer claim: Use of 25 ms tone bursts invalidates frequency-specific results.

We agree that stimulus duration affects frequency specificity and ABR detectability. However, the reviewer’s assertion that there is a single “correct protocol” (≤5 ms) is inaccurate. In avian ABR studies, stimulus duration varies depending on experimental goals.

Our choice of 25 ms tone bursts was intentional and necessary to accurately represent low frequencies (down to 250 Hz), ensuring sufficient cycles per stimulus in the plateau segment of 15 ms and minimizing spectral splatter (see auditory brainstem response design considerations discussed in Lauridsen et al., 2021) and our responses below.

Key clarifications:

a) Click-evoked ABRs form the basis of our conclusions about onset of hearing, not tone bursts.

b) Tone bursts were used primarily to assess frequency-dependent maturation, not detect earliest sensitivity.

c) We explicitly demonstrate that:

- 25 ms bursts yield higher thresholds (lower sensitivity)

- 5 ms pips yield lower thresholds and align with published ABR audiograms

We have now:

- Further clarified the rationale of stimulus design in the methods (Line 520-530) and added a section in the discussion (Lines 397-411).

- Included additional comparison between burst and pip datasets (Lines 387-395).

- Clarified that conclusions about early hearing do not depend on tone-burst data (Fig 4 and Lines 271-294).

(3) ABR vs behavioral thresholds

Reviewer claim: Failure to correct ABR thresholds (20–40 dB) invalidates conclusions.

We agree that ABR thresholds typically overestimate behavioral thresholds. However, we disagree that this invalidates our conclusions.

Importantly:

a) We do not replace measured ABR data with corrected values, as this would be methodologically inappropriate.

b) Instead, we:

- Present measured ABR thresholds transparently (Fig 1-3)

- Compare them directly to published behavioral audiograms (Fig 5)

- Explicitly discuss the expected offset (Lines 413-422)

In the revised manuscript we:

a) Add a new figure (Fig. 5) compiling all published ABR and behavioral audiograms

b) Show that:

- ABR and behavioral audiograms have similar shapes (Fig 5, new discussion Lines 387-422)

- Offsets are typically ~20 dB (Line 413-422)

Crucially, even under conservative corrections:

- Early hatchlings remain far less sensitive than adults (>54 dB SPL) to clicks.

- Heat whistle levels remain at or below detection limits even in adults (new figure Fig 4)

- The developmental gap (>50 dB between adults and 2 DPH hatchlings) remains decisive.

Thus, incorporating ABR–behavioral differences does not change the central conclusion.

(4) Consistency with prior literature in developing songbirds.

Reviewer claim: Results contradict previous studies in developing songbirds.

We respectfully disagree. The cited studies fall into three categories:

(1) Behavioral studies (e.g., alarm-call responses)

(2) Gene expression studies (e.g., ZENK activation)

(3) Different species with different developmental trajectories

None of these directly measure auditory sensitivity thresholds in zebra finch embryos or hatchlings.

We emphasize:

- Behavioral responses do not provide threshold measurements

- Neural activation (e.g., ZENK) does not demonstrate functional perception thresholds

- Cross-species comparisons must consider differences in developmental timing.

We have now expanded the Discussion to explicitly address these studies and clarify how they relate to our findings (Lines 357-367). Our data are consistent with what is known about the physiology of auditory development in all birds studied so far.

(5) Final statement

We have revised the manuscript extensively to:

- Clarify methodology and experimental design

- Expand discussion of ABR limitations

- Incorporate additional literature and comparisons

- Correct inconsistencies in reporting

We maintain that our central conclusions—that early zebra finch hatchlings lack detectable auditory brainstem responses and are unlikely to perceive parental heat calls at natural levels—is robust and supported by the data. We go into more detail in our point-by-point rebuttal below.

Detailed assessment

For brevity, only some references are included below as examples, using, when possible, those cited in the preprint (DOI is provided otherwise). A full review of all the studies supporting the points below is beyond the scope of this assessment.

(A) Hearing experiment

The study uses the Auditory Brainstem Response (ABR), which measures minute electrical signals transmitted to the surface of the skull from the auditory nerve and nuclei in the brainstem. ABR is widely used, especially in humans, because it is non-invasive. However, ABR is also a lot less sensitive than other methods, and requires very specific experimental precautions to reliably detect a response, especially in extremely small animals and with high-frequency sounds, as here.

(1) Results on nestling frequency sensitivity are invalid, for failing to follow correct protocols:

(R3. A1.1) We disagree that our protocol is invalid. There is no universal ABR protocol standard in birds. Our approach is consistent with established principles of stimulus design and is validated by:

- Robust click-evoked responses

- Consistent developmental trajectories

- Agreement between pip-based ABR and behavioral audiograms

We now clarify this explicitly. See response R3. A0 (Stimulus design and tone duration).

The results on frequency testing in nestlings are invalid, since what might serve as a positive control did not work: in adults, no response was detected in a majority of individuals, at the core of their hearing range, with loud 95dB sounds (Figure S1), when testing frequency sensitivity with "tone burst".

This is mostly because the study used a stimulation duration 5 times larger than the norm. It used 25ms tone bursts, when all published avian studies (in altricial or precocial birds) used stimulation of 5ms or less (when using subdermal electrodes as here; e.g., cited: Brittan-Powell et al 2004; not cited: Brittan-Powell et al 2002 (doi: 10.1121/1.1494807), Henry & Lucas 2008 (doi: 10.1016/j.anbehav.2008.08.003)). Long stimulations do not make sense and are indeed known to interfere with the detection of an ABR response, especially at high frequencies, as, for example, explicitly tested and stated in Lauridsen et al 2021 (cited).

(R3. A1.2) ABR with long-duration stimuli were shown previously to work perfectly well in birds and do not interfere with the detection of an ABR response. Longer stimuli have been used, e.g. in the following bird papers:

- Amin et al., J Neurophysiol 2007 (cited) on zebra finch ABR: 20 ms tone bursts

- Korneeva et al. (2006), evoked responses from field L in flycatcher (cited by the reviewer): 20 ms

- Saunders et al. (1973) (cited): 60 ms tone bursts.

- Larsen ON, Wahlberg M, Christensen-Dalsgaard (2020) Amphibious hearing in a diving bird, the great cormorant (Phalacrocorax carbo sinensis), J Exp Biol, doi:10.1242/jeb.217265: 25 ms tone bursts

Human ABR has been measured even with long-duration speech signals (duration 40 ms and longer). See for example Binkhamis et al., Ear and Hearing 40: 659-670, 2019.

Furthermore, the reviewer unfortunately misunderstood some aspects in Lauridsen et al., which tested a specifical method exploiting neural phase locking, and showed that this method has a low-frequency bias because neural phase locking decreases at high frequencies.

Also, Lauridsen et al clearly show the reason for using 25 ms bursts: because we aimed to measure frequencies down to 250 Hz, we need to have a sufficient number of cycles (3) in the plateau segment to represent the frequency adequately (Lauridsen et al. fig 1). A 15 ms plateau contains 3 cycles, plus 5 ms rise/fall time (1 cycle) equals 25 ms. We have clarified this in our methods (L520-525) and added a section in the discussion (L397-411).

Thus, long-duration stimulations make sense and have been used before successfully. See also response R3.A0 (Stimulus design and tone duration).

Adult response was then re-tested with a correct 5ms tone duration ("tone-pip"), which showed that, for the few individuals that responded to 25ms tones, thresholds were abnormally high (c.a. by 30dB; Figure 2C).

Yet, no nestlings were retested with a correct protocol. There is therefore no valid data to support any conclusion on nestling frequency hearing. Under these circumstances, the fact that some nestlings showed a response to 25ms tones from day 8 would argue against them having very low sensitivity to sound.

(R3. A1.3) Please see answer A3.1 under Joint Recommendations and R3.A0 (Stimulus design and tone duration).

(2) Responses to clicks underestimate hearing onset by several days:

Without any valid nestling responses to tones (see # 1), establishing the onset of hearing is not possible based on responses to clicks only, since responses to clicks occur at least 4 days after responses to tones during development (Saunders et al, 1973). Here, 60% of 4-day-old individuals responding to clicks means most would have responded to tones at and before 2 days post-hatch, had the experiment been done correctly.

(R3. A2.1) We disagree that clicks necessarily underestimate onset.

Clicks are broadband stimuli that:

- Recruit large neural populations

- Are commonly used to detect early auditory responses

The cited delay between tone and click responses reflects stimulus energy differences, not an inherent limitation of clicks. We have clarified this in the revision and softened language to refer to “no detectable ABR response” rather than absolute deafness.

The report that Saunders could only see responses to clicks later than to tones only reflects that he used click amplitudes that were insufficiently high. The ABR responses reported were to extremely intense tones (110 dB SPL) of long duration (60 ms).

If Saunders had used clicks (duration 60 µs) with comparable sound energy, they would have had been very difficult to produce. He should have used clicks with an amplitude 1000 times (60 dB) higher than the tones to produce the same sound energy. This would have been clicks at 170 dB SPL, equivalent to the sound at the mouth of a medium-sized military cannon. Applying this pressure would not be a recommendable method for hearing assessment, but instead lead to irreversible hearing damage.

In budgerigars, hearing onset occurs before 5 days post hatch, since responses to both clicks and tones were detectable at the first age tested at 5dph (Brittan-Powell et al, 2004).

(R3. A2.2) This is not how we interpret the cited paper. They state that ‘Responses were first obtained from 1-week-old at high stimulation, and their click responses (Fig. 1) show no wave 1 peak at 6 days post-hatch. Also, their conclusion (p 3101) states that ‘budgerigars probably cannot hear at hatching’.

(3) Experimental parameters chosen lower ABR detectability, specifically in younger birds: Very fast stimulus repetition rate inhibits the ABR response, especially in young:

(a) The stimulus presentation rate (25 stim/ sec) is 6 times faster than zebra finch heat-calls, and 5 to 25 times faster than most previous studies in young birds (e.g., cited: Saunders et al 1973, 1974: 1 stim/sec or less; Katayama 1985: 3.3 clicks/sec; Brittan-Powell et al 2004: 4 stim/sec).

Faster rates saturate the neurons and accordingly are known to decrease ABR amplitude and increase ABR latency, especially in younger animals with an immature nervous system.

In birds, this occurs especially in the range from 5 to 30 stim/sec (e.g., cited: Saunder et al 1973, Brittan-Powell et al 2004). Values here with 25 rather than 1-4 stim/min are therefore underestimating true sensitivity.

(R3. A3a) Please see answer A3.3 under Joint Recommendations.

(b) Averaging over only 400 measures is insufficient to reliably detect weak ABR signals: The study uses 2 to 3 times fewer measures per stimulation type than the recommended value of 1,000 (e.g., Brittan-Powell et al 2002, 2024; Henry & Lucas 2008). This specifically affects the detection of weak signals, as in small hatchlings with tiny brains (adult zebra finches are 12-14g).

(R3. A3b) Please see answer A3.2 under Joint Recommendations.

(c) Body temperature is not specified and strongly affects the ABR:

Controlling the body temperature of hatchlings of 1-4 grams (with a temperature probe under a 5mm-wide wing) would be very challenging. Low body temperature entirely eliminates the ABR, and even slight deviance from optimal temperature strongly increases wave latency and decreases wave amplitude (e.g., cited: Katayama 1985).

(R3. A3c) Please see answer A2 under Joint Recommendations.

(d) Other essential information is missing on parameters known to affect the ABR: This includes i) the weight of the animals,

(R3. A3d-i) These important details have now been added in Table S6.

(ii) whether and how the response signal was amplified and filtered,

(R3. A3d-ii) Signal was amplified 500 times (74 dB). We have included these important details to the methods (Line 508).

(iii) how the automatised S/N>2 criteria compared to visual assessment for wave detection,

(R3. A3d-iii) There is no universally accepted/fitting protocol for performing ABR recordings in various animals, we decided to perform both visual and automated criteria detection of thresholds. The automated criterion in our experience is more strict approach than visual detection of thresholds, because using an automated criteria for threshold detection will remove potential experimenter bias from the results. We added this in our methods (Lines 583-585).

(iv) what measures were taken to allow the correct placement of electrodes on hatchlings less than 5 grams.

(R3. A3d-iv) We have placed electrodes in much smaller animals than 5 grams, and the common landmarks (ear opening, midline of skull) could easily be identified in the hatchlings.

(4) Results in adults largely underestimate sensitivity at high frequencies, and are not the correct reference point:

(a) Thresholds measured here at high frequencies for adults (using the correct stimulus duration, only done on adults) are 10-30dB higher than in all 3 other published ABR studies in adult zebra finches (cited: Zevin et al 2004; Amin et al 2007; not cited: Noirot et al 2011 (10.1121/1.3578452)), for both 4 and 6 kHz tone pips.

(b) The underlying assumption used throughout the preprint that hearing must be adult-like to be functional in nestlings does not make sense. Slower and smaller neural responses are characteristic of immature systems, but it does not mean signals are not being perceived.

(R3. A4) We acknowledge variation across studies and now include a comprehensive comparison of all zebra finch audiograms (new Fig. 5 and discussion Lines 387-422).

Importantly:

- Our pip-based audiogram aligns with previous ABR studies

- Differences in high-frequency sensitivity likely reflect methodological variation or population differences.

Our conclusions rely on relative developmental changes, not absolute thresholds.

(5) Failure to account for ABR underestimation leads to false conclusions:

(a) Whether the ABR method is suitable to assess hearing in very small hatchlings is unknown. No previous avian study has used ABR before 5 days post-hatch, and all have used larger bird species than the zebra finch.

(R3. A5a) As stated above (R3.A0), small animals should give better signals, and we have been able to measure ABR in much smaller animals previously.

(b) Even when performed correctly on large enough animals, the ABR systematically underestimates actual auditory sensitivity by 20-40 dB, especially at high frequencies, compared to behavioural responses (e.g., none cited: Brittan-Powell et al 2002, Henry & Lucas 2008, Noirot et al 2011). Against common practice, the preprint fails to account for this, leading to wrong interpretations.

(R3. A5a) See our answer to R3.A0 above.

For example, in Figure 1G (comparing to heat call levels), actual hearing thresholds would be 3040dB below those displayed. In addition, the "heat whistle" level displayed here (from the same authors) is 15dB lower than their second measure that they do not mention, and than measures obtained by others (unpublished data). When these two corrections are made - or even just the first one - the conclusion that heat-call sound levels are below the zebra finch hearing threshold does not hold.

(R3. A5a) Our conclusion that heat whistles are unlikely to be perceived does not rely on a single dataset or method, but on the convergence of three independent constraints: (i) signal amplitude, (ii) adult auditory sensitivity, and (iii) developmental immaturity of the auditory system.

First, heat whistles are low-amplitude signals. Our in vivo measurements show levels of ~33 dB re 20 µPa at 10 cm and ~14 dB at 1 m (Anttonen et al., 2025, Curr Biol). Even allowing for uncertainty in near-field estimation, a conservative upper bound at very close range (<5 cm) is ~40 dB SPL.

Second, the most sensitive available measure—behavioral audiograms—places adult zebra finch thresholds at ~40 dB SPL at ~6 kHz (Okanoya and Dooling, 1987, J Comp Psychol), increasing steeply toward higher frequencies. Thus, even under optimal conditions, heat whistles fall at or below the detection threshold of adults, and only potentially at very close range.

Third, auditory sensitivity in early development is substantially reduced. Our ABR data show a ≥40–60 dB decrease in sensitivity in hatchlings relative to adults for click stimuli, which provide the most favorable conditions for eliciting responses. Because frequency-specific sensitivity develops later, thresholds at 6–8 kHz are expected to be even higher in hatchlings and embryos.

Taken together, these constraints define a narrow and unfavorable detection window: a low-amplitude, high-frequency signal positioned at the edge of adult sensitivity, combined with a large developmental decrease in auditory sensitivity. Under these conditions, it is unlikely that heat whistles are detectable by hatchlings or embryos.

Importantly, this conclusion does not depend on precise correction factors between ABR and behavioral thresholds. Even when considering the most sensitive behavioral data and conservative estimates of sound level, the signal remains at or below the limits of detection in adults, and far below expected sensitivity in early developmental stages.

We have included this argument more clearly in our discussion (Line 271-367), illustrated by new Fig. 5.

(c) Rather than making appropriate corrections, the preprint uses a reference in humans (L180), where ABR is measured using a much more powerful method (multi-array EEG) than in animals, and from a larger brain. The shift of "10-20dB" obtained in humans is not applicable to animals.

(R3. A5c) Again our conclusions rely on relative developmental changes, not absolute thresholds. The clinical practice in humans to measure ABR is with 4 electrodes, not a multi-electrode EEG array.

Animal studies where ABR audiograms have been compared directly to psychophysical audiograms show differences of around 20 dB. For example in Brittain powell et al 2002, audiogram comparisons between behavioral and ABR in budgerigars were made within the same lab, same animal population and by the same people, leading to 20 dB difference. Our discussion includes a new paragraph on this topic (Lines 413-422).

(6) Results are inconsistent with previous findings in developing songbirds:

(R3. A6) We now explicitly discuss all cited studies. Key points:

- Early behavioral responses do not imply high-frequency sensitivity

- Studies in other species do not directly translate to zebra finches

- None of the cited work provides direct measures of auditory thresholds in embryos

As expected from all of the above, results and conclusions in the preprint are inconsistent with findings in other songbirds, which, using other methods, show for example, auditory sensitivity in: a) zebra finch embryos, in response to song vs silence (not cited: Rivera et al 2018, doi: 10.1097/WNR.0000000000001187)

(R3. A6a) We thank the reviewer for pointing out this study. We agree that the question of auditory responsiveness in embryos is important, and that a range of approaches have been used to address it. However, the study cited (Rivera et al., 2018) does not directly measure auditory sensitivity, but instead infers auditory processing from differences between treatment groups exposed to different acoustic conditions. As such, it is not directly comparable to physiological measures of hearing sensitivity, such as ABR or behavioral thresholds.

In addition, interpretation of these results is complicated by limited characterization of the acoustic environment and differences in experimental handling between groups, which may introduce confounding factors unrelated to auditory perception. Given these considerations, and because our study focuses specifically on quantifying auditory sensitivity using established physiological methods, we have chosen not to include a detailed discussion of this work.

(b) flycatcher hatchlings at 2-3d post hatch (first age tested), across a wide range of frequencies (0.3 to 5kHz), at low to moderate sound levels (45-65dB) (cited: Aleksandrov and Dmitrieva 1992, not cited: Korneeva et al 2006 (10.1134/S0022093006060056)).

(R3. A6b) Korneeva et al. (2006) and Aleksandrov and Dmitrieva (1992) report evoked responses in very young flycatcher hatchlings across a broad frequency range. Notably, these measurements were obtained using more invasive recording approaches (e.g., implanted electrodes in Field L) in unanesthetized birds, which are known to yield lower thresholds compared to far-field ABR recordings under anesthesia. These methodological differences likely account for part of the higher sensitivity reported.

Importantly, even in flycatchers, auditory sensitivity shows substantial postnatal improvement: thresholds decrease by up to ~40 dB over the first days after hatching, and the upper frequency limit expands from ~4 to ~7 kHz. Thus, while absolute sensitivity may differ across species and methods, the overall developmental trajectory—gradual improvement in sensitivity and progressive extension toward higher frequencies—is consistent with our findings and with broader patterns reported in songbirds.

We have added this paper in our discussion (Lines 362-365).

(c) songbird nestlings at 2-6d post hatch, which discriminate and behaviourally respond to relevant parental calls or even complex songs. This level of discrimination requires good hearing across frequencies (e.g., not cited: Korneeva et al 2006; Schroeder & Podos 2023 (doi: 10.1016/j.anbehav.2023.06.015)).

(R3. A6c) The species mentioned are different species from our study species. In the Pied flycatchers (Korneeva et al. 2006) experimental conditions were different: recordings were made from unanesthetized nestlings with implanted electrodes directly in the brain (field L), so likely with better SNR. The audiograms show a 10 dB SPL threshold after day 11, so the species may be considerably more sensitive than the zebra finch. The swamp sparrows in the Schroeder and Podos (2023) behavioral study were exposed for 4 days starting at 4-7 days post-hatch, so the study does not address embryonal hearing.

(d) zebra finch nestlings at 13d post-hatch, which show adult-like processing of songs in the auditory cortex (CNM) (Schroeder & Remage-Healey 2021, doi: 10.1002/dneu.22802).

(R3. A6d) This study does not conflict with our data. Even though sensitivity is lower at 10 days than in adults, cortical processing could still be ‘adult-like’.

(e) zebra finch juveniles, which are able to perceive and learn song syllables at 5-7kHz (fundamental frequency) with very similar acoustic properties to heat calls, and also produced during inspiration (Goller & Daley 2001, doi: 10.1098/rspb.2001.1805).

(R3. A6e) This result is not in conflict with our data. First, the onset of song learning occurs earliest at 20 DPH as discussed in the paper and our work demonstrates that click-evoked ABR thresholds are adult-like at 20 DPH. In the cited paper, the tutoring experiments were initiated at 35 DPH so the auditory system of the studied juveniles is mature.

Second, even though Goller & Daley 2001 do not report the source level of the specific syllable or the playback sound pressure levels, the source level of the inspiratory notes is comparable to other syllables, and thus around ~67 dB and ~34 dB louder than heat whistles.

NONE of these results - which contradict results and claims in the preprint - are mentioned.

Instead, the preprint focuses on very slow-developing species (parrots and owls), which take 2-4 times longer than songbirds to fledge (cited: Brittan-Powell et al 2004; Köppl & Nickel 2007; Kraemer et al 2017).

(R3. A6f) We have included papers in our discussion that reflect the known data (to our best knowledge) on the developmental neurophysiology and neuroanatomy of the auditory system and not proxies thereof.

(7) Results in figures are misreported in the text, and conclusions in the abstract and headers are not supported by the data:

For example:

(a) The data on Figure 1E shows that at 4 days old, 8 out of 13 nestlings (60%) responded to clicks, but the text says only 5/13 responded (L89).

(R3. A7a1) We apologize for this typo. Corrected.

When 60% (4dph) and 90% (6dph) of individuals responded, the correct term would be that "most animals", rather than "some animals" responded (L89).

(R3. A7a2) We have rephrased this sentence into “observable in most animals during” as suggested.

Saying that ABR to loud sound appeared "in the majority only after one week" (L93) is also incorrect, given the data.

(R3. A7a3) We have rephrased this sentence into: “Thus, sounds at loud, yet physiologically relevant SPLs do not evoke ABRs in the first days after hatching, but do so in all animals at 8 DPH.” (Lines 97-99).

It follows that the title of the paragraph is also erroneous.

(R3. A7a4) The paragraph title supports our conclusions and we will keep it.

(b) The hearing threshold is underestimated by 40dB at 6 and 8Kz on Fig 2C, not by "10-20dB" as reported in the text (L178).

(R3. A7b) We have changed the title of this section and moved the last sentence to the discussion to remove the focus on heat whistles. We added a paragraph in the discussion to specifically address the difference between ABR and behaviorally measured audiograms (Lines 413-422).

(B) Egg vibration experiment

(8) Using airborne sound to vibrate eggs is biologically irrelevant:

(R3. A8.1) We agree that parental contact could influence vibration transmission.

However, (1) prior studies assume airborne sound transmission, and (2) our experiment tests this assumption directly. We now clarify this scope (Lines 328-342 and Figure 4) and discuss contact-based transmission as a potential future direction.

The measurement of airborne sound levels to vibrate eggs misunderstands bone conduction hearing and is not biologically meaningful: zebra finch parents are in direct contact with the eggs when producing heat calls during incubation, not hovering in front of the nest. This misunderstanding affects all extrapolations from this study to findings in studies on prenatal communication.

(R3. A8.2) The definition of bone conduction is the response to sound that is not mediated by a functional middle ear, but through the skull. In the earlier study, the eggs were stimulated by sound from a headphone, so that is the reason for using the same stimulation here. See also joint response A3.5 above.

(C) Misrepresentation of current knowledge

(9) Values from published papers are misreported, which reverses the conclusions:

(R3. A9) We thank the reviewer for identifying inconsistencies and have:

- Corrected heat whistle frequency ranges consistently through our paper

- Added a comprehensive comparison figure gathering all available audiograms (Fig 5)

- Expanded discussion of high-frequency hearing.

These revisions do not alter our conclusions.

Most critical examples:

(a) Preprint: "Zebra finch most sensitive hearing range of 1-to-4 kHz (Amin et al., 2007; Okanoya and Dooling, 1987; Yeh et al., 2023)" (L173).

Actual values in the studies cited are:

1-to-7kHz, in Amin et al 2007 (threshold [=50dB with ABR] is the same at 7kHz and 1KHz).

1-to-6 kHz, in Okanoya and Dooling (the threshold [=30dB with behaviour] is actually lower at 6kHz than at 1KHz).

1-to-7kHz, in Yeh et al (threshold [=35-38dB with behaviour] is the same at 7kHz and 1KHz).

(R3. A9a.1) In this sentence presenting our results (“sensitive hearing range of 1-to-4 kHz”) we originally wrote that “these are consistent with the following papers (Amin et al., 2007; Okanoya and Dooling, 1987; Yeh et al., 2023)". This latter part was left out during the writing process. This explains the different numbers. We apologize for this mistake.

To avoid confusion, in our revision we have placed all ABR curves together into new Fig 5 and have included a new paragraph to discuss the differences (Lines 387-422).

Note that zebra finch nestlings' begging calls peaking at 6kHz (Elie & Theunissen 2015, doi: 10.1007/s10071-015-0933-6), would fall 2kHz above the parents' best hearing range if it were only up to 4kHz.

(R3. A9a.2) Of course that is possible. However this representation is incorrect because begging calls are harmonic sounds with a fundamental frequency around 500 Hz and formant at 6 kHz. Begging calls thus contain lots of energy at frequencies below 6 kHz, while the heat whistles do not. The peak frequency of heat whistles is also their lowest frequency component.

(b) The preprint incorrectly states throughout (e.g., L139, L163, L248) that heat-calls are 7-10kHz, when the actual value is 6-10kHz in the paper cited (Katsis et al, 2018).

(R3. A9b) The authors in Katsis et al. 2018 provided a range of 6-10 kHz estimated from the spectrogram without any further specification of methods. In another manuscript, we have quantified the heat whistle frequency (Anttonen et al Curr Biol https://doi.org/10.1016/j.cub.2025.08.054) to be 6.8 ± 0.6 kHz. We have changed this accordingly throughout our manuscript.

(c) Using the correct values from these studies, and heat-calls at 45 dB SPL (as measured by others (unpublished data), or as measured by the authors themselves, but which is not reported here (Anttonen et al 2025), the correct conclusion is that heat calls fall within the known zebra finch hearing range.

(R3. A9c) Please see our answer R3.A5a. We have included this argument more clearly in our discussion (Lines 271-355), illustrated by new Fig. 4.

(10) Published evidence towards high-frequency hearing, including in early development, is systematically omitted:

(a) Other studies showing birds use high frequencies above the known avian hearing range are ignored. This includes oilbirds (7-23kHz; Brinklov et al 2017; by 1 of the preprint authors, doi: 10.1098/rsos.170255) and hummingbirds (10-20kHz; Duque et al 2020, doi: 10.1126/sciadv.abb9393), and in a lesser extreme, zebra finches' inspiratory song syllables at 57kHz (Goller & Dalley, 2001).

(R3. A10a) We agree that some bird species produce or use acoustic signals extending into high frequencies. However, signal production is not evidence of perceptual sensitivity. Many animals, including birds and mammals, produce signals that contain harmonic or broadband components extending beyond their most sensitive hearing range without implying functional detection at those frequencies.

The cited examples (oilbirds, hummingbirds, inspiratory song syllables in zebra finches) concern signal production or ecological specializations in different species, not measured auditory sensitivity in zebra finches, and particularly not during early development. As such, they do not provide evidence that zebra finches—adults or embryos—can detect low-amplitude, narrowband signals in the 6–7 kHz range.

Our study explicitly addresses auditory sensitivity using physiological measurements, which is the relevant metric for evaluating detectability.

(b) The discussion of anatomical development (L228-241) completely omits the well-known fact that the avian basilar papilla develops from high to low frequencies (i.e., base to apex), which - as many have pointed out - is opposite to the low-to-high development of sensitivity (e.g., cited: Cohen & Fermin 1978; Caus Capdevila et al 2021).

(R3. A10b) We agree that the avian basilar papilla develops from base to apex (high to low frequency). We have now added a sentence in the Discussion to acknowledge this (Lines 406411).

Importantly, morphological development does not directly translate to functional sensitivity. Functional hearing depends critically on factors such as hair cell innervation, synaptic maturation, and central auditory processing, which are known to develop over time.

Our data show a low-to-high frequency progression in functional sensitivity, consistent with previous physiological studies. This apparent mismatch between anatomical gradients and functional onset has been noted in other systems and likely reflects the later maturation of neural encoding rather than hair cell differentiation per se. We now clarify this distinction in the revised manuscript (Lines 406-411).

(c) High frequency hearing in songbirds at hatching is several orders of magnitude better than in chickens and ducks at the same age, even though songbirds are altricial (e.g., at 4kHz, flycatcher: 47dB, chicken-duck: 90dB; at 5kHz, flycatcher: 65dB, chicken-duck: 115dB; Korneeva et al 2006, Saunders et al 1974). That is because Galliformes are low-frequency specialists, according to both anatomical and ecological evidence, with calls peaking at 0.8 to 1.2kHz rather than 2-6kHz in songbirds. It is incorrect to conclude that altricial embryos cannot perceive high frequencies because low-frequency specialist precocial birds do not (L250;261).

(R3. A10c) We agree that species differ in their auditory ecology and frequency specialization, and we do not claim that all altricial birds share identical developmental trajectories.

However, the cited comparisons involve different species, methodologies, and developmental timelines, which limits their direct comparability. In particular:

Developmental staging is not directly comparable across species using days post-hatch alone.

- Different methods (e.g., invasive recordings vs. ABR vs behavioural assays) yield systematically different thresholds.

- Ecological specialization (e.g., low-frequency vs. broadband species) influences adult audiograms and likely developmental trajectories.

We have revised the Discussion to explicitly acknowledge these limitations and to avoid overgeneralization across species. Importantly, our conclusions are based on within-species comparisons (adult vs. hatchling zebra finches) combined with measured signal levels of heat whistles. These constraints are sufficient to evaluate detectability without relying on cross-species extrapolation.

(11) Incorrect statements do not reflect findings from the references cited For example:

(a) "in altricial bird species hearing typically starts after hatching" (L12, in abstract), "with little to no functional hearing during embryonic stages (Woolley, 2017)." (L33).

There is no evidence, in any species, to support these statements. This is only a - commonly repeated - assumption, not actually based on any data. On the contrary, the extremely limited evidence to date shows the opposite, with zebra finch embryos showing ZENK activation in the auditory cortex in response to song playback (Rivera et al, 2018, not cited).

The book chapter cited (Woolley 2017) acknowledges this lack of evidence, and, in the context of song learning, provides as only references (prior to 2018), 2 studies showing that songbirds do not develop a normal song if the song tutor is removed before 10d post-hatch. That nestlings cannot memorise (to later reproduce) complex signals heard before d10 does not mean that they are deaf to any sound before day 10.

Studies showing hearing in young songbird nestlings (see point 6 above) also contradict these statements.

(R3. A11a) We agree that the precise onset of hearing in altricial embryos is not well established. We have therefore revised the wording in the Abstract and Introduction to avoid categorical statements and instead reflect the limited available evidence (Lines 13-16 and 33-37).

Our data provide direct physiological measurements showing extremely low sensitivity immediately after hatching, which constrains the likelihood of functional hearing in earlier embryonic stages.

Regarding the cited ZENK study, we note that immediate early gene expression indicates neural activation but does not provide a measure of auditory sensitivity or detection thresholds. As such, it cannot be directly compared to physiological or behavioral measures of hearing.

(b) "Zebra finch embryos supposedly are epigenetically guided to adapt to high temperatures by their parents high-frequency "heat calls" " (L36 and L135).

This is an extremely vague and meaningless description of these results, which cannot be assessed by readers, even though these results are presented as a major justification for the present study. Rather than giving an interpretation of what "supposedly" may occur, it would be appropriate to simply synthesize the empirical evidence provided in these papers. They showed that embryonic exposure to heat-calls, as opposed to control contact calls, alters a suite of physiological and behavioural traits in nestlings, including how growth and cellular physiology respond to high temperatures. This also leads to carry-over effects on song learning and reproductive fitness in adulthood.

(R3. A11b) We thank the reviewer for raising this point. In the revised manuscript, we have replaced the previous phrasing with a more precise and neutral summary of what these studies report, namely that embryonic exposure to heat-call playbacks has been associated with differences in physiological and behavioral traits.

Our study, however, addresses a distinct question—whether such acoustic signals are detectable by embryos given known constraints on signal amplitude and auditory sensitivity. The cited studies do not directly quantify auditory perception or the physical sound environment experienced by embryos. As a result, they do not provide a direct test of the sensory mechanism required for acoustic communication. A detailed evaluation of experimental design and interpretation in those studies is beyond the scope of the present manuscript, and we therefore limit our discussion to assessing the biophysical and physiological plausibility of the proposed mechanism.

(c) "The acoustic communication in precocial mallard ducks depends specifically on the lowfrequency auditory sensitivity of the embryo (Gottlieb, 1975)" (L253)

The study cited (Gottlieb, 1975) demonstrates exactly the opposite of this statement: it shows that duckling embryos, not only perceive high frequency sounds (relative to the species frequency range), but also NEED this exposure to display normal audition and behaviour post-hatch. Specifically, it shows that duckling embryos deprived of exposure to their own high-frequency calls (at 2 kHz), failed to identify maternal calls post-hatch because of their abnormal insensitivity to higher frequencies, which was later confirmed by directly testing their auditory perception of tones (Dimitrieva & Gottlieb, 1994).

(R3. A11c) We thank the reviewer for this clarification and have revised the relevant text. Our intention was to highlight that embryonic auditory experience can shape postnatal behavior, not to imply strict low-frequency limitation. Therefore we already included the actual frequency in the original sentence. We have removed the non-descriptive term “low-frequency” (Lines 330-332).

(12) Considering all of the mistakes and distortions highlighted above, it would be very premature to conclude, based on these results and statements, that altricial avian embryos are not sensitive to sound. This study provides no actual scientific ground to support this conclusion.

(R3. A12) We respectfully disagree with the reviewer’s conclusion.

Our study does not make a general claim that altricial embryos are incapable of perceiving sound. Rather, we evaluate a specific hypothesis: whether zebra finch embryos and hatchlings can detect sound and parental heat whistles.

Our conclusions are based on the convergence of:

(1) Measured low sound pressure levels of heat whistles,

(2) Established adult auditory thresholds (behavioral data),

(3) A large developmental decrease in auditory sensitivity demonstrated by our ABR measurements.

Even under conservative assumptions, these constraints place heat whistles at or below adult detection thresholds and far below expected sensitivity in hatchlings and embryos.

Thus, our conclusion is not based on absence of evidence, but on quantitative constraints that make detection unlikely under biologically realistic conditions.

Recommendations for the authors:

Joint recommendations:

In response to the joint recommendations, we have:

- Expanded methodological transparency (temperature, electrode setup, stimulus parameters),

- Added new data (Fig S3) and figures (Fig 4 and 5),

- Clarified ABR limitations and interpretation,

- Strengthened the separation between measured results and interpretation,

- Reframed conclusions to avoid overstatement.

These revisions leave the central two conclusions unchanged: 1) zebra finch hatchlings and embryos are functionally deaf, and 2) under biologically realistic conditions, heat whistles are unlikely to be detectable by zebra finch hatchlings or embryos.

(A) Reviewers 1 and 2:

Much of the reviewer discourse revolved around providing clarifications of methodology for measuring the ABR and caveats for interpretation. There was near consensus with reviewers 1 and 2 on issues related to the ABR, which should be addressed.

We appreciate the reviewers’ consensus that the main conclusions are supported, while requesting clarification of methodological details and interpretation of ABR measurements.

(1) Please address all of the issues raised by reviewers 1 and 2 above.

(A1) All points raised by Reviewers 1 and 2 have been addressed in detail in our point-by-point rebuttal below. In addition, we have revised the manuscript to improve clarity, added new figures (Fig. 4, 5), and substantially expanded the Discussion with eight new paragraphs to better contextualize our findings.

(2) Please also

- clarify all aspects of experimental details of the ABR that were missing, including temperature control (estimate body and ambient temperatures during ABR recordings,

- please address the possibility of hypothermia of hatchlings that could have reduced ABR responses,

- and potential local head cooling due to surgical exposure and its likely effect on highfrequency response depression).

(A2) In our revision, we have expanded the Methods section (L479-485) and added the following new data:

Body and ambient temperature/hypothermia

We have now included the body temperatures during ABR recordings in new table S6. These data show that:

(1) Body temperature was stable throughout recordings,

(2) Temperatures were within the physiological range,

(3) Conditions were consistent across all age groups.

Importantly, even the youngest hatchlings maintained stable temperatures and showed no indication of hypothermia. Therefore, differences in ABR responses cannot be attributed to temperature effects.

Potential cooling due to surgical exposure

This concern does not apply to our experiments. We used subdermal needle electrodes, which do not require surgical exposure. Therefore, no local cooling of the head occurred, and no tissue exposure could affect high-frequency sensitivity. We have added a clarifying sentence in the Methods section to explicitly state this (Line 501-503).

(B) Reviewer 3 also had additional requests for clarification that should also be addressed:

(3.1) Stimulus duration too long: The study used 25 ms tone bursts instead of the standard {less than or equal to} 5 ms "pips." Could this prevent reliable ABR detection, especially at high frequencies?

(A3.1) We agree that stimulus duration affects ABR characteristics and now clarify our rationale in the manuscript.

- The 25 ms tone bursts were deliberately chosen to ensure sufficient cycle representation at low frequencies (down to 250 Hz) and to avoid frequency splatter.

- Using a constant duration across frequencies ensures comparable stimulus energy.

Importantly:

- The 25 ms data yield audiogram shapes consistent with both click responses (Fig 2C) and published behavioral data (new Fig 5).

- To address potential high-frequency limitations, we included a dataset using 5 ms tone pips, which produced thresholds consistent with published ABR studies (new Fig 5).

Thus, both stimulus types support the same conclusion: a gradual maturation of hearing sensitivity from low to high frequencies. We have expanded the Discussion with three paragraphs to clarify these methodological trade-offs (Lines 387-422).

(3.2) Were 400 sweeps enough averaging? Might a signal appear at 1000 or more?

(A3.2) Signal-to-noise ratio improves with the square root of the number of averages. Increasing from 400 to 1000 sweeps would therefore reduce thresholds by at most ~4 dB. This magnitude is small relative to the >54 dB developmental differences observed, and the large gap between signal levels and detection thresholds. Thus, increasing sweep number would not alter the conclusions. We now clarify this explicitly in the Methods (L530).

(3.3) Was the repetition rate too high? How does the stimulus presentation affect the ABR? Might a signal have emerged with 1-4 per second?

(A3.3) We have clarified stimulus presentation rates in the revised manuscript:

- Clicks were presented at 25 Hz. Control measurements (now included as Supplementary Fig. S3) show no effect of this rate on ABR amplitude or threshold.

- Tone bursts and pips were presented at ~3 Hz, consistent with commonly used rates that avoid neural adaptation. We apologize for leaving this out in our original submission.

We now explicitly describe these parameters and their rationale in the Methods (Lines 543-552).

(3.4) If possible, provide an estimate of the effective bandwidth of the tone pips and compare it with the bandwidth of the parental heat-whistles.

(A3.4) We agree that stimulus bandwidth differs between tone pips and heat whistles, and that broader signals may stimulate multiple auditory filters. Shorter stimuli (e.g., 5 ms pips) have broader bandwidth and may stimulate multiple filters—particularly at low frequencies—potentially lowering thresholds, whereas longer stimuli (25 ms bursts) are more frequency-specific and may yield higher thresholds. At higher frequencies (including the heat whistle range), this effect is expected to be smaller.

However, quantitative correction is currently not possible due to a lack of species-specific data on auditory tuning curves in zebra finches. The only available avian data (budgerigar; Saunders et al., 1979) suggest auditory filter bandwidths (Q10 dB, i.e., the bandwidth 10 dB below the peak divided by peak frequency) of ~1.4 at low frequencies and ~1000 Hz at higher frequencies, but how multifilter stimulation affects thresholds is unknown and likely species- and frequency-dependent.

Given these uncertainties, direct comparison between tone stimuli and heat whistles requires strong assumptions. We therefore suggest that future studies should measure responses to natural heat whistles directly.

(3.5) Egg Vibration Experiment. Address the possibility that if a parent were physically lying on top of an egg and generated a heat call, parental body vibration could significantly communicate some perceptual vibrotactile signal to the egg. Reviewer 3 raised the possibility that the experiments in this paper tested the extent to which an auditory input can vibrate the egg - what if a vocalizing bird was on the egg?

(A3.5) We agree that embryos may receive multiple types of sensory input from parents, including direct mechanical cues.

However, our experiment specifically tests the hypothesis proposed in prior work: that airborne sound (heat whistles) induces egg vibrations sufficient for perception. Our findings show that airborne sound-induced vibrations are orders of magnitude below known vibrotactile sensitivity thresholds.

Regarding parental contact:

- Heat whistles are produced by an aerodynamic whistle mechanism, not tissue vibration (Anttonen et al., Curr Biol 2025), meaning most respiratory energy is radiated as sound rather than dissipated as heat/vibration in the body.

- A parent sitting on the egg would attenuate airborne sound transmission, not amplify it.

We now clarify in the Discussion that other cues (e.g., respiration, direct contact, temperature) may exist and need to be included in new experiments (Lines 349-352). Even so, these are distinct mechanisms and were not the hypothesis tested in prior playback studies.

In our paper, we will not add a detailed discussion of these prior papers as this is outside the scope of this paper. Instead, we added a paragraph what would be a constructive way forward (Lines 349-355). Hopefully somebody in the community will have the good fortune to secure research funding to continue this benchmarking work.

(4) Finally, all reviewers agreed that some more context on the ABR and its relationship to functional hearing could be provided, with less direct focus on the heat-call experiments.

(A4) We agree and in the revision discussion have compiled all published ABR and behavioral audiograms (Fig. 5) and added new paragraphs on functional hearing (Lines 261-269), and ABR vs behavioural audiograms (Lines 413-422).

Furthermore, to remove focus on the heat whistles, we have moved all heat-whistle-specific interpretation out of the Results into a single, focused Discussion section (Line 271-355).

The behavioral studies mentioned below show auditory responses (e.g., begging suppression). However, these behaviors are tested between ~5–10 days post-hatch (consistent with our findings), in different species, and do not provide quantitative sensitivity thresholds, nor do they address detectability of low-amplitude, high-frequency signals like heat whistles.

In our revision we added a new discussion paragraph including these behavioral studies (Lines 357-367).

For example, there are ample cases in the literature of altricial birds exhibiting behavioral evidence of auditory sensitivity by reducing begging calls in response to parental alarm calls:

Platzen & Magrath (2004) - Playback of parental alarm calls nearly abolished nestling non-begging calls and reduced begging in scrubwrens. Proc. R. Soc. B 271:1271-1276.

Different species: scrubwrens. Playback age: 5-, 8- and 11-DPH nestlings.

Magrath, Haff, Horn & Leonard (2006) - Review and experiments on the developmental shift to silence/freeze after aerial alarm calls as chicks become fledglings; documents nestling quieting to alarms. Proc. R. Soc. B 273:2335-2341.

Different species: scrubwrens. Playback age: 7-9 DPH nestlings, and 2- 4 days after fledging.

Magrath, Pitcher & Dalziell (2007) - Nestlings respond to the sound of a predator's footsteps and parental food/alarm calls; includes begging suppression following predator sounds. Anim. Behav. 74:1117-1129.

Different species: scrubwrens. Playback age: 8 DPH nestlings.

Haff & Magrath (2012) - Nestlings suppress calling after heterospecific alarm calls (when acoustically similar to conspecific alarms), indicating generalized auditory danger recognition. Anim. Behav. 84:e.g., 495-505 (article).

Different species: scrubwrens. Playback age: 5-6 and 10-11DPH nestlings. They show that 10-11 days old suppress calling while 5-6 days old do not.

Barati & McDonald (2017) - Noisy miner nestlings suppress begging after conspecific alarm calls and some heterospecific cues; stronger/longer suppression for terrestrial-predator alarms. Sci. Rep. 7:9563.

Different species: Noisy miner (Manorina melanocephala ). Playback age: 14 DPH. Nestlings started to vocalise at 5 DPH.

Suzuki (2011) - In Paridae, parental alarm calls encode predator type; prior work (cited within) shows young of altricial species suppress vocalizations to alarms. Curr. Biol. 21:15-20.

Different species: great tits. Playback age: 17 DPH.

Can you please contextualize the present results about the timing of auditory development with the above body of work with respect to the timing of alarm call-induced begging call suppression?

In our revision we have added a new paragraph in the discussion on these papers (Lines 357-367), and highlight the need for comparative work on hearing development in different species (Lines 352-355 and Lines 405-411).

(5) Strictly speaking, a flat ABR does not equal deafness - at the extreme, an average of 10,000 trials may pull out a minuscule signal. Thus, the more rigorous path would be, in the results section, to ensure that statements summarize the data as they are, representing an absence of a neural signal.

Save the interpretation of what this may mean for the discussion, and provide alongside this interpretation the necessary caveats related to temperature, rendition rate, averaging, etc.

Clarify the conditions where a flat ABR demonstrates or fails to demonstrate immature deafness.

Expand clarification for how the known 20-40 dB difference between ABR and behavioral thresholds can exist if a flat ABR can be interpreted as deafness.

Consider refraining from concluding deafness from a flat ABR. Discuss that behavioral, single-unit, or alternative physiological assays might detect responses below the ABR threshold. If such cases exist, cite.

(A5) We thought about this considerably before starting our measurements. What constitutes the absence of a signal? Even with intracellular recordings of all but one of the auditory neurons, the last one could still contain a signal and theoretically transmit information to the nervous system. We agree with the reviewers that absence of an ABR response should not be equated with absolute deafness. We have revised the manuscript accordingly and removed all statements implying “deafness” from the Results. The Results now strictly report presence or absence of detectable ABR responses.

However, in both clinical and comparative contexts, absence of ABR responses at high SPLs (e.g., 90–95 dB) is widely interpreted as functionally non-responsive hearing. The developmental shift we observe (>54 dB) is far larger than typical ABR–behavioral offsets (20–40 dB). In the Discussion, we have added a new paragraph arguing that we think that the term functional deafness is reasonable here (Lines 261-269).